Application of isovaleryl spiramycin compound or composition thereof in preparation of medicament for treating immune disorder

文档序号：176869 发布日期：2021-11-02 浏览：18次中文

阅读说明：本技术 异戊酰螺旋霉素类化合物或其组合物在制备治疗免疫失调的药物中的应用 (Application of isovaleryl spiramycin compound or composition thereof in preparation of medicament for treating immune disorder ) 是由姜恩鸿夏明钰王恒赵小峰于 2021-04-29 设计创作，主要内容包括：本发明属于药物应用领域,具体地说,本发明提供了异戊酰螺旋霉素类化合物或其组合物在制备治疗免疫失调相关疾病的药物中的应用。异戊酰螺旋霉素化合物选自异戊酰螺旋霉素Ⅰ或其衍生物、异戊酰螺旋霉素Ⅱ或其衍生物、异戊酰螺旋霉素Ⅲ或其衍生物；异戊酰螺旋霉素组合物选自异戊酰螺旋霉素Ⅰ或其衍生物、异戊酰螺旋霉素Ⅱ或其衍生物、异戊酰螺旋霉素Ⅲ或其衍生物中两种或两种以上的组合,或者可利霉素。本发明的异戊酰螺旋霉素化合物或其组合物可以作为免疫调节剂,改善机体的免疫能力,对多种免疫失调相关的疾病、哮喘、炎症具有疗效,具有社会意义。(The invention belongs to the field of medicine application, and particularly provides an application of isovaleryl spiramycin compounds or compositions thereof in preparation of medicines for treating immune disorder-related diseases. The isovaleryl spiramycin compound is selected from isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof; the isovaleryl spiramycin composition is selected from isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a combination of two or more of the isovaleryl spiramycin II or the derivative thereof, or colimycin. The isovaleryl spiramycin compound or the composition thereof can be used as an immunomodulator to improve the immunity of organisms, has curative effects on various diseases related to immune disorder, asthma and inflammation, and has social significance.)

1. Application of isovaleryl spiramycin compounds or compositions thereof in preparing medicaments for treating diseases related to immune dysfunction.

2. The use according to claim 1, wherein the isovalerylspiramycin compound is selected from isovalerylspiramycin I or a derivative thereof, isovalerylspiramycin II or a derivative thereof, isovalerylspiramycin III or a derivative thereof; the isovaleryl spiramycin composition is selected from isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a combination of two or more of the isovaleryl spiramycin II or the derivative thereof, or colimycin.

3. Use according to claim 1 or 2, characterized in that the isovalerylspiramycin-like compound or a composition thereof increases the activity of at least one immune cell against a pathogen, a cancer cell, an abnormal or mutated cell; and/or promoting the production of immunologically active ingredients;

preferably, the isovaleryl spiramycin compound or combination thereof increases phagocytosis by macrophages;

preferably, the isovalerylspiramycin compound or composition thereof increases the activity of T lymphocytes against cancer cells.

4. The use according to any one of claims 1 to 3, wherein the isovaleryl spiramycin or combination thereof promotes proliferation and differentiation of immune cells, alters the proportion of subpopulations of immune cells;

preferably, the isovalerylspiramycin compound or composition thereof induces the conversion of macrophages of type M2 to macrophages of type M1; inhibit M2 type macrophage, and increase M1 type macrophage ratio.

5. The use of any one of claims 1 to 4, wherein the isovalerylspiramycin-like compound or composition thereof promotes the migration of immune cells to a site of inflammation;

preferably, the isovaleryl spiramycin compound or combination thereof promotes migration of neutrophils to a site of inflammation.

6. The use according to any one of claims 2 to 5, wherein the immune disorder-related disease comprises a disease caused by hypoimmunity or lack of immunity, a disease caused by immune hyperactivity, a disease caused by immune disorder, a disease caused by immune monitoring dysfunction;

preferably, the diseases caused by hypoimmunity or lack of immunity include common cold, cough, pharyngitis, gastritis, enteritis, pneumonia, bronchitis, pulmonary tuberculosis, rhinitis, otitis media, hepatitis, cancer, mastitis, skin infection, nephritis sexual function diseases, AIDS, upper respiratory tract infection, and single prismatic cell deficiency;

preferably, the diseases caused by immune dysfunction include pollen allergy, allergic dermatitis, measles, asthma, intractable headache, toothache, pinkeye, acne, constipation, hypertension, hyperlipidemia, heart disease, and apoplexy;

preferably, the diseases caused by immune disorders include lupus erythematosus, dermatomyositis, rheumatism and rheumatoid diseases, pernicious anemia, aplastic anemia, myasthenia gravis, psoriasis, ichthyosis, behcet's disease and diabetes;

preferably, diseases resulting from immune monitoring dysfunction include cancer and tumors; cancers and tumors include hematopoietic tumors of the lymphatic system, leukemia, hematopoietic tumors of the myeloid system, tumors of mesenchymal origin, tumors of the central and peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, follicular thyroid cancer and kaposi's sarcoma, bladder cancer, breast cancer, colon cancer, mesothelioma, kidney cancer, liver cancer, lung cancer, head and neck cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, lymphatic cancer, cervical cancer, colon cancer, thyroid cancer, prostate cancer, skin cancer, oral cancer.

7. The isovaleryl spiramycin compound or the composition thereof is applied to the preparation of the medicine for treating asthma;

preferably, the application in preparing the medicament for treating induced asthma;

preferably, the isovalerylspiramycin compound or composition thereof inhibits airway goblet cell proliferation;

preferably, the isovalerylspiramycin-like compound or composition thereof inhibits the expression of Muc5ac mucin.

8. Use of a compound or composition of isovalerylspiramycin for the manufacture of a medicament for the treatment of inflammation;

preferably, the isovaleryl spiramycin compound or the composition thereof inhibits the production of inflammatory cytokine IL-6;

preferably, the isovalerylspiramycin compound or composition thereof inhibits the production of NO in a cell;

preferably, the isovalerylspiramycin-like compound or composition thereof inhibits the production of IL-4 factor and/or IL-1 β factor in a cell.

9. An immunomodulator comprising at least one of isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof, or a calicheamicin.

10. A pharmaceutical composition for treating diseases related to immune dysfunction, which is characterized by comprising at least one of isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof or kelimycin serving as a first pharmaceutical active ingredient and a pharmaceutically acceptable carrier;

preferably, the composition further comprises a second pharmaceutical active ingredient;

preferably, said second pharmaceutically active ingredient is selected from the group consisting of related drugs for the treatment of immune disorders;

preferably, the first pharmaceutically active ingredient and the second pharmaceutically active ingredient are separate formulations or are combined into one formulation.

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of isovaleryl spiramycin compounds or a composition thereof in preparing medicines for treating immune disorder.

Background

Immunoregulation refers to the recognition and elimination of antigenic foreign bodies by the body, and the maintenance of physiological dynamic balance and relatively stable physiological functions. Immunomodulation refers to the interaction between immune cells and immune molecules in the immune system, as well as with other systems such as the neuroendocrine system, such that the immune response maintains the body at the most appropriate level in the most appropriate form. Immunomodulation is achieved by means of the immune system (Immunesystem).

Immunity is a rejection reaction of the body, and is affected by many genes, proteins and cells. Immune disorders cause a number of diseases including allergies (allergies, immune complex type, delayed type immune diseases, cytotoxic type immune diseases), immunodeficiency (AIDS, etc.), and impaired immune system.

Because of the immune deficiency of tumor patients, the chemotherapy and radiotherapy of tumor often further aggravate the immune deficiency, and reduce the anti-tumor and anti-infection immunity of the patients. The tumor immunotherapy using immunomodulators should improve the immune function of patients, prevent damage to the immune system from chemotherapy and radiotherapy, and enhance the efficacy of chemotherapy, radiotherapy and surgical therapies. With the continuous development of immunomodulators and tumor immunotherapy, etc. become the standard forms of tumor therapy.

The colimycin (Carrimycin), also called Bitespiramycin (Bitespiramycin) and Shengmiamycin (Shengjimycin) is a novel antibiotic which is formed by cloning 4 ' -isovaleryl transferase group (4 ' -o-acyl-transferase) of a carbon mycin producing strain into the spiramycin producing strain through transgenic technology by the cooperation of the institute of biotechnology of Chinese medical college and the applicant, directionally acylating 4 ' -OH of the spiramycin and adding isovaleryl side chain to the 4 ' -position, wherein 4 ' -position isovaleryl spiramycin is used as a main component.

The kelimycin is composed of a plurality of spiramycin derivatives, the total content of the main active ingredients isovaleryl spiramycin (I + II + III) is not less than 60%, the total content of acylated spiramycin is not less than 80%, and the kelimycin is an acceptable pharmaceutical composition in pharmacy. The central structure is a 16-membered lactone ring which is connected with a molecule of forosamine, a molecule of mycaminose and a molecule of mycaminose, and the main components of the isovaleryl spiramycin I, II and III are structurally different from spiramycin in that a group connected to the 4' position of the mycaminose is isovaleryl instead of hydroxyl. The structural schematic diagram of the main component of the kelimycin is shown as a formula (1), and does not represent conformation; contains more than ten components. The composition standard of the prior finished product of the colimycin is that the content of isovaleryl spiramycin III is more than or equal to 30 percent, the sum of the proportions of isovaleryl spiramycin I, II and III is more than or equal to 60 percent, the content of total acylated spiramycin is more than or equal to 80 percent, and the sum of other unknown components is less than or equal to 5 percent.

Wherein, when R ═ H, R ═ COCH₂CH(CH₃)₂Is isovaleryl spiramycin I; when R is COCH₃，R′＝COCH₂CH(CH₃)₂Isovaleryl spiramycin II; when R is COCH₂CH₃，R′＝COCH₂CH(CH₃)₂Is isovaleryl spiramycin III.

The colimycin belongs to 16-membered macrolide antibiotics, and has an active group of carboxyl, alkoxy, epoxy, keto and aldehyde group and a pair of conjugated C ═ C, and the molecular weight is about 884-982. The kelimycin is easily soluble in most organic solvents such as esters, acetone, chloroform, alcohols and the like, is slightly soluble in petroleum ether and is insoluble in water; the molecular structure contains two dimethylamino groups, is alkalescent and is easy to dissolve in an acidic aqueous solution; has a "negative solubility" property in which the solubility decreases with increasing temperature.

Preliminary in vivo and in vitro pharmacodynamic tests show that the medicine is not only used for most G⁺The bacteria have good antibacterial activity, and can be used for treating part G^-The bacteria also have a certain effect, each technical index is obviously superior to azithromycin, erythromycin, acetylspiramycin and midecamycin, the antibacterial activity to mycoplasma pneumoniae is strongest, the antibacterial activity to erythromycin drug-resistant bacteria, gonococcus, pneumococcus, staphylococcus aureus, pseudomonas aeruginosa, bacillus influenzae, haemophilus influenzae, bacteroides fragilis, legionella, multirow bacillus and clostridium perfringens is certain, and the antibacterial activity to clinical erythromycin drug-resistant staphylococcus aureus only has little cross-resistance. The colimycin is mainly used for treating gram-positive bacteria infectious diseases, particularly upper respiratory tract infection, and possibly urinary system infection and the like.

At present, no specific report of the use of the kelimycin for regulating immunity exists, and no report of the role of the kelimycin in resisting infection and tumors through an immune regulation mechanism exists.

The present invention has been made in view of this situation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides the application of the isovaleryl spiramycin compound or the composition thereof in preparing the medicine for treating the diseases related to immune disorder, can improve the immunity of an organism, and has important economic and social benefits.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention provides the application of isovaleryl spiramycin compounds or compositions thereof in preparing medicaments for treating diseases related to immune disorder.

The isovaleryl spiramycin compound is selected from isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, and isovaleryl spiramycin III or a derivative thereof; the isovaleryl spiramycin composition is selected from isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a combination of two or more of the isovaleryl spiramycin II or the derivative thereof, or colimycin.

In a further aspect, the isovalerylspiramycin-like compound or composition thereof increases the activity of at least one immune cell against a pathogen, cancer cell, abnormal or mutant cell; and/or promoting the production of immunologically active ingredients.

Immune cells refer to cells involved in or associated with an immune response, including lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, and the like. Immune cells can be classified into various types, and various immune cells play an important role in the human body. Immune cells (immunecells) are commonly referred to as leukocytes, including innate lymphocytes, various phagocytic cells, and lymphocytes that recognize antigens and produce specific immune responses. Lymphocytes are the core of the immune response. According to the difference in the origin, morphological structure, surface marker and immune function of lymphocytes, they can be classified into T cells, B cells and NK cells. T lymphocytes circulate in the thymus along with blood, mature by the action of thymic hormones and the like, while B cells differentiate and mature in bone marrow. When stimulated by antigen, T lymphocyte is transformed into lymphoblast, then transformed into sensitized T lymphocyte to participate in cellular immunity, and the immune function is mainly to resist intracellular infection, tumor cell, variant cell and the like; b lymphocyte is transformed into plasmablast, then transformed into plasma cell, and produces and secretes immunoglobulin (antibody) to participate in humoral immunity, and the function of B lymphocyte is to produce antibody, present antigen and secrete intracellular factor to participate in immune regulation; NK cells spontaneously exert cytotoxic effects independent of antigen stimulation, and have the effect of killing target cells.

Preferably, the isovaleryl spiramycin or combination thereof enhances phagocytosis by macrophages;

preferably, the isovalerylspiramycin-like compound or composition thereof increases the activity of T lymphocytes against cancer cells.

In a further scheme, the isovaleryl spiramycin compound or the composition thereof promotes the proliferation and differentiation of immune cells and changes the proportion of immune cell subgroups;

preferably, the isovaleryl spiramycin compound or the composition thereof induces the conversion of M2 type macrophages to M1 type macrophages; inhibit M2 type macrophage, and increase M1 type macrophage ratio.

In a further scheme, the isovaleryl spiramycin compound or the composition thereof promotes immune cells to migrate to an inflammation site;

preferably, the isovalerylspiramycin-like compound or composition thereof promotes migration of neutrophils to the site of inflammation.

Further, the immune disorder-related diseases include diseases caused by hypoimmunity or lack of immunity, diseases caused by immune hyperactivity, diseases caused by immune disorders, and diseases caused by immune monitoring disorders;

A second object of the present application is to provide the use of isovaleryl spiramycin or a composition thereof in the preparation of a medicament for the treatment of asthma;

preferably, the application in preparing the medicine for treating induced asthma.

Preferably, the isovalerylspiramycin compound or composition thereof inhibits airway goblet cell proliferation;

preferably, the isovalerylspiramycin-like compound or composition thereof inhibits the expression of Muc5ac mucin.

A third object of the present application is to provide the use of isovaleryl spiramycin, or a composition thereof, in the preparation of a medicament for the treatment of inflammation.

Preferably, the isovaleryl spiramycin compound or the composition thereof inhibits the production of inflammatory cytokine IL-6;

preferably, the isovalerylspiramycin compound or composition thereof inhibits the production of NO in a cell;

preferably, the isovalerylspiramycin-like compound or composition thereof inhibits the production of IL-4 factor and/or IL-1 β factor in a cell.

It is a fourth object of the present application to provide an immunomodulator comprising at least one of isovaleryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof, or a calicheamicin.

A fifth object of the present application is to provide a pharmaceutical composition for treating diseases associated with immune disorders, comprising at least one of isovaleryl spiramycin i or a derivative thereof, isovaleryl spiramycin ii or a derivative thereof, isovaleryl spiramycin iii or a derivative thereof, or colimycin as a first pharmaceutically active ingredient, and a pharmaceutically acceptable carrier.

A sixth object of the present application is to provide a pharmaceutical composition for treating diseases associated with immune disorders, comprising at least one of isovaleryl spiramycin i or a derivative thereof, isovaleryl spiramycin ii or a derivative thereof, isovaleryl spiramycin iii or a derivative thereof, or colimycin as a first pharmaceutical active ingredient, and a second pharmaceutical active ingredient selected from drugs associated with immune disorders;

preferably, the first pharmaceutically active ingredient and the second pharmaceutically active ingredient are separate formulations or are combined into one formulation.

The second pharmaceutically active ingredient may be selected from interferon, BCG polysaccharide nucleic acid, thymosin, Brevibacterium, lentinan, pachyman, ganoderan feces, Tremella polysaccharide, Schizophyllum commune polysaccharide, zymosan, interferon, interleukin-2, transfer factor, levamisole, isoprinosine, hydroxynonanpurine: ginsenoside, astragalus polysaccharide, medlar carbendazim, epimedium polysaccharide, total paeony glycoside, certain compound Chinese medicinal preparations and the like, sodium diethyldithiocarbamate, polymyxin, polyinosinic acid, tylosin, carboxyamido-propylpyridine, maleic anhydride vinyl ether and the like.

After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. in the invention, at least one of valeryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof and colimycin has the application of treating diseases related to immune dysregulation, so that the phagocytosis of macrophages is enhanced; enhancing the activity of T lymphocytes against cancer cells; can induce the conversion of M2 type macrophage to M1 type macrophage; inhibiting M2 type macrophage, and increasing M1 type macrophage ratio; can promote migration of immune cells to inflammatory sites.

2. The isovaleryl spiramycin compound or the composition thereof has curative effect on the immunotherapy of tumors, and the trial feedback of more than 30 tumor patients is as follows: relieving inflammation focus, relieving or eliminating pain, eliminating edema, restoring body temperature, and relieving cough and asthma; relates to tumors including brain glioma, pancreatic cancer, colon cancer, non-small cell lung cancer, breast cancer, cervical cancer and the like.

3. In the application, in a classical OVA-induced asthma model, the kelimycin can obviously relieve asthma symptoms of mice, and inhibit the hyperplasia of airway goblet cells and the expression of Muc5ac mucin of the mice. The results suggest that kelimycin has potential in the treatment of asthma; by combining the sterilization advantages of the kelimycin, the kelimycin can possibly obtain better clinical treatment effect on patients with bacterial infection and asthma.

4. In the application, at least one of valeryl spiramycin I or a derivative thereof, isovaleryl spiramycin II or a derivative thereof, isovaleryl spiramycin III or a derivative thereof and colimycin has the function of treating inflammation, and can inhibit the production of inflammatory cytokines IL-6, inhibit the production of NO in cells and inhibit the production of IL-4 factors and/or IL-1 beta factors in cells.

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:

FIG. 1is a control group in the evaluation of the ability of corrigents to phagocytose chicken erythrocytes by macrophages in Experimental example 2;

FIG. 2is a group of rokitamycin in Experimental example 2, which evaluated the ability of rokitamycin to phagocytose chicken erythrocytes by macrophages;

FIG. 3is a group of itraconazole for evaluation of ability of corrigents to phagocytose chicken red blood cells by macrophages in Experimental example 2;

FIG. 4 is a diagram of the flow-through cell clustering of abdominal cells of a mouse in Experimental example 3;

FIG. 5 shows the results of peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for three days at 50mg/kg to construct a peritoneal inflammation model;

FIG. 6 shows the results of the peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for seven days at 50mg/kg to construct an abdominal inflammation model;

FIG. 7 shows the results of peritoneal neutrophil (Gr-1 and CD11b double positive cells) detection in a second batch of C57BL/6 mice, which are continuously gavaged for three days at 50mg/kg to construct a peritoneal inflammation model;

FIG. 8 shows the results of the measurement of the ratio of CD4+ and CD8+ cells in peripheral blood, in a second batch of C57BL/6 mice, which were continuously gavaged for three days at 50mg/kg to construct a celiac inflammation model;

FIG. 9 is a bar graph of the proportion of CD4+/CD3+ and CD8+/CD3+ cells in the peripheral blood of FIG. 8;

FIG. 10 shows the results of the measurement of the ratio of CD3+ cells in peripheral blood, in a second batch of C57BL/6 mice, which were subjected to continuous gavage for three days at a dose of 50mg/kg, and a peritoneal inflammation model was constructed;

FIG. 11 is a bar graph of the proportion of CD3+ cells in the peripheral blood of FIG. 10;

FIG. 12 is a photograph of subcutaneous tumors of mice taken in groups ranging from large to small;

fig. 13 is a scatter plot of tumor weights and statistical tests (compared to control) are performed, P < 0.05.

FIG. 14 shows that the level of TNF- α was measured after the RAW cells were treated with the drug for 1 hour and then induced to differentiate into M1 type cells;

FIG. 15 shows that iNOS levels were detected by first exposing RAW cells to a drug for 1 hour and then inducing differentiation of RAW cells to M1 type;

in FIGS. 14 and 15, NC (RAW cells, without any treatment); PC1(RAW cells plus LPS + INF-. gamma., induced differentiation of RAW cells into M1 type macrophages); keli (RAW cells were first supplemented with colimycin, then LPS + INF- γ); yiqu (RAW cells were first added with itraconazole, then LPS + INF- γ); statistical tests were performed, P <0.05, P <0.01, P < 0.001.

FIG. 16 shows that the drug was added to RAW cells for 1 hour, and then induced to differentiate into M2 type, and the level of Arg-1 was detected; drawing notes: NC (RAW cells, without any treatment); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli (RAW cells plus first colimycin plus then IL-4); yiqu (RAW cells were added itraconazole first, then IL-4); statistical tests were performed, P <0.05, P <0.01, P < 0.001.

FIG. 17 shows that cytokines are added to induce RAW cells to differentiate into M1 type macrophages, and then corresponding drugs are added to detect the expression of TNF- α;

FIG. 18 shows that cytokines are added to induce RAW cells to differentiate into M1-type macrophages, and then corresponding drugs are added to detect iNOS expression;

FIG. 19 shows that cytokines are added to induce RAW cells to differentiate into M2-type macrophages, and corresponding drugs are added to detect Arg-1 expression;

in FIGS. 17-19, NC (RAW cells, without any treatment); PC1(RAW cells plus LPS + INF-. gamma., induced differentiation of RAW cells into M1 type macrophages); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli ((LPS + INF- γ added to RAW cells first, inducing RAW cells to differentiate into M1-type macrophages, then calicheamicin), Yiqu (LPS + INF- γ added to RAW cells first, inducing RAW cells to differentiate into M1-type macrophages, then itraconazole), and statistical tests were performed, P <0.05, > P <0.01, > P < 0.001.

FIG. 20 shows the differentiation of RAW cells into M2-type macrophages induced by cytokine, and the detection of TNF- α expression by the corresponding drugs

FIG. 21 shows that cytokines are added to induce RAW cells to differentiate into M2-type macrophages, and then the corresponding drugs are added to detect the expression of iNOS

FIG. 22 shows that the cytokine is added to induce RAW cells to differentiate into M2 type macrophages, and the corresponding drugs are added to detect the expression of Arg-1

In fig. 20-22: NC (RAW cells, without any treatment); PC1(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli ((initial addition of IL-4 to RAW cells, induced differentiation of RAW cells into M2-type macrophages, and then calicheamicin)), Yiqu (initial addition of IL-4 to RAW cells, induced differentiation of RAW cells into M2-type macrophages, and then itraconazole), and statistical tests were performed, P <0.05, P < 0.001.

FIGS. 23 to 24 are schematic diagrams of mouse airway epithelial goblet cells in the placebo group in Experimental example 7;

FIGS. 25 to 26 are schematic diagrams of mouse airway epithelial goblet cells of the treatment group of calicheamicin in Experimental example 7;

FIGS. 27-28 are schematic diagrams of mouse airway epithelial goblet cells of a healthy control group in Experimental example 7;

FIG. 29 is the results of the effect of ISP I and LPS on the viability of BV2 cells in Experimental example 8; wherein A is the action result of ISP I on BV2 cell viability, B is the action result of LPS on BV2 cell viability;

FIG. 30 is a graph showing the effect of ISP I and LPS in Experimental example 8 on the amount of NO produced in BV2 cells.

FIG. 31 shows the effect of ISP I and LPS on IL-6 in BV2 cells in Experimental example 8.

It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1 Isovalerylspiramycin I or II or III or a lincomycin tablet

Specification: 200mg/350mg

Tablet core prescription:

the preparation process comprises the following steps:

preparation of the tablet core: the main medicine and the auxiliary materials respectively pass through 10Sieving with 0 mesh sieve, mixing isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III, microcrystalline cellulose and 1/2 sodium carboxymethyl starch, and adding 5% polyvidone K₃₀Preparing soft material from water solution, granulating with 18 mesh sieve, and drying wet granules at 60 deg.C for 2 hr under ventilation condition; drying, granulating with 18 mesh sieve, adding 1/2 prescription amount of carboxymethyl starch sodium and magnesium stearate, mixing, and tabletting with 11mm diameter shallow concave punch to obtain tablet core containing medicine with weight of 350mg and hardness of 6.5 kg.

Preparing a coating solution: weighing the required opadry II (white), adding the required amount of water into a liquid preparation container, adding the water in several times, reducing the stirring speed after all the water is added, eliminating the vortex, and continuing stirring for 30min to obtain the product.

Preparation of film-coated tablets: and (3) putting the tablet cores into a coating pot, determining coating conditions, coating at the speed of a main engine of 20r/min, the air inlet temperature of 40 ℃, the air outlet temperature of 30 ℃, the spraying pressure of 0.02Mpa and the spraying flow of 1ml/min, and continuously spraying for 1.5h after the constant speed until the surfaces of the tablet cores are smooth and uniform in color and luster, and the tablets are qualified according with the inspection standard of film coating. The weight of the coating is increased by about 5 percent.

Example 2 Isovalerylspiramycin I or II or III (calculated as 10000 tablets)

Prescription:

1000g of raw powder of isovaleryl spiramycin I, isovaleryl spiramycin II or isovaleryl spiramycin III

Low-substituted hydroxypropylcellulose (5%) 92.5g

55.5g sodium carboxymethyl starch (3%)

Magnesium stearate (1%) 18.5g

Total weight of starch-weight of other raw and auxiliary materials

Total weight of 1850g

The preparation process comprises the following steps: weighing a proper amount of starch, diluting to a concentration of 15%, and heating to paste to prepare an adhesive; respectively sieving the main material isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III, the auxiliary material starch, the low-substituted hydroxypropyl cellulose, the sodium carboxymethyl starch and the magnesium stearate with a 100-mesh sieve, and weighing the required main material and the auxiliary material according to the prescription amount; mixing isovaleryl spiramycin I, starch and low-substituted hydroxypropyl cellulose uniformly, pasting starch with the concentration of 15% of starch to prepare a soft material, granulating by using a 14-mesh sieve, drying at 50-60 ℃, controlling the water content to be 3-5%, grading by using the 14-mesh sieve, adding sodium carboxymethyl starch and magnesium stearate, mixing, and determining the content of granules; calculating the weight of the tablets according to the content of the particles, tabletting (phi 9mm shallow concave punch), and detecting the difference of the weight of the tablets; and packaging after the inspection is qualified.

Example 3 Isovalerylspiramycin I or II or III capsules (calculated as 10000 capsules)

Prescription:

1000g of raw powder of isovaleryl spiramycin I, isovaleryl spiramycin II or isovaleryl spiramycin III

Starch 1080-Isovalerylspiramycin I raw powder weight

Medicinal No. 3 capsule 1000 granules

Liquid paraffin 50ml

The preparation process comprises the following steps: respectively weighing the main material isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III and the auxiliary material medicinal starch according to the process formula amount, and filling the materials into a mixer for fully mixing for 1.5 to 2 hours; the data obtained by sampling and detecting the content is basically consistent with theoretical data (the weight of each capsule is about 0.105g), the qualified pharmaceutical No. 3 capsules and the mixed raw materials to be filled are respectively filled into a filling machine for filling according to the operation requirements of a full-automatic capsule machine, the filled capsules are subjected to difference detection (within +/-10 percent and less than 0.3g), the dissolution rate meets the requirements, the capsules meeting the requirements after detection are put into a polishing machine, liquid paraffin is added for polishing for 15-20 minutes, and then the capsules are taken out for finished product packaging box detection.

Example 4 Isovalerylspiramycin I or II or III dry syrups (calculated as 10000 bags)

Prescription:

1250g of isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder

Citric acid (0.5%) 15g

Sucrose gross weight-other raw and auxiliary materials

A total weight of about 5000g

About 1g of pigment (curcumin)

The preparation process comprises the following steps: the method comprises the steps of crushing isovaleryl spiramycin I, isovaleryl spiramycin II or isovaleryl spiramycin III raw powder, citric acid and cane sugar into particles 85% passing 300 meshes and 15% passing 180 meshes by using a high-speed airflow crusher respectively, weighing the crushed fine powder according to the prescription amount, fully mixing for 1-1.5 hours, measuring the content of the fine powder, calculating the filling amount (the theoretical filling amount is 500mg per bag), filling the mixture into a bagging machine, filling aluminum foil paper, subpackaging according to the operation requirements of the bagging machine, wherein the filling amount difference is within +/-5%, and carrying out inspection and external packaging after the packaging is qualified.

Example 5 Isovalerylspiramycin I or II or III granules (calculated as 10000 bags)

Prescription:

1250g of isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder

20000g powdered sugar

Dextrin 9000g

5％PVP-K₃₀Proper amount of

The preparation process comprises the following steps: isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder, sugar powder and dextrin are sieved by a 120-mesh sieve, the isovaleryl spiramycin I, the sugar powder and the dextrin are weighed according to the prescription amount and are uniformly mixed, and the uniformly mixed materials are mixed by 5 percent of PVP-K₃₀Making the mucilage into soft material, granulating with swing type granule, drying at 70 deg.C, grading, inspecting, and packaging.

Example 6 Isovalerylspiramycin I, isovalerylspiramycin II or isovalerylspiramycin III lyophilized powder for injection

Weighing 500mg of isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder, uniformly mixing the isovaleryl spiramycin I or isovaleryl spiramycin II or isovaleryl spiramycin III raw powder with equimolar adipic acid, dissolving the mixture in 5ml of water to obtain a light yellow clear solution, wherein the pH value of the solution is between 4.6 and 5.6. Adding mannitol 40mg as lyophilized proppant, rapidly freezing at low temperature for 9 hr, freeze drying to obtain yellowish loose block, and dissolving in 10ml sterile water before use.

Example 7 lyophilized powder for injection of Colimycin

Weighing 500mg of the colimycin, uniformly mixing the colimycin with equimolar adipic acid, and dissolving the mixture in 5ml of water to obtain a light yellow clear solution, wherein the pH value is between 4.6 and 5.6. Adding mannitol 40mg as lyophilized proppant, rapidly freezing at low temperature for 9 hr, freeze drying to obtain yellowish loose block, and dissolving in 10ml sterile water before use.

Experimental example 1: routine detection of mouse blood after treatment with colimycin

Purpose of the experiment: and evaluating the toxic and side effects of the long-term use of the kelimycin on the mice. The toxic and side effects of the long-term use of the kelimycin on the mice are verified by detecting the conventional parameters of the blood of the experimental mice.

Reagent: normal saline, tween 80, PEG400, and the like; consumable material: EDTA anticoagulant tubes, forceps, etc.

Experimental mice: the strain is Balb/c, the week age is 8-12, the source is: achievement of great achievements, quantity: each group had 2 mice.

The experimental steps are as follows: (1) grouping and treating: a) physiological saline group: same volume as experimental group, p.o.; b) group of kelimycin: 50mg/kg, p.o.; c) group of itraconazole: 50mg/kg, p.o.; d) the administration is continued for five days; (2) the mouse picks eyeballs to take blood, the blood is put into an EDTA anticoagulation tube, and the mixture is gently inverted and mixed, so that no coagulation phenomenon is caused, and the volume is more than 300 ul. (3) Immediately sending samples at normal temperature, and immediately detecting, wherein the detection is completed by a GLP center in a high new area.

And (3) recording an experiment: after the administration of the drug, the drug has no obvious toxic and side effects, and the body weight is not reduced.

The experimental results are as follows: the conventional blood results of normal mice after administration show that the total number of leucocytes, neutrophils, lymphocytes and the like have no obvious change. As shown in table 1.

TABLE 1

And (4) surface note: blood from mice after treatment with colimycin was routinely tested. The detection indexes are marked on the leftmost side in the table, and the Units represent detection index Units; ctrl is a control group, and the group values can be used as normal references; itraconazole group was used as positive reference.

Results and analysis: the fact that the kelimycin has no obvious toxic and side effects on the blood system under the condition of continuous administration for five days is proved.

Experimental example 2: assessment of ability of kelimycin to phagocytose chicken erythrocytes by macrophages

Purpose of the experiment: and (3) detecting whether the kelimycin can enhance the function of normal mouse macrophage, wherein the main detection index is the phagocytosis of the macrophage. Since there is no standard reagent for the research of immunology by the corrigent, according to the report, itraconazole has the effect of promoting macrophage polarization and enhancing macrophage phagocytosis, and therefore, itraconazole is selected as a positive control in the experiment.

Reagent: normal saline, 6% chicken red blood cells, methanol, acetone, Giemsa dye solution and the like; consumable material: 1ml syringe, common glass slide, gauze, petri dish, etc.

Experimental mice: the strain is Balb/c, the week age is 8-12, the source is: achievement of great achievements, quantity: each group had 2 mice.

The experimental steps are as follows: (1) grouping administration: a) physiological saline group: same volume as experimental group, p.o.; b) group of kelimycin: 50mg/kg, p.o.; c) group of itraconazole: 50mg/kg, p.o.; the administration is continued for five days; (2) each mouse was injected with 1ml of chicken red blood cells and the mice were sacrificed after waiting 30 min. (3) Injecting 1ml of normal saline into abdominal cavity, massaging to make it uniformly distributed, and making the mouse lie prostrate for 5 min. (4) The abdominal cavity of the mouse is cut open, the needle head is removed by a 1ml syringe, the abdominal cavity cleaning solution is sucked out and dripped on the glass slides, two drops are dripped on each glass slide, and the equal volume is ensured as much as possible. (5) Placing into a culture dish with wet gauze, transferring to a 37 ℃ incubator, and incubating for 30 min. (6) After incubation, the cells were rinsed in normal saline to remove nonadherent cells (pre-warmed in normal saline), and air dried. (7) Fixing with 1:1 acetone formaldehyde solution (pre-cooling at-20 deg.C). (8) Dyeing with liquid A for 45s, then adding liquid B for 4min, blowing slightly to dye into ripple shape, mixing the two, washing with distilled water, and air drying. (9) Random field of view by microscope, photographed and counted, and calculated the percent phagocytosis (10) percent phagocytosis calculation formula number of phagocytic cells/total number of macrophages × 100%

Results and analysis:

after the mouse ascites cells are stimulated by itraconazole or the colimycin, macrophages with phagocytosis phagocytose chicken erythrocytes (with megakaryocytes) or chicken erythrocytes with more peripheral aggregation. Fig. 1is a control group, fig. 2is a clarithromycin group, and fig. 3is an itraconazole group.

Both the Keli (Keli) and itraconazole (Yiqu) groups had some degree of enhancement in the ability of macrophages to phagocytose chicken red blood cells compared to the placebo group (NC); however, no statistically significant difference was observed between the group of clarithromycin and the group of itraconazole.

Overall, the individual components are not significantly different. The experiment has inevitable subjective intervention of experiment operators, and quantitative judgment is not suitable for small differences; therefore, the macrophage is used for carrying out phagocytosis test on the fluorescent microsphere, and a flow method is used for detection, so that human subjective factors are reduced.

Experimental example 3: macrophage in vitro fluorescent microbead phagocytosis experiment-flow detection

Reagent: fluorescent microbeads, serum-free media, and the like; consumable material: 5ml syringe, 1.5ml EP tube, etc.; the instrument comprises the following steps: BDfortessa flow meter.

Experimental mice: the strain is Balb/c, the week age is 8-12, the source is: nanjing model animal center, quantity: each group had 2 mice.

The experimental steps are as follows: (1) grouping administration: a) same volume as experimental group, p.o.; b) group of kelimycin: 50mg/kg, p.o.; c) group of itraconazole: 50mg/kg, p.o.; the administration is continued for five days; (2) abdomen contracting water cells: the mice were sacrificed, the superficial skin of the abdominal cavity of the mice was carefully cut open without cutting the inner layer, about 4ml of serum-free medium was injected into the inside with a 5ml syringe without pulling out the needle, the abdominal part of the mice was continuously massaged to mix the medium, and the medium was aspirated and repeated for 3 times. (3) And centrifuging to obtain abdominal cavity cells. (4) Taking about 1x 10^6 cells to be re-suspended by 1ml of culture medium, adding 10ul of fluorescent microbeads, uniformly mixing, and placing 1.5ml of EP tube in a 37 ℃ incubator for 40 min. (5) Washing with PBS twice, washing off unphaged microbeads, detecting with a flow machine, and analyzing with large macrophages.

The experimental results are as follows: flow results showed that macrophage phagocytosis was significantly enhanced in one mouse each of the kelimycin group (Keli) and the itraconazole group (Yiqu) compared to the placebo group (NC).

FIG. 4 is a histogram of fluorescence intensity of peritoneal macrophages, a flow cytometric clustering graph of mouse peritoneal cells, and it can be seen that the cell clustering is obvious, because the size of macrophages is bigger than that of other cells. The results suggest that the group of clarithromycin and itraconazole can enhance the phagocytic ability of mouse macrophages in a certain "unknown" state of the mouse. It is necessary to enlarge the sample size for verification and to exclude whether the result is due to accidental factors.

Experimental example 4: detection of influence of kelimycin on neutrophil function

Purpose of the experiment: and detecting whether the kelimycin can enhance the inflammatory chemotactic migration capability of the mouse neutrophil, wherein the method is mainly used for detecting the proportion of the mouse neutrophil in the abdominal cavity in a flow mode by constructing a mouse abdominal cavity inflammation model, and the detection indexes are CD11b and Gr-1. Itraconazole was used as a positive control in this experiment.

Reagent: sterile PBS, fMLP, sterile HBSS, Gr-1-APC flow antibody, CD11b-FITC flow antibody, etc.; consumable material: pipette, rubber band, scissors, forceps, 1ml syringe, 15ml centrifuge tube, flow tube, etc.

Experimental mice: strain: c57BL/6 mice, week old: 8-12, source: achievement of great achievements, quantity: the first three days of dosing 3 mice each per group and 7 days of dosing 4 mice each per group. The second replicate experiment was dosed with 7 mice each for three days.

The experimental steps are as follows:

(1) grouping administration: d) physiological saline group: same volume as experimental group, p.o.; e) group of kelimycin: 50mg/kg, p.o.; f) group of itraconazole: 50mg/kg, p.o.; continuous administration for three days and seven days; (2) and (3) preparing fMLP, namely preparing 100 nMLP, diluting with PBS, using the solution as the solution is prepared, and placing the solution on ice after the solution is prepared. (3) And (3) intraperitoneal injection: each mouse was intraperitoneally injected with 100uL100nM of precooled fMLP. (4) Abdomen contracting water cells: after 4 hours of injection, the mice were sacrificed, the four limbs were fixed, the abdominal skin was cut open, a small opening in the peritoneum was cut open, the peritoneum was fixed with a rubber band and a clip, the precooled HBSS were sucked with a straw to repeatedly wash the abdominal cavity, the motion was gentle, approximately 8-10 ml of ascites was collected, and they were placed on ice. (5) Centrifuge at 1000rpm for 5min and carefully discard the supernatant. (6) If red blood cells are present, they are resuspended in 1ml of red blood cell lysate and lysed on ice for 3-5 min. (7) Centrifuge at 1000rpm for 5min, discard the supernatant, and wash once with 3ml PBS. (8) Add 500uLPBS to resuspend. (9) Taking 10^6 cells, incubating the flow antibody at room temperature in the dark: CD11b-FITC and Gr-1-APC. (10) The cells were washed once with PBS, resuspended, placed on ice, protected from light, and tested on the machine.

The experimental results are as follows: the results of evaluating the neutrophil migration ability of the mouse celiac inflammation model by the kelimycin are shown in fig. 5-7. FIGS. 5-7 show the flow measurement of the ratio of neutrophils (Gr-1 and CD11b double positive cells) in the mouse abdominal inflammation model with different administration times and different batches. FIG. 5 shows the results of peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for three days at 50mg/kg to construct a peritoneal inflammation model; FIG. 6 shows the results of the peritoneal neutrophilic granulocytes (Gr-1 and CD11b double positive cells) detection in a first batch of C57BL/6 mice which are continuously gazed for seven days at 50mg/kg to construct an abdominal inflammation model; FIG. 7 shows the results of peritoneal neutrophil (Gr-1 and CD11b double positive cells) detection in a second batch of C57BL/6 mice, which were continuously gavaged for three days at 50mg/kg to construct a peritoneal inflammation model.

FIGS. 8-11 show the detection of T lymphocytes in peripheral blood after three days after the administration of the drug to construct a mouse abdominal inflammation model, and the detection of the proportion of CD3+, CD4+ and CD8+ cells by flow cytometry.

The kelimycin and the itraconazole can remarkably promote the migration of the neutrophils to an inflammation part in a mouse body, and the result in a specific mouse body is particularly obvious; three days compared to seven days, no further enhancement of the effect was found with the continuous administration of seven days. The kelimycin and itraconazole can remarkably promote the increase of total T cells (CD3 positive cells) in mice, wherein the CD4 and CD8 positive cells are increased, but the itraconazole performs better.

Experimental example 5: immunomodulatory testing of colimycin in tumor conditions

Purpose of the experiment: whether the kelimycin has the immunoregulation function under the tumor condition is discussed. As people research on immunity finds that malignant melanoma is a tumor with better immunogenicity, an anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) antibody and an anti-PD-1 antibody are developed by researching a molecular mechanism of T cell activation and how to mobilize an immune system to resist the tumor. Therefore, we tried to investigate whether or not the kelimycin has an immunomodulatory function under tumor conditions by establishing a B16F10 mouse malignant melanoma subcutaneous tumor model.

Experimental reagent consumables:

reagent: B16F10 melanoma cells, 1640 medium, FBS; consumable material: cell culture related consumables

Experimental mice: line C57BL/6, week old 8-12, source: nanjing model animal center, quantity: each group had 6 mice

The experimental steps are as follows: (1) culturing B16F10 melanoma cells in vitro, approximately up to 10cm in dish 5, and harvesting cells in log phase; (2) the cells were washed 3 times with PBS and adjusted to 1X 10 cell concentration⁷Per ml; (3) the right shoulder of the mice was inoculated subcutaneously, 100ul per mouse, i.e., 1X 10⁶(ii) individual cells; (4) mice were observed daily and tumor size was measured, approximately at 5 days, with the average tumor size of the mice reaching around 100, and the group dosing was started and the first data was recorded. The grouping scheme is as follows: a) physiological saline group: same volume as experimental group, p.o.; b) group of kelimycin: 50mg/kg, p.o.; c) group of itraconazole: 50mg/kg, p.o.; d) RJ-5 group: 30mg/kg, p.o. (RJ-5 is an immunomodulatory drug synthesized by the laboratory, and is used herein as a distinction fromPositive control of itraconazole); (5) mice were dosed daily and tumor size and mouse body weight were recorded every other day, and tumor volume was calculated as: v (mm3) is 0.5 × length (mm) × width (mm) 2; (6) the mice were sacrificed the last day, the tumors were stripped, weighed, photographed, and the mice were left with 4% paraformaldehyde for pathology. (7) Since the mice in the calicheamicin group were in worse condition than the other groups and were close to dying on the last day, no relevant immunization data were received, only subcutaneous tumors.

And (3) recording an experiment:

one of the groups administered with the clarithromycin on day 4 died, one of the control group administered on day 6 died, 2 of the groups administered with the clarithromycin on day 9 died (subcutaneous tumors of the mice were retained immediately after the death of the mice), and the remaining 3 of them were also poor (close to the state of dying). (itraconazole group had a small tumor, suspected of being drug-independent, that the tumor was not long, and was rejected at statistical time RJ-5 group tumors were uniform in size overall and one was randomly rejected at statistical time.)

The experimental results are as follows:

the kelimycin, itraconazole and RJ-5 are considered to have the effect of inhibiting the growth of subcutaneous tumors of B16. On the indexes of tumor size, tumor weight, tumor inhibition rate and the like, the kelimycin is superior to itraconazole and RJ-5. The results are shown in tables 2 and 3.

TABLE 2

Tumor weight (g)	Control	Kelimycin	Itraconazole	RJ-5
					1	3.03	3.29	1.8	2.49
2	4.66	1.73	3.17	2.27
					3	2.32	2.24	2.71	3.75
4	3.8	1.05	1.17	1.9
					5	3.65	1.88	2.37	3.22
Mean value of	3.49	2.04	2.25	2.73
					Tumor inhibition Rate (%)	0	41.6	35.7	21.9

And (4) surface note: tumor weight comparison in mice. Mice were sacrificed the last day to strip subcutaneous tumors, weights were taken, and the average was calculated.

The tumor inhibition rate is calculated by the formula: the average tumor weight (g) of the administration groups/the average tumor weight (g) of the administration groups multiplied by 100 percent

TABLE 3

Body weight (g)	Control	Kelimycin	Itraconazole	RJ-5
					1	25.7	18	19.2	21
2	25.8	17.3	20.3	20.6
					3	19	20.7	18.6	18.4
4	24.7	16.3	14.9	20.1
					5	22	18.4	22.1	25.2
			20.5	23.2
					Mean value of	23.4	18.1	19.3	21.4

And (4) surface note: body weights of mice were recorded on the last day. The weight of each mouse was taken as the total body weight of the tumor containing mice before tumor detachment after sacrifice.

Results and analysis: the kelimycin is superior to itraconazole and RJ-5 in the indexes of tumor size, tumor weight, tumor inhibition rate and the like; its tumor-inhibiting mechanism in vivo needs further study.

Experimental example 6: effect of Colimycin on macrophage differentiation

Experimental background and purpose: macrophages can be divided into two main categories: classically activated macrophages (M1) are characterized by increased expression of the major histocompatibility complex MHC class ii, increased Nitric Oxide (NO), increased levels of reactive oxygen species and proinflammatory cytokines such as Tumor Necrosis Factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6). Another class is alternatively activated macrophages (M2), also known as selectively activated macrophages, which are a class of macrophages with immunosuppressive activity that increase interleukin-4 (IL-4) levels and increase the expression of interleukin-10 (IL-10) and Arginase (Arg) under multiple stimuli, resulting in increased cell proliferation and collagen production. Polarization of M1 cells has a protective effect on human health when encountered with infection and cancer. Therefore, the purpose of this experiment was to examine whether or not the differentiation of macrophages was affected by colimycin, and further to investigate the potential immunomodulatory effects of colimycin.

Experimental reagent consumables: reagent: RAW246.7 cell line, 1640 culture medium, FBS, RNA extraction kit, reverse transcription kit, SYBR fluorescence quantitative kit. Cytokines: IL-4, INF- γ, LPS; consumable material: cell culture related consumables.

The experimental steps are as follows:

the first scheme is as follows: whether the drug would promote or inhibit the polarization process of RAW246.7 cells was investigated.

(1) The kelimycin and itraconazole were stored in DMSO at 10mM concentration stock. (2) Culturing RAW246.7 cell line in vitro, collecting cells growing in logarithmic phase, spreading in 6-well plate according to density of 1 × 10^6 cells per well, and treating cells with 20uM of colimycin and 20uM of itraconazole without adding drugs. (3) After 1h of action, each treatment was subdivided into 2 groups, one group was supplemented with LPS (200ng/ml) and IFN-. gamma. (20ng/ml), the other group was supplemented with IL-4(20ng/ml), and cells were harvested after 12h of culture. (4) Extracting total RNA of cells, carrying out reverse transcription to obtain cDNA, and detecting the RNA level of a corresponding index.

Scheme II: to investigate whether the drug has an effect on already polarized cells.

(1) The kelimycin and itraconazole were stored in DMSO at 10mM concentration stock. (2) The RAW246.7 cell line was cultured in vitro, plated in the same manner, and the cells were treated with the corresponding cytokines (LPS + IFN-. gamma.or IL-4) for 12 h. (3) After 12h, cells that had been polarized to M1 or M2 were added with either colimycin (20uM) or itraconazole (20uM) and allowed to react for another 12 h. (4) Collecting cells, extracting total RNA of the cells, carrying out reverse transcription to obtain cDNA, and detecting the RNA level of a corresponding index.

The experimental results are as follows:

the first scheme is as follows: whether or not Cochinomycin promotes or inhibits differentiation of RAW246.7 cells was investigated

FIG. 14RAW cells were treated with drug for 1 hour, and then induced to differentiate into M1 type cells, and the level of TNF-. alpha.was measured. Drawing notes: NC (RAW cells, without any treatment); PC1(RAW cells plus LPS + INF-. gamma., induced differentiation of RAW cells into M1 type macrophages); keli (RAW cells were first supplemented with colimycin, then LPS + INF- γ); yiqu (RAW cells were first added with itraconazole, then LPS + INF- γ); statistical tests were performed, P <0.05, P <0.01, P < 0.001.

FIG. 15 shows that iNOS levels were detected by first exposing RAW cells to a drug for 1 hour and then inducing differentiation of RAW cells to M1 type;

Analysis of a result of the protocol:

the kelimycin can increase the expression of TNF-alpha and iNos and inhibit the expression of Arg-1. Suggesting that the kelimycin may promote the differentiation and function of M1 type macrophage.

Scheme II: A. to investigate whether the kelimycin has the transdifferentiation effect on differentiated macrophage

FIG. 17 shows the differentiation of RAW cells into M1-type macrophages induced by cytokine, and the detection of TNF-alpha expression by the corresponding drugs

FIG. 18 shows that cytokines are added to induce RAW cells to differentiate into M1-type macrophages, and then corresponding drugs are added to detect iNOS expression

FIG. 19 shows that the cytokine is added to induce RAW cells to differentiate into M2 type macrophages, and the corresponding drugs are added to detect the expression of Arg-1

In FIGS. 17-19, NC (RAW cells, without any treatment); PC1(RAW cells plus LPS + INF-. gamma., induced differentiation of RAW cells into M1 type macrophages); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli ((LPS + INF- γ added to RAW cells first, inducing RAW cells to differentiate into M1-type macrophages, then calicheamicin), Yiqu (LPS + INF- γ added to RAW cells first, inducing RAW cells to differentiate into M1-type macrophages, then itraconazole), and statistical tests were performed, P <0.05, > P <0.01, > P < 0.001.

The experimental results are as follows: consistent with the results obtained on the RAW246.7 cell line, the expression of TNF- α and iNOS induced by LPS + INF- γ was not further enhanced by colimycin, but was able to suppress the expression level of Arg-1 in M1-type macrophages, suggesting that the potential role of colimycin in enhancing the function of M1-type macrophages is.

Scheme II: b to investigate whether the kelimycin has transdifferentiation effect on differentiated macrophage

FIG. 20 shows the differentiation of RAW cells into M2-type macrophages induced by cytokine, and the detection of TNF- α expression by the corresponding drugs

FIG. 21 shows that cytokines are added to induce RAW cells to differentiate into M2-type macrophages, and then the corresponding drugs are added to detect the expression of iNOS

FIG. 22 shows that the cytokine is added to induce RAW cells to differentiate into M2 type macrophages, and the corresponding drugs are added to detect the expression of Arg-1

In legends 20-22: NC (RAW cells, without any treatment); PC1(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); PC2(RAW cells plus IL-4, induced differentiation of RAW cells into M2 type macrophages); keli ((initial addition of IL-4 to RAW cells, induced differentiation of RAW cells into M2-type macrophages, and then calicheamicin)), Yiqu (initial addition of IL-4 to RAW cells, induced differentiation of RAW cells into M2-type macrophages, and then itraconazole), and statistical tests were performed, P <0.05, P < 0.001.

The experimental results are as follows: the kelimycin can obviously improve the expression level of TNF-alpha and iNos in M2 type macrophages, is obviously stronger than the expression level of TNF-alpha and iNos induced by LPS + INF-gamma, is better than itraconazole, and can obviously inhibit the expression level of Arg-1 in M2 type macrophages.

Initial conclusion of RAW cell experimental results:

and (4) combining the results to prompt: 1) the kelimycin has potential of inhibiting M2 type macrophages and improving the functions of M1 type macrophages; 2) the kelimycin has extremely strong function of inducing the conversion of M2 type macrophages to M1 type macrophages. Previous studies have shown that M2-type macrophages contribute to inflammation and tumorigenesis, with positive correlation between tumor immune escape, especially multiple myeloma. Therefore, the significance of the role of the kelimycin in the treatment of the tumor in which M2 type macrophages are involved is worthy of further research and exploration.

Experimental example 7 exploration on the effect of kelimycin on OVA-induced asthma disease in mice

Mice: in the experiment, 15 mice of Balb/c of the Softsto SPF level, which are 6-8 weeks old and 20-28g female mice, were purchased, and 10 mice of the 10 mice were used to induce asthma: 5 were used as placebo, 5 were treated with colimycin and 5 were used as healthy controls. Each group had 5 mice.

Reagent: preparing a sensitizing intraperitoneal injection (15-20 g/mouse): 100g/100ul egg white protein solution was added with 100ul Alum adjuvant for injection. Asthma-inducing aerosolized liquid: 5% egg white protein in physiological saline solution.

And (3) recording an experiment: continuous sensitization from 2019, 9, 12 months: the mice in the model group were injected with 200 ul/mouse of sensitizing injection into the abdominal cavity at d1, d7, and d14, and the healthy control group was injected with an equal amount of PBS solution. Induction of asthma: onset of induction of the disease starts from 10 months and 4 days in 2019: the mice were orally administered with the kelimycin (50mg/kg) 0.5h before nebulization, continuously induced at d21-d27, the mice of the experimental group were nebulized with the inducing solution by nebulization, and the mice of the control group were administered with the same amount of nebulized normal saline once a day for 30 mins. The last nebulization was given in 2019, 10, 11 days. Materialization of mice in 2019, 10 months and 4 days (induction of asthma attack): in the atomization process, the symptoms of the mice in the treatment group of the kelimycin, such as itch on the head and face, piloerection, tachypnea, nodding breathing, dysphoria, abdominal muscle twitch and the like, are obviously better than those of the mice in the placebo group. The healthy control group mice had no symptoms described above. Atomizing in 2019, 10 and 11 days, and after 24h, namely, in 2019, 10 and 12 days, cervical dislocation and killing the old, taking the lung of the mouse, and carrying out alveolar lavage: after the neck of the mouse is dislocated and dies, the mouth is fixed by a rubber band, the mouse is cut from the abdomen and is cut upwards to the point of the sternum (the sternum can be completely taken out to expose the heart and the lung without damaging the heart and the lung), the esophagus and the blood vessel below the lung are cut off and can be completely cut off, the joint of the heart and the left lung is clamped by a hemostatic clamp, the skin and the muscle of the neck of the mouse are cut off to expose the bronchus, the upper part of the bronchus is clamped by the hemostatic clamp, the mouse is injected by 1-3ml PBS, and then the mouse is sucked back after waiting for several seconds, and the recovery rate reaches 50% -90%; then the connecting part of the right lung and the heart is clamped by a hemostatic clamp, polyformaldehyde is injected from the trachea through an indwelling needle to fix the left lung, and then the left lung is cut off and put into a tube for storage. Preparing lung paraffin slices: after alveolar lavage, the left lung was removed, fixed with 10% formaldehyde, embedded in paraffin, sectioned and then stained with hematoxylin-eosin to observe inflammatory cell infiltration and mucus plug formation, and stained with iodic acid-Schiff to observe goblet cell formation.

The experimental results are as follows: in the atomization process, the symptoms of the mice in the treatment group of the kelimycin, such as itch on the head and face, piloerection, tachypnea, nodding breathing, dysphoria, abdominal muscle twitch and the like, are obviously better than those of the mice in the placebo group. The healthy control group mice had no symptoms described above. The generation of H & E staining and iodic acid-schiff staining (observation of goblet cells, asthma-responsive cells) of the lungs of the experimental mice. Airway epithelial goblet cell proliferation and mucus high reserve significantly increased in mice of the placebo group (figures 23 and 24); airway epithelial goblet cell proliferation was not evident in mice treated with colimycin (figures 25 and 26); there was no significant difference from the healthy control mice (fig. 27 and 28).

And (4) conclusion: in a classical OVA-induced asthma model, the kelimycin can remarkably relieve asthma symptoms of mice, and inhibit airway goblet cell hyperplasia and Muc5ac mucin expression of the mice. The results suggest that kelimycin has potential in the treatment of asthma; by combining the sterilization advantages of the kelimycin, the kelimycin can possibly obtain better clinical treatment effect on patients with bacterial infection and asthma.

Drawing notes: the alleviative effect of the kelimycin on OVA-induced asthma diseases. HE staining mainly observes the accumulation and infiltration of inflammation (lymphocytes) in the bronchi and around the blood vessels. And thickening of the vessel wall. PAS staining was primarily observed for ring-forming hyperplastic goblet cells in the bronchi (PAS staining is reddish). Note: goblet cells are the main functions in the tracheal tissue to secrete mucin and mucus, which are secreted in large quantities during asthma attacks to cause airway obstruction, resulting in dyspnea. FIGS. 23-24 are asthma model groups; FIGS. 25-26 are asthma model kelimycin treatment groups; FIGS. 27-28 show that the goblet cells (dark purple indicated by arrows, i.e., hyperplastic goblet cells) proliferated significantly in the normal control and model groups, while the administration and control groups showed little or no goblet cells. Infiltration of lymphocytes and inflammatory cells (remission relative to the model group) the control group had little infiltration and the thickening of the vessel wall was not significant.

Combining the above results, the immunoregulatory function of the kelimycin is summarized as follows:

1) the kelimycin has the effects of inhibiting M2 type macrophages and improving the functions of M1 type macrophages;

2) the kelimycin has extremely strong function of inducing the conversion of M2 type macrophages to M1 type macrophages. Previous studies have shown that M2-type macrophages contribute to inflammation and tumorigenesis, with positive correlation between tumor immune escape, especially multiple myeloma. Therefore, the significance of the role of the kelimycin in the treatment of the tumor in which M2 type macrophages are involved is worthy of further research and exploration.

3) In a classical OVA-induced asthma model, the kelimycin can remarkably relieve asthma symptoms of mice, and inhibit airway goblet cell hyperplasia and Muc5ac mucin expression of the mice. The results suggest that kelimycin has potential in the treatment of asthma; by combining the sterilization advantages of the kelimycin, the kelimycin can possibly obtain better clinical treatment effect on patients with bacterial infection and asthma.

Experimental example 8

In the experimental example, Lipopolysaccharide (LPS) is used as an induction activator, and a BV2 cell line is used as an in vitro model cell of macrophage to establish an in vitro inflammation model. The effect of isovalerylspiramycin I (ISP I), the major active ingredient of kelimycin, on IL-6 production was examined.

1. Test materials and reagents:

cell lines: mouse microglia BV2 cells were purchased from national laboratory cell resources sharing platform (Beijing)

Isovalerylspiramycin I (Shenyang Cogeneration Co., Ltd.), lipopolysaccharide (LPS 055: B5L6529), trypsin, penicillin, streptomycin, dimethyl sulfoxide (DMSO), and methyl thiazole blue (MTT) were all purchased from Sigmachemical (St. Louis, MO, USA), DMEM medium was purchased from Gibco chemical (GrandIsland, NY, USA), extra-grade fetal bovine serum was purchased from Lonsera, N.sub.H., NO detection kit (Biyuntan Biotech), and ELISA detection kit (Shanghai Aibixin Biotech).

The instruments used in the experimental examples were conventional instruments in the prior art.

2. Test method

2.1 cell culture

BV2 cells were cultured in DMEM medium containing 10% FBS at 37 ℃ in 5% CO₂Cultured in an incubator. When the cell is cultured to the density of about 90%, passage and subsequent experiments can be carried out.

2.2 cell growth inhibition assay

The MTT method was used to examine the effect of ISP I on BV2 cell activity. MTT (tetramethylazoazolium salt) is a yellow dye that can accept hydrogen ions. The MTT method detects the cell activity according to the following principle: succinate dehydrogenase and cytochrome c exist in mitochondria of living cells, tetrazole ring of MTT is cracked under the catalysis of the succinate dehydrogenase and the cytochrome c to generate formazan crystal with blue-purple color, the DMSO or the triple liquid can dissolve the crystal, and the absorbance value is detected at the wavelength of 492nm/630nm, so that the activity of the cells can be detected.

BV2 cells were seeded in 96-well plates at a density of 1.6X 10⁵cell/ml, 100 mul/well, six multiple wells per group, and adding medicine after normal culture for 24 hours. ISP I with different concentrations is added in addition to the negative control group, and the culture is continued for a prescribed time. The culture medium was aspirated off, sterile PBS was added for washing once, PBS was aspirated off, 100. mu.l of the prepared MTT was added to each well, and incubation was continued for 4 h. Adding 100 μ l of triple liquid, culturing for 12h, shaking with a micro-oscillator for 3-5min, measuring the star light value (A) at 630nm with a microplate reader, and calculating the inhibition rate of ISP I on BV2 cells according to the following formula.

Inhibitoryratio(％)＝(A₆₃₀，control-A_{630，control})/(A₆₃₀，control-A_630，blank)×100

2.3Griess method for detecting NO content

BV2 cells at 1X 10⁵And inoculating the cells into a 24-well plate, culturing by adopting a DMEM culture solution containing 10% FBS, continuously culturing for 24 hours, and continuously culturing the cells for 6 hours by replacing the cells with a serum-free culture solution. 250 μ l of ISP I was added to the corresponding wells at final concentrations of 20 μ M, 10 μ M, and 5 μ M for pretreatment, and 1 hour later, LPS at a final concentration of 10 μ g/ml was added to the corresponding wells for induction treatment. Standing for 5% CO₂And after culturing in 37 ℃ culture boxes for 24 hours respectively, taking the supernatant and storing at-20 ℃ for NO detection.

The determination of NO was carried out as specified, the absorbance was determined at 540nm and the corresponding NO content was calculated using a standard curve.

2.4 detection of cytokines by ELISA kit

1) Preparing all required reagents and standards; 2) taking out the microporous plate from the sealed bag restored to room temperature, putting the unused lath back into the aluminum foil bag, and sealing again; 3) the standard substance, the experimental sample or the quality control substance with different concentrations are respectively added into corresponding holes, and each hole has 100 mu L. The reaction wells were sealed with a sealing plate gummed paper and incubated at room temperature for 2 h. 4) The plate was washed with a wash bottle by aspirating off the liquid in the plate. Wash solution was added at 400. mu.L per well and the plate wash was then blotted. The operation was repeated 3 times. Every time the plate is washed, the residual liquid is sucked away as much as possible, which can help to obtain good experimental results. After the last washing, please suck all the liquid in the plate or invert the plate, and pat all the residual liquid on the absorbent paper; 5) add 100. mu.L of detection antibody to each well. Sealing the reaction hole with sealing plate gummed paper, and incubating at room temperature for 2 hours; 6) repeating the plate washing operation of the step 4; 7) mu.L of diluted streptavidin-HRP was added to each well and incubated for 20 minutes at room temperature. Attention is paid to light protection; 8) repeating the plate washing operation of the step 4; 9) add 100. mu.L of chromogenic substrate to each well and incubate for 20 minutes at room temperature. Attention is paid to light protection; 10) by adding 50. mu.L of stop solution to each well, the color of the solution in the well changes from blue to yellow. If the color of the solution is changed to green or the color change is inconsistent, the microporous plate is tapped to uniformly mix the solution; 11) within 30 minutes after the addition of the stop solution, the absorbance value at 450nm was measured using a microplate reader, and 540nm was set as a calibration wavelength. 12) And (3) calculating the result: the corrected absorbance values (OD450-OD540), the duplicate well readings for each standard and sample were averaged and then the average zero standard OD value was subtracted. Curves can be generated by plotting the logarithm of the concentration of the standard substance and the corresponding OD value, and the best fit line can be determined by regression analysis.

2.5 statistical treatment

Statistical software SPSS26.0 is used for data analysis, Excel2016 is used for data summarization, GraphPad is used for drawing a chart, metering data are expressed in the form of mean plus or minus standard deviation (mean plus or minus SD), data among groups are compared by adopting one-factor variance analysis, and differences with P <0.05 have statistical significance.

3. Test results

3.1 Effect of ISP I and LPS on BV2 cell viability

After BV2 cells were treated for 24h with ISP I at different concentrations, the MTT assay showed: compared with the untreated group, the cell viability of the 2.5 mu M, 5 mu M and 10 mu M groups has no obvious difference; after BV2 cells were treated with LPS at different concentrations for 24h, the MTT assay showed: compared with the untreated group, the cell viability of the groups of 0.01. mu.g/ml, 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml has no obvious difference. As shown in FIG. 29 (A is the effect of ISP I on the activity of BV2 cells and B is the effect of LPS on the activity of BV2 cells.)

3.2ISP I inhibits LPS-induced NO production

And detecting the level of NO in cell supernatant when LPS with different concentrations acts on cells, wherein the result shows that 0.01-10 mu g/ml of LPS can induce NO generation. The effect of ISP I on the NO production amount in cell supernatant is detected, and the result shows that ISP I can inhibit NO production induced by LPS in a concentration-dependent manner. As shown in FIG. 30, in A, when the LPS concentration was 0.01. mu.g/ml, 0.1. mu.g/ml, 1. mu.g/ml, 10. mu.g/ml, the NO release amount increased with the increase in the concentration as compared with the control group. P <0.05, p <0.001 vs 0. In B, when the concentration of ISP I was 2.5. mu.M, 5. mu.M and 10. mu.M, the concentration of NO produced in ISP I decreased in a concentration-dependent manner, indicating that ISP I can decrease the amount of NO induced by LPS. # p <0.05, # p <0.01 vsLPS group, # p <0.001 vs blank group.

3.3ISP I inhibits LPS-induced IL-6 production

And (3) detecting the level of IL-6 in cell supernatant when LPS with different concentrations acts on cells, wherein the result shows that 0.01-10 mu g/ml of LPS can induce the generation of IL-6. The ELISA method detects the influence of ISP I on the IL-6 production amount in cell supernatant, and the result shows that 5 mu M and 10 mu MISP I can obviously inhibit the IL-6 production induced by LPS. As shown in FIG. 31, in A, the concentration of LPS was 0.01. mu.g/ml, 0.1. mu.g/ml, 1. mu.g/ml and 10. mu.g/ml, respectively, and the amount of IL-6 produced by the cells was increased. P <0.05, p <0.01 vs.0. mu.g/ml group. In B, when ISP I concentration was 2.5. mu.M, 5. mu.M and 10. mu.M, IL-6 concentration was decreased in an ISP I concentration-dependent manner, indicating that ISP I can decrease LPS-induced IL-6. LPS group p <0.05, p < 0.001.

And 4, conclusion: the main active ingredient isovaleryl spiramycin I (ISP I) of the kelimycin can inhibit the generation of inflammatory cytokines IL-6 and NO induced by LSP.

Experimental example 9ELISA assay for the Effect of Colimycin on inflammatory factors in various tissues and organs of mice

Kunming mice for experiments are purchased from the center of experimental animals of Jiangsu university, and a mouse IL-1 beta ELISA kit (Samerfei (88-7013-88)), a mouse IL-4ELISA kit (Samerfei (88-7044-88)), and other experimental instruments and reagents are all conventional instruments and reagents.

1. Grouping and administration of mice

And (3) kelimycin: the dissolving method comprises adding 0.48ml polyethylene glycol 400 into some of the kelimycin, adding 2.4 μ l Tween 80, shaking, mixing, adding 1.92ml distilled water (adding 200 μ l each time, shaking, and mixing), and making into 1.44mg/ml, 2.88mg/ml, and 5.76mg/ml concentrations respectively.

Azithromycin: dissolving with small amount of anhydrous ethanol, adding water to make the content of anhydrous ethanol 10%, and making into 1.82 mg/ml.

Kunming mice, male, 18-20g size, 144, mice weighed approximately 24g after laboratory acclimation feeding, and were randomly divided into 6 groups: the normal group, model group, low group (30mg/kg), medium group (60mg/kg), high group (120mg/kg), azithromycin group (37.9 mg/kg). Each component was divided into 8 time points: 0h, 0.5h, 2.5h, 4.5h, 12h, 24h, 48h and 72 h. 3 mice per time point. The normal group of mice is not dosed and is not injected with bacteria, the model group is dosed with bacteria and is not dosed with kelimycin and azithromycin by gavage (500 microliter), the dosage of the first day is doubled, the subsequent normal dosing is carried out every day, and the model group is dosed with solvent with the same volume. Mice were sacrificed in batches at different time points after dosing.

Establishing a model: the concentration of Staphylococcus aureus was determined according to the in vitro test report, and after the concentration was determined, it was resuspended in physiological saline to a concentration of 3X 108CFU/ml, and injected into the tail vein at 24 g/100. mu.l. Administration was started one hour after injection.

2. Preparation of samples

The mouse eyeballs were bled and sacrificed, tissue and organs were harvested and weighed on an electronic balance, 50mg of each tissue was weighed and added to a 1.5ml EP tube, followed by 1ml of pre-cooled PBS, magnetic beads and homogenate (300Hz, 30s) in a homogenizer. After standing on ice for 30 minutes, it was centrifuged at 4 ℃ by a centrifuge (10000g, 10min), and the supernatant was taken as a test sample.

3. Experimental methods the experimental procedures were performed exactly according to the ELISA kit instructions and are briefly described as follows:

preparation of reagents: PBS (pH 7.35), Tween 20, PBS solution containing 0.05% Tween 20 was prepared as washing solution. (if crystals form in the buffer concentrate, heat it gently until completely dissolved). 1. Coating buffer (1 ×): PBS (10 fold) was diluted 1:10 in deionized water. 2. Capture antibody: the capture antibody (250x) was diluted 1:250 in coating buffer (1 x). 3.5xELISA/ELISPOT dilutions: the concentrated dilutions (5x) were diluted 1:5 in deionized water. 4. And (3) standard substance: recombinant mouse il-1 β standard, dissolved in distilled water, the volume of distilled water added is noted on the label of the standard vial. Standard solutions were prepared 10-30 minutes in advance and mixed thoroughly to ensure complete uniform dissolution (concentration of recombinant standard 1000 pg/ml). The standards were freshly prepared, used immediately, and not stored. 5. Detecting an antibody: the detection antibody (250X) was diluted 1:250 in ELISA/ELISPOT dilutions (1X). 6. Enzyme: HRP concentrate (100X) was diluted 1:100 in ELISA/ELISPOT dilutions (1X).

The experimental steps are as follows: 1. corning TMCostarTM9018ELISA plates were coated in coating buffer in an amount of 100. mu.L of capture antibody per well (diluted as described in point 1 of reagent preparation). The ELISA plates were sealed and incubated overnight at 4 ℃. 2. Remove the solution from the wells and wash 3 times with more than 250 microliters of buffer, leave a soak time (1 minute) in each wash step to improve the wash effect, wipe dry with absorbent paper, remove residual solution. 3. Add 200. mu.l ELISA/ELISPOT dilutions (1X) per well and incubate for 1 hour at room temperature. 4. Standards were prepared 30 minutes in advance. 5. The washing solution is pumped and washed at least once. 6. 100ul of standard, sample, blank wells were added with ELISA/ELISPOT dilutions (1X). 7. Plates were incubated at room temperature for 2 hours. 8. The detection antibody is prepared. 9. And (4) sucking air and cleaning according to the step 2, and repeatedly washing for 3-5 times. 10. To all wells 100 μ l/well diluted detection antibody was added. 11. Plates were incubated for 1 hour at room temperature. 12. HRP was prepared. 13. And (4) sucking air and cleaning according to the step 2, and repeatedly washing for 3-5 times. 14. 100ul of diluted HRP was added to each well. 15. The plates were incubated for 30min at room temperature. 16. Aspiration and rinsing was performed according to step 2, ensuring that 1 to 2 minutes of soaking was left before aspiration, and rinsing was repeated 5-7 times. 17. 100ul of TMB solution was added to each well. 18. Incubate for 15 minutes at room temperature. 19. Add 50. mu.L of stop buffer to each well. 20. Plates were read at 450 nm. 21. And (4) collecting and processing data.

And (3) test results: the results of the action of kelimycin on IL-4 factor and IL-1. beta. in various tissues and organs of mice are shown in tables 4 and 5, respectively.

TABLE 4

TABLE 5

Note: p < 0.05; p < 0.01; p < 0.001; p < 0.0001.

And (4) experimental conclusion: the kelimycin has the obvious effect of reducing the IL4 factor in the lung, the kidney, the liver and the spleen, and has more obvious effect in the liver and the spleen; the kelimycin has obvious IL-1 beta factor reducing effect in small intestine, lung, spleen, liver and kidney, especially in small intestine and lung.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

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Application of isovaleryl spiramycin compound or composition thereof in preparation of medicament for treating immune disorder

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