阅读说明：本技术 甘油磷酸胆碱在制备预防和治疗结核病药物中的应用 (Application of glycerophosphorylcholine in preparing medicine for preventing and treating tuberculosis ) 是由 戈宝学 杨华 王菲 郭欣娅 于 2021-08-27 设计创作，主要内容包括：本发明提供了甘油磷酸胆碱(GPC)在制备预防和治疗结核病药物中的应用。本发明验证了GPC通过增强巨噬细胞中抗菌肽的表达,从而抑制结核分枝杆菌的胞内存活和体内存活,抑制了结核性肉芽肿的恶化,且无细胞毒性,可作为新的有效代谢分子用于制备预防和治疗结核病的药物,为治疗耐多药结核病提供新的策略。(The invention provides an application of Glycerophosphorylcholine (GPC) in preparing a medicament for preventing and treating tuberculosis. The invention verifies that GPC inhibits the intracellular survival and in-vivo survival of mycobacterium tuberculosis and inhibits the deterioration of tuberculous granuloma by enhancing the expression of antibacterial peptide in macrophages, has no cytotoxicity, can be used as a new effective metabolic molecule for preparing a medicament for preventing and treating tuberculosis, and provides a new strategy for treating multi-drug resistant tuberculosis.)

1. The application of glycerophosphorylcholine in preparing medicine for preventing and treating tuberculosis is disclosed.

2. Application of glycerophosphorylcholine in preparing medicine for inhibiting tuberculous granuloma deterioration is provided.

3. The application of glycerophosphorylcholine in preparing medicine for promoting the killing and eliminating action of macrophage on Mtb.

4. Application of glycerophosphorylcholine in preparing medicine for promoting the production of macrophage antibacterial peptide.

5. The use according to claim 4, wherein the related genes of the antibacterial peptide comprise one or more of Hamp, Defb3, Defb4 and Cathelicidin.

6. A medicament for preventing and treating tuberculosis, characterized by comprising glycerophosphorylcholine as a main active ingredient.

7. The medicament of claim 6, further comprising a pharmaceutically acceptable carrier or excipient.

8. The medicament of claim 6, wherein the medicament, when administered, performs the following functions:

a. reducing the amount of bacteria loaded in the Mtb-infected lung;

b. remarkably enhancing the expression of macrophage antibacterial peptide; and/or

c. Inhibiting tuberculous granuloma deterioration.

Technical Field

The invention relates to the field of biological medicines, in particular to application of glycerophosphorylcholine in preparation of a medicine for preventing and treating tuberculosis.

Background

Tuberculosis (TB) is a serious infectious disease seriously harming the health of people, is one of ten causes of death worldwide and is also a main cause of death caused by a single infection source. About one fourth of the world population is Latent Tuberculosis infectors (LTBI), and once the immunity of the organism is low, LTBI develops into active Tuberculosis, becomes a new Infection source and infects more people. In 2019, about 1,000 million people suffer from TB, 145 million people die from TB, and nearly 50 million people suffer from rifampicin-resistant tuberculosis (RR-TB), wherein 78% of people suffer from multi-drug resistant tuberculosis (MDR-TB), and especially the widely drug resistant tuberculosis is a new incurable disease all over the world. The three countries with the greatest global burden are india (27%), china (14%) and russian federation (8%). The current epidemic situation of tuberculosis in China is still severe, and is one of 30 tuberculosis high-load countries in the world. The tuberculosis prevention and treatment work in China still faces a plurality of problems and challenges, wherein multi-drug resistant tuberculosis is a difficult point in the current tuberculosis control, and new treatment drugs and means are urgently needed to be developed so as to realize the urgent requirements of the country for reducing the morbidity and mortality of the tuberculosis.

Metabonomics adopts a high-throughput and high-precision analysis technology to carry out accurate quantitative and qualitative analysis on metabolites, potential biomarkers with statistical significance are screened out from the metabolites, and metabolic information is organically combined with information of body reaction, regulation factors and the like, so that researchers can better understand pathological changes of diseases and metabolic characteristics of pathogens, and effective basis is provided for researches such as intervention of related metabolic pathways or artificial addition of metabolites with a protective effect. More and more researches show that the manual addition of protective metabolic molecules can directly influence the outcome of a plurality of diseases, and the artificial addition of protective metabolic molecules can be possibly used as a new treatment method for clinical application. However, the metabolic products have no relevant research and report on the progress of tuberculosis diseases and the application of the metabolic products in the prevention and treatment of tuberculosis.

Granuloma is a typical pathological feature of Mycobacterium tuberculosis (Mtb) infection and is a major defense mechanism for host inhibition of Mtb. Granulomas caused by Mtb, centered as caseous necrosis, contain multinucleated macrophages, surrounded by epithelioid cells and many other cells to form tubercular nodules. Mtb is wrapped by various cells layer by layer, is in a latent infection state, and is propagated at random to promote granuloma to worsen and form active tuberculosis. During the formation of granuloma, the energy metabolism and the substance metabolism of bacteria and cells are drastically changed. Which metabolic molecules or pathways play a key role in regulating and controlling granuloma functions become the focus of attention at present, and are to be further deeply analyzed to provide strategy basis for preventing and radically treating tuberculosis.

In the early stage, through comparative metabonomics analysis of lung and peripheral blood of a mouse infected with granuloma by Mtb, the content of Glycerophosphorylcholine (GPC) in the lung granuloma and the peripheral blood of the mouse is remarkably reduced, which indicates that GPC may participate in the generation and development process of tuberculous granuloma, but the action and mechanism of the GPC in the development process of the granuloma are not clear, and particularly the application in the preparation of a medicine for preventing and treating tuberculosis is not reported.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides the application of glycerophosphorylcholine in preparing the medicine for preventing and treating tuberculosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the first aspect of the invention provides the application of glycerophosphorylcholine in preparing a medicament for preventing and treating tuberculosis.

In a second aspect, the present invention provides the use of glycerophosphorylcholine in the manufacture of a medicament for the inhibition of the progression of tuberculous granuloma.

In a third aspect, the invention provides the use of glycerophosphorylcholine in the manufacture of a medicament for promoting the killing and clearance of Mtb by macrophages.

The fourth aspect of the invention provides the application of glycerophosphorylcholine in preparing a medicament for promoting the production of macrophage antibacterial peptide.

Further, the related genes of the antibacterial peptide comprise one or more of Hamp, Defb3, Defb4 and Cathelicidin.

The fifth aspect of the present invention is to provide a medicament for the prevention and treatment of tuberculosis, which comprises glycerophosphorylcholine as a main active ingredient.

Further, the medicine also comprises a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" refers, inter alia, to substances which are suitable for use in humans without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The "carrier or excipient" includes one or more of binder, filler, diluent, tabletting agent, lubricant, disintegrating agent, coloring agent, flavoring agent, and humectant.

Further, the above-mentioned drugs, after administration, can exert the following functions:

a. reducing the amount of bacteria loaded in the Mtb-infected lung;

b. remarkably enhancing the expression of macrophage antibacterial peptide; and/or

c. Inhibiting tuberculous granuloma deterioration.

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

the invention verifies that GPC inhibits the intracellular survival and in-vivo survival of Mtb and inhibits the deterioration of granuloma by enhancing the expression of antibacterial peptide in macrophage, has no cytotoxicity, can be used as a new effective metabolic molecule for preparing a medicament for preventing and treating tuberculosis, and provides a new strategy for treating multi-drug resistant tuberculosis.

Drawings

FIG. 1 shows the result of measurement of the amount of pulmonary bacteria in H37 Rv-infected mice after control or GPC treatment in one embodiment of the present invention;

FIG. 2 is the results of HE and acid fast staining of lungs after treatment of H37Rv infected mice with control or GPC in one embodiment of the invention;

FIG. 3 shows the result of the measurement of the bacterial load of the Mycobacteria marinum infected adult fish after the control or GPC treatment in accordance with an embodiment of the present invention;

FIG. 4 shows HE and acid fast staining results of control or GPC treatment of Mycobacteria marinum infected adult fish according to one embodiment of the present invention;

FIG. 5 is the intracellular CFU results of macrophage infection with H37Rv after control or GPC treatment in one embodiment of the invention;

FIG. 6 shows the expression of the antimicrobial peptide gene of macrophage infected H37Rv after control or GPC treatment in accordance with one embodiment of the present invention;

FIG. 7 shows the OD of HEK293, SH-SY5Y, and HePG2 cells treated with different concentrations of GPC according to one embodiment of the present invention₅₇₀And (4) light absorption value.

Detailed Description

The invention discovers that GPC can obviously inhibit the intracellular and in-vivo survival of Mtb and can promote macrophage to kill and eliminate Mtb, thereby providing the application of GPC in preparing the drugs for preventing and treating tuberculosis. The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

The effect of GPC on tuberculosis was analyzed using the C57BL/6 mouse infection model, and the specific experimental procedures and results are as follows:

female C57BL/6 mice, 6 per group, 6 at 6-8 weeks were infected with H37Rv strain, approximately 200 CFU/mouse, by the nasal drip route. After 3 weeks of infection, the control group was added 1% DMSO in the drinking water, the GPC group was added GPC (1%) in the drinking water, and the mice were sacrificed 4 weeks after administration. The effect of GPC on tuberculosis was analyzed by examining the following indices:

(1) visceral lotus amount: a part of lungs were collected under aseptic conditions, homogenized by PBS, diluted 10-fold, inoculated on 7H10 agar medium, cultured at 37 ℃ for 4 weeks, and Mtb growth was observed to calculate the lung load, and as a result, the lung load infected with H37Rv was found to be significantly reduced in mice that had been administered GPC (FIG. 1).

(2) And (3) lung histopathological detection: lungs were collected under sterile conditions, and a portion of lung tissue was fixed with 4% paraformaldehyde, dehydrated, waxed, embedded, sliced, stained with H & E and acid-fast stain, and examined by microscope for pathological changes in lung tissue, resulting in a significant reduction in pathological changes in lung, immune cell infiltration and lung tissue damage in mice receiving GPC compared to controls (fig. 2).

The above results indicate that GPC can significantly improve the host's ability to resist Mtb.

Example 2

In this example, the effect of GPC on mycobacterial disease, particularly granuloma, was analyzed using a zebrafish infection model, with the following specific experimental procedures and results:

wild zebra fish (AB strain) is obtained from the China zebra fish resource center, is raised in a circulating culture system, is transferred to a toxicology system to carry out infection experiments, and ensures that the standard conditions of zebra fish raising and infection are that the water temperature is about 28 ℃, the pH is about 7.4, and the conductivity is about 1,500 mu S. Adult zebrafish were anesthetized with 0.1% tricaine and then infected with mycobacterium marinum by intraperitoneal injection at a bacterial load of about 200CFU, after 1 week, the control group was orally filled with DMSO, and the GPC group was orally filled with GPC at 0.1 mg/Fish. Sacrificed 1 week after administration, and the effect of GPC on the development of granuloma development in adult fish of mycobacterium marinum infected zebrafish was analyzed by the following criteria:

(1) the bacterial load: after the zebra fish after infection administration is subjected to terminal anesthesia in 0.5% tricaine, the zebra fish is homogenized in PBS, diluted by 10 times and inoculated on 7H10 agar medium, the zebra fish is cultured at 37 ℃ for 1-2 weeks, the growth of strains of different groups is observed, the number of viable bacteria CFU is calculated, and the adult fish of the oral perfusion GPC group is found to have lower bacterial load (figure 3).

(2) And (3) histopathological detection: the zebra fish is subjected to terminal anesthesia in 0.5% tricaine, the whole fish is fixed in 4% paraformaldehyde solution for 72 hours, decalcification, wax immersion, embedding and slicing are carried out, H & E and acid-fast staining are carried out, the pathological change of the whole fish, mainly the granulomatosis of kidney and liver tissues, is observed by a microscope, and the result shows that the quantity of the granulomatosis of the adult fish in the oral perfusion GPC group is obviously reduced, and the pathological change is also obviously relieved (figure 4).

The results show that GPC can significantly improve the host's resistance to mycobacteria, and reduce the formation and progression of granulomas.

Example 3

This example uses an in vitro macrophage infection model to analyze the effect of GPC on survival of Mtb in macrophage cells, and the specific experimental procedures and results are as follows:

primary macrophages of wild mice abdominal cavities are taken and cultured for 4 hours at 37 ℃ by using complete 1640 culture medium (containing 10% FBS + 1% penicillin-streptomycin), the 1640-free culture medium is replaced after the cells are attached to the walls, GPC is added to enable the final concentration to be 1mM, H37Rv is infected after 12 hours, and MOI is 2. After 3 hours of infection, the supernatant was removed, the cells were washed twice with PBS to remove extracellular bacteria, and the number of entering cells was counted by CFU. After another portion of the cells were washed, they were cultured for 24 hours in 1640 medium containing DMSO or 1mM GPC, and the survival of intracellular bacteria was examined by CFU counting. As a result, GPC was found to significantly promote intracellular clearance of Mtb by macrophages, see fig. 5.

Example 4

In this example, a macrophage infection model was used to analyze the effect of GPC on macrophage bactericidal function, and the specific experimental procedures and results are as follows:

wild type mouse peritoneal macrophages were taken, treated with DMSO control or GPC at a final concentration of 1mM for 12 hours, and then given H37Rv infection with MOI 2. After 0, 3 and 6 hours of infection, respectively, removing supernatant, using 1ml TRIZOL to crack cells, extracting RNA, detecting the change of expression levels of antibacterial peptide related genes Hamp, Defb3, Defb4 and Cathelicidin by qRT-PCR, and determining the influence of GPC on the antibacterial peptide of macrophages. As a result, GPC was found to significantly promote expression of Hamp, Defb3, Defb4, Cathelicidin genes, as shown in FIG. 6.

Example 5

In this example, the effect of GPC on cytotoxicity was analyzed using HEK293, SH-SY5Y, HePG2 cell models, and the specific experimental procedures and results were as follows:

culturing HEK293, SH-SY5Y, and HePG2 cell lines in vitro, adding GPC (0, 0.001, 0.01, 0.1, 1,5, 10, 30, 100, 1000 and 10000 μ M) at different concentrations when the cell density reaches 80%, treating for 24h, and determining OD for cytotoxicity with MTT kit₅₇₀The absorbance values were determined and no significant toxicity of GPC was found on all three cell lines, see FIG. 7.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.