阅读说明：本技术 一种新型靶点Mob1及靶向小分子管花苷B在制备抗炎产品中的应用 (Novel target Mob1 and application of targeted small molecular angionoside B in preparation of anti-inflammatory product ) 是由 曾小斌 肖凌云 姚杰 葛岚岚 缪雨阳 欧宝如 于 2021-02-02 设计创作，主要内容包括：本发明提供了一种新型靶点Mob1及靶向小分子管花苷B在制备抗炎产品,特别是制备治疗与M1型巨噬细胞分化相关的炎症性疾病药物、食品或保健品中的应用。本发明首次发现,Mob1蛋白对于巨噬细胞M1型分化具有显著抑制作用,而天然小分子化合物管花苷B能够与Mob1蛋白直接结合,改变其构象,稳定其结构,抑制Mob1蛋白在巨噬细胞M1型分化中的降解,从而提高Mob1蛋白的水平,发挥抗炎作用。本发明拓宽了抗炎药物的作用靶点,为抗炎药物研发提供了新的思路；还提供了活性优秀、靶点明确的活性小分子--管花苷B,为抗炎产品的开发提供了新的药物选择。(The invention provides application of a novel target Mob1 and a targeted small molecular angionoside B in preparation of an anti-inflammatory product, in particular to preparation of a medicine, a food or a health-care product for treating inflammatory diseases related to M1 macrophage differentiation. The invention discovers for the first time that the Mob1 protein has a remarkable inhibiting effect on macrophage M1 type differentiation, while a natural small molecular compound, namely angionoside B, can be directly combined with the Mob1 protein to change the conformation, stabilize the structure and inhibit the degradation of the Mob1 protein in macrophage M1 type differentiation, so that the level of the Mob1 protein is improved, and the anti-inflammatory effect is exerted. The invention widens the action target of the anti-inflammatory drug and provides a new idea for the research and development of the anti-inflammatory drug; also provides an active small molecule, namely the tubular flower glycoside B with excellent activity and definite target spot, and provides a new drug choice for the development of anti-inflammatory products.)

The application of the Mob1 protein in preparing medicines, foods or health products for treating inflammatory diseases related to M1 type macrophage differentiation is characterized in that the medicines, foods or health products comprise medicines which take the Mob1 protein as a target point and inhibit the expression of M1 type macrophage inflammatory factors.

2. The use according to claim 1, wherein the inflammatory disease associated with macrophage differentiation of type M1 comprises acute liver injury, acute lung injury, acute kidney injury, sepsis, endotoxemia, inflammatory bowel disease, non-alcoholic fatty liver disease, arthritis, diabetes, obesity, atherosclerosis, Alzheimer's disease, and chronic obstructive pulmonary disease.

3. The use of claim 2, wherein the acute liver injury comprises acute viral liver injury, acute pharmaceutical liver injury, acute alcoholic liver injury, and ischemia-reperfusion liver injury.

4. The use of claim 2, wherein the acute lung injury comprises acute lung injury caused by viruses, bacteria, fungi, parasites, endotoxins, drugs, trauma, smoke and noxious gas inhalation, embolism, ischemia.

5. The use of claim 2, wherein the acute kidney injury comprises acute renal function decline caused by bacteria, viruses, drugs, trauma, endotoxins, embolisms, and ischemia.

6. The use according to claim 2, wherein the inflammatory bowel disease comprises ulcerative colitis and Crohn's disease.

7. The use according to claim 1, wherein the pharmaceutical, food or health product is in the form of a solid or liquid formulation; the solid preparation is tablets, capsules, granules, pills, dripping pills, mixture or powder; the liquid preparation can be injection, oral liquid, soft capsule, and aerosol.

The application of the Mob1 protein in screening and treating inflammatory diseases related to M1 type macrophage differentiation is characterized in that the Mob1 protein is used as a target point to screen and screen drugs for inhibiting the expression of M1 type macrophage inflammatory factors.

9. A method for screening a candidate drug for treating inflammatory diseases related to M1 type macrophage differentiation is characterized in that a Mob1 protein is used as a target point to screen a drug for inhibiting the expression of an M1 type macrophage inflammatory factor.

10. The application of the tubular anthocyanin B in preparing medicines, foods or health products for treating inflammatory diseases related to M1 type macrophage differentiation is characterized in that the tubular anthocyanin B is a natural small molecular compound capable of targeting Mob1 protein; the effective components of the medicine, food or health care product comprise tubular flower glycoside B with the structure as shown in the formula or medicinal salt, solvate, hydrate and prodrug thereof:

Technical Field

The invention relates to the technical field of biological medicines, in particular to a novel target Mob1 and application of targeted small molecular angionoside B in preparation of an anti-inflammatory product.

Background

Inflammation is an important protective mechanism of the body against external damage. Although normal inflammation can help the body to remove foreign matters and resist external environment stimulation, excessive or continuous inflammatory reaction can cause body injury and promote the occurrence and development of various diseases. For example, in sepsis, the balance between pro-inflammatory and anti-inflammatory responses in the body is disrupted, the inflammatory response is uncontrolled, and the release of large amounts of inflammatory cytokines leads to a systemic inflammatory response, which in turn leads to multiple organ failure. In atherosclerosis, macrophage-mediated inflammatory responses span the entire process from fatty streak formation to plaque rupture. In viral pneumonia, SARS-CoV-2, like SARS-CoV, causes severe lung injury primarily through acute inflammatory reactions, and the intensity of a patient's post-infection cytokine storm correlates with the severity of the disease. The anti-inflammatory drugs currently available in clinic mainly include glucocorticoids (such as dexamethasone), non-steroidal anti-inflammatory drugs (such as aspirin), immunosuppressants, inflammatory factor antagonists (such as the TNF α antagonist infliximab), and the like, and the side effects and the effectiveness of the existing drugs are receiving more and more attention. Dexamethasone can effectively inhibit inflammatory reaction of organisms, but has multiple physiological functions besides anti-inflammatory action, and side effects are generated. The inflammatory factor antagonists can only target specific inflammatory factors, and may not have good effects on diseases mediated by various inflammatory factors. Therefore, effective, safe and non-side-effect anti-inflammatory drugs remain the main direction of drug development.

Macrophages are sensors in inflammatory responses and play an important role in the regulation of inflammatory responses, thereby affecting the outcome of inflammation-related diseases. Macrophages are heterogeneous and can be polarized under different stimuli to pro-inflammatory M1 and anti-inflammatory M2, with the interplay between the two phenotypes throughout the development, progression and regression of inflammation-related diseases. Among them, M1 type macrophages are generally induced by Th1 type cytokines such as interferon-gamma and lipopolysaccharide, activate Toll-like receptors and IFN pathway polarization. The M1 type macrophage can induce Th1 type cellular immune response, mediate inflammatory reaction, release proinflammatory factors such as TNF-alpha, interleukin-1, interleukin 6, interleukin 12, interleukin 18 and interleukin 23, release proinflammatory mediator NO, highly express cyclooxygenase 2, and highly express partial chemokines such as CXCL1, CXCL9, CXCL10 and the like. M1 type macrophages can phagocytose foreign pathogens, play a role in the initiation of the inflammatory response, but also cause tissue inflammatory responses, and damage tissue cells. For example, in the initial phase of sepsis, infection of the body causes macrophages to become polarized to M1 type, causing an excessive inflammatory response, inducing systemic inflammatory response syndrome and resulting in organ failure. High expression of inflammatory factors secreted by macrophages M1 is detected in unstable angina patients, myocardial infarction patients, and in vulnerable plaques, the increase of macrophages M1 is more likely to cause severe acute atherosclerotic vascular accidents. The M1 type differentiation of macrophage is regulated, so that the compound preparation has broad-spectrum efficacy on the treatment of inflammatory diseases, and a new idea is provided for the development of medicaments for the inflammatory diseases.

The Mob1(Mob Kinase Activator 1) protein comprises two homologous proteins Mob1a and Mob1b, which are highly homologous in humans. The Mob1 protein was originally found in the Hippo signaling pathway and is one of the core members of this pathway. In the classical Hippo signaling pathway, Mst1/2 kinase is phosphorylated and activated by an upstream signaling molecule, and phosphorylates a downstream kinase, Lats1/2, activated Lats1/2 is combined with Mob1 protein, and further phosphorylates effector protein Yap of the Hippo signaling pathway, so that the effector protein Yap is retained in cytoplasm and degraded through a ubiquitination pathway, and the Hippo signaling pathway is maintained in a closed state. The non-phosphorylated Yap is activated and can enter the nucleus to play the function of transcriptional regulation. The existing research shows that the classical Hippo-Yap signal channel is highly conserved in evolution and plays an important role in regulating cell proliferation and apoptosis and controlling organ development. In recent years, more and more researches show that the Hippo signaling pathway also has various non-classical regulatory functions, such as no abnormal cell proliferation or tumorigenic phenotype is found when Mst1/2 is knocked out conditionally in bone marrow hematopoietic cells or immune cells, and the non-classical Hippo signaling pathway is inactivated to cause serious immune dysfunction. Other functions of Mob1 protein, including its role in inflammation and its relationship to Hippo signaling pathways, have not been reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel anti-inflammatory target Mob1 protein for regulating macrophage M1 type differentiation; the invention also provides a small molecular compound tubuloside B (TubB) capable of regulating the target spot, and the compound has the advantages of simple structure, high safety and obvious inflammation inhibition effect.

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

in a first aspect, the invention provides an application of Mob1 protein in preparing a medicament, food or health-care product for treating inflammatory diseases related to M1 type macrophage differentiation; the medicine, food or health product comprises a medicine which takes Mob1 protein as a target point and inhibits the expression of M1 type macrophage inflammatory factor.

Further, the above-mentioned inflammatory diseases associated with macrophage differentiation of M1 type include, but are not limited to, acute liver injury, acute lung injury, acute kidney injury, sepsis, endotoxemia, inflammatory bowel disease, nonalcoholic fatty liver, arthritis, diabetes, obesity, atherosclerosis, alzheimer's disease, and chronic obstructive pulmonary disease.

Further, the acute liver injury includes various acute viral liver injuries, acute drug-induced liver injuries, acute alcoholic liver injuries, ischemia-reperfusion liver injuries and the like.

Further, the acute lung injury includes acute lung injury caused by various viruses, bacteria, fungi, parasites, endotoxins, drugs, trauma, smoke and toxic gas inhalation, embolism, ischemia, and the like.

Furthermore, the acute kidney injury includes acute renal function decline caused by various bacteria, viruses, medicines, wounds, endotoxins, embolism, ischemia and other factors.

Further, the inflammatory bowel disease includes chronic inflammatory bowel diseases such as ulcerative colitis and crohn's disease.

Furthermore, the dosage form of the medicine, food or health care product is a solid preparation or a liquid preparation; the solid preparation is tablet, capsule, granule, pill, dripping pill, mixture or powder; the liquid preparation can be injection, oral liquid, soft capsule, and aerosol.

In a second aspect, the invention provides application of the Mob1 protein in screening and treating inflammatory diseases related to M1 type macrophage differentiation, and the Mob1 protein is used as a target point to screen and screen drugs for inhibiting the expression of M1 type macrophage inflammatory factors.

In a third aspect, the invention provides a method for screening a candidate drug for treating inflammatory diseases related to M1 type macrophage differentiation, which screens a drug for inhibiting the expression of M1 type macrophage inflammatory factor by taking Mob1 protein as a target point.

In a fourth aspect, the invention provides an application of tubular flower glycoside B in preparing a medicament, food or health-care product for treating inflammatory diseases related to M1 type macrophage differentiation; the tubular flower glycoside B is a natural small molecular compound capable of targeting Mob1 protein; the effective components of the medicine, food or health product comprise tubular flower glycoside B with the structure as shown in the following formula or medicinal salt, solvate, hydrate and prodrug thereof:

further, the above-mentioned medicament, when administered, has at least one of the following functions:

a. inhibiting degradation of Mob1 protein in macrophage M1-type differentiation;

b. inhibiting the expression of inflammatory factors secreted by M1 type macrophages.

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

the invention discovers for the first time that the Mob1 protein has a remarkable inhibiting effect on macrophage M1 type differentiation, while a natural small molecular compound, namely angionoside B, can be directly combined with the Mob1 protein to change the conformation, stabilize the structure and inhibit the degradation of the Mob1 protein in macrophage M1 type differentiation, so that the level of the Mob1 protein is improved, and the anti-inflammatory effect is exerted. The invention widens the action target of the anti-inflammatory drug and provides a new idea for the research and development of the anti-inflammatory drug; also provides active small molecules with excellent activity and definite target, and provides a new drug choice for the development of anti-inflammatory products.

Drawings

FIG. 1 shows that Mob1 protein can inhibit macrophage M1-type differentiation in one embodiment of the present invention; among them, FIG. 1(A) shows the change of the Mob1 protein level of RAW264.7 macrophage upon differentiation of M1 type after LPS and IFN-. gamma.induction; (B) shows the change in Mob1 knockdown of Mob1 protein levels in the cell line; (C) (D) shows the effect of Mob1 knockdown on the expression levels of the macrophage inflammatory factors Il-1 β and Il6 genes; (E) shows changes in the level of Mob1 protein in the Mob1 overexpressing cell line; (F) (G) shows the effect of Mob1 overexpression on the expression level of IL-1. beta. and IL6 genes, macrophage inflammatory factors

FIG. 2 shows that tubular flower glycoside B can target Mob1 type protein, inhibiting its degradation in inflammatory state in one embodiment of the present invention; wherein (a) shows the effect of angionoside B on macrophage RAW264.7 cell viability; (B) shows the effect of angioside B on the Hippo signalling pathway in macrophages under LPS and IFN- γ treatment; (C) shows the effect of angioside B on the nuclear entry of Yap protein; (D) shows the effect of tubular flower glycoside B on the expression level of Mob1 protein gene; (E) shows the influence of the tubular anthocyanin B on the degradation speed of the Mob1 protein after the treatment of the imine cyclohexanone CHX by Western blot detection; (F) shows the effect of tubular flower glycoside B on the autofluorescence of recombinant Mob1a protein; (G) shows the effect of tubular flower glycoside B on the circular dichroism spectrum of recombinant Mob1a protein; (H) shows the effect of tubular flower glycoside B on the thermal stability of recombinant Mob1a protein; (I) shows the effect of tubular flower glycoside B on the heat stability of intracellular Mob1 protein;

FIG. 3 shows that angiogases B have anti-inflammatory activity to inhibit macrophage differentiation of M1 type in one embodiment of the present invention; wherein (A) shows the effect of angioside B on NO release from macrophage RAW264.7 under LPS and IFN-gamma treatment; (B-E) shows the effect of angioside B on the expression levels of various inflammatory factor genes by macrophages under LPS and IFN-gamma treatment; (F) shows the effect of angioside B on macrophage iNOS and Cox2 protein levels;

FIG. 4 shows that tubular flower glycoside B can inhibit LPS induced acute liver injury and lung injury in mice, and reduce inflammatory secretion; wherein (A) shows the effect of angioside B on serum IL-1 β levels in mice; (B) (C) shows the effect of angioside B on mouse serum ALT and AST levels; (D) the HE staining picture of mouse liver is obtained; (E) images of HE staining of mouse lungs.

Detailed Description

The invention discovers that the Mob1 protein can inhibit M1 type differentiation of macrophages and inhibit inflammatory reaction of the macrophages. Macrophages differentiate towards M1 type under stimulation by LPS and IFN- γ, with a significant reduction in Mob1 protein levels. The expression level of the inflammatory factor of macrophages is increased under the stimulation of LPS and IFN-gamma after the Mob1 protein is knocked down, and the expression level of the inflammatory factor of the macrophages is reduced under the stimulation of LPS and IFN-gamma after the Mob1 protein is over-expressed. And the phosphorylation of the Yap protein is not influenced after the Mob1 protein is knocked down, which indicates that the anti-inflammatory effect exerted by the Mob1 protein is independent of the classical Hippo-Yap signal pathway. Therefore, the Mob1 protein is a new target for exerting anti-inflammatory efficacy by regulating macrophage M1 type polarization.

By screening the small molecule compound targeting Mob1, the invention also provides a natural small molecule compound tubular anthocyanin B capable of targeting Mob1 protein and exerting anti-inflammatory efficacy, which has the following structural formula (I):

the tubular flower glycoside B (Tubulioside B) belongs to phenethyl alcohol glycoside compounds, can be separated from traditional Chinese medicine cistanche, has the main reported activities in two aspects of nerve protection and oxygen free radical removal at present, and has no report on the anti-inflammatory activity. According to the invention, screening is carried out by taking Mob1 protein as an anti-inflammatory target, and the discovery that angioflorin B can be directly combined with Mob1 protein, so that the protein structure of the angioflorin B is stabilized, the degradation of the angioflorin B in the differentiation process of macrophage M1 type is inhibited, the protein level of Mob1 is increased, the effect of inhibiting the expression of inflammatory factors is exerted, and the angioflorin B has no obvious toxic effect on cells and is high in safety concentration. The acute inflammatory injury model induced by LPS is further proved that the angionoside B can inhibit the release of inflammatory factors and relieve the acute inflammatory injury of tissues such as liver, kidney and the like. Therefore, the tubular anthocyanin B can be used as a lead compound, is beneficial to the development of products for treating inflammation-related diseases, and has wide (clinical) application prospect.

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

In this example, the effect of Mob1 protein on macrophage M1 type differentiation and inflammatory factor expression level was studied, the regulatory effect of small molecular angionoside B on Mob1 was studied, and the anti-inflammatory activity was explored, and the specific operating method and results are as follows:

first, experiment method

1.1 cell culture and cell viability assay

RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. The effect of tubular flower glycoside B on cell viability was determined using the method of CCK 8. The cells were cultured at 1X 10⁴The density of each well was inoculated into a 96-well plate and incubated for 24 hours, followed by the addition of different concentrations of tubular anthocyanin B, control wells with the same amount of DMSO solvent as a control, and incubation was continued for 24 hours. After the treatment, the medium was removed, a DMEM medium containing 10% CCK-8 solution was added thereto, and after further incubation for 3 hours, absorbance at 450nm was measured. Cells in Western blot and qPCR experiments were 2X 10⁵The density of each well was plated in 6-well plates, as before. To induce M1-type macrophage polarization, 10ng/ml LPS and 20ng/ml IFN-. gamma.were added to the cell culture medium as stimulators simultaneously with angioside B.

1.2Western blot detection of protein levels

The drug-treated cells were lysed with a lysis solution containing protease and phosphatase inhibitors. The isolation of the nucleoplasm of the cells was carried out using a commercial kit according to the instructions. Proteins of different molecular weights were separated on 8-15% SDS-PAGE gels and then transferred to PVDF membranes by wet transfer. After blocking the membrane with 5% skim milk for 1 hour, incubation with the specific primary antibody solution for 1 hour at room temperature or overnight at 4 ℃ followed by 3 washes to remove unbound primary antibody. The membrane was then incubated with HRP-conjugated secondary antibody for 1 hour and washed three more times to remove unbound secondary antibody. The target protein content is detected with a chemiluminescent reagent.

1.3qPCR detection of Gene expression levels

RNA from cells was extracted using a commercial kit and an equivalent amount of RNA was reversed to cDNA using a reverse transcription kit. Specific primers are designed for different genes, and a commercial qPCR premix is adopted for carrying out fluorescent quantitative PCR analysis. Calculation of Gene expression level normalization with reference Gene Gapdh (2)^-ΔΔCTAnalytical method).

1.4Mob1 knockdown and overexpression cell line construction

Knockdown of Mob1 protein was performed in RAW264.7 cells by lentivirus-based CRISPR/Cas9 technology. Two sets of sgrnas (KD1: Mob1a sgRNA1+ Mob1b sgRNA1, KD2: Mob1a sgRNA2+ Mob1b sgRNA2) were designed for the gene sequences of Mob1a and Mob1b, these sgrnas were ligated to the lentiCRISPR v2 vector, respectively, the plasmid mixture was co-transfected with the packaging plasmid into HEK293T cells, virus packaging was performed, cell supernatants were collected at 24 and 48 hours and filtered to remove cell debris, mixed with 8 μ g/ml polybrene, and added to RAW264.7 cells. 48 hours after infection, resistant cells were selected using puromycin at 3. mu.g/ml. Cells transfected with lentiCRISPR v2 vector (Cas9) served as controls. The effect of intracellular Mob1 knockdown was confirmed using Western blot.

The over-expression of Mob1 protein was achieved using pLVX-3Flag plasmid, the full-length cDNA of Mob1a was cloned into pLVX-3Flag vector, and lentivirus infection and cell line selection were performed as described above. Cells transfected with pLVX-3Flag vector (pLVX) served as control. The expression level of Mob1 in cells was confirmed by Western blot and qPCR.

1.5 fluorescence Spectroscopy

The tryptophan, tyrosine and other residues in the protein can emit fluorescence to form inherent fluorescence of the protein; and the small molecules bound to the protein can change the microenvironment near the chromophoric residue, resulting in fluorescence quenching of the protein. The recombinant Mob1a protein was dissolved in 50mM PB (pH7.6) at a concentration of 0.1 mg/ml. Tuniloside B was added to the Mob1a protein solution and fluorescence spectra were recorded using a Biotek Synergy H1 fluorescence microplate reader. The excitation wavelength of the protein was set at 280nm, and the detection range of the emission wavelength was set at 300-450 nm.

1.6 circular dichroism

The absorption of left and right circularly polarized light by structures such as protein peptide bonds is different, resulting in a difference in polarization of a polarized light vector, resulting in circular dichroism of proteins, and the secondary structure of proteins can be measured by circular dichroism of proteins. Recombinant Mob1a protein was dissolved in 50mM PB (pH7.6) at a concentration of 0.1mg/ml, and angioside B was added to the Mob1a protein solution. A Chirascan circular dichroism instrument is used for detecting the circular dichroism spectrum of Mob1a protein, the wavelength is set to be 190-260nm, the detection path is 1mm, the scanning speed and the bandwidth are respectively fixed to be 0.5s and 1nm, each sample is detected for 3 times, and the base line is deducted for average treatment.

1.7 thermomigration experiments

The SYPRO Orange probe is capable of binding to the exposed hydrophobic region of the denatured protein and generating fluorescence, thereby monitoring the denaturation temperature of the protein, when hydrophobic residues are exposed during the denaturation of the protein. Recombinant Mob1a protein was dissolved in 50mM PB (pH7.6) at a concentration of 0.5mg/ml, and angioside B was added to the Mob1a protein solution, and a SYPRO Orange probe (1:500 dilution) was added. The dissolution profile of Mob1a protein was measured in a Roche Light Cycler 480 fluorescent quantitative PCR instrument, the temperature was set to increase from 37 ℃ to 99 ℃ (1 ℃/min), and the fluorescent signal was measured. The melting temperature Tm of the protein is determined by calculating the peak of the first derivative of the protein solubilization curve d (RFU)/dT.

For the intracellular protein thermomigration experiment, cells in a 10cm dish (about 80% confluency) were collected, resuspended in PBS containing protease inhibitor cocktail, lysed by repeated freeze-thawing in liquid nitrogen three times, an appropriate amount of lysate was added to tubiflorin B, and incubated for 30 minutes. The incubated lysates were heated at the indicated temperatures and centrifuged to remove heat-denatured insoluble proteins, and the remaining protein levels were detected using Western blot.

1.8 animal experiments

Male C57BL/6 mice (weighing 20-25g) at 6 to 8 weeks were used to construct an acute inflammatory injury model. Mice were housed under standard experimental conditions (day-night cycle 12 hours, 18-22 ℃) and fed standard pelleted feed and water. After 1 week of acclimation, mice were randomized into 6 groups (7 mice per group): a control group, a model group, a tubular flower glycoside B low dose group (10mg/kg), a tubular flower glycoside B medium dose group (20mg/kg), a tubular flower glycoside B high dose group (40mg/kg), and a positive control methylprednisolone group (Met, 2.5 mg/kg). Each treatment group was treated by administration (intraperitoneal injection) 3 days earlier, and the control group and the model group were given the same dose of physiological saline. After the administration, the model group and the treatment group were subjected to a molding treatment by intraperitoneal injection of 20mg/kg LPS, except for the control group. After 20 hours, all mice were sacrificed under anesthesia. Whole blood was collected and centrifuged at 3000g for 15 minutes to separate serum for biochemical analysis. Serum IL-1 beta, ALT levels, AST levels were detected using a commercial kit. Samples for histopathological examination were cut into small pieces, fixed in neutral formalin for 24 hours, dehydrated, embedded in paraffin, cut into 10 μm pieces, H & E stained, and photographed under a microscope.

Second, experimental results

2.1Mob1 protein can inhibit macrophage M1 type differentiation

As shown in fig. 1A, the protein level of Mob1 was significantly decreased in macrophage RAW264.7 induced by 10ng/ml LPS and 20ng/ml IFN- γ, while there was a significant increase in the protein level of Mob1 after administration of proteasome inhibitor MG132, indicating that Mob1 protein levels were significantly decreased upon macrophage differentiation to M1 type and that the ubiquitin-proteasome pathway was involved in degradation of Mob1 protein. The cell line with stably knocked-down Mob1 is constructed by using CRISPR/Cas9 and lentivirus transfection technology, as shown in FIG. 1B, the protein level of Mob1 is stably reduced in two cell lines of KD1 and KD2, and after the Mob1 is knocked down, the phosphorylation level of Yap is not obviously changed under the action of LPS and IFN-gamma. As shown in FIGS. 1C and 1D, the mRNA expression level of the inflammatory factor genes Il-1 beta and Il6 was significantly increased by Mob1 knocking down macrophages under the action of LPS and IFN-gamma. A Flag-Mob1 protein overexpression cell line is constructed by using a lentivirus transfection technology, as shown in figure 1E, Flag-Mob1 is obviously overexpressed in the cell line, and under the action of LPS and IFN-gamma, the mRNA expression levels of inflammatory factor genes Il-1 beta and Il6 are obviously reduced (figures 1F and 1G). The above results indicate that Mob1 protein is able to inhibit M1-type differentiation of macrophages, and that this effect is independent of Yap protein, an effector of Hippo signaling pathway.

2.2 Tulipside B is able to target Mob1 type protein, inhibiting its degradation in inflammatory states

As shown in FIG. 2A, angionoside B at 100. mu.g/ml had no significant effect on the viability of the macrophage RAW 264.7. As shown in FIG. 2B, angioside B was able to significantly increase the level of Mob1 in macrophages after LPS and IFN- γ treatment, but had no significant effect on the levels of other proteins of the Hippo signaling pathway. In addition, angioside B also did not affect the nuclear entry of Yap protein (fig. 2C). Further studies showed that tubular anthocyanin B did not affect the gene expression level of Mob1 in macrophages (fig. 2D), suggesting that tubular anthocyanin B acts by affecting post-transcriptional regulation of Mob1 protein. As shown in fig. 2E, by inhibiting protein synthesis by CHX, tubular flower glycoside B was found to prolong the half-life of Mob1 protein, suggesting that tubular flower glycoside B can stabilize the structure of Mob1, reducing its degradation. Protein fluorescence spectrum (fig. 2F), circular dichroism spectrum (fig. 2G) and thermal migration (fig. 2H and 2I) experiments show that the tubular floroside B can reduce the autofluorescence of Mob1a protein, change the content of alpha helix of protein, increase the thermal stability of protein, and confirm that tubular floroside B can be directly combined with Mob1 protein. The above results indicate that, in macrophages, Mob1 protein is able to act independently of Yap protein; the tubular flower glycoside B can be directly combined with Mob1 protein to inhibit the degradation of the tubular flower glycoside B.

2.3 Tunicaoside B has anti-inflammatory activity for inhibiting macrophage M1 type differentiation, and its anti-inflammatory action is closely related to Mob1 protein

The angioflorin B can obviously inhibit NO release of macrophage RAW264.7 after LPS and IFN-gamma treatment (figure 3A), reduce the expression level of various inflammatory genes (figures 3B-3E), and reduce the levels of M1 differentiation marker proteins iNOS and Cox2 (figure 3F), which indicates that the angioflorin B has obvious anti-inflammatory activity. The anti-inflammatory activity of tubular flower glycoside B was found to be greatly reduced in Mob1 knockdown cells using the Mob1 knockdown cell line (fig. 1B) (fig. 1C and 1D), suggesting that this anti-inflammatory activity of tubular flower glycoside B is closely related to its regulation of stability of Mob1 protein.

2.4 Tulisosine B can inhibit acute liver injury and lung injury of mice caused by LPS, and reduce inflammation secretion

Using the acute systemic inflammation model in mice created by LPS (20mg/ml) injection, it was found that after treatment with angionoside B, the level of inflammatory factor Il-1 β in mouse serum was significantly reduced (fig. 4A), in addition, serum liver injury indicators ALT and AST levels were significantly reduced compared to the model group (fig. 4B and 4C), and inflammatory injury of mouse liver and lung was alleviated as observed from pathological sections (fig. 4D and 4E).

In summary, the Mob1 protein is an important regulatory protein in macrophage M1 type differentiation, and the function is independent of the classical Hippo-Yap signaling pathway; the small molecular compound angionoside B can directly target Mob1 protein and inhibit the degradation of the Mob1 protein, thereby exerting anti-inflammatory activity.

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. Any equivalent modifications and substitutions to those skilled in the art are also 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.