Application of four-ingredient Tibetan cat milk soup in preparation of medicine for regulating bile acid metabolic disorder

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of four-ingredient Tibetan cat milk soup in preparation of a medicine for regulating bile acid metabolic disorder. By exploring the influence of the four-ingredient Tibetan cat milk soup on bile acid metabolism related to rheumatoid arthritis, the four-ingredient Tibetan cat milk soup has the advantages that the bile acid metabolism is effectively regulated and controlled by increasing the content ratio of free bile acid to combined bile acid and the content ratio of secondary bile acid to primary bile acid; the four-ingredient Tibetan cat milk soup has the effects of improving and protecting intestinal barriers and continuously regulating and controlling immune balance of the organism, provides a new method and a favorable basis for treating bile acid disorder, and also widens the application range of the four-ingredient Tibetan cat milk soup at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of a four-flavor Tibetan cat milk soup in preparing a medicine for regulating bile acid metabolism disorder. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory cell infiltration, abnormal synovial proliferation and bone erosion. During the occurrence and development of RA, bile acid metabolism disorders are prone to occur, resulting in immune imbalance, aggravating inflammatory reactions and bone destruction in the joints, and causing a vicious cycle of disease development.

[0003] Traditional drug treatments for RA mainly use glucocorticoids, nonsteroidal anti-inflammatory drugs or immunosuppressants, etc. These drugs can relieve inflammation and pain symptoms, but they have adverse reactions such as digestive tract damage and metabolic disorders, and are not suitable for long-term use. For example, methotrexate, as an immunosuppressant, can achieve the therapeutic effect of RA with long-term use. However, the inventors found that methotrexate may be able to improve RA in the early stage, but in the case of long-term use, due to the hepatotoxicity of the drug, the bile acid metabolism disorder caused by rheumatoid arthritis cannot be effectively controlled. In the long run, the bile acid metabolism disorder will inevitably recur.

[0004] Four-flavor Tibetan cat milk soup is a commonly used prescription in Tibetan medicine for the treatment of RA. It is recorded in the Tibetan medical classic "Four Medical Classics" and has been previously explored by the inventor for the treatment of RA. A full understanding of the interaction between Four-flavor Tibetan cat milk soup and bile acid metabolism is of great significance for its understanding and development. Summary of the invention

[0005] In order to solve the above-mentioned technical problems, the present invention provides the use of Four-flavor Tibetan Cat Milk Decoction in the preparation of drugs for regulating bile acid metabolism disorders, explores the effect of Four-flavor Tibetan Cat Milk Decoction on bile acid metabolism associated with rheumatoid arthritis, and provides new ideas for the development and utilization of Four-flavor Tibetan Cat Milk Decoction.

[0006] The technical solution of the present invention is as follows:

[0007] Application of Four-flavor Tibetan Cat Milk Decoction in the preparation of drugs for regulating bile acid metabolism disorders.

[0008] Preferably, the bile acid metabolism disorder refers to the bile acid metabolism disorder caused by rheumatoid arthritis.

[0009] Preferably, the regulation is related to at least one of the ratio of free bile acid to conjugated bile acid content and the ratio of secondary bile acid to primary bile acid content.

[0010] Preferably, said regulation is associated with regulating the activity of bile salt hydrolase.

[0011] Preferably, the regulation is related to pathological changes in joints.

[0012] Preferably, the regulation plays a role in regulating bile acid metabolism by increasing the ratio of free bile acid to conjugated bile acid content and the ratio of secondary bile acid to primary bile acid content.

[0013] Preferably, the regulation plays a role in regulating bile acid metabolism by increasing the activity of bile salt hydrolase.

[0014] Preferably, the regulation includes increasing the expression level of at least one of FXR and TGR5 bile acid receptor proteins in ileal tissue.

[0015] Preferably, the regulation includes increasing the expression level of at least one of IL-4, IL-10, Foxp3, and GATA3 proteins in colon tissue.

[0016] Preferably, the regulation comprises reducing at least one of the expression levels of IFN-γ, IL-17, ROR-γt and T-bet proteins in colon tissue.

[0017] The beneficial effects of the present invention are:

[0018] (1) Siwei Tibetan Cat Milk Decoction effectively regulates bile acid metabolism by regulating the ratio of free bile acid to conjugated bile acid, the ratio of secondary bile acid to primary bile acid, bile salt hydrolase activity, and the expression levels of bile acid receptors and specific proteins in colon tissue, thereby improving and protecting the intestinal barrier and continuously regulating the body's immune balance;

[0019] (2) Four-flavor Tibetan cat milk soup can be used to prepare drugs for regulating bile acid metabolism disorders. The discovery of this new use provides a new method and favorable basis for the treatment of bile acid disorders, and also broadens the application scope of Four-flavor Tibetan cat milk soup. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Effects of Four-flavor Tibetan Cat Milk Decoction on body weight, paw swelling and arthritis score in CIA rats, (A) body weight, (B) paw thickness and (C) arthritis score;

[0021] Figure 2 The effect of Four-flavor Tibetan Cat Milk Decoction on pathological changes of knee joint tissues in CIA rats;

[0022] Figure 3Pathological changes of ileum tissues of rats in each group, (A) H&E staining (B) Pathological scoring (C) Villus length (D) Crypt depth (E) Muscle layer length (F) Goblet cell number (G) Mucosal layer length;

[0023] Figure 4 The expression of tight junction proteins in ileum tissue of rats in each group;

[0024] Figure 5 The changes of bile acid levels in rats of each group, (A) total bile acid content (B) free bile acid content (C) conjugated bile acid content (D) ratio of free bile acid to conjugated bile acid content (E) secondary bile acid content (F) primary bile acid content (G) ratio of secondary bile acid to primary bile acid content;

[0025] Figure 6 PCA score diagram of bile acid in each group;

[0026] Figure 7 OPLS-DA score diagram of bile acid in rats of each group;

[0027] Figure 8 is the bile salt hydrolase activity of rats in each group;

[0028] Fig. 9 The expressions of FXR and TGR5 proteins and their mRNA in the ileum of rats in each group, (A) protein expression of FXR and TGR5 (B) quantitative analysis of FXR and TGR5 protein expression (C) mRNA expression of TGR5 (D) mRNA expression of FXR;

[0029] Fig.10 The expression of immune cytokines in colon tissue of rats in each group;

[0030] Fig.11 The expression of specific transcription factor proteins in colon tissue immune cells of rats in each group;

[0031] Fig.12 The mRNA expression of immune cell-specific transcription factors in the colon tissue of rats in each group: (A) ROR-γt mRNA expression level (B) Foxp3 mRNA expression level (C) T-bet mRNA expression level (D) GATA3 mRNA expression level;

[0032] Compared with Control, ## P<0.01; compared with the CIA group, * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0034] Example 1

[0035] 1. Experimental Animals

[0036] SPF grade SD rats, male, 6-8 weeks old, weighing 200±20g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The rats were housed in a sterile animal room and maintained under constant temperature (22±2°C), constant humidity (55±5%), and 12h light / 12h dark conditions. During this period, the rats were given free access to food and water to adapt to the environment for one week.

[0037] 2. Experimental Methods

[0038] 2.1 Preparation of the Four-flavor Tibetan Cat Milk Soup in the Examples of the Present Invention

[0039] Weigh the medicinal materials according to the mass ratio of Tibetan cat milk: Gentiana macrophylla: Berberis bark: Terminalia chebula = 12:10:6.5:3, mix and grind, and pass through a 20-mesh sieve to obtain a powder raw material;

[0040] The powdered raw material is added with water at a weight-to-volume ratio of 1:15, and reflux extraction is performed twice, each extraction time being 2 hours. The powdered raw material is filtered, and the filtrates are combined to obtain the four-flavor Tibetan cat milk soup.

[0041] 2.2 Establishment of animal model of rheumatoid arthritis

[0042] The pathological characteristics of collagen-induced arthritis model (CIA) are similar to those of human RA and can be used for immune mechanism research. In the present invention, the CIA rat model is selected as the RA animal model for research.

[0043] 2.2.1 Preparation of modeling reagent (type II collagen emulsion)

[0044] Prepare 2 mg / mL type II collagen solution. Use a homogenizer to mix and emulsify the collagen solution and incomplete Freund's adjuvant in an ice-water bath in equal volumes until the emulsion does not loosen when in contact with water. 2.2.2 Establishment of CIA rat model

[0045] Ten rats were randomly selected as the blank group (Control), and the remaining rats were used to establish the CIA model. A microinjection needle was used to intradermally inject 200 μL of the above modeling reagent at the base of the tail of the remaining rats for the first immunization, and the day was defined as day 0; a second booster immunization was performed three weeks later, and 100 μL of the modeling reagent was injected intradermally at the base of the tail. One week after the second immunization, the degree of joint inflammation was judged according to the redness, swelling, inflammation and limited activity of the rat's limbs, and the arthritis index (AI) was scored. The standard for successful model establishment was an AI score of >6 points, which was considered a CIA rat. The scoring criteria are shown in Table 1.

[0046] Table 1 Joint inflammation scoring criteria

[0047]

[0048] 2.2.3 Animal grouping and drug administration

[0049] The blank group was used as the control group, and the rats with successful modeling were randomly divided into 5 groups (n=10). That is, the model group (CIA), the low-dose group of Four-flavor Tibetan Cat Milk Decoction (L-SXD, 1417.5mg / kg / d), the medium-dose group of Four-flavor Tibetan Cat Milk Decoction (M-SXD, 2835mg / kg / d), the high-dose group of Four-flavor Tibetan Cat Milk Decoction (H-SXD, 5670mg / kg / d), and the methotrexate group (MTX, 0.9mg / kg, twice / week) were used as positive controls. The administration method was oral administration, and the Control and CIA groups were given equal amounts of normal saline, respectively, for a total of 4 weeks, and the living conditions of the rats in each group were observed every day.

[0050] 2.3 Index detection

[0051] 2.3.1 Observation and recording of arthritis symptoms in rats

[0052] Drug treatment began on the 28th day of the experiment. During the treatment, the body weight of the rats was recorded every 3 days and the diet and mental state of the rats were observed. The degree of swelling of the toe joints of each group of rats was measured with a vernier caliper, and the arthritis index was scored and recorded by three fixed experimenters.

[0053] 2.3.2 Specimen collection and processing

[0054] After the last administration, the rat body weight, toe swelling and AI score were recorded. Then, the fresh feces of rats in each group were collected by strict aseptic operation and placed in 1.5 mL cryotubes. After sealing, they were quickly placed in liquid nitrogen for quick freezing. After being completely frozen, the samples were transferred to a -80 °C low-temperature refrigerator for storage. They were later used for the determination of bile acid (BAs) targeted metabolomics.

[0055] Each group of rats was anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta of each group of rats using a blood collection needle. One part of the blood samples was used for subsequent flow cytometry experiments, and the other part was centrifuged and the supernatant was obtained to obtain serum samples, which were placed in a -80°C refrigerator for later use.

[0056] The knee joints of rats were taken, the bone tissues were separated, and then fixed with 4% paraformaldehyde and stored at room temperature for future use.

[0057] The rat ileum tissue was cut and divided into two parts. One part was placed in a 1.5 mL centrifuge tube and stored at -80°C; the other part was fixed with 4% paraformaldehyde solution and stored at room temperature for later use.

[0058] 2.3.3 Histopathological analysis of knee joint

[0059] Take the fixed knee bone tissue sample under "2.3.2", decalcify it with ethylenediaminetetraacetic acid (EDTA), dehydrate it with gradient ethanol solution after acupuncture without resistance, permeabilize it with xylene, then embed it in paraffin and cut it into paraffin slices. Then, stain it with hematoxylin and eosin (H&E). Finally, observe the slices under a microscope and take photos for record. Pathological scoring analysis was performed according to the scoring criteria, as shown in Table 2.

[0060] Table 2 H&E staining scoring criteria

[0061]

[0062] 2.3.4 Ileal histopathological analysis

[0063] The fixed ileum tissue was dehydrated, embedded, and sliced ​​before being stained with H&E, sealed with neutral gum, and finally observed under a microscope and photographed. The pathological score of the ileum tissue after H&E staining was performed, and the length of villi, crypt depth, muscle layer length, goblet cell number, and mucosal layer length of rat ileum tissue were analyzed with ImageJ software. The ileum tissue pathological scoring criteria are shown in Table 3.

[0064] Table 3 Scoring criteria for ileum tissue

[0065]

[0066] 2.3.5 Immunohistochemical staining of rat ileum tissue

[0067] Take the paraffin sections of ileum tissue under "2.3.4" and perform dewaxing and hydration treatments; then put them in a boiled sodium citrate buffer solution to restore antigens for 10 minutes, then return to room temperature, and repeat twice; put the sections after the above treatment in a 3% H2O2 solution to block the activity of endogenous peroxidase; after PBS washing, wipe the surface moisture of the sections, and add blocking solution to the sections for blocking treatment; wash away the blocking solution, add primary antibody overnight (dilution ratio of 1:200), wash the sections with PBS after the end, add the corresponding secondary antibody and incubate for 1 hour at a ratio of 1:100; after the incubation, rinse with PBS, add DAB colorimetric solution, wash with water, and then counterstain with hematoxylin, and then undergo differentiation, dehydration, sealing, etc. to obtain sections. The sections were placed under a microscope for observation and photographed. The positive expression of ileum immunohistochemical staining was analyzed with Image J software.

[0068] 2.3.6 Bile acid metabolism analysis

[0069] 2.3.6.1 Sample preparation

[0070] Weigh 25 mg of fecal samples from each group of rats in a homogenizer tube using a balance, add 1000 μL of pre-cooled extract containing internal standard to mix, the extract is prepared in the ratio of methanol: acetonitrile: water = 2:2:1, add a few small steel balls, and then place in a vortex instrument to mix for 30 seconds. Next, grind with a grinder for 4 minutes at a frequency of 35 Hz. Then perform ultrasonic treatment on an ice water bath for 5 minutes, repeat twice; pre-cool the centrifuge in advance, place the sample under low temperature conditions and let it stand for 1 hour, and then place it in a centrifuge for centrifugation at a speed of 12000 rpm for 15 minutes. After the centrifugation is completed, transfer the supernatant to another new EP tube and centrifuge again under the same conditions. Finally, take the supernatant into a sample injection bottle for subsequent analysis.

[0071] 2.3.6.2UHPLC-MS / MS detection method

[0072] (1) Chromatographic conditions

[0073] BAs were separated by ultra-high performance liquid chromatography (UHPLC), model Vanquish (Thermo Fisher Scientific), and the chromatographic column used was Waters ACQUITY UPLC BEH C18 (150×2.1mm, 1.7μm). The mobile phase A was 5mmol / L ammonium acetate aqueous solution, and the mobile phase B was acetonitrile solution. During the separation process, the temperature of the sample tray was set to 4°C, and the column oven temperature was set to 45°C. The injection volume was set to 1μL each time, and the flow rate was 1.0mL / min.

[0074] (2) Mass spectrometry

[0075] A high-resolution mass spectrometer was used for mass spectrometry analysis in parallel reaction monitoring (PRM) mode. The mass spectrometer model was Orbitrap Exploris 120, and the mode was negative ion mode. It was equipped with an electrospray ion source, and the parameters were set as follows: the spray voltage was +3500 / -3200V, the capillary temperature was set to 320°C, the sweep gas (N2) flow rate was set to 0, the sheath gas (N2) flow rate was set to 40, the auxiliary gas (N2) flow rate was 15, and the auxiliary gas (N2) temperature was 350°C.

[0076] 2.3.7 Bile salt hydrolase (BSH) assay

[0077] The contents of the rat ileum tissue were removed with sterile forceps, and then the activity of BSH in the feces of each group of rats was measured according to the operating method in the instructions of the enzyme-linked immunosorbent assay kit.

[0078] 2.3.8 Determination of FXR and TGR5 protein expression in ileum tissue

[0079] According to the Western blotting method, the expressions of BAs receptors FXR and TGR5 in the ileum tissue of rats in each group were detected.

[0080] 2.3.9 Determination of FXR and TGR5 gene expression in ileum tissue

[0081] According to the qRT-PCR method, the expression of BAs receptor FXR and TGR5 mRNA in the ileum tissue of rats in each group was detected. The primer sequences used in the experiment are shown in Table 4.

[0082] Table 4 qRT-PCR primer sequences

[0083]

[0084] 2.3.10 Immunohistochemical staining of rat colon tissue

[0085] Take the fixed colon tissue under "2.3.4", make paraffin sections, and then perform immunohistochemical staining according to the experimental method of "2.3.5". Finally, observe the obtained sections under a microscope and take photos for record. Use Image J software to analyze the positive expression of colon immunohistochemical staining.

[0086] 2.3.11 Detection of transcription factor protein expression in immune cells of rat colon tissue by western blot

[0087] The colon tissues were taken out from a -80°C refrigerator, and the expression of immune cell transcription factor proteins in the colon tissues of rats in each group was detected according to the Western blotting experimental method.

[0088] 2.3.12 qRT-PCR detection of transcription factor mRNA expression in immune cells of rat colon tissue

[0089] The colon tissue was taken out from a -80°C refrigerator, and the expression of immune cell transcription factor mRNA in the colon tissue of each group of rats was detected according to the qRT-PCR experimental method. The primer sequences used in the experiment are shown in Table 5.

[0090] Table 5 qRT-PCR primer sequences

[0091]

[0092] 2.3.13 Determination of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN)

[0093] The detection kits were used to determine the levels of ALT, AST, Cr and BUN in the serum of rats in each group.

[0094] 2.3.14 Statistical analysis

[0095] The images were analyzed using Image J software. GraphPad Prism 8.0 software was used for data statistics and analysis, and the measured results were expressed as mean ± standard deviation. The differences between the groups were statistically analyzed by T test, and P < 0.05 indicated that the difference was statistically significant.

[0096] 3. Experimental results

[0097] 3.1 Comparison of joint symptoms in rats of each group

[0098] Figure 1 The effect of four-flavor Tibetan cat milk soup on body weight, paw swelling and arthritis score of CIA rats (n=10). Figure 1As shown in A, before administration, there was no significant difference in the body weight of rats in each group (P>0.05). During the entire treatment process, the body weight of rats in each group showed a trend of gradual increase. Among them, the rats in the Control group had normal appetite and rapid weight gain during the treatment period. However, the rats in the CIA group had poor appetite, limited exercise, and relatively slow weight gain, which was significantly different from the Control group (P<0.01). On the 9th day of SXD treatment, compared with the CIA group, the body weight of rats in the H-SXD group increased significantly, which was statistically significant (P<0.01). On the 12th day of treatment, the appetite of rats in each treatment group began to recover, the limited exercise was relieved to a certain extent, and the body weight increased, which was significantly different from the CIA group (P<0.05 or P<0.01). This shows that the treatment of Four-flavor Tibetan Cat Milk Decoction can improve the pathological state of CIA rats.

[0099] like Figure 1 As shown in B, after the second immunization, the toes of rats in the CIA group were significantly swollen, which was significantly different from that in the Control group (P<0.01). Before the administration of SXD, there was no significant difference in the degree of paw swelling between rats in each treatment group and the CIA group (P>0.05). Starting from the 37th day, the treatment with Four-flavor Tibetan Cat Milk Decoction could significantly improve the swelling of rat toes, and the degree of improvement was dose-dependent.

[0100] like Figure 1 As shown in C, the joints of rats in the Control group were normal, and the AI ​​score was 0. Except for the Control group, the rats in the other groups showed obvious joint swelling, and the arthritis index increased significantly. After 4 weeks of administration, the joint swelling of rats in the SXD and MTX treatment groups was effectively relieved, and the arthritis index was significantly reduced, which was statistically significant compared with the CIA group (P<0.05 or P<0.01). Throughout the treatment process, the therapeutic effects of the H-SXD group and the MTX group were comparable.

[0101] 3.2 Comparison of pathological changes in the knee joints of rats in each group

[0102] Figure 2 The effect of four-flavor Tibetan cat milk soup on the pathological changes of knee joint tissues in CIA rats (n=10). Figure 2As shown in the results, the knee joint structure of rats in the Control group was normal, the synovial tissue did not have abnormal proliferation, there was no obvious inflammatory cell infiltration, and the surface of the articular cartilage was smooth and undamaged. However, the knee joints of rats in the CIA group showed obvious pathological structural changes, narrow joint cavity, rough joint surface, abnormal synovial proliferation accompanied by a large number of inflammatory cell infiltration, severe cartilage tissue destruction, and the difference in histopathological scores was statistically significant compared with the Control group (P<0.05 or P<0.01). After 28 days of drug intervention, the above pathological changes were alleviated to varying degrees compared with the CIA group, especially the H-SXD and MTX groups had the best treatment effects, the articular cartilage surface was smooth, there was no abnormal synovial proliferation, and the inflammatory cell infiltration was significantly reduced. The above results suggest that the Four-flavor Tibetan Cat Milk Decoction has a significant improvement effect on collagen-induced joint inflammation in CIA rats.

[0103] 3.3 Effect of ileum histopathological changes in CIA rats

[0104] Figure 3 The pathological changes of ileum tissues of rats in each group (n=10) are shown in Figure 2. Figure 3 A. The results showed that there was no pathological structural change in the ileum of rats in the Control group, while the ileum of rats in the CIA group was severely damaged, with shorter villi, damaged crypt structure, a large number of inflammatory factors infiltrating the muscle layer and mucosal layer, a reduced number of goblet cells, and thinning of the intestinal wall. Compared with the Control group, the histopathological score was significantly different (P<0.01). In contrast, the ileum of rats in the L-SXD group still showed irregular arrangement of epithelial cells, shorter villi, damaged mucosal layer, fewer goblet cells, and a small amount of inflammatory factor infiltration; the ileum of rats in the M-SXD group was somewhat relieved, the villi length increased, and the crypt structure was relatively intact; the ileum structure of rats in the L-SXD and MTX groups was significantly reduced, with only less villi damage, intact crypts, mucosal layers, and muscle layers, no obvious infiltration of inflammatory factors, and a significant increase in the number of goblet cells. Figure 3 The results showed that SXD had a certain protective effect on the intestinal morphological and structural damage in collagen-induced arthritis rats.

[0105] 3.4 Comparison of tight junction protein expression in ileum tissue of rats in each group

[0106] The present invention locates the expression of ZO-1, Occludin and Claudin-1 proteins in the intestine by immunohistochemistry and performs semi-quantitative analysis thereof. Figure 4 The expression of tight junction proteins in the ileum of rats in each group (n=10). Figure 4As shown in the figure, the ZO-1, Occludin and Claudin-1 proteins in the ileum tissue of rats in the Control group were brown, and their positive staining signals were strongly expressed and evenly distributed, indicating that the three tight junction proteins were abundant in the ileum tissue. In contrast, the brown positive staining of ZO-1, Occludin and Claudin-1 in the ileum tissue of rats in the CIA group was significantly reduced, unevenly distributed and interrupted. After SXD intervention, the positive signal expression was slightly weakened, but compared with the CIA group, the positive signal was significantly enhanced, and the effect of high-dose Chinese medicine was the most obvious. The positive expression of ZO-1, Occludin and Claudin-1 in the ileum tissue of rats in the MTX group showed an upward trend. Quantitative analysis and statistics using Image J software showed that the expressions of ZO-1, Occludin and Claudin-1 in the CIA group were significantly lower than those in the Control group (P<0.01), while the expressions of ZO-1, Occludin and Claudin-1 in the M-SXD, H-SXD and MTX groups were significantly higher than those in the CIA group (P<0.05 or P<0.01).

[0107] 3.5 Bile acid metabolism analysis

[0108] 3.5.1 Changes in fecal bile acid levels in rats of each group

[0109] The content and composition of BAs in rat feces were analyzed. Figure 5As shown (n=10). Compared with the Control group, the total bile acid (TBA) content of rats in the CIA group was significantly increased, and the difference was statistically significant (P<0.01), indicating that CIA rats had BAs metabolic disorder. After SXD drug treatment, the TBA content decreased, but the difference was not statistically significant (P>0.05). The results of the free bile acid content determination showed that there was no significant difference in the free bile acid content of rats in each group. Correspondingly, the conjugated bile acid content of rats in each group showed obvious changes. Among them, the conjugated bile acid content of rats in the CIA group increased significantly, which was statistically significant compared with the Control group (P<0.01). After treatment, the conjugated bile acid content of each drug group was significantly reduced, which was statistically significant compared with the CIA group (P<0.01). Among them, the conjugated bile acid content of the H-SXD group decreased significantly, which was close to that of the Control group. The ratio of free bile acid to conjugated bile acid also showed significant changes. The ratio of the CIA group was significantly reduced, and there was a significant difference compared with the Control group (P<0.05), while the ratios of each drug-treated group increased. Among them, the positive (MTX) group and the H-SXD group increased more significantly, and there was a significant difference compared with the CIA group (P<0.01). In the detection of primary bile acid and secondary bile acid content, the secondary bile acid content of the CIA group was significantly reduced, and there was a very significant difference compared with the Control group (P<0.01), while the secondary bile acid content of each drug-treated group increased. Compared with the CIA group, the MTX group had a significant difference (P<0.05), and the H-SXD group had a very significant difference (P<0.01). For primary bile acid, the content of primary bile acid in CIA group increased significantly, which was significantly different from that in Control group (P<0.01). The content of primary bile acid in each medication group decreased, among which the decrease in H-SXD group was particularly obvious, which was significantly different from that in CIA group (P<0.01). Figure 5The results also showed that the ratio of secondary bile acid to primary bile acid content also changed significantly. Compared with the Control group, the ratio of the two in the CIA group decreased significantly, with a very significant difference (P<0.01). The ratio of the two in each drug-treated group increased, among which the ratio of the H-SXD group increased most significantly, close to the Control group, and compared with the CIA group, there was a very significant difference (P<0.01). Under normal circumstances, bile acid metabolism disorders will lead to Th17 / Treg immune imbalance, macrophage polarization and inflammatory factor secretion, leading to the continuous development of inflammation in the body, and then the persistence and deterioration of RA symptoms. After treating CIA rats with Four-Ingredient Tibetan Cat Milk Decoction, the bile acid metabolism disorder was significantly improved. Two key indicators of bile acid metabolism: the ratio of free bile acid to conjugated bile acid content, and the ratio of secondary bile acid to primary bile acid content both increased. The improvement in the high-dose group was better, close to that in the blank group, indicating that Four-Ingredient Tibetan Cat Milk Decoction has a clear effect in improving the bile acid metabolism disorder caused by rheumatoid arthritis, thereby restoring immune balance and improving the clinical symptoms of RA.

[0110] 3.5.2 PCA analysis of fecal bile acid in rats of each group

[0111] After obtaining the fecal BAs concentration data of each group of rats, they were sorted and statistically analyzed using various multivariate models. Figure 6 This is the score diagram of the PCA analysis results of fecal BAs in each group tested by the institute (n=6). In the PCA score diagram, the horizontal axis PC1 represents the score of the first-ranked principal component, and the vertical axis PC2 represents the score of the second principal component. Each scattered point in the figure represents a sample tested, and its different colors represent that it is in different groups, and it can be found that the samples are basically within the 95% confidence interval. The study found that the sample points of the Control group and the CIA group were far apart, and the PCA score difference was significant, indicating that there was a significant difference in BAs between the Control group and the CIA group. After SXD drug treatment, although the sample points of each group were still far away from the Control group, they were closer to the Control group than the CIA group, indicating that after SXD treatment, the BAs changes were close to the Control group, with a certain callback effect, which could regulate BAs metabolic disorders to a certain extent. The regulatory effect of the MTX group was similar to that of the L-SXD group, and the regulatory effect on BAs changes was weaker.

[0112] 3.5.3 OPLS-DA analysis of fecal bile acid in rats of each group

[0113] Since the above two statistical analysis methods are affected by some related variables, it is difficult to achieve better visualization effects. Therefore, in order to make the analysis results more accurate, we used the OPLS-DA method to conduct an in-depth analysis of the experimental results. This method can filter out orthogonal variables that are not directly related to the categorical variables, thereby more accurately analyzing the differences between metabolite groups.

[0114] Figure 7 The following is a score chart of the OPLS-DA analysis results of fecal BAs in each group of the study (n=6). In the figure, the horizontal axis T score[1] represents the predicted principal component score, which shows the difference between the groups, and the vertical axis Orthogonal T score[1] represents the orthogonal component score, which shows the difference within the group. The study found that the sample points of the Control group and the CIA group were far apart, and the distinction was extremely significant. Almost all samples were within the 95% confidence interval, indicating that CIA rats had BAs metabolic disorders. Compared with the CIA group, the BAs metabolic profiles in the feces of rats in each drug treatment group showed a more obvious separation from the CIA group, and the separation degree between the H-SXD group and the CIA group was the highest. The results showed that SXD had a positive regulatory effect on BAs metabolism in CIA rats and could effectively reverse the situation of BAs metabolic disorders.

[0115] 3.5.4 Analysis of bile salt hydrolase activity in rats of each group

[0116] Bile salt hydrolase (BSH) is a type of microbial enzyme secreted by intestinal flora, which plays an important role in BAs metabolism. It not only converts bile salts into deaminocholic acid by hydrolyzing amino acids (such as glycine or taurine) in bile salts, but also affects the metabolism of BAs by generating secondary BAs. The ileum is one of the key sites of BAs circulation, involving the absorption, recycling and transformation of BAs. Its BSH activity is as follows: Figure 8 As shown in the results (n=10), compared with the Control group, the BSH activity in the ileum of rats in the CIA group was significantly reduced (P<0.01), resulting in a decrease in the proportion of secondary BAs and free BAs, and the reabsorption of BAs was inhibited. Compared with the CIA group, the BSH activity in the ileum of rats in the SXD group was significantly increased, and the difference was statistically significant (P<0.01 or P<0.05). The results showed that SXD regulates the activity of BSH, thereby affecting the metabolic pathway of BAs, and helps maintain the dynamic balance of BAs in the body.

[0117] 3.5.5 Expression of intestinal bile acid receptors in rats of each group

[0118] FXR and TGR5 are important receptors of BAs, which not only play a key role in regulating the metabolic process of BAs, but also can regulate the inflammatory response of the immune system through different pathways. Therefore, the present invention used Western blotting and qRT-PCR techniques to detect the expression of BAs receptors in the ileum tissue of each group of rats. Fig. 9 The expression of FXR and TGR5 proteins and their mRNA in the ileum of rats in each group (n=10). Fig. 9 As shown in the figure, compared with the Control group, the FXR and TGR5 protein expression in the ileum of rats in the CIA group was significantly decreased, and their mRNA levels were also significantly decreased, and the differences were statistically significant (P<0.01 or P<0.05). Compared with the CIA group, the FXR and TGR5 protein expression and mRNA expression in the ileum of rats in the SXD group showed a significant upward trend, among which the FXR and TGR5 protein and mRNA expression in the M-SXD and H-SXD groups increased more significantly, and the differences were statistically significant (P<0.01 or P<0.05).

[0119] 3.6 Expression of immune cytokines in colon tissue of rats in each group

[0120] In the pathogenesis of RA, immune cells, especially T cells, play a key role in the initiation and maintenance of inflammation and the imbalance of the immune system. There are abundant microorganisms and immune cells in the colon tissue. Intestinal immune dysfunction may induce systemic immune inflammatory response and aggravate the development of RA. The study detected the expression of immune cytokines in the colon tissue of rats in each group (n = 10). Fig.10 As shown. Compared with the Control group, the protein expressions of IFN-γ and IL-17 in the colon tissue of rats in the CIA model group were significantly increased (P<0.01), while the protein expressions of IL-4 and IL-10 were significantly decreased (P<0.01), indicating that immune imbalance occurred in the colon tissue of CIA rats. Compared with the CIA group, the protein expressions of IFN-γ and IL-17 immune factors in the colon of rats in the SXD group gradually decreased, while the protein expressions of IL-4 and IL-10 immune factors gradually increased, among which the increases in the M-SXD and H-SXD groups were more significant, and the differences were statistically significant (P<0.05 or P<0.01).

[0121] 3.7 Expression of transcription factors in immune cells of colon tissue of rats in each group

[0122] ROR-γt, Foxp3, T-bet and GATA3 are key transcription factors of immune cells, regulating the development and function of different types of immune cells, and are crucial to the homeostasis of the immune system. Specifically, ROR-γt and T-bet regulate Th17 cells and Th1 cells, respectively, to secrete pro-inflammatory factors, while Foxp3 and GATA3 regulate Treg and Th2 cells, respectively, to secrete anti-inflammatory factors. Fig.11 The expression of specific transcription factor proteins in colon tissue immune cells of rats in each group (n=10). Fig.11 As shown in the figure, compared with the Control, the expression levels of ROR-γt and T-bet proteins in the colon tissue of rats in the CIA group were significantly increased (P<0.01), and the expression levels of Foxp3 and GATA3 proteins were significantly decreased (P<0.01), indicating that the differentiation ratios of Th17 / Treg and Th1 / Th2 cells in the colon tissue of CIA rats were imbalanced. Compared with the CIA group, after drug treatment, the expression levels of ROR-γt and T-bet proteins in the colon tissue of rats in the SXD group were significantly decreased (P<0.05 or P<0.01), and the expression levels of Foxp3 and GATA3 proteins were significantly increased (P<0.05 or P<0.01), and the differences were statistically significant.

[0123] To further study the effect of SXD on specific transcription factors of immune cells in colon tissue of CIA rats, the mRNA levels of the above four transcription factors were detected by qRT-PCR (n=10). Fig.12 As shown in the results, compared with the Control group, the mRNA level of ROR-γt in the colon tissue of CIA rats was significantly increased (P<0.01), the mRNA level of Foxp3 was significantly decreased (P<0.01), the mRNA level of T-bet was significantly increased (P<0.01), and the mRNA level of GATA3 was significantly decreased (P<0.01). After SXD treatment, the mRNA level of ROR-γt in the colon tissue of rats in each group was significantly decreased (P<0.01), the mRNA level of Foxp3 was significantly increased (P<0.01), the mRNA level of T-bet was significantly decreased (P<0.01), and the mRNA level of GATA3 was significantly increased (P<0.01), and the differences were statistically significant. The results of the mRNA level study were consistent with the results of its protein expression level.

[0124] 3.8 ALT, AST, Cr and BUN levels in serum of rats in each group

[0125] ALT and AST are two important indicators for evaluating liver function. If the ALT and AST levels in serum increase, it means that the liver function is impaired; Cr and BUN are important indicators for evaluating kidney function. If the levels of these two indicators in serum increase, it means that the kidney function is impaired. The results in Table 6 show that compared with the Control group, the levels of ALT, AST, Cr, and BUN in the serum of rats in the MTX group increased significantly, which was statistically significant (P<0.05 or P<0.01), while the four indicators in the CIA group and the SXD dose groups did not show significant fluctuations, and there was no significant difference compared with the Control group. Therefore, long-term administration of MTX is prone to liver and kidney damage, indicating that it has certain liver and kidney toxicity; SXD does not have these safety issues.

[0126] Table 6 ALT, AST, Cr and BUN levels in serum of rats in each group

[0127]

[0128] The collagen-induced arthritis model (CIA) is one of the commonly used animal models for studying RA. This model can induce the animal body to produce an autoimmune response against type II collagen in articular cartilage by immunizing animals with heterologous type II collagen, resulting in rheumatoid arthritis symptoms such as joint inflammation, cartilage destruction, bone resorption, and pannus formation. The present invention adopts this animal model, and the model group animals show obvious joint pathological changes, accompanied by intestinal inflammatory pathological changes and intestinal barrier damage; moreover, the model animals also show bile acid metabolism disorders, such as: the ratio of free bile acid to bound bile acid content, the ratio of secondary bile acid to primary bile acid content are significantly decreased, the activity of bile salt hydrolase decreases, the expression of bile acid receptors such as FXR and TGR5 in ileum tissue decreases, the expression of proteins such as IFN-γ and IL-17 in colon tissue increases significantly, the protein expression of IL-4 and IL-10 in colon tissue decreases significantly, the expression level of ROR-γt and T-bet protein in colon tissue increases significantly, and the expression level of Foxp3 and GATA3 protein decreases significantly, indicating that the Th17 / Treg and Th1 / Th2 cell differentiation ratios in the model rat body are imbalanced. These pathological manifestations show that the model rats have problems with disorder in bile acid metabolism-immune balance, which leads to further aggravation of systemic inflammatory symptoms. The experimental results of the present invention show that the addition of four Tibetan cat milk soup and the increase in dosage can effectively regulate the bile acid metabolism disorder of CIA model rats, improve the ratio of free bile acid to conjugated bile acid content, the ratio of secondary bile acid to primary bile acid content, increase the activity of bile salt hydrolase, increase the expression of bile acid receptors such as FXR and TGR5 in ileum tissue, significantly reduce the expression of IFN-γ, IL-17 and other proteins in colon tissue, significantly increase the protein expression of IL-4 and IL-10 in colon tissue, significantly reduce the expression level of ROR-γt and T-bet protein in colon tissue, and significantly increase the expression level of Foxp3 and GATA3 protein, showing a significant effect of repairing the balance of bile acid metabolism in the intestine. On this basis, the addition of four Tibetan cat milk soup and the increase in dosage can effectively repair the intestinal barrier, improve the intestinal microecology, regulate the body's immune balance, inhibit the continuous occurrence and development of inflammation, thereby blocking the vicious cycle of RA inflammation and immune imbalance, and comprehensively improving RA symptoms.

[0129] Methotrexate was used as a positive control drug. Although it also has a certain improvement effect on bile acid metabolism disorders, the analysis showed that the regulatory effect of methotrexate on bile acid changes was relatively limited and did not reach the effect of the high-dose group of Four-flavor Tibetan Cat Milk Decoction. In addition, methotrexate has high hepatotoxicity and kidney toxicity, and it is easy to cause liver and kidney damage. Therefore, methotrexate is not suitable for bile acid metabolism disorders caused by RA.

[0130] In summary, the addition of four-flavor Tibetan cat milk soup can effectively regulate the bile acid disorder caused by rheumatoid arthritis, thereby regulating the body's immunity, inhibiting the continuous development of RA, and improving the intestinal barrier damage caused by RA.

Claims

1. Application of Four-flavor Tibetan Cat Milk Decoction in the preparation of drugs for regulating bile acid metabolism disorders.

2. The use according to claim 1, characterized in that The bile acid metabolism disorder refers to the bile acid metabolism disorder caused by rheumatoid arthritis.

3. The use according to claim 1, characterized in that The regulation is related to at least one of the ratio of free bile acid to conjugated bile acid content and the ratio of secondary bile acid to primary bile acid content.

4. The use according to claim 3, characterized in that The regulation plays a role in regulating bile acid metabolism by increasing the ratio of free bile acid to conjugated bile acid content and the ratio of secondary bile acid to primary bile acid content.

5. The use according to claim 1, characterized in that The regulation is related to repairing intestinal pathological changes and intestinal barrier damage.

6. The use according to claim 1, characterized in that The regulation is associated with regulating the activity of bile salt hydrolase.

7. The use according to claim 1, characterized in that The regulation plays a role in regulating bile acid metabolism by increasing the activity of bile salt hydrolase.

8. The use according to claim 1, characterized in that The regulation includes increasing the expression level of at least one of FXR and TGR5 bile acid receptor proteins in ileal tissue.

9. The use according to claim 1, characterized in that The regulation includes increasing the expression level of at least one of IL-4, IL-10, Foxp3, and GATA3 proteins in colon tissue.

10. The use according to claim 1, characterized in that The regulation includes reducing at least one of the expression levels of IFN-γ, IL-17, ROR-γt and T-bet proteins in colon tissue.

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