Application of xanthoceras sorbifolia 4-flavor decoction in anti-inflammation and detumescence of RA rats

Abstract

The invention provides an application of xanthoceras sorbifolia 4-flavor decoction in anti-inflammation and detumescence of RA rats. According to the present invention, the determination is performed according to the comparison of the toe and ankle swelling degree and the AI score of each group of rats at different time, the pathological change of the ankle joint of the rats, and the experimental results of the inhibition conditions of the RF, the ASO, the CRP, the TNF-a, the IL-6 and the MMP-9 factors in the serum of the rats; the xanthoceras sorbifolia-4-flavor decoction can significantly inhibit the increase of RF, CRP, TNF-a and IL-6 in serum. Wherein the ankle joints of the rats in the normal group do not have definite inflammatory cell infiltration and vascular nebula generation; model group rats find out synovial growth, nebula generation and large-scale inflammatory cell infiltration, and CIA rat ankle joint in-vivo observation finds out; the positive group still has synovial hyperplasia and inflammatory cells; in low dose and medium dose administration, only slight synovial hyperplasia, formation of a small amount of small blood vessels and generation of inflammatory cells exist, and in a high dose group, the pathological structure that the synovial hyperplasia is reduced and the inflammatory cells are reduced is improved to a certain extent.

Description

Technical Field

[0001] The invention relates to the field of Mongolian medicine Xanthoceras sorbifolia-4-flavor decoction, and in particular to application of Xanthoceras sorbifolia-4-flavor decoction in anti-inflammatory and detumescent effects in RA rats. Background Art

[0002] Rheumatoid arthritis (RA) is a combination of diseases. A chronic, debilitating systemic inflammation. Rheumatoid arthritis is highly disabling due to inflammation, and existing drugs cannot fully meet the needs of patients.

[0003] The four-flavor soup of Xanthoceras sorbifolia is composed of Xanthoceras sorbifolia, Terminalia chebula, Gentiana macrophylla, and Phellodendron amurense. The medicinal parts are the dried wood of the stems or branches, the dried mature fruits, the dried roots, and the dried bark. In the "Selection of Common Prescriptions of Mongolian Medicine" published in 1975, it is recorded that the compatibility ratio of Xanthoceras sorbifolia is 5 qian, Terminalia chebula 2 qian, Gentiana macrophylla root 2 qian, and Phellodendron amurense is decocted in water and taken warm. No relevant research on this prescription has been found in modern literature reports, but the research on single herbs Xanthoceras sorbifolia, Terminalia chebula, Gentiana macrophylla, and Phellodendron amurense is relatively rich, mainly in chemistry, pharmacology, and clinical research, which provides valuable ideas for clinical and basic scientific research.

[0004] The polyphenolic compounds and terpenoids in the chebula fruit in the 4-flavor soup have a good anti-rheumatoid arthritis activity. Two different doses of the polyphenol component chebula fruit (80, 160 mg kg -1 ) can significantly inhibit the expression of TNF-α and IL-6 pro-inflammatory factors, cyclooxygenase-2 and matrix metalloproteinase-3 in CIA mice after continuous administration, showing effective anti-inflammatory activity. After continuous administration of Terminalia chebula extract for 7 days, it can significantly reduce the expression of MCP-1 in the serum and joint tissues of model animals, thereby exerting anti-inflammatory activity. How to effectively extract the effective ingredients of the anti-inflammatory and detumescent effect of the Four-flavor Decoction on RA rats is a technical problem to be solved at present. Summary of the invention

[0005] The invention aims to provide the application of Xanthoceras sorbifolia-4-flavor decoction in the anti-inflammatory and detumescent effects on RA rats.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention relates to the application of Xanthoceras sorbifolia-4-flavor decoction in the anti-inflammatory and detumescent effect on RA rats, and the anti-inflammatory and detumescent effect on RA rats is determined by comparing the swelling degree of toes and ankles and AI scores of rats in different groups at different times, the pathological changes of the ankle joints of rats, and the inhibition of RF, ASO, CRP, TNF-a, IL-6 and MMP-9 factors in the serum of rats;

[0008] The specific steps are:

[0009] Step 1, Preparation:

[0010] (1.1) Preparation of drugs and reagents: Wenguanmu-4-flavor decoction (Sendeng-4), referred to as WGM-4 (SD 4), was provided by Horqin Left Middle Banner Mongolian Hospital; Tripterygium wilfordii glycosides tablets (Positive) were provided by Liaoning Xinshanyuan Pharmaceutical Co., Ltd.; Bovine Type II collagen (10 mg, 2 mg / ml, 5 ml; 20022); Complete Freund's Adjuvant (Incomplete Freund's Adjuvant, 5 ml; Sendeng-4 (SD-4) Wenguanmu-4-flavor decoction, referred to as WGM-4 (SD-4), was provided by Horqin Left Middle Banner Mongolian Hospital; Tripterygium wilfordii glycosides tablets (Positive) were provided by Liaoning Xinshanyuan Pharmaceutical Co., Ltd.; Bovine Type II collagen (10 mg, 2 mg / ml, 5 ml, 20022) and Complete Freund's Adjuvant (Incomplete Freund's Adjuvant, 5 ml; Freund's Adjuvant, 5ml, 7002, were all provided by Beijing Bolide Biological Company. Anhydrous ethanol (100092683), xylene (10023418) and neutral gum (10004160) were provided by Sinopharm Biochemical Company; enzyme-linked immunosorbent assay (ELISA) for rat anti-streptolysin O (ASO), rat C-reactive gene (CRP), rat rheumatoid factor (RF), rat interleukin 6 (IL-6), rat matrix metalloproteinase 9 (MMP-9), rat tumor necrosis factor a (TNF-a) were provided by Jiangsu Jingmei Biotechnology Company. HE staining solution, separation solution and blue night, including Jinkelong; staining solution (g1005) and separation solution (Jinkelong;), as well as ethylene glycol ether acetate (E808814-2.5L), were all provided by MacLean Company.

[0011] (1.2) Preparation of instruments: Midea Electric Appliances provided the intelligent decoction pot, Tianjin Leiborui Instrument Machinery Equipment Co., Ltd. provided it; Nikon Eclipse E100 oven, provided by Nikon of Japan; Nikon Eclipse e100 upright optical photography microscope, provided by Nikon of Japan; Nikon DS-U3 imaging system, provided by Thermo Fisher Scientific Group; Vario-skan LUX multifunctional microplate reader, provided by Nikon Group of Japan; DS-U3 microscope, low-speed centrifuge and high-speed refrigerated centrifuge provided by Nikon Group.

[0012] (1.3) Preparation of experimental animals:

[0013] The number of Wister rats provided by Liaoning Changsheng Biotechnology Co., Ltd. reached 72, including 24 model rats and 48 formal experimental rats. The experimental process strictly followed the 3R principle and complied with the norms of animal ethics.

[0014] Step 2, preparation of drug

[0015] (2.1) Sample preparation

[0016] The 4-flavor decoction of Wenguanmu was purchased from the Mongolian Hospital of Horqin Left Middle Banner. It was extracted according to the extraction method and then decocted in water (the optimal process conditions of Wenguanmu-4-flavor decoction are decocting time of 35 minutes, adding water of 40 times, and soaking time of 30 minutes) to make an extract. The Tripterygium wilfordii polyglycoside tablets produced by Liaoning Xinshanyuan Pharmaceutical Co., Ltd. need to be ground into powder before taking, and then stirred evenly with warm water.

[0017] (2.2) Preparation of type II collagen emulsifier

[0018] According to the requirements for emulsion preparation, a volume (2.5 mL) of incomplete adjuvant was mixed in a 1:1 ratio, placed in a syringe, and then a collagen solvent (final content 2 mg / mL) in a 0.05 M acetic acid aqueous solution was added, and an electric high-speed stirrer (with a blade with a diameter of 5 mm or less) was used to stir at a low speed, and then the collagen solvent was dripped in. The emulsion was continuously stirred in an ice bath until a drop of the emulsion was dripped into a beaker of water and the emulsion was found to solidify into a mass at the bottom of the water and would not separate, which proved that the prepared emulsion was stable and the preparation was completed for use.

[0019] (2.3) Serum and tissue collection Serum:

[0020] Collect the blood in a 37°C water bath (or room temperature) and let it stand for 1 hour to allow coagulation and stratification. Then, transfer the blood to a clean centrifuge tube, centrifuge at 3000rpm for 10 minutes at room temperature, and then centrifuge at 12000rpm for 10 minutes at 4°C. Remove the supernatant and divide it into 1.5mL centrifuge tubes, which can be frozen at -80°C for later use. Toes: The collected toes are placed in 4% tissue cell fixative and stored in the dark. Toes: The collected toes are placed in 4% tissue cell fixative and stored in the dark.

[0021] Step 3: Establishment of collagen-induced arthritis model in rats

[0022] According to the CIA model, bovine type II collagen can be emulsified with complete Freund's adjuvant and sterilized at the base of the rat's tail using a 75% ethanol cotton ball. The mixed emulsion is then injected subcutaneously at a ratio of 0.2 mL / rat on day 0 of the primary immunization. Seven days later, 0.15 ml of the emulsion is injected subcutaneously at the base of the tail of each rat to boost immunization. During the injection, the needle should be inserted 2 cm from the base of the tail until the needle tip is 0.5 cm from the base of the tail, and make sure to wipe completely after each injection to avoid leakage of the emulsion. The needle should be inserted obliquely upward along the position of the rat's tail, and the rat should be injected 3 cm from the base of the tail during the second immunization to enhance immunity. 2.3 Treatment and evaluation of collagen arthritis.

[0023] Step 4, Test

[0024] (4.1) Animal grouping and drug administration

[0025] According to the clinical equivalent dose commonly used in 70kg adults, the dosing group was divided into three groups: low, medium and high, with the dosage of each group being 0.0475g / kg, 0.1425g / kg and 0.285g / kg respectively. These drugs are all from the extract of the 4-flavor decoction of the crown wood. The positive group was the Tripterygium wilfordii polyglycoside tablets, with a dosage of 0.009g / kg; in the normal group and the model group, the same volume of normal saline was taken every day for 21 consecutive days. The degree of swelling of the left hind ankle joint of each rat was scored and recorded every 7 days until the end of the experiment.

[0026] (4.2) Measurement of rat paw swelling

[0027] In this experiment, the thickness of the left / right foot was measured with a vernier caliper. The model was measured once before immunization and then once every 7 days, and the degree of joint swelling was calculated.

[0028] (4.3) Clinical scoring and assessment of arthritis

[0029] In this experiment, a 0-4 joint scoring method was used to measure the severity of swelling in the main joints of the hind limbs. 0 points indicate health (no swelling or redness), 1 point indicates mild redness or swelling of the ankle and wrist joints, 2 points indicate moderate redness or swelling, 3 points indicate severe redness or swelling of the paws (including the fingertips), and 4 points indicate the most severe inflammation of the limbs (including multiple major joints), with significant restrictions on movement. After the second immunization, daily records will be recorded and assessments will be conducted every 7 days. Finally, the sum of the scores of the rat's two feet will be used as the score for each joint.

[0030] Step 5, Enzyme-linked Immunosorbent Assay (ELISA)

[0031] The serum obtained in the above step was taken out from the -80° refrigerator, stored at room temperature for 20-30 minutes, and then centrifuged for 15 minutes (2000 rpm) to collect the serum supernatant. According to the instructions of the ELISA kit, the absorbance was tested using an enzyme marker to determine the content of RF, ASO, CRP, IL-6, MMP-9 and TNF-a cytokines.

[0032] Step 6, histopathological HE staining

[0033] The sections were de-molded to water state in order with ethylene glycol ether acetate (Ⅰ) for 6h, 37℃, 37℃, 37℃, room temperature for 10-15min, room temperature for 10-15min, 100% Ⅰ ethanol for 10min, 100% Ⅱ ethanol for 10min, 95% ethanol for 10min, and 90% ethanol for 10min to complete the de-molding process. Soak in 80% ethanol for 10 minutes and then rinse with water. Stain the sections in hematoxylin stain for 8-10min, then rinse with water, differentiate with differentiation solution, and finally return to blue with blue solution to complete hematoxylin staining. Soak the sections in 85% and 95% gradient ethanol for 5 minutes, respectively, and then wash them in eosin stain for 8-10min to obtain the best dye effect. The slices were placed in anhydrous ethanol II for 5 min, II for 5 min, III for 5 min, xylene I for 5 min and xylene II for 5 min respectively. After a 5-minute washing process, the slices became clear and transparent, and then sealed with neutral gum.

[0034] The present invention has the following advantages:

[0035] (1) The anti-inflammatory and detumescent effect of the 4-flavor decoction of the present invention on RA rats is based on the principle that 48 Wistar rats were divided into a normal control group, a CIA (collageninduced arthritis) model group, a positive group (tripterygium wilfordii polyglycosides tablets), and a drug-administered group (4-flavor decoction extract of the 4-flavor decoction) of large, medium, and small doses. The experimental period was 21 days. The ELISA (Enzyme Linked Immunosorbent Assay) method was used to determine the levels of TNF-a (Tumor Necrosis Factor-a), IL-6 (Interleukin-6), RF (Radio Frequency), MMP-9 (Recombinant Human Matrixmetalloproteinase-9), CRP (C-reactive protein), and ASO (American Standards Organizat ion) in the serum of each group; and the HE staining method was used to observe the effects of each group of drugs on the pathological morphology of the toes of the rats in the group.

[0036] (2) The Xanthoceras sorbifolia-4-flavor decoction of the present invention can significantly reduce synovial hyperplasia and the number of inflammatory cells, thereby effectively improving the pathological changes of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a demonstration diagram of the in vivo experiment;

[0038] Figure 2 This is a comparison of the swelling of the toes and ankles of both hind limbs of rats;

[0039] Figure 3 It is a comparison chart of the swelling degree of toes and ankles and AI scores of rats in each group at different times;

[0040] Figure 4 It is the effect diagram of pathological changes in the ankle joint of rats;

[0041] Figure 5 This is a graph showing the effect of the Four-flavor Decoction of the Xanthoceras sorbifolia on inhibiting RF, ASO, CRP, TNF-a, IL-6, and MMP-9 in serum. DETAILED DESCRIPTION

[0042] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0043] Example

[0044] This example relates to the application of the 4-flavor decoction for anti-inflammatory and detumescent effects in RA rats. The decoction is judged to have anti-inflammatory and detumescent effects on RA rats by comparing the swelling degree of toes and ankles and AI scores of rats in each group at different times, pathological changes in the ankle joints of rats, and the inhibition of RF, ASO, CRP, TNF-a, IL-6, and MMP-9 factors in the serum of rats. Figure 1 As shown, the specific steps are:

[0045] Step 1, Preparation:

[0046] (1.1) Preparation of drugs and reagents: Wenguanmu-4-flavor decoction (Sendeng-4), referred to as WGM-4 (SD 4), was provided by Horqin Left Middle Banner Mongolian Hospital; Tripterygium wilfordii glycosides tablets (Positive) were provided by Liaoning Xinshanyuan Pharmaceutical Co., Ltd.; Bovine Type II collagen (10 mg, 2 mg / ml, 5 ml; 20022); Complete Freund's Adjuvant (Incomplete Freund's Adjuvant, 5 ml; Sendeng-4 (SD-4) Wenguanmu-4-flavor decoction, referred to as WGM-4 (SD-4), was provided by Horqin Left Middle Banner Mongolian Hospital; Tripterygium wilfordii glycosides tablets (Positive) were provided by Liaoning Xinshanyuan Pharmaceutical Co., Ltd.; Bovine Type II collagen (10 mg, 2 mg / ml, 5 ml, 20022) and Complete Freund's Adjuvant (Incomplete Freund's Adjuvant, 5 ml; Freund's Adjuvant, 5ml, 7002, were all provided by Beijing Bolide Biological Company. Anhydrous ethanol (100092683), xylene (10023418) and neutral gum (10004160) were provided by Sinopharm Biochemical Company; enzyme-linked immunosorbent assay (ELISA) for rat anti-streptolysin O (ASO), rat C-reactive gene (CRP), rat rheumatoid factor (RF), rat interleukin 6 (IL-6), rat matrix metalloproteinase 9 (MMP-9), rat tumor necrosis factor a (TNF-a) were provided by Jiangsu Jingmei Biotechnology Company. HE staining solution, separation solution and blue night, including Jinkelong; staining solution (g1005) and separation solution (Jinkelong;), as well as ethylene glycol ether acetate (E808814-2.5L), were all provided by MacLean Company.

[0047] (1.2) Preparation of instruments: Midea Electric Appliances provided the intelligent decoction pot, Tianjin Leiborui Instrument Machinery Equipment Co., Ltd. provided it; Nikon Eclipse E100 oven, provided by Nikon of Japan; Nikon Eclipse e100 upright optical photography microscope, provided by Nikon of Japan; Nikon DS-U3 imaging system, provided by Thermo Fisher Scientific Group; Vario-skan LUX multifunctional microplate reader, provided by Nikon Group of Japan; DS-U3 microscope, low-speed centrifuge and high-speed refrigerated centrifuge provided by Nikon Group.

[0048] (1.3) Preparation of experimental animals:

[0049] The number of Wister rats provided by Liaoning Changsheng Biotechnology Co., Ltd. reached 72, including 24 model rats and 48 formal experimental rats. The experimental process strictly followed the 3R principle and complied with the norms of animal ethics.

[0050] Step 2, preparation of drug

[0051] (2.1) Sample preparation

[0052] The 4-flavor decoction of Wenguanmu was purchased from the Mongolian Hospital of Horqin Left Middle Banner. It was extracted according to the extraction method and then decocted in water (the optimal process conditions of Wenguanmu-4-flavor decoction are decocting time of 35 minutes, adding water of 40 times, and soaking time of 30 minutes) to make an extract. The Tripterygium wilfordii polyglycoside tablets produced by Liaoning Xinshanyuan Pharmaceutical Co., Ltd. need to be ground into powder before taking, and then stirred evenly with warm water.

[0053] (2.2) Preparation of type II collagen emulsifier

[0054] According to the requirements for emulsion preparation, a volume (2.5 mL) of incomplete adjuvant was mixed in a 1:1 ratio, placed in a syringe, and then a collagen solvent (final content 2 mg / mL) in a 0.05 M acetic acid aqueous solution was added, and an electric high-speed stirrer (with a blade with a diameter of 5 mm or less) was used to stir at a low speed, and then the collagen solvent was dripped in. The emulsion was continuously stirred in an ice bath until a drop of the emulsion was dripped into a beaker of water and the emulsion was found to solidify into a mass at the bottom of the water and would not separate, which proved that the prepared emulsion was stable and the preparation was completed for use.

[0055] (2.3) Serum and tissue collection Serum:

[0056] Collect the blood in a 37°C water bath (or room temperature) and let it stand for 1 hour to allow coagulation and stratification. Then, transfer the blood to a clean centrifuge tube, centrifuge at 3000rpm for 10 minutes at room temperature, and then centrifuge at 12000rpm for 10 minutes at 4°C. Remove the supernatant and divide it into 1.5mL centrifuge tubes, which can be frozen at -80°C for later use. Toes: The collected toes are placed in 4% tissue cell fixative and stored in the dark. Toes: The collected toes are placed in 4% tissue cell fixative and stored in the dark.

[0057] Step 3: Establishment of collagen-induced arthritis model in rats

[0058] According to the CIA model, bovine type II collagen can be emulsified with complete Freund's adjuvant and sterilized at the base of the rat's tail using a 75% ethanol cotton ball. The mixed emulsion is then injected subcutaneously at a ratio of 0.2 mL / rat on day 0 of the primary immunization. Seven days later, 0.15 ml of the emulsion is injected subcutaneously at the base of the tail of each rat to boost immunization. During the injection, the needle should be inserted 2 cm from the base of the tail until the needle tip is 0.5 cm from the base of the tail, and make sure to wipe completely after each injection to avoid leakage of the emulsion. The needle should be inserted obliquely upward along the position of the rat's tail, and the rat should be injected 3 cm from the base of the tail during the second immunization to enhance immunity. 2.3 Treatment and evaluation of collagen arthritis.

[0059] Step 4, Test

[0060] (4.1) Animal grouping and drug administration

[0061] According to the clinical equivalent dose commonly used in 70kg adults, the dosing group was divided into three groups: low, medium and high, with the dosage of each group being 0.0475g / kg, 0.1425g / kg and 0.285g / kg respectively. These drugs are all from the extract of the 4-flavor decoction of the crown wood. The positive group was the Tripterygium wilfordii polyglycoside tablets, with a dosage of 0.009g / kg; in the normal group and the model group, the same volume of normal saline was taken every day for 21 consecutive days. The degree of swelling of the left hind ankle joint of each rat was scored and recorded every 7 days until the end of the experiment.

[0062] (4.2) Measurement of rat paw swelling

[0063] In this experiment, the thickness of the left / right foot was measured with a vernier caliper. The model was measured once before immunization and then once every 7 days, and the degree of joint swelling was calculated.

[0064] (4.3) Clinical scoring and assessment of arthritis

[0065] In this experiment, a 0-4 joint scoring method was used to measure the severity of swelling in the main joints of the hind limbs. 0 points indicate health (no swelling or redness), 1 point indicates mild redness or swelling of the ankle and wrist joints, 2 points indicate moderate redness or swelling, 3 points indicate severe redness or swelling of the paws (including the fingertips), and 4 points indicate the most severe inflammation of the limbs (including multiple major joints), with significant restrictions on movement. After the second immunization, daily records will be recorded and assessments will be conducted every 7 days. Finally, the sum of the scores of the rat's two feet will be used as the score for each joint.

[0066] Step 5, Enzyme-linked Immunosorbent Assay (ELISA)

[0067] The serum obtained in the above step was taken out from the -80° refrigerator, stored at room temperature for 20-30 minutes, and then centrifuged for 15 minutes (2000 rpm) to collect the serum supernatant. According to the instructions of the ELISA kit, the absorbance was tested using an enzyme marker to determine the content of RF, ASO, CRP, IL-6, MMP-9 and TNF-a cytokines.

[0068] Step 6, histopathological HE staining

[0069] The sections were de-molded to water state in order with ethylene glycol ether acetate (Ⅰ) for 6h, 37℃, 37℃, 37℃, room temperature for 10-15min, room temperature for 10-15min, 100% Ⅰ ethanol for 10min, 100% Ⅱ ethanol for 10min, 95% ethanol for 10min, and 90% ethanol for 10min to complete the de-molding process. Soak in 80% ethanol for 10 minutes and then rinse with water. Stain the sections in hematoxylin stain for 8-10min, then rinse with water, differentiate with differentiation solution, and finally return to blue with blue solution to complete hematoxylin staining. Soak the sections in 85% and 95% gradient ethanol for 5 minutes, respectively, and then wash them in eosin stain for 8-10min to obtain the best dye effect. The slices were placed in anhydrous ethanol II for 5 min, II for 5 min, III for 5 min, xylene I for 5 min and xylene II for 5 min respectively. After a 5-minute washing process, the slices became clear and transparent, and then sealed with neutral gum.

[0070] Experimental Results

[0071] 1. Comparison of joint inflammation

[0072] (1) Comparison of swelling of toes and ankles of rats’ hind limbs

[0073] After 21 days of model experiment, compared with the control group, the swelling of the toes and ankles of the rats in the model group increased significantly, while the swelling of the toes and ankles of the rats in the positive group and the drug-treated group (WGM-4 flavor decoction) decreased significantly after 21 days. Among them, the swelling of the medium-dose drug-treated group decreased the most on day 14 (P < 0.01). Figure 2 and Figure 3 .

[0074] (2) Comparison of joint inflammation scores in rats

[0075] Comparing the control group and the model group, the inflammation score increased significantly after 21 days (P < 0.0001), indicating that the inflammatory response was significantly improved. After 21 days of administration, the AI ​​values ​​of the middle, low and high groups decreased significantly (P < 0.05, P < 0.01). Figure 3 .

[0076] 2. Effect of inflammatory factors on serum levels in rats

[0077] Through ELISA technology, the content of RF, ASO, CRP, IL-6, MMP-9 and TNF-a factors in the serum of CIA rats can be measured to further verify the function and effect. The results showed that the 4-flavor decoction of wenguanmu could significantly inhibit the increase of RF, CRP, TNF-a and IL-6 in serum, among which ASO and MMP-9 did not show obvious therapeutic effects. However, the test results showed that the low-dose and positive group drugs of wenguanmu-4-flavor decoction had significant inhibitory effects, as shown in Figure 2. Figure 5 .

[0078] 3 Effects of pathological changes in the ankle joint of rats

[0079] The anti-inflammatory and detumescent effects of the 4-flavor decoction on RA rats were analyzed by pathological tissue section technology. After 21 days of successful model experiments, the drug was administered. The results showed that there was no clear inflammatory cell infiltration and pannus formation in the ankle joints of the normal group of rats, while synovial growth, pannus formation and large-scale inflammatory cell infiltration were found in the model group of rats. Observation within the ankle joints of CIA rats found that the positive group still had synovial hyperplasia and inflammatory cells, while the drug-administered group had only mild synovial hyperplasia, a small amount of small blood vessel formation and the production of inflammatory cells at low and medium doses; at high doses, synovial hyperplasia was reduced, and the pathological structure of reduced inflammatory cells was improved to a certain extent. Pathological section experiments confirmed that the 4-flavor decoction can significantly reduce synovial hyperplasia and the number of inflammatory cells, thereby effectively improving the pathological structure of RA disease, and its efficacy is better than that of Tripterygium wilfordii polyglycosides tablets, see Figure 4 shown.

[0080] According to the experimental verification results of the above-mentioned embodiment, the mechanism of the anti-inflammatory and detumescent effect of the 4-flavor decoction on RA rats was explored: the 4-flavor decoction can significantly inhibit the increase of RF, CRP, TNF-a and IL-6 in serum. Among them, no clear inflammatory cell infiltration and pannus formation were found in the ankle joints of the rats in the normal group; synovial growth, pannus formation and large-scale inflammatory cell infiltration were found in the rats in the model group, and in vivo observation of the ankle joints of CIA rats found that the positive group still had synovial hyperplasia and inflammatory cells; in the low and medium doses, there was only slight synovial hyperplasia, a small amount of small blood vessel formation and the production of inflammatory cells, while in the high-dose group, the pathological structure of reduced synovial hyperplasia and reduced inflammatory cells was improved to a certain extent, as shown in the figure. Figure 5 shown.

[0081] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. The application of the four-flavor decoction of Rhizoma Coptidis in the anti-inflammatory and detumescent effect in RA rats, characterized in that: The anti-inflammatory and detumescent effects of the drug on RA rats were determined by comparing the swelling of the toes and ankles and AI scores of each group of rats at different times, the pathological changes in the ankle joints of rats, and the inhibition of RF, ASO, CRP, TNF-a, IL-6, and MMP-9 factors in the serum of rats.

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