A pharmaceutical composition for treating rheumatoid arthritis and use thereof

Abstract

The application provides a pharmaceutical composition for treating rheumatoid arthritis, which is composed of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid in a weight ratio of (4.5-7.5):(0.4-1.8):(1.8-4.3):(22-40). The pharmaceutical composition can significantly inhibit the proliferation of fibroblast-like synoviocytes, reduce the secretion of inflammation-related factors (TNF-alpha, IL-1beta, IL-6 and COX-2), regulate the expression of TNF-alpha / NF-kappaB signal pathway-related proteins, and has a significant curative effect on rheumatoid arthritis.

Description

TECHNICAL FIELD

[0001] The application relates to a pharmaceutical composition for treating rheumatoid arthritis and application thereof, and belongs to the technical field of traditional Chinese medicine. BACKGROUND

[0002] Rheumatoid arthritis (RA) is an autoimmune disease, and its main pathological features are synovial hyperplasia and inflammation, pannus formation, and cartilage destruction. RA has the characteristics of long disease course and persistent aggravation. RA belongs to the category of "Bi disease" in traditional Chinese medicine. Traditional Chinese medicine believes that the causes of RA are mainly deficiency of healthy qi, unstable external defense, imbalance of yin and yang of viscera, internal invasion of evil, and blockage of phlegm turbidity and blood stasis. Traditional Chinese medicine has a long history of treating Bi disease, and has accumulated some effective prescriptions. Shaoyao Gancao Decoction is a famous prescription for relieving pain in various generations of doctors, and is included in the "Ancient Classical Prescription Catalogue (First Batch)" issued by the State Administration of Traditional Chinese Medicine and the State Drug Administration in 2018. Clinical studies have shown that Shaoyao Gancao Decoction has definite curative effect in the treatment of RA. Experimental studies have confirmed that Shaoyao Gancao Decoction has good anti-inflammatory, analgesic and immunoregulatory effects, and its anti-inflammatory mechanism is related to the regulation of the NF-κB related signal pathway and the down-regulation of the levels of TNF-α and other inflammatory factors.

[0003] At present, the western medicines used in clinical treatment of RA mainly include non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids (GCs), biological agents and plant medicines. These drugs have definite curative effects on relieving symptoms and improving the quality of life, but some serious and even dangerous adverse reactions still need to be solved. SUMMARY

[0004] In order to solve the problems existing in the prior art, the application provides a pharmaceutical composition for treating rheumatoid arthritis and application of the pharmaceutical composition in preparation of a medicine for treating rheumatoid arthritis.

[0005] As one aspect of the application, the application provides a pharmaceutical composition for treating rheumatoid arthritis, which is composed of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid.

[0006] Preferably, the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid in the pharmaceutical composition is (4.5-7.5):(0.4-1.8):(1.8-4.3):(22-40).

[0007] Further preferably, the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid in the pharmaceutical composition is (5-7):(0.5-1.5):(2.2-3.8):(26-37).

[0008] More preferably, the mass ratio of paeoniflorin, liquiritin, glycyrrhizic acid and glycyrrhetic acid in the pharmaceutical composition is (5.5-6.5):(0.7-1.2):(2.6-3.4):(28-35).

[0009] Most preferably, the mass ratio of paeoniflorin, liquiritin, glycyrrhizic acid and glycyrrhetic acid in the pharmaceutical composition is 6:1:3:31.5.

[0010] In a specific embodiment, the pharmaceutical composition is added with common pharmaceutically acceptable excipients and prepared into conventional dosage forms, including tablets, capsules, granules, pills, powders, oral solutions or injections, according to conventional preparation processes. The pharmaceutically acceptable excipients include fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases and the like. The fillers include starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose and the like; the disintegrants include starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose and the like; the lubricants include magnesium stearate, sodium dodecyl sulfate, talc, silicon dioxide and the like; the suspending agents include polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl cellulose and the like; the binders include starch paste, polyvinylpyrrolidone, hydroxypropyl methyl cellulose and the like.

[0011] As a second aspect of the present application, the present application provides use of the pharmaceutical composition in the preparation of a medicament for treating rheumatoid arthritis.

[0012] Preferably, the pharmaceutical composition is used in the preparation of a medicament for anti-inflammation and / or inhibition of synovial membrane hyperplasia.

[0013] Preferably, the pharmaceutical composition is used in the preparation of a medicament for reducing secretion of inflammation-related factors TNF-α, IL-1β, IL-6 and COX-2, and / or regulating expression of proteins related to TNF-α / NF-κB signaling pathway.

[0014] The structural formulas of paeoniflorin, liquiritin, glycyrrhizic acid and glycyrrhetic acid are as follows:

[0015]

[0016] The paeoniflorin, liquiritin, glycyrrhizic acid and glycyrrhetic acid can be commercially available or prepared according to the prior art.

[0017] The method and dosage of using the pharmaceutical composition depend on various factors, such as the age, weight, health condition, diet and duration and severity of the disease of the patient.

[0018] Advantages of the present application:

[0019] The present application research shows that the combination of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid achieves a synergistic effect and achieves remarkable results in treating rheumatoid arthritis.

[0020] The pharmaceutical composition of the present application is composed of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid in a specific ratio, which can significantly inhibit the proliferation of fibroblast-like synoviocytes, reduce the secretion of inflammation-related factors (TNF-α, IL-1β, IL-6 and COX-2), regulate the expression of TNF-α / NF-κB signaling pathway-related proteins, and has a significant effect on rheumatoid arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The results of the effect of the pharmaceutical composition of the present application on the expression of TNF-α / NF-κB pathway-related proteins in MH7A cells induced by IL-1β. DETAILED DESCRIPTION

[0022] The present application is further described below through specific examples, and the present application is not limited to the following examples. Within the scope of the present application or without departing from the content, spirit and scope of the present application, changes, combinations or substitutions of the present application are obvious to those skilled in the art, and are included within the scope of the present application.

[0023] Example 1

[0024] Composition: The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid is 6:1:3:31.5.

[0025] According to the proportion of each raw material, tablets are prepared according to the conventional preparation process.

[0026] Example 2

[0027] Composition: The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid is 5.6:1.1:2.7:34.5.

[0028] According to the proportion of each raw material, capsules are prepared according to the conventional preparation process.

[0029] Example 3

[0030] Composition: The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid is 6.4:0.8:3.3:28.5.

[0031] According to the proportion of each raw material, granules are prepared according to the conventional preparation process.

[0032] Example 4

[0033] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 5.8:1.2:2.8:33.

[0034] According to the proportion of each raw material, the conventional preparation process is used to prepare the pill.

[0035] Example 5

[0036] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 6.2:0.9:3.2:30.

[0037] According to the proportion of each raw material, the conventional preparation process is used to prepare the oral liquid.

[0038] Example 6

[0039] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 5.2:1.4:2.3:36.

[0040] According to the proportion of each raw material, the conventional preparation process is used to prepare the injection.

[0041] Example 7

[0042] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 6.9:0.6:3.6:27.

[0043] According to the proportion of each raw material, the conventional preparation process is used to prepare the tablet.

[0044] Example 8

[0045] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 4.8:1.5:2.1:37.

[0046] According to the proportion of each raw material, the conventional preparation process is used to prepare the pill.

[0047] Example 9

[0048] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 7.2:0.4:4:25.

[0049] According to the proportion of each raw material, the conventional preparation process is used to prepare the injection.

[0050] Example 10

[0051] Composition: the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 4.6:1.7:1.9:39.

[0052] According to the proportion of each raw material, the conventional preparation process is used to prepare the capsule.

[0053] Example 11

[0054] Composition: The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid is 7.4:0.5:4.2:23.

[0055] According to the proportion of each raw material, granules were prepared according to the conventional preparation process.

[0056] Example 12 Drug composition screening experiment

[0057] 1 Experimental cell

[0058] Human rheumatoid arthritis fibroblast-like synoviocytes (MH7A) were purchased from Guangzhou Jinniu Biological Technology Co., Ltd. (JN0977-B) and cultured in a 37°C, 5% CO2 incubator.

[0059] 2 Experimental drugs and drug solution preparation

[0060] 2.1 Experimental drugs

[0061] Paeoniflorin, Chengdu Desite Biological Technology Co., Ltd., purity: 98% (HPLC), catalog number: DS0070;

[0062] Glycyrrhizin, Chengdu Desite Biological Technology Co., Ltd., purity: 98% (HPLC), catalog number: DG0009;

[0063] Glycyrrhizic acid, Chengdu Desite Biological Technology Co., Ltd., purity: 98% (HPLC), catalog number: DG0006;

[0064] Glycyrrhetinic acid, Chengdu Desite Biological Technology Co., Ltd., purity: 98% (HPLC), catalog number: DG0007;

[0065] Animal-free recombinant human IL-1β, Proteintech, batch number: HZ-1164.

[0066] 2.2 Experimental drug solution preparation

[0067] Paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid combination solution: According to the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid (6:1:3:31.5), the following combination solutions were prepared: ① Paeoniflorin + glycyrrhizin (6:1); ② Paeoniflorin + glycyrrhetinic acid (6:31.5); ③ Paeoniflorin + glycyrrhizin + glycyrrhetinic acid (6:1:31.5); ④ Paeoniflorin + glycyrrhizic acid + glycyrrhetinic acid (6:3:31.5); ⑤ Paeoniflorin + glycyrrhizin + glycyrrhizic acid + glycyrrhetinic acid (6:1:3:31.5). The specific preparation scheme is shown in Table 1.

[0068] Table 1 Preparation of different component combination solutions

[0069]

[0070] IL-1β: IL-1β powder was dissolved with PBS to prepare a 100 μg / mL drug solution as a stock solution, which was aliquoted into 200 μL centrifuge tubes, 5 μL per tube, and stored at -20°C. When used, the appropriate amount of stock solution was diluted to 10 ng / mL with complete medium.

[0071] 3 Main experimental reagents

[0072] Thiazole blue (MTT), Biotryl, batch number: BN30793;

[0073] Human tumor necrosis factor alpha (TNF-α) ELISA kit, KOT Biological, batch number: KT0121-A.

[0074] The specific experimental operation includes:

[0075] 1 The effect of different component combination solutions on IL-1β-induced MH7A cell proliferation

[0076] MH7A cells were seeded in a 96-well plate at a density of 1 × 10 4 cells / well, and blank groups (no cell groups), control groups (no drug groups, 0.1% DMSO medium), IL-1β groups (20 ng / mL), IL-1β+(peony glycoside+licorice glycoside 6:1), IL-1β+(peony glycoside+glycyrrhetinic acid 6:31.5), IL-1β+(peony glycoside+licorice glycoside+glycyrrhetinic acid 6:1:31.5), IL-1β+(peony glycoside+licorice acid+glycyrrhetinic acid 6:3:31.5), IL-1β+(peony glycoside+licorice glycoside+licorice acid+glycyrrhetinic acid 6:1:3:31.5) were set up, with 6 replicate wells in each group. The dosing amount of each drug group was 100 μL. The MTT method was used to explore the inhibitory effect of the above drugs on IL-1β-induced MH7A cell growth.

[0077] The above test results were statistically processed: the measurement data were expressed in the form of mean ± standard deviation SPSS22.0 software was used for statistical analysis. If it conforms to the normal distribution and the variance is equal, one-way analysis of variance is used, otherwise non-parametric test is used. P<0.05 is considered to have statistical significance. The results are shown in Table 2.

[0078] Table 2 The effect of different component combination solutions on IL-1β-induced MH7A cell proliferation n=6, 490 nm

[0079]

[0080] Note: compared with the control group ***P<0.001; compared with IL-1β group # P<0.05, ## P<0.01, ### P<0.001.

[0081] According to the statistical data in Table 1, it is shown that the proliferation of MH7A cells is obviously increased after being induced and stimulated by 20 ng / mL IL-1β (P<0.001 compared with the control group). After the IL-1β-induced MH7A cells are given the mixed solution of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid in different proportions, the mixed solution of paeoniflorin+glycyrrhizin+glycyrrhetic acid (6:1:31.5), paeoniflorin+glycyrrhizic acid+glycyrrhetic acid (6:3:31.5) and paeoniflorin+glycyrrhizin+glycyrrhizic acid+glycyrrhetic acid (6:1:3:31.5) has obvious inhibitory effect on the proliferation of IL-1β-induced MH7A cells (P<0.05, P<0.01 or P<0.001), and the inhibitory effect of the mixed solution of paeoniflorin+glycyrrhizin+glycyrrhizic acid+glycyrrhetic acid (6:1:3:31.5) is the best.

[0082] 2Effect of different component combination solution on TNF-α level in supernatant of IL-1β-induced MH7A cells

[0083] The MH7A cells are inoculated in a 24-well plate at a density of 1×10 5 Each well has a liquid volume of 1 mL, and the blank group (cell-free group), the control group (drug-free group, 0.1% DMSO medium), the IL-1β group (20 ng / mL), the IL-1β+(paeoniflorin+glycyrrhizin 6:1) group, the IL-1β+(paeoniflorin+glycyrrhetic acid 6:31.5) group, the IL-1β+(paeoniflorin+glycyrrhizin+glycyrrhetic acid 6:1:31.5) group, the IL-1β+(paeoniflorin+glycyrrhizic acid+glycyrrhetic acid 6:3:31.5) group and the IL-1β+(paeoniflorin+glycyrrhizin+glycyrrhizic acid+glycyrrhetic acid 6:1:3:31.5) group are set up, and each group has 6 replicate wells. The grouping and dosing treatment are performed for 24 h. The cell supernatant is collected, the cells are taken out of the incubator, and each well of the drug-containing culture is sucked into a 1.5 mL centrifuge tube, centrifuged at 3000 rpm and 4°C for 20 min, and the supernatant is collected and stored in a-80°C refrigerator. The content of TNF-α in the supernatant is detected by an ELISA kit.

[0084] The above determination results are statistically processed: the measurement data are all in the form of mean ± standard deviation SPSS22.0 software is used for statistical analysis. If it conforms to normal distribution and the variance is equal, single-factor analysis of variance is used, otherwise non-parametric test is used. P<0.05 is considered to have statistical significance. The results are shown in Table 3.

[0085] Table 3 Effect of different component combinations on TNF-a level in IL-1β-induced MH7A cell supernatant n = 6, 450 nm

[0086]

[0087] Note: Compared with the control group ** P < 0.01; compared with the IL-1β group # P < 0.05, ## P < 0.01.

[0088] According to the data in Table 3, the content of TNF-a in the supernatant of MH7A cells significantly increased after being induced by 20 ng / mL IL-1β (P < 0.01). After the IL-1β-induced MH7A cells were given different ratios of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid mixed solutions, the content of TNF-a in the paeoniflorin + glycyrrhizic acid + glycyrrhetic acid (6:3:31.5) and paeoniflorin + glycyrrhizin + glycyrrhizic acid + glycyrrhetic acid (6:1:3:31.5) mixed solutions significantly decreased (P < 0.05 or P < 0.01), and the content of TNF-a in the paeoniflorin + glycyrrhizin + glycyrrhizic acid + glycyrrhetic acid (6:1:3:31.5) mixed solution decreased most significantly, and the anti-inflammatory activity was best.

[0089] In summary, the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid in the mixed solution was selected as 6:1:3:31.5, which was used as the final composition solution (referred to as "PLGGS") for subsequent experiments.

[0090] Example 13 Pharmacodynamic effect of the pharmaceutical composition on IL-1β-induced MH7A cells and mechanism of action

[0091] 1 Experimental cells

[0092] Human rheumatoid arthritis fibroblast-like synoviocytes (MH7A) were purchased from Guangzhou Jnieuo Biotechnology Co., Ltd. (JN0977-B) and cultured in a 37°C, 5% CO2 constant temperature incubator.

[0093] 2 Experimental drugs and preparation of drug solutions

[0094] 2.1 Experimental drugs

[0095] Paeoniflorin, Chengdu Desite Biotechnology Co., Ltd., purity: 98% (HPLC), product number: DS0070;

[0096] Glycyrrhizin, Chengdu Desite Biotechnology Co., Ltd., purity: 98% (HPLC), product number: DG0009;

[0097] Glycyrrhizic acid, Chengdu Desite Biotechnology Co., Ltd., Purity: 98% (HPLC), Catalog No: DG0006;

[0098] Glycyrrhetinic acid, Chengdu Desite Biotechnology Co., Ltd., Purity: 98% (HPLC), Catalog No: DG0007;

[0099] Animal-free Recombinant Human IL-1β, Proteintech, Batch No: HZ-1164.

[0100] 2.2 Preparation of experimental drug solution

[0101] Paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid composition solution (referred to as "PLGGS"): according to the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid (6:1:3:31.5), 6.14 mg of paeoniflorin, 1.02 mg of glycyrrhizin, 3.07 mg of glycyrrhizic acid and 32.26 mg of glycyrrhetinic acid powder were precisely weighed, 200 μL of DMSO was added, and a composition mother liquor containing 30.72 ng / mL of paeoniflorin, 5.12 ng / mL of glycyrrhizin, 15.36 ng / mL of glycyrrhizic acid and 161.28 ng / mL of glycyrrhetinic acid was prepared.

[0102] IL-1β: IL-1β powder was dissolved in PBS to prepare a drug solution of 100 μg / mL as a stock solution, which was aliquoted into 200 μL centrifuge tubes, 5 μL per tube, and stored at -20°C. When used, the appropriate amount of stock solution was diluted to 10 ng / mL with complete medium.

[0103] 3 Main experimental reagents

[0104] Thiazolyl blue (MTT), Borysai, Batch No: BN30793;

[0105] Human interleukin 6 (IL-6) ELISA kit, KOTBT, Batch No: KT0049-A;

[0106] Human tumor necrosis factor alpha (TNF-α) ELISA kit, KOTBT, Batch No: KT0121-A;

[0107] Human interleukin 1β (IL-1β) ELISA kit, KOTBT, Batch No: KT0179-A;

[0108] Human cyclooxygenase 2 (COX-2) ELISA kit, KOTBT, Batch No: KT0564-A;

[0109] Anti-TNF alpha Rabbit antibody, Abeam, Batch No: ab183218;

[0110] Anti-IKK alpha+IKK beta Rabbit antibody, Abeam, Lot: ab178870l;

[0111] Phospho-IKKa / b (Ser176 / 180) (16A6) Rabbit mAb, CST, Lot: 2697T;

[0112] COX2 Rabbit Polyclonal antibody, Proteintech, Lot: 12375-1-AP;

[0113] Anti-NF-kB p65 Rabbit antibody, Abeam, Lot: ab16502;

[0114] Anti-IKB alpha Rabbit antibody, Abeam, Lot: ab76429;

[0115] Phospho-IkBa (Ser32) (14D4) Rabbit mAb, CST Lot: 2859T;

[0116] Anti-NF-kB p65 (phospho S276) Rabbit antibody, Abeam, Lot: ab194726;

[0117] Beta Tubulin Rabbit Polyclonal antibody, Proteintech, Lot: 10068-1-AP;

[0118] HRP-conjugated Goat Anti-Rabbit Ig (H+L) (Goat Anti-Rabbit HRP), Proteintech, Lot: SA00001-2;

[0119] Super sensitive ECL chemiluminescence kit, new sail, Lot: P10100.

[0120] The specific experimental operation includes:

[0121] 1. Composition concentration screening

[0122] MH7A cells were seeded at 1 x 10 4The density of 1 cell / hole was inoculated in 96-well plates, and blank groups (no cell group), control groups (no drug group, 0.1% DMSO medium), different concentrations of PLGGS groups (see Table 4) were set up, each group was set up 6 duplicate holes, and was placed in a 37℃, 5% CO2 cell incubator for 24h. Then, different concentrations of PLGGS were added to each hole for treatment, 20μL of 5mg / mL MTT solution was added to each hole after 24h, and was gently shaken, incubated at 37℃ for 4h, the culture medium in the hole was aspirated, 150μL of DMSO was added to each hole, and was placed on a shaking bed for low-speed shaking for 10min. The absorbance value (OD value) of each hole was determined by an enzyme-labeled instrument at 490nm, and the cell viability of different drug concentrations was calculated.

[0123] Table 4: PLGGS series drug concentration (μg / mL)

[0124] PLGGS Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Concentration 7 Concentration 8 Paeoniflorin 0.12 0.24 0.48 0.96 1.92 3.84 7.68 15.36 Liquiritin 0.02 0.04 0.08 0.16 0.32 0.64 1.28 2.56 Glycyrrhizic acid 0.06 0.12 0.24 0.48 0.96 1.92 3.84 7.68 Glycyrrhetinic acid 0.63 1.26 2.52 5.04 10.08 20.16 40.32 80.64

[0125] The above determination results were statistically processed: the measurement data were expressed in the form of mean ± standard deviation SPSS22.0 software was used for statistical analysis. If it conforms to normal distribution and variance is equal, single factor analysis of variance is used, otherwise non-parametric test is used. P<0.05 is considered to have statistical significance. The results are shown in Table 5.

[0126] Table 5: Effect of PLGGS on MH7A cell viability n=6, 490nm

[0127]

[0128] Note: compared with the control group ** P<0.01, *** P<0.001.

[0129] According to the statistical data in Table 5, the viability of MH7A cells was inhibited after being treated with different concentrations of paeonol, glycyrrhizin, glycyrrhizic acid and glycyrrhetic acid for 24 hours, showing a dose-dependent manner. The mixed solution of 1.92 μg / mL paeonol, 0.32 μg / mL glycyrrhizin, 0.96 μg / mL glycyrrhizic acid and 10.08 μg / mL glycyrrhetic acid had a significant inhibitory effect on the viability of MH7A cells (P<0.01 or P<0.001). Therefore, the mixed solution containing 0.24 μg / mL paeonol, 0.04 μg / mL glycyrrhizin, 0.12 μg / mL glycyrrhizic acid and 1.26 μg / mL glycyrrhetic acid was selected as the low concentration group of PLGGS, the mixed solution containing 0.48 μg / mL paeonol, 0.08 μg / mL glycyrrhizin, 0.24 μg / mL glycyrrhizic acid and 2.52 μg / mL glycyrrhetic acid was selected as the medium concentration group of PLGGS, and the mixed solution containing 0.96 μg / mL paeonol, 0.16 μg / mL glycyrrhizin, 0.48 μg / mL glycyrrhizic acid and 5.04 μg / mL glycyrrhetic acid was selected as the high concentration group of PLGGS for subsequent experiments.

[0130] 2Effect of the composition on IL-1β-induced proliferation of MH7A cells

[0131] The MH7A cells were inoculated in a 96-well plate at a density of 1×10 4 The blank group (no cell group), the control group (no drug group, 0.1% DMSO medium), the IL-1β group (20 ng / mL), the IL-1β (20 ng / mL)+PLGGS low concentration group, the IL-1β (20 ng / mL)+PLGGS medium concentration group and the IL-1β (20 ng / mL)+PLGGS high concentration group were set up, with 6 replicate wells in each group. The MTT method was used to explore the effect of PLGGS on IL-1β-induced proliferation of MH7A cells. The concentrations of the components in the low, medium and high concentrations of PLGGS are shown in Table 6.

[0132] Table 6. Low, medium and high concentrations of PLGGS (μg / mL)

[0133] PLGGS Low concentration group Medium concentration group High concentration group Paeoniflorin 0.24 0.48 0.96 Liquiritin 0.04 0.08 0.16 Glycyrrhizic acid 0.12 0.24 0.48 Glycyrrhetinic acid 1.26 2.52 5.04

[0134] The above determination results were statistically processed: the measurement data were expressed in the form of mean ± standard deviation SPSS22.0 software was used for statistical analysis. If it conforms to the normal distribution and the variance is equal, one-way ANOVA is used, otherwise non-parametric test is used. P<0.05 is considered to have statistical significance. The results are shown in Table 7.

[0135] Table 7. Effect of PLGGS on IL-1β-induced proliferation of MH7A cells n=6, 490 nm)

[0136]

[0137]

[0138] Note: Compared with the control group *** P<0.01; compared with the IL-1β group # P<0.05, ## P<0.01.

[0139] According to the data in Table 7, it is shown that the proliferation of MH7A cells is obviously increased after being induced and stimulated by 20 ng / mL IL-1β (P<0.001 compared with the control group). After the IL-1β-induced MH7A cells are given different concentrations of PLGGS, the medium concentration group and the high concentration group have obvious inhibitory effect on the proliferation of IL-1β-induced MH7A cells (P<0.05 or P<0.01).

[0140] 3. Effect of the composition on the levels of TNF-α, IL-6, IL-1β and COX-2 in the supernatant of IL-1β-induced MH7A cells

[0141] The MH7A cells are inoculated in a 24-well plate at a density of 1×10 5 Each well has a liquid volume of 1 mL, and blank groups (cell-free groups), control groups (without drug groups, 0.1% DMSO medium), IL-1β groups (20 ng / mL), IL-1β (20 ng / mL)+PLGGS low concentration groups, IL-1β (20 ng / mL)+PLGGS medium concentration groups, IL-1β (20 ng / mL)+PLGGS high concentration groups are set, and each group has 6 replicate wells. The grouping is given a drug treatment for 24 h. The cell supernatant is collected, the cells are taken out of the incubator, and the drug-containing culture medium in each well is sucked into a 1.5 mL centrifuge tube, centrifuged at 3000 rpm and 4°C for 20 min, the supernatant is collected, and stored in a-80°C refrigerator. The levels of TNF-α, IL-6, IL-1β and COX-2 in the supernatant are detected according to the ELISA kit instructions.

[0142] The above determination results are statistically processed: the measurement data are expressed in the form of mean ± standard deviation SPSS22.0 software is used for statistical analysis. If it conforms to the normal distribution and the variance is equal, single factor analysis of variance is used, otherwise non-parametric test is used. P<0.05 is considered to have statistical significance. The results are shown in Tables 8-9.

[0143] Table 8. Effect of PLGGS on the levels of TNF-α and IL-6 in the supernatant of IL-1β-induced MH7A cells n = 6, 450 nm

[0144]

[0145]

[0146] Note: Compared with the control group ** P < 0.01; compared with the IL-1β group # P < 0.05, ## P < 0.01. Table 9 Effect of PLGGS on IL-1β, COX-2 levels in supernatant of MH7A cells induced by IL-1β n = 6, 450 nm

[0147]

[0148] Note: Compared with the control group ** P < 0.01; compared with the IL-1β group # P < 0.05, ## P < 0.01.

[0149] According to the statistical data in Tables 8 and 9, the contents of TNF-α, IL-6, IL-1β and COX-2 in the supernatant of MH7A cells were significantly increased after being induced by 20 ng / mL IL-1β (P < 0.01). After the IL-1β-induced MH7A cells were given different concentrations of PLGGS, the IL-1β level in the supernatant of cells in the low-concentration group was significantly reduced (P < 0.05), and the contents of TNF-α, IL-6, IL-1β and COX-2 in the supernatant of cells in the medium-concentration and high-concentration groups were significantly reduced (P < 0.05 or P < 0.01).

[0150] 4 Effect of the composition on the expression of TNF-α / NF-κB pathway-related proteins in IL-1β-induced MH7A cells

[0151] The MH7A cells were induced by 20 ng / mL IL-1β, and then were given 0.5 mL of the composition at different concentrations, and the control group was given 0.5 mL of the culture medium. After 24 h, the cells were collected, and the expression of TNF-α / NF-κB pathway-related proteins was detected by Western blotting. 5The density of 1 x 106 cells / well was inoculated in 6-well plates, with a liquid volume of 2 mL per well, and blank groups (cell-free groups), control groups (without drug groups, 0.1% DMSO medium), IL-1β groups (20 ng / mL), IL-1β (20 ng / mL) + PLGGS low concentration groups, IL-1β (20 ng / mL) + PLGGS medium concentration groups, and IL-1β (20 ng / mL) + PLGGS high concentration groups were set. The groups were administered for 24 h, and after adding the lysis solution and lysis on ice for 30 min, the supernatant was centrifuged to detect the total protein concentration. SDS-PAGE gel electrophoresis separation was performed, the membrane was transferred, 5% BSA was blocked, IKK, p-IKK, IκBα, p-IκBα, NF-κB p65, NF-κB p-p65, TNF-α, COX-2 and β-Tubulin were anti, and the secondary antibody was incubated, and then ECL was developed. Image J software was used to analyze the gray value of the protein band, and the results of six repeated independent experiments were taken.

[0152] The above determination results were statistically processed: the measurement data were in the form of mean ± standard deviation SPSS22.0 software was used for statistical analysis. If it conforms to the normal distribution and the variance is equal, one-way analysis of variance is used, otherwise non-parametric test is used. P<0.05 is considered to have statistical significance. The results are shown in Tables 10-11 and Figure 1 .

[0153] Table 10 Effect of PLGGS on IL-1β-induced expression of TNF-α / NF-κB pathway-related proteins in MH7A cells n=6

[0154]

[0155] Note: compared with the control group ** P<0.01; compared with the IL-1β group # P<0.05, ## P<0.01. Table 11 Effect of PLGGS on IL-1β-induced expression of TNF-α / NF-κB pathway-related proteins in MH7A cells n=6

[0156] Grouping p-IκBα / IκBα TNF-α / β-Tubulin p-IKK / IKK Control group 1.0000±0.21597 1.0000±0.26091 1.0000±0.83432 IL-1β group 1.9730 ± 0.27434 ** ]] 1.7705 ± 0.23612 ** ]] 3.5524 ± 0.55923 ** ]] Low concentration group 1.6625±0.34259 1.3100 ± 0.26466 # ]] 2.2951 ± 0.52306 ## ]] Medium concentration group 1.3838 ± 0.44907 # ]] 1.2848 ± 0.3377 ## ]] 1.6925 ± 0.56715 ## ]] High concentration group 1.0876 ± 0.50696 ## ]] 1.1567 ± 0.31202 ## ]] 1.8433 ± 0.62512 ## ]]

[0157] Note: compared with the control group ** P<0.01; compared with the IL-1β group # P<0.05, ## P<0.01.

[0158] According to the statistical data in Tables 10 and 11, the expression levels of TNF-α, p-IKK, p-IκBα, NF-κB p-p65 and COX-2 proteins in the MH7A cells were significantly increased after being stimulated by 20 ng / mL IL-1β (P < 0.01). After the IL-1β-induced MH7A cells were given different concentrations of PLGGS, the expression levels of TNF-α and p-IKK proteins in the cells of the low-concentration group were significantly decreased (P < 0.05 or P < 0.01), the expression levels of TNF-α, p-IKK, p-IκBα, NF-κB p-p65 and COX-2 proteins in the cells of the medium-concentration group were significantly decreased (P < 0.05 or P < 0.01), and the expression levels of TNF-α, p-IKK, p-IκBα and NF-κB p-p65 proteins in the cells of the high-concentration group were significantly decreased (P < 0.01).

[0159] According to the above test results, the fibroblast-like synoviocytes (MH7A) were cultured in vitro, and stimulated by IL-1β to simulate local inflammation. After being treated by the composition, the proliferation of the MH7A cells caused by inflammation can be inhibited, and the inflammatory response can be reduced. The mechanism is mainly related to the regulation of the TNF-α / NF-κB signaling pathway and the inhibition of the expression of inflammatory-related factors.

Claims

1. A pharmaceutical composition for treating rheumatoid arthritis, characterized in that, The pharmaceutical composition comprises paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid; the mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid in the pharmaceutical composition is (4.5~7.5):(0.4~1.8):(1.8~4.3):(22~40).

2. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid in the pharmaceutical composition is (5~7):(0.5~1.5):(2.2~3.8):(26~37).

3. The pharmaceutical composition according to claim 2, characterized in that, The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid in the pharmaceutical composition is (5.5~6.5):(0.7~1.2):(2.6~3.4):(28~35).

4. The pharmaceutical composition according to claim 3, characterized in that, The mass ratio of paeoniflorin, glycyrrhizin, glycyrrhizic acid and glycyrrhetinic acid in the pharmaceutical composition is 6:1:3:31.

5.

5. The pharmaceutical composition according to any one of claims 1-4, characterized in that, The pharmaceutical composition is formulated with commonly used pharmaceutically acceptable excipients and prepared into tablets, capsules, granules, pills, powders, oral liquids, or injections according to conventional pharmaceutical processes.

6. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for treating rheumatoid arthritis.

7. The application as described in claim 6, characterized in that, The pharmaceutical composition is used in the preparation of anti-inflammatory and / or synovial hyperplasia-inhibiting drugs.

8. The application as described in claim 6, characterized in that, The pharmaceutical composition is used in the preparation of drugs that reduce the secretion of inflammatory factors TNF-α, IL-1β, IL-6 and COX-2, and / or regulate the expression of proteins related to the TNF-α / NF-κB signaling pathway.

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