Use of a pharmaceutical composition for the preparation of a medicament for the treatment of rheumatoid arthritis

Abstract

The application discloses application of a medicine composition in preparation of a medicine for treating rheumatoid arthritis (RA), and belongs to the field of RA treating medicines. The medicine composition is composed of swertiamarin (SW) and neochlorogenic acid (NCA) and is screened from two active ingredients in Danteng Yimu Decoction. With the help of animal model and cell model experiments, the action mechanism of the medicine composition in treating RA is explored, the related symptoms and indexes of collagen-induced arthritis (CIA) rats can be effectively improved, joint swelling can be relieved, arthritis inflammation index can be reduced, inflammatory factor release can be inhibited, joint bone quality can be protected, arthritis inflammation infiltration and cartilage destruction can be reduced, and the medicine safety is good. The composition can effectively inhibit the inflammatory factor release and migration ability of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Animal experiments and cell experiments prove that the combination of neochlorogenic acid and swertiamarin (SW+NCA) can be effectively applied to clinical treatment of RA and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of drugs for the treatment of rheumatoid arthritis (RA), and specifically relates to the use of a pharmaceutical composition in the preparation of drugs for the treatment of RA. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune inflammatory disease that primarily affects joints and surrounding soft tissues. Persistent synovial hyperplasia caused by the pathological activation, proliferation, and migration of fibroblast-like synoviocytes (FLS) is one of its important pathological manifestations. Due to its long course and high disability rate, RA can severely impact patients' work ability and quality of life, causing significant physiological, psychological, and economic impacts on patients and their families.

[0003] Currently, clinical treatment for rheumatoid arthritis (RA) centers on disease-modifying antirheumatic drugs (DMARDs), with methotrexate (MTX) being the preferred anchoring agent. Other treatments include leflunomide, biologics, and glucocorticoids. While these drugs can control inflammatory symptoms and slow disease progression to some extent, they still have significant clinical limitations: traditional synthetic DMARDs have adverse reactions such as hepatotoxicity, nephrotoxicity, bone marrow suppression, and gastrointestinal reactions, and long-term tolerability is poor; biologics and targeted therapies are expensive and pose risks such as increased infection risk and inducing autoimmune reactions, with some patients experiencing poor or no response, failing to meet the clinical needs of RA patients for long-term, safe treatment.

[0004] Rheumatoid arthritis (RA) falls under the categories of "arthritis" and "bi syndrome" in Traditional Chinese Medicine (TCM). TCM treatment of RA has garnered significant attention due to its advantages of being "universal, simple, inexpensive, and effective." The 2023 "Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis Based on Syndrome Differentiation" and the 2024 "Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis" affirmed the role of herbal medicines and prepared TCM formulas such as Tripterygium wilfordii preparations, total glucosides of Paeonia lactiflora, and sinomenine in RA treatment. TCM treatment for RA has a relatively high safety profile; its rational application on top of conventional Western medicine treatments does not increase adverse events and may even reduce toxicity. However, the complex composition of TCM compound formulas hinders standardized development into new drugs, thus failing to meet the diverse needs of clinical patients. Summary of the Invention

[0005] To address the above-mentioned problems, the present invention provides a pharmaceutical composition for treating RA, the pharmaceutical composition comprising swertiamarin (SW) and neochlorogenic acid (NCA).

[0006] Furthermore, the molar ratio of SW to NCA is 1:1 to 4.

[0007] Furthermore, the molar ratio of SW to NCA is 1:2.

[0008] The use of any of the above-described pharmaceutical compositions in the preparation of a drug for treating RA.

[0009] Furthermore, the applications include reducing ankle swelling, improving foot swelling, improving arthritis index scores, and increasing weight.

[0010] Furthermore, the application includes reducing serum inflammatory factor levels.

[0011] Furthermore, the application includes inhibiting bone destruction.

[0012] Furthermore, the application includes inhibiting joint inflammation infiltration and cartilage destruction.

[0013] Furthermore, the application includes therapeutic effects on RA-FLS cells.

[0014] Furthermore, the therapeutic effect on RA-FLS cells includes reducing the levels of interleukin-1β (IL-1β), IL-6, and matrix metalloproteinase-1 (MMP-1).

[0015] Furthermore, the therapeutic effects of the RA-FLS cells include inhibiting cell migration.

[0016] The beneficial effects of this invention are as follows: Although there are several publicly available patents related to traditional Chinese medicine compounds for the treatment of rheumatoid arthritis (RA), these still cannot meet the diverse needs of clinical patients. The complex active ingredients in traditional Chinese medicine compound formulas hinder standardized development into new drugs.

[0017] This invention validates the efficacy and safety of SW+NCA in treating RA by implementing a combination of SW and NCA (SW+NCA) in a rat model of collagen-induced arthritis (CIA) and RA-FLS cells. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Effects of NCA+SW on the appearance of ankle joints in CIA rats; Figure 2 Effects of NCA+SW on paw edema, arthritis index score, and body weight in CIA rats ( (n=6) Figure 3 Effects of NCA+SW on the levels of inflammatory factors in CIA rats (n=6) Figure 4 The effect of NCA+SW on micro-computed tomography (micro-CT) reconstruction of the ankle joint in CIA rats; Figure 5 Effects of NCA+SW on ankle bone mineral density and related parameters in CIA rats ( (n=3) Figure 6 Effects of NCA+SW on HE staining and Safranin-Fast Green staining of ankle joints in CIA rats; Figure 7 Effects of NCA+SW on HE staining of liver and kidney in CIA rats; Figure 8 Effects of NCA+SW on the expression of IL-1β, IL-6, and MMP-1 in RA-FLS cells (n=3) Figure 9 Effect of NCA+SW on 24h migration rate of RA-FLS cells ( (n=3). Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0021] Example 1: Therapeutic effect of SW+NCA on CIA rats 1. Grouping, intervention, and sampling of experimental animals 1.1 Laboratory animals: A rat model of CIA was established using male 7-8 week old SD rats. The rats were housed in the SPF-grade animal experimental center of Guangzhou University of Chinese Medicine, with room temperature maintained at (23±2℃) and humidity maintained at (55%±5%), and subjected to alternating light and dark changes for 12 hours for 7 days of acclimatization feeding.

[0022] 1.2 Construction of CIA animal models The first immunization was performed on day 0 (an equal volume mixture of incomplete Freund's adjuvant (4 mg / mL) and bovine type II collagen solution (2 mg / mL) was emulsified and injected subcutaneously into the tail of the rats, resulting in a dose of 0.2 mL). A second immunization was performed on day 7 (an equal volume mixture of incomplete Freund's adjuvant (4 mg / mL) and bovine type II collagen solution (2 mg / mL) was emulsified and injected subcutaneously into the tail of the rats, resulting in a dose of 0.2 mL). Model formation was observed on day 14.

[0023] 1.3 Grouping and Administration Methods Grouping: Rats were randomly divided into 6 groups: blank group (NC group), model group (CIA group), swertiamarin group (SW group), neochlorogenic acid group (NCA group), neochlorogenic acid and swertiamarin combination group (SW+NCA group), and positive control drug group (MTX group). Except for the blank group, all other groups were subjected to CIA modeling.

[0024] Administration methods: Rats in each group were given the corresponding drug intervention starting on day 14 after the second immunization. The NC and CIA groups were given an equal volume of physiological saline once daily; the SW group was given 100 mg / kg SW solution once daily; the NCA group was given 50 mg / kg NCA solution once daily; the SW+NCA group was given a mixture of 100 mg / kg SW solution and 50 mg / kg NCA solution once daily; the MTX group was given 0.5 mg / kg MTX solution twice a week, with an equal volume of physiological saline for the remaining 5 days. All administration was intraperitoneal injection, and continued for 3 weeks.

[0025] Twenty-one days after administration, blood was collected from the abdominal aorta under isoflurane inhalation anesthesia. After standing at 4°C for 1 hour, the blood was centrifuged at 3000 r·min⁻¹ (centrifugation radius 18 cm) for 15 minutes at 4°C. The supernatant was aliquoted into EP tubes and stored at -80°C. The rats subsequently died of hemorrhagic shock. Their ankle joints, livers, and kidneys were collected and fixed in 4% formalin at room temperature.

[0026] 2 Experimental Methods 2.1 Arthritis scoring to assess the severity of arthritis The condition of rat joints was observed and recorded starting 14 days after the second immunization. Paw swelling was observed every 3 days. The degree of inflammation in the rats' limbs and paws was scored according to the arthritis scoring criteria, recorded on a scale of 0 to 4. The cumulative score was the arthritis index score for each rat. The specific scoring method is shown in Table 1. Table 1. Arthritis Index Scoring Criteria 2.2 Detection of serum inflammatory factor levels Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammation-related cytokines tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-17A in rat serum.

[0027] According to the ELISA kit instructions (purchased from Shanghai Jianglai Biotechnology Co., Ltd.), CIA rat plasma was centrifuged at 1000g for 20 min, the supernatant was collected, and the sample was diluted appropriately. 100 μL of the sample or different concentrations of standards were added to each well of the ELISA plate, and the plate was incubated at 37℃ for 60 min. The liquid was discarded, and 100 μL of biotinylated antibody working solution was added to each well, and the plate was incubated at 37℃ for 60 min. The liquid was discarded, and 300 μL of 1× washing buffer was added to each well. The plate was allowed to stand for 1 min, the washing buffer was discarded, and the plate was patted dry on absorbent paper. The washing process was repeated 3 times. 100 μL of enzyme conjugate working solution was added to each well, and the plate was incubated at 37℃ for 30 min. The liquid was discarded, and the plate was washed. 90 μL of substrate was added to each well, and the plate was incubated at 37℃ in the dark for 15 min. The ELISA plate was removed, and 50 μL of stop solution was added directly to each well. The OD value of each well was measured at 450 nm, and the concentration of the sample index was calculated based on the OD value.

[0028] 2.3 Micro-CT scan of the ankle joint The right hind limb ankle joint of rats was fixed with 4% paraformaldehyde. The rat ankle joint was scanned and reconstructed in three dimensions using the SkyScan-1276 X-ray micro-CT system. Scanning parameters: resolution 16 μm, voltage 70 kV, current 47 μA. A semi-quantitative scoring method was used to assess bone destruction in the micro-CT scan bone tissue. CTAn software was used to analyze the bone mineral density (BMD) and related parameters of the rat ankle joint, with the talus being the analysis site.

[0029] 2.4 Pathological sections of ankle joint and visceral tissues Ankle joint and visceral tissues were immersed in 4% paraformaldehyde for 48 hours, rinsed with PBS, and decalcified with 13% ethylenediaminetetraacetic acid (EDTA) solution at 4°C. The decalcification solution was changed every 4-5 days until a pin could easily pierce the bone. After rinsing with PBS, the tissues were dehydrated using a gradient alcohol dehydrator. The paraffin-soaked tissues were then embedded in an embedding machine, the paraffin blocks were trimmed, and sections were prepared, each approximately 8µm thick. The sections were placed on a slide, flattened at 4°C, and then lifted with a glass slide and baked in a 60°C oven. After the water-dried paraffin melted, the tissues were removed and stored at room temperature.

[0030] 2.5 HE staining of ankle joint and visceral tissues HE staining of ankle joint and visceral tissues: 1. Dewaxing paraffin sections to hydration: Immerse sections sequentially in xylene I for 10 min → xylene II for 10 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → 95% ethanol for 2 min → 90% ethanol for 2 min → 85% ethanol for 2 min → 75% ethanol for 2 min → wash with distilled water. 2. Mayer's hematoxylin staining of cell nuclei: Immerse sections in Harris hematoxylin staining for 2 min, rinse with warm water for 10 min, differentiate with 75% hydrochloric acid ethanol for 1 min, and rinse with tap water for 30 s. 3. Eosin staining of cytoplasm: Immerse sections in eosin staining solution for 2 min, and rinse with tap water for 30 s. 4. Dehydration and mounting: Immerse the sections sequentially in 85% ethanol for 2 min → 95% ethanol I for 2 min → 95% ethanol II for 2 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → xylene I for 10 min → xylene II for 10 min to dehydrate and clear. Remove the sections from the xylene and allow them to dry slightly before mounting with neutral resin. 5. Microscopic examination and image acquisition and analysis. 6. Staining results: Cell nuclei are blue, cytoplasm is red.

[0031] 2.6 Safranin-Fixel staining of the ankle joint Safranin-Fix Green Staining of Ankle Joints: 1. Dewaxing Paraffin Sections to Water: Immerse sections sequentially in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min → anhydrous ethanol I for 10 min → anhydrous ethanol II for 5 min → anhydrous ethanol III for 5 min → 95% ethanol for 5 min → 90% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min → rinse with running water for 5 min → rinse with distilled water for 5 min. 2. Safranin O Staining: Immerse sections in Safranin O staining solution for 10 min, then rinse with running water for 5 min. 3. Fast Green Staining: Immerse sections in Fast Green staining solution for 2 min, then rinse with running water for 30 s. 4. Dehydration and Mounting: Differentiate sections sequentially in 1% glacial acetic acid for 1 min, then dehydrate and clear them in 95% ethanol I for 2 min → anhydrous ethanol I for 2 min → anhydrous ethanol II for 2 min → xylene I for 10 min → xylene II for 10 min. Remove sections from xylene, allow them to dry slightly until clear, then mount with neutral resin. 5. Microscopic examination, image acquisition and analysis.

[0032] 2.7 Statistical Methods Statistical analysis was performed using SPSS 27.0, and the experimental data were visualized using Graphpad Prism 10.0 software. For single-sample data, the Shapiro-Wilk test was used to determine the normality of quantitative data. Data conforming to a normal distribution were represented as mean ± standard deviation. The statement indicates that a homogeneity of variance test should be performed. If the variances are homogeneous, Bonferroni analysis in One-way ANOVA should be used for comparisons between multiple groups. If the variances are unequal, Dunnett's T3 analysis in One-way ANOVA should be used for comparisons between multiple groups. For quantitative or ordinal data that do not conform to a normal distribution, the median (interquartile range) (M(P)) should be used. 25 -P 75 This indicates that the nonparametric Kruskal-Wallis rank-sum test was used for comparisons among multiple groups, and the nonparametric Mann-Whitney U rank-sum test was used for comparisons between two groups. For repeated measures data, repeated measures ANOVA was used for those conforming to a normal distribution and homogeneity of variance, while generalized estimating equations were used for those not conforming to a normal distribution and homogeneity of variance. P <0.05 indicates a statistically significant difference.

[0033] 3 Experimental Results 3.1 SW+NCA can reduce ankle swelling in CIA rats A CIA animal model was established using SD rats to simulate the pathological manifestations of RA. Compared with the NC group, the CIA group rats showed significant ankle swelling and limited mobility, indicating successful model establishment; while the SW+NCA and MTX groups showed significant improvement in ankle swelling and mobility, suggesting that SW+NCA can improve paw edema in CIA rats, with better results than the SW and NCA groups. Figure 1 ).

[0034] 3.2 SW+NCA can improve paw edema, arthritis index score, and body weight in CIA rats. The Shapiro-Wilk test showed that the foot edema, arthritis index scores, and weight data followed a normal distribution; therefore, the mean ± standard deviation was used. The data are represented in Tables 2, 3, and 4. Among them, the scores for foot edema and arthritis index do not satisfy homogeneity of variance, so the generalized estimation equation is used for analysis. The weight data satisfy homogeneity of variance, so repeated measures ANOVA is used for analysis.

[0035] Analysis results of the generalized estimation equation suggest ( Figure 2 (Tables 5 and 6) Both time and grouping had a significant effect on paw edema and arthritis index scores in rats (P < 0.001). The paw edema thickness and arthritis index score of CIA model rats were significantly higher than those of NC group (P < 0.001). NCA+SW and MTX could significantly improve the paw edema thickness (P < 0.05) and arthritis index score increase (P < 0.01 or P < 0.001) caused by the disease model, and the effect was more obvious than that of SW group and NCA group.

[0036] Repeated measures equation analysis results suggest ( Figure 2 (Tables 5 and 6) Both time and grouping had a significant effect on rat body weight (P < 0.001). The body weight of CIA model rats was significantly lower than that of NC group rats (P < 0.001). SW+NCA and MTX could improve the weight loss caused by the disease model, but there was no statistical difference between them and CIA group.

[0037] Table 2. Effect of SW+NCA on paw edema thickness in CIA rats (mm). (n=6) Table 3 Effects of SW+NCA on Arthritis Index Scores in CIA Rats ( (n=6) Table 4. Effects of SW+NCA on body weight in CIA rats (g, (n=6) Table 5 Model Effect Test Table 6 Paired Comparisons of Marginal Means Remark: P <0.05, P <0.01, P <0.001, compared with the NC group, the difference was statistically significant; # P <0.05, ## P <0.01, ### P <0.001, compared with the CIA group, the difference was statistically significant.

[0038] 3.3 SW+NCA can reduce serum inflammatory factor levels in CIA rats. The level of inflammatory cytokines in rat serum was detected using ELISA. Figure 3 (Table 7) The data conform to the overall distribution and homogeneity of variance, and the mean ± standard deviation is used. The results were expressed as follows: Bonferroni analysis in One-way ANOVA was used for comparisons among multiple groups. Compared with the NC group, the serum levels of TNF-α, IL-1β, IL-6, and IL-17A in the CIA group were significantly increased ( ). P <0.001 indicates successful model establishment. The levels of the aforementioned cytokines decreased to varying degrees after intervention with different drugs. Among them, the levels of the aforementioned inflammatory factors in the SW+NCA and MTX groups were significantly different from those in the CIA group. P <0.05 or P <0.01 or P <0.001, and there was no statistically significant difference compared to the NC group ( P >0.05). The SW and NCA groups showed a certain inhibitory trend against TNF-α, IL-6, and IL-17A. P <0.05 or P<0.001), but not as significant as the SW+NCA group; neither the SW group nor NCA showed statistical significance in inhibiting IL-1β levels. P >0.05).

[0039] Table 7 Effects of SW+NCA on the levels of inflammatory factors in CIA rats ( (n=6) Remark: P <0.05, P <0.01, P <0.001, compared with the NC group, the difference was statistically significant; # P <0.05, ## P <0.01, ### P <0.001, compared with the CIA group, the difference was statistically significant.

[0040] 3.4 SW+NCA can inhibit bone destruction in CIA rats Combining 2D and 3D images of rat ankle joint reconstructed by micro-CT ( Figure 4 One-way ANOVA was used to compare the skeletal microstructural parameters of each group. Figure 5 Table 8 shows that the data conform to a normal distribution, expressed as mean ± standard deviation ( ). The results indicate that Bonferroni correction was used for comparisons of BS / BV, Tb.Sp, and BMD between groups satisfying homogeneity of the equation, and Dunnett's T3 test was used for BV / TV with unequal variances. Compared with the NC group, the BV / TV and BMD of rats in the CIA group were significantly lower ( P <0.01), BS / BV and Tb.Sp were significantly increased (P<0.01 or P<0.001), indicating that CIA induced significant bone loss and bone microstructure destruction. After intervention with different drugs, the above indicators were improved to varying degrees. Among them, the BV / TV, BS / BV, and BMD of rats in the SW+NCA group and MTX group were significantly different from those in the CIA group (P<0.05 or P<0.01), and Tb.Sp in the SW+NCA group was also significantly decreased (P<0.01). P <0.05), the bone microstructure parameters of both groups tended to be at the level of the NC group. The SW group had a significant improvement effect on BV / TV ( P <0.05, but the improvement in BS / BV, Tb.Sp and BMD did not reach statistical significance ( P >0.05); in the NCA group, only BS / BV was significantly reduced ( P <0.05, and there were no statistically significant differences in other indicators ( P >0.05). The above results indicate that SW+NCA can effectively alleviate bone loss in CIA rats and protect the integrity of bone microstructure, with better effects than SW or NCA alone.

[0041] Table 8 Effects of SW+NCA on ankle BMD and related parameters in CIA rats ( (n=3) Remark: P <0.05, P <0.01, P <0.001, compared with the NC group, the difference was statistically significant; # P <0.05, ## P <0.01, ### P <0.001, compared with the CIA group, the difference was statistically significant.

[0042] 3.5 Effects of SW+NCA on HE staining and Safranin-Fast Green staining of ankle joints in CIA rats Figure 6 HE staining and Safranin-Fix Green (SO / FG) staining of the ankle joints of rats in each group were shown. The CIA group showed more obvious inflammatory infiltration and cartilage destruction in the ankle joint than the NC group. SW+NCA and MTX could inhibit joint inflammation and cartilage destruction, and the effect was better than SW or NCA.

[0043] 3.6 Effects of NCA+SW on HE staining of liver and kidney in CIA rats Figure 7 HE-stained pathological sections of liver and kidney tissues from rats in each group were shown. The liver and kidney tissues were structurally intact, the hepatocytes were neatly arranged, and the glomeruli and renal tubules were normal in morphology with no obvious lesions, suggesting that the combined use of SW and NCA did not cause organic damage to the liver and kidneys of CIA rats.

[0044] Example 2: Therapeutic effect of SW+NCA on fibroblast synovial cells in human rheumatoid arthritis 1. Experimental cells RA-FLS cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd., catalog number: FH0699. Cells were cultured in high-glucose DMEM medium + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin solution (double antibiotics) at 37℃ and 5% CO2. Cells from passages 4 to 10 were used as experimental subjects. Cell status and density were observed before each procedure; generally, passage, cryopreservation, and seeding were performed when the cell density reached 80%. Cell starvation and different conditioned media were prepared using medium containing 1% FBS before the experiment.

[0045] 2. Cell grouping and intervention RA-FLS cells were randomly divided into 6 groups: NC group (1% FBS medium), TNF-α group (TNF-α 20 ng / mL). -1 The following groups were selected for their respective media: SW group (TNF-α 20 ng·mL⁻¹ + 60 μM SW + 1% FBS medium), NCA group (TNF-α 20 ng·mL⁻¹ + 60 μM NCA + 1% FBS medium), SW+NCA group (TNF-α 20 ng·mL⁻¹ + 40 μM SW + 20 μM NCA + 1% FBS medium), and MTX group (TNF-α 20 ng·mL⁻¹ + 10 μM MTX + 1% FBS medium).

[0046] 3. Experimental Methods 3.1 RT-qPCR detection of cellular inflammatory cytokine and matrix metalloproteinase levels After collecting cells from each group, total RNA was extracted using the Trizol method, and the RNA content and purity (A260 / A280 = 1.9–2.1) were measured. cDNA was synthesized according to the reverse transcription kit instructions. Subsequently, RT-qPCR amplification was performed, with a total reaction volume of 10 μL. The amplification program was set as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 34 s, for a total of 40 cycles. β-actin (β-actin) was used as an internal control gene, and 2... -ΔΔCt The relative expression levels of IL-1β, IL-6, and MMP-1 mRNA were calculated using the following method. Primers were purchased from Sangon Biotech (Shanghai) Co., Ltd., and their sequences are shown in Table 9.

[0047] Table 9 Primer Sequences 3.2 Scratch assay to detect the migration ability of RA-FLS cells Draw 3-5 even horizontal lines on the back of the 6-well plate using a marker. The concentration during the logarithmic growth phase is approximately 1×10⁻⁶. 6 cells·mL -1Cells were prepared into a suspension and evenly seeded into 6-well plates, then incubated at 37°C in a 5% CO2 incubator using standard methods. After the cells grew into a monolayer, a straight line (perpendicular to the horizontal line on the back of the cell plate) was drawn in the center of each well using a 10 μL sterile micropipette tip. The cells were rinsed 2-3 times with PBS to remove the central adherent cells, and this was recorded as 0 h. Each group was then cultured in the corresponding conditioned medium at 37°C in a 5% CO2 incubator. The healing of the scratches was observed under an inverted microscope at 0 h and 24 h post-scratching, and photographs were taken. ImageJ software was used to measure the scratch width at three random locations at the same time point for each group of cells. The cell migration distance was calculated as: migration rate = (0 h scratch area - 24 h scratch area) / 0 h scratch area.

[0048] 3.3 Statistical Methods Statistical analysis was performed using SPSS 27.0, and the experimental data were visualized using Graphpad Prism 10.0 software. Data were expressed as mean ± standard deviation. This indicates that comparisons among multiple groups were performed using one-way ANOVA, and pairwise comparisons were performed using the LSD method. P <0.05 indicates a statistically significant difference.

[0049] 4. Results 4.1 SW+NCA reduces IL-1β, IL-6, and MMP-1 levels in RA-FLS cells. The effects of SW+NCA on the levels of IL-1β, IL-6, and MMP-1 in RA-FLS cells were detected by RT-qPCR. Figure 8 Compared with the NC group, the relative expression levels of IL-1β, IL-6, and MMP-1 mRNA in the TNF-α group were significantly increased. P <0.001), indicating successful model establishment; compared with the TNF-α group, the relative expression levels of the above mRNAs in the SW+NCA group were significantly decreased (IL-1β, IL-6 group). P <0.001, MMP-1 group P <0.01), indicating that SW+NCA can inhibit the expression of inflammatory factors and matrix proteases in RA-FLS cells, and the effect is better than SW or NCA alone.

[0050] 4.2 SW+NCA inhibits the migration ability of RA-FLS cells The effect of SW+NCA on the 24-h migration rate of RA-FLS cells was detected by scratch assay. Figure 9 The TNF-α group showed a significantly higher cell migration rate at 24 hours compared to the NC group. P<0.01), SW+NCA can significantly inhibit the massive cell migration induced by TNF-α modeling ( P <0.01), indicating that SW+NCA can inhibit the migration ability of RA-FLS cells, and the effect is better than SW or NCA alone.

[0051] The present invention has been further described above with reference to specific embodiments. However, it should be understood that the specific description herein should not be construed as limiting the nature and scope of the present invention. Various modifications made to the above embodiments by those skilled in the art after reading this specification are all within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating rheumatoid arthritis, characterized in that, The pharmaceutical composition consists of swertiamarin and neochlorogenic acid.

2. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of swertiamarin to neochlorogenic acid is 1:1 to 4.

3. The pharmaceutical composition according to claim 2, characterized in that, The molar ratio of swertiamarin to neochlorogenic acid is 1:

2.

4. The use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a medicament for treating rheumatoid arthritis.

5. The application according to claim 4, characterized in that, The applications include reducing ankle swelling, improving foot swelling, improving arthritis index scores, and increasing weight.

6. The application according to claim 4, characterized in that, The application includes reducing serum inflammatory factor levels.

7. The application according to claim 4, characterized in that, The applications include inhibiting bone destruction.

8. The application according to claim 4, characterized in that, The applications include inhibiting joint inflammation infiltration and cartilage destruction.

9. The application according to claim 4, characterized in that, The applications include therapeutic effects on fibroblast synovial cells in rheumatoid arthritis.

10. The application according to claim 9, characterized in that, The therapeutic effects on fibroblast synovial cells in rheumatoid arthritis include reducing the levels of inflammatory factors in fibroblast synovial cells and inhibiting their migration ability.

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