Use of a diterpenoid compound in the preparation of a medicament for the treatment of rheumatoid arthritis

Abstract

The application belongs to the technical field of medicine, and discloses application of a diterpenoid compound in preparation of an anti-rheumatoid arthritis drug, wherein the diterpenoid compound is Paniculoside III; and the combination of the Paniculoside III and triptolide has a reduced-toxicity and synergistic effect on the anti-rheumatoid arthritis of triptolide, and greatly improves the efficacy and safety of triptolide.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of a diterpenoid compound in the preparation of drugs for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by morning stiffness, joint pain, and swelling. Its pathogenesis involves chronic inflammation of the synovial membrane and erosive destruction of the joints, leading to deformities and even loss of function. Currently, clinical treatment for RA primarily relies on nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and biologics. However, long-term use often results in adverse reactions such as gastrointestinal issues, liver and kidney damage, and immunosuppression. Furthermore, the treatment cycle is lengthy, and patient compliance is poor, leading to unsatisfactory clinical efficacy. Therefore, the clinical treatment of RA remains a bottleneck that modern medicine urgently needs to overcome. Exploring novel, highly effective, and low-toxicity treatments or combination therapies from traditional Chinese medicine and other natural remedies has significant scientific and clinical value.

[0003] Tripterygium wilfordii is a plant belonging to the genus Tripterygium in the family Celastraceae. Tripterygium wilfordii The dried root of *H. hookeriana* (H. hookeriana f.), widely used in the treatment of rheumatoid arthritis (RA) since 1969. Triptolide (TP), an epoxy diterpene lactone compound extracted from *Tripterygium wilfordii*, has been proven to be one of the active ingredients responsible for its immunosuppressive and anti-inflammatory effects, with definite clinical efficacy. However, its clinical application is limited due to potential hepatotoxicity, nephrotoxicity, and bone marrow suppression. To reduce TP toxicity and improve safety and efficacy, current research mainly focuses on changing dosage forms and routes of administration, as well as multi-drug combination therapy. Combination therapy is based on the theory of modern chemical drug combination therapy, combining active monomers with synergistic effects to reduce toxicity and increase efficacy. Paniculoside III (P), a diterpene compound, is a representative diterpene compound in *Pteris vittata* and is abundant in *Pteris vittata*.

[0004] In summary, there is an urgent need to provide an application of diterpenoids in the preparation of anti-rheumatoid arthritis drugs, combining P with TP to enhance anti-rheumatoid arthritis activity while systematically reducing the toxic side effects of TP, thus providing a safer and more effective traditional Chinese medicine intervention strategy for rheumatoid arthritis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an application of diterpenoid compounds in the preparation of anti-rheumatoid arthritis drugs. By combining the diterpenoid compound Paniculoside III with triptolide, the toxicity of triptolide in anti-rheumatoid arthritis drugs is reduced and the efficacy is enhanced, which greatly improves the efficacy and safety of triptolide.

[0006] The technical solution of the present invention is as follows: The use of a diterpenoid compound in the preparation of an anti-rheumatoid arthritis drug, wherein the diterpenoid compound is Paniculoside III, and the diterpenoid compound is used in combination with triptolide.

[0007] Preferably, the application includes the use of diterpenoid compounds to reduce the toxicity and enhance the efficacy of triptolide in the preparation of anti-rheumatoid arthritis drugs.

[0008] The present invention also provides a combination formulation of antirheumatoid arthritis drugs, comprising diterpenoid compounds and triptolide.

[0009] Preferably, the chemical structural formula of Paniculoside III is: .

[0010] Preferably, the mass ratio of the diterpenoid compound to triptolide is (120~500):1.

[0011] More preferably, the mass ratio of the diterpenoid compound to triptolide is (247~371):1.

[0012] Preferably, the combination formulation further includes a pharmaceutically acceptable carrier or excipient.

[0013] Technical effects of the present invention: 1. This invention combines the diterpenoid compound Paniculoside III (P) with triptolide (TP) to reduce the toxicity and enhance the efficacy of triptolide in treating rheumatoid arthritis, thus significantly improving the efficacy and safety of triptolide.

[0014] 2. In the treatment of rheumatoid arthritis, the combined use of P and TP in different proportions of this invention has shown significantly better efficacy than the TP group alone, producing a synergistic effect and providing a more efficient traditional Chinese medicine intervention plan for clinical practice.

[0015] 3. The experiment showed that the combined use of different proportions of drugs was superior to the TP monotherapy group in treating rheumatoid arthritis (general condition of rats; paw swelling and arthritis score; serum levels of IL-6, TNF-α, and IL-1β; synovial tissue pathology; protein expression levels of JAK1, STAT3, p-JAK1, and p-STAT3 in synovial tissue; mRNA expression levels of IL-6, TNF-α, and IL-17A in synovial tissue), demonstrating a significant synergistic effect. Furthermore, the combined use of different proportions of drugs improved rat organ indices, liver and kidney injury indicators, liver and kidney tissue pathology, and Fe in rat liver and kidney tissue. 2+ The levels of GSH, MDA, and the expression levels of SLC7A11 and GPX4 proteins and their mRNA in liver and kidney tissues showed a synergistic toxicity reduction effect superior to that of TP alone. Attached Figure Description

[0016] Figure 1 The swelling of the right paw of rats in each group; Figure 2 Pathological sections of synovial tissue from each group of rats (×200) (blue arrow: inflammatory cell infiltration; red arrow: synovial tissue hyperplasia or angiogenesis; green arrow: changes in synovial cell arrangement and morphology). Figure 3 Western blot was used to detect the expression of JAK1, STAT3, p-JAK1 and p-STAT3 proteins in the synovial tissue of the ankle joint in each group. Figure 4 The pathological changes in liver tissue of each group during liver and kidney tissue pathology examination (×200) (green arrow: hepatocyte nuclear lysis; yellow arrow: amorphous, eosinophilic clumps; black arrow: lymphocyte infiltration; light blue arrow: hepatic sinusoidal dilatation; red arrow: neutrophil infiltration). Figure 5 The histopathological changes of kidney tissue in each group during liver and kidney tissue pathology examination (Group M is ×100, and the rest are ×200) (Blue arrow: brownish-yellow needle-shaped or rhomboid crystals in the kidney, which are urate crystals; Green arrow: edema of renal tubular epithelial cells; Yellow arrow: renal tubular lumen filled with homogeneous, unstructured, light red amyloid deposits; Black arrow: lymphocyte infiltration; Light blue arrow: neutrophil infiltration; Red arrow: crescent-shaped bodies). Figure 6 Western blot was used to detect the expression of SLC7A11 and GPX4 proteins in liver tissues of different groups; Figure 7 Western blot was used to detect the expression of SLC7A11 and GPX4 proteins in kidney tissues of different groups. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 scope of protection of the present invention.

[0018] Unless otherwise specified in the following examples, the implementation conditions are generally those of routine experiments, and the raw materials are all commercially available or prepared by conventional methods in the art.

[0019] I. Experimental Reagents and Animals 1. Experimental reagents Paniculoside Ⅲ (P) (CAS No. 60129-65-9) is available through regular purchase.

[0020] Bovine type II collagen (Chondrex, USA, catalog number: 20022); Freund's incomplete adjuvant (Chondrex, USA, catalog number: 7002); JAK1 mouse antibody (Proteintech, catalog number: 66466-1-Ig); p-JAK1 rabbit antibody (Affinity Biosciences, catalog number: AF2012); p-STAT3 mouse antibody (Proteintech, catalog number: 60479-1-Ig); STAT3 rabbit antibody (Proteintech, catalog number: 10253-2-AP); GAPDH mouse antibody (Proteintech, catalog number: 60004-1-Ig); goat anti-rabbit IgG-HRP (Proteintech, catalog number: RGAR001); goat anti-mouse IgG-HRP (Proteintech, catalog number: RGAM001). SLC7A11 and GPX4 rabbit antibodies (Proteintech, catalog numbers: 32384-1-AP, 30388-1-AP). Other materials and reagents were obtained through routine purchase unless otherwise specified.

[0021] 2. Laboratory animals SPF-grade male SD rats, weighing 180–220 g and aged 5–6 weeks, were used. The animals were housed at a room temperature of 23±2 °C and a humidity of 50%–60%. After one week of acclimatization, a rat model of CIA was established.

[0022] II. Experimental Methods 1. Experimental grouping, modeling, and drug administration Based on the conversion of the human oral dose of TP, the routine administration dose of TP in rats was determined to be 18.62 μg / kg•d. -1The standard rat dosage of P was determined to be 2.3 mg / kg / day through preliminary rat experiments. -1 In the combined use trial of TP and P in this invention, the dosage of TP was maintained at 18.62 μg / kg•d. -1 The dosage of P remains unchanged at its usual dosage (2.3 mg / kg / day). -1 Based on this, the ratios were increased by a gradient (1x, 2x, 3x) to obtain the TP:P 1:1 group, the TP:P 1:2 group, and the TP:P 1:3 group, respectively.

[0023] Rats were randomly divided into a normal control group (C), a model group (M), and a positive control group (Y) using methotrexate (1 mg / kg•d). -1 ), TP group (TP) (18.62 μg / kg•d) -1 ), TP:P 1:1 group (18.62 μg / kg•d) -1 2.3 mg / kg•d -1 ), TP:P 1:2 group (18.62 μg / kg•d) -1 4.6 mg / kg•d -1 ), TP:P 1:3 group (18.62 μg / kg•d) -1 6.9 mg / kg•d -1 Six rats were placed in each group. After successful model establishment, the above-mentioned dosage was administered for 21 days. All rats were housed in the same environment with free access to water and food. Their body weight was measured daily, and the dosage was adjusted according to their body weight.

[0024] Model establishment: After one week of adaptive feeding, a rat model of collagen-induced arthritis (CIA) was established. An emulsion (concentration C=1 mg / mL) was prepared by thoroughly mixing bovine type II collagen solution with an equal volume of Freund's incomplete adjuvant under ice bath conditions. Rats were initially immunized by intradermal injection of 0.2 mL of the emulsion at the base of their tails, followed by a booster injection of 0.1 mL of the emulsion seven days later. Rats in the normal control group were intradermally injected with the same volume of physiological saline at the base of their tails. The two modeling events were performed one week apart.

[0025] III. Experimental Test Items and Results (I) Study on the synergistic effect of TP and P in treating rheumatoid arthritis 1. General condition observation After successful modeling, rats in all groups exhibited reduced appetite and activity, lethargy, and swollen paws with limited joint movement. Some rats gnawed at the swollen areas due to pain. After the drug administration ended, the normal group rats were in good spirits and active. The model group rats remained swollen. In all drug-treated groups, appetite returned, activity levels were basically normal, and paw swelling significantly decreased. The swelling of the right paw in each group is shown in the figure. Figure 1 .

[0026] 2. Rat paw swelling and arthritis scoring Before the first modeling, before the second modeling, after successful modeling, one week after drug administration, two weeks after drug administration, and three weeks after drug administration, the paw volume of rats was measured, paw swelling was calculated, and arthritis scores were assigned. Paw swelling (mL) = Average paw volume after modeling - Average paw volume before modeling. Arthritis scoring criteria: 0 points - no swelling or redness; 1 point - redness and swelling of the tarsal or ankle joints; 2 points - swelling and redness from the ankle to the tarsal; 3 points - redness and swelling from the ankle to the metatarsal joints; 4 points - redness and swelling around the ankle, tarsal, and metatarsal bones. An arthritis score greater than 4 points indicates successful modeling. The score for a single limb of the rat cannot exceed 4 points, and the total score cannot exceed 16 points.

[0027] The results of paw swelling in each group are shown in Table 1. The paw swelling in the model group was significantly higher than that in the normal group after successful modeling, and it continued to worsen over time, indicating that arthritis was successfully induced. The paw swelling in all drug-treated groups was significantly lower than that in the model group after successful modeling and at all subsequent time points, indicating that drug administration can alleviate the arthritis swelling in RA rats.

[0028] Table 1 Comparison of paw swelling in rats of different groups (mL)

[0029] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with M, 3) indicates P<0.05, 4) indicates P<0.01.

[0030] The arthritis scores are shown in Table 2. The arthritis scores in the model group showed no improvement from the time the model was successfully established until the end of the experiment, confirming that RA arthritis symptoms were successfully induced and persisted. From the first week of administration, the arthritis scores in all treatment groups were significantly lower than those in the model group, indicating that administration could rapidly and effectively relieve overall arthritis symptoms. Different ratios of TP:P combination therapy were superior to TP monotherapy, with the TP:P 1:3 group showing a lower arthritis score at three weeks of administration, demonstrating relatively better efficacy.

[0031] Table 2 Comparison of arthritis scores in rats of different groups (points)

[0032] Note: Compared with M, 3) indicates P<0.05, and 4) indicates P<0.01.

[0033] 3. Serum ELISA detection Before the last administration, rats were fasted for at least 12 hours but allowed free access to water. One hour after the last administration, the rats were anesthetized, and blood was collected from the abdominal aorta. After blood collection, the rats were left to stand at room temperature for 1 hour, and then incubated at 4 ℃ and 5000 r•min. -1 Centrifuge for 10 min, separate the serum, and store at -80 ℃. Serum samples were then analyzed using ELISA to detect the levels of interleukin-6 (IL-6), rat tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β).

[0034] The results are shown in Table 3. Compared with the normal group, the serum levels of IL-6, TNF-α, and IL-1β in the model group rats were significantly increased, indicating that the rat RA model was successfully established. Compared with the model group, the levels of inflammatory factors in the Y group, TP group, and each TP:P combination treatment group were significantly reduced. Among them, with the increase of dose, the levels of inflammatory factors in each TP:P combination treatment group showed a decreasing trend, indicating that each TP:P combination treatment group had a synergistic effect superior to TP alone, and the efficacy was positively correlated with the dose, effectively improving the RA symptoms in rats.

[0035] Table 3. Serum levels of IL-6, TNF-α, and IL-1β in rats of each group

[0036] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with M, 3) indicates P<0.05, 4) indicates P<0.01.

[0037] 4. Pathological examination of synovial tissue Fixed ankle synovial tissue was collected, stained with hematoxylin and eosin (HE), and mounted with neutral resin. The morphological changes of the synovial tissue in each group of ankle joints were observed under an optical microscope.

[0038] See results Figure 2 In the normal group, the synovial cells were regularly arranged, the interstitial fibrous tissue within the synovium was loosely arranged, and there was no inflammatory cell infiltration. In the model group, the synovial cells showed structural integrity disruption, degeneration and necrosis, irregular arrangement of the interstitial fibrous tissue within the synovium, and extensive inflammatory cell infiltration, with significant synovial tissue hyperplasia and vascular proliferation. In the positive drug group, mild synovial tissue hyperplasia was observed in some areas, while the synovial cells in most areas were normally arranged, with a small amount of inflammatory cell infiltration. In the TP group, the synovial cells were regularly arranged, with a small amount of inflammatory cell infiltration and tissue hyperplasia. Different ratios of TP:P combined therapy groups showed mild inflammatory cell infiltration, no significant vascular proliferation, and mild synovial tissue hyperplasia.

[0039] 5. Western Blot analysis of ankle synovial tissue The protein concentration in the supernatant of ankle synovial tissue was measured using a BCA protein concentration assay kit. Based on the results, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After transfer, the membrane was blocked with a 5% skim milk solution prepared with 1×TBST buffer (1×Tris buffered saline containing Tween-20). At 4 ℃, the membrane was incubated overnight with primary antibodies JAK1 mouse antibody, p-JAK1 (phosphorylated JAK1) rabbit antibody, STAT3 rabbit antibody, p-STAT3 (phosphorylated STAT3) mouse antibody (dilution ratio 1:1000), and GAPDH mouse antibody (dilution ratio 1:2000). Then, the membrane was incubated at room temperature with goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP secondary antibodies (dilution ratio 1:3000) for 1 h. After development, the gray values ​​of each band were quantitatively analyzed using gray value software. The relative expression levels of JAK1 mouse antibody, p-JAK1 rabbit antibody, STAT3 rabbit antibody, and p-STAT3 were represented by the ratio of their gray values ​​to GAPDH.

[0040] See results Figure 3 As shown in Table 4, compared with the normal group, the phosphorylation levels of JAK1 and STAT3 in the model group were significantly increased; compared with the model group, the protein phosphorylation levels in each treatment group were significantly decreased. Different ratios of TP:P combined with other drugs all showed a synergistic effect superior to TP alone, especially the TP:P 1:3 group, which showed the best reduction in JAK1 and STAT3 phosphorylation levels in the synovial tissue of the rat ankle joint.

[0041] Table 4. Relative expression levels of JAK1, STAT3, p-JAK1, and p-STAT3 proteins in the synovial tissue of rats in each group.

[0042] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with M, 3) indicates P<0.05, 4) indicates P<0.01.

[0043] 6. RT-qPCR detection of ankle synovial tissue RNA was extracted from the synovial tissue of the ankle joint of rats in each group using the TRNzol reagent method. The concentration and purity of the extracted RNA were determined, and its integrity was detected by 1.5% agarose gel electrophoresis. The mRNA was reverse transcribed using a template, followed by PCR amplification using cDNA as a template. Finally, quantitative analysis was performed using real-time quantitative PCR, and the relative gene expression level was determined by... To calculate.

[0044] The results are shown in Table 5. Compared with the normal group, the relative expression levels of IL-6, TNF-α, and IL-17A mRNA in the model group were significantly increased. Compared with the model group, the relative expression levels of IL-6, TNF-α, and IL-17A mRNA in each treatment group were significantly reduced. The TP:P combination treatment groups at different ratios all showed a synergistic effect superior to TP alone, especially the TP:P 1:3 group, which significantly reduced the relative expression levels of IL-6, TNF-α, and IL-17A mRNA in the synovial tissue of rat ankle joints.

[0045] Table 5. Relative mRNA expression levels of IL-6, TNF-α, and IL-17A in synovial tissue of rats in each group.

[0046] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with M, 3) indicates P<0.05, 4) indicates P<0.01.

[0047] (II) Study on the combined toxicity reduction effect of TP and P

[0048] 7. Organ Index Measurement After blood collection from each group of rats, the liver, kidneys, spleen, and thymus were removed using sterile instruments. The rats were then rinsed with physiological saline to remove surface blood, and any remaining liquid was blotted dry with filter paper before weighing. The weight was recorded using a 0.01% balance, and the organ index was calculated based on the rat's body weight. Organ index = organ mass (mg) / body weight (g) × 100%.

[0049] The results are shown in Table 6. All organ indices decreased due to the modeling effect, indicating non-specific changes. The kidney index in the TP:P combined treatment group recovered to levels comparable to the normal group compared to the TP group, suggesting that TP and P combined in a certain ratio showed a trend of reducing nephrotoxicity caused by TP alone. Compared to the normal group, the spleen and thymus indices in the model group were significantly increased, indicating that the RA model successfully induced hyperplasia or edema of rat immune organs. The spleen and thymus indices in all treatment groups were significantly lower than those in the model group, and the thymus index in the combined treatment group recovered to levels comparable to the normal group. This indicates that the TP:P combined treatment group can effectively inhibit the abnormal enlargement of immune organs induced by the RA model.

[0050] Table 6 Organ indices of rats in each group

[0051] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0052] 8. Liver and kidney damage marker detection Serum samples were collected and tested for alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Crea) using a fully automated biochemical analyzer.

[0053] The results are shown in Table 7. Compared with the normal group, the liver and kidney damage indicators in the TP group were significantly higher than those in the normal group and all other groups, indicating that the drug has significant liver and kidney toxicity at this dose. The liver damage indicators (ALT, AST) and kidney damage indicators (BUN, Crea) in the TP and P combination therapy group were significantly lower than those in the TP group, indicating that the combined therapy reduced the impact on liver and kidney damage indicators and significantly alleviated the liver and kidney biochemical damage caused by TP alone.

[0054] Table 7 Comparison of serum ALT, AST, BUN, and Crea levels in different groups of rats

[0055] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0056] 9. Liver and kidney histopathological examination Fixed liver and kidney tissues were collected, stained with hematoxylin and eosin (HE), mounted with neutral resin, and the histomorphological changes of the liver and kidneys were observed under an optical microscope.

[0057] The results of liver histopathological changes in each group are shown in the figure. Figure 4 In the normal group, the liver lobules and lamina were arranged regularly and neatly, with no obvious dilation or compression of the sinusoids; there were no obvious abnormalities in the portal areas between adjacent liver lobules; and no obvious inflammatory changes were observed. In the model group, the liver lamina were disordered; hepatocyte degeneration, edema, and mild necrosis were observed in the surrounding area. In the Y group, the liver lamina were slightly disordered, with no obvious hepatocyte degeneration or edema. In the TP group, the liver lamina were disordered; hepatocyte degeneration, edema, and partial hepatocyte necrosis were observed, with obvious inflammatory cell infiltration. In the TP:P 1:1 group, hepatocyte shrinkage, disordered liver lamina arrangement, and sinusoidal dilation were observed. In the TP:P 1:2 group, lymphocyte infiltration was observed, with a small amount of hepatocyte degeneration and edema. In the TP:P 1:3 group, no obvious hepatocyte degeneration or edema was observed.

[0058] The results of the renal histopathological changes in each group are shown in the figure. Figure 5In the normal group, the renal tissue of rats showed normal glomeruli and tubular epithelial cell structure, without degeneration or edema, and no congestion, inflammatory cell infiltration, or fibrosis in the renal interstitium. In the model group, significant proliferation of parietal epithelial cells of the glomerular capsule was observed in multiple areas of the kidney tissue, forming crescent-shaped bodies around the glomeruli; lymphocytes and neutrophils were also observed infiltrating the surrounding area. In the Y group, glomeruli were evenly distributed in the cortex, with uniform cell number and matrix within the glomeruli. In the TP group, the renal glomeruli were significantly atrophied, with degeneration of the tubular epithelial cells, significant interstitial congestion, and localized inflammatory cell infiltration. In the TP:P 1:1 group, the renal glomeruli were still atrophied, some tubular epithelial cells showed degeneration and edema, and localized interstitial congestion was observed. In the TP:P 1:2 and TP:P 1:3 groups, the glomerular structure was normal, with only mild interstitial congestion observed; the number of cells within the glomeruli was uniform.

[0059] 10. Colorimetric determination of Fe in rat liver and kidney tissues 2+ GSH and MDA content Weigh 0.1 g of liver and kidney tissue samples and add 1 mL of 1×PBS (each 1 mL of PBS should contain 10 μL of benzyl sulfonyl fluoride PMSF). Homogenize the homogenized liver and kidney tissues using a tissue homogenizer. Centrifuge the homogenized liver and kidney tissues at 4 ℃ and 12000 rpm for 10 min, and collect the supernatant. Quantify BCA protein in the supernatant and then detect Fe in rat liver and kidney tissues using a colorimetric method. 2+ Content of reduced glutathione (GSH) and malondialdehyde (MDA).

[0060] The results are shown in Tables 8 and 9. Compared with the normal group, GSH levels in the liver and kidney tissues of other groups were significantly decreased, and Fe... 2+ And MDA levels were significantly elevated. Compared with the TP group, Fe in each TP and P combination therapy group was significantly higher. 2+ MDA levels decreased significantly, while GSH levels increased significantly. This indicates that different ratios of TP and P combined administration effectively increased GSH levels in liver and kidney tissues and decreased Fe levels. 2+ The levels of MDA showed a trend of dose-dependent reduction in hepatotoxicity and nephrotoxicity caused by TP alone.

[0061] Table 8. Fe in liver tissue of rats in each group 2+ Comparison of GSH and MDA levels

[0062] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0063] Table 9. Fe in the kidney tissue of rats in each group 2+ Comparison of GSH and MDA levels

[0064] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0065] 11. Western Blot analysis of liver and kidney tissues Refer to the aforementioned Western Blot method for detecting ankle synovial tissue. Primary antibodies: rabbit-derived antibodies SLC7A11 (dilution ratio 1:1000) and GPX4 (dilution ratio 1:1000), and mouse-derived antibody GAPDH (dilution ratio 1:2000); Secondary antibodies: goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (dilution ratio 1:4000).

[0066] The results of liver tissue examination are shown below. Figure 6 The results of kidney tissue examination are shown in Table 10 and below. Figure 7 According to Table 11, the expression of SLC7A11 and GPX4 proteins in the liver and kidney tissues of rats in the TP group was significantly lower than that in the normal group, while the protein levels in the other drug-treated groups were significantly higher than those in the TP group, indicating that the combined use of TP and P can effectively upregulate the expression of SLC7A11 and GPX4 proteins.

[0067] Table 10. Expression levels of SLC7A11 and GPX4 proteins in liver tissue of rats in each group.

[0068] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0069] Table 11. Expression levels of SLC7A11 and GPX4 proteins in the kidney tissue of rats in each group.

[0070] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0071] 12. RT-qPCR detection of liver and kidney tissues Following the aforementioned method for detecting ankle synovial tissue using RT-qPCR, the mRNA expression levels of SLC7A11 and GPX4 in rat liver and kidney tissues were detected. The results of the liver and kidney tissue detection are shown in Tables 12 and 13, respectively.

[0072] The mRNA expression of SLC7A11 and GPX4 in the liver and kidney tissues of rats in the TP group was significantly lower than that in the normal group. Compared with the TP group, the mRNA levels in other drug-treated groups were significantly increased, indicating that the combination of TP and P can effectively upregulate the mRNA levels of SLC7A11 and GPX4, showing a synergistic toxicity reduction effect superior to TP alone.

[0073] Table 12. mRNA expression levels of SLC7A11 and GPX4 in liver tissue of rats in each group.

[0074] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0075] Table 13. mRNA expression levels of SLC7A11 and GPX4 in kidney tissue of rats in each group

[0076] Note: Compared with C, 1) indicates P<0.05, 2) indicates P<0.01; compared with TP, 3) indicates P<0.05, 4) indicates P<0.01.

[0077] The above experiments demonstrate that the combined use of P and TP not only ensures the therapeutic effect on RA but also reduces the toxicity of TP and improves the safety of medication.

[0078] 13. Conclusion The above experimental results demonstrate that the combined use of P and TP not only ensures the therapeutic effect on RA but also reduces the toxicity of TP, thereby improving drug safety. This invention effectively enhances the efficacy and safety of TP, and has significant clinical application value and market prospects for RA.

[0079] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a diterpenoid compound in the preparation of an antirheumatoid arthritis drug, characterized in that: The diterpenoid compound is Paniculoside III, and Paniculoside III is used in combination with triptolide.

2. The application according to claim 1, characterized in that: The applications include the use of Paniculoside III to reduce the toxicity and enhance the efficacy of triptolide in the preparation of anti-rheumatoid arthritis drugs.

3. A combination formulation of drugs for treating rheumatoid arthritis, characterized in that: Including Paniculoside III and Tripterygium wilfordii.

4. The application according to claim 1 or the combination formulation according to claim 3, characterized in that: The chemical structural formula of Paniculoside III is as follows: 。 5. The application according to claim 1 or the combination formulation according to claim 3, characterized in that: The mass ratio of Paniculoside III to Tripterygium wilfordii is (120~500):

1.

6. The application or combination formulation according to claim 5, characterized in that: The mass ratio of Paniculoside III to Tripterygium wilfordii is (247~371):1.

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