Use of oridonin in preparation of a medicine for preventing and / or treating rheumatoid arthritis synovitis
Oridonin A blocks glycolysis flux in synovial cells by covalently binding to glyceraldehyde-3-phosphate dehydrogenase, activating antioxidant signaling pathways, and inhibiting inflammatory pathways, thus solving the treatment challenge of synovitis in rheumatoid arthritis and achieving highly effective and safe drug efficacy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU UNIV
- Filing Date
- 2026-04-14
- Publication Date
- 2026-05-29
Smart Images
Figure CN122097339A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of oridonin in the preparation of drugs for the prevention and / or treatment of synovitis in rheumatoid arthritis. Background Technology
[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic erosive arthritis. Synovitis is its core pathological change, mainly manifested as abnormal thickening of the synovial lining, massive infiltration of inflammatory cells, and pannus formation, which in turn leads to the destruction of articular cartilage and bone tissue, ultimately causing joint deformity and loss of function. Fibroblast-like synovial cells (FLSs) play a key role in the progression of RA synovitis. They undergo metabolic reprogramming in the inflammatory microenvironment, exhibiting high glycolytic characteristics similar to tumor cells, abnormally proliferating, migrating, invading, and secreting large amounts of inflammatory factors and matrix metalloproteinases, forming a vicious positive feedback loop of "metabolism-inflammation-oxidative stress," continuously driving synovial lesions and joint damage.
[0003] Currently, the main drugs used in clinical treatment of rheumatoid arthritis (RA) include nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, disease-modifying antirheumatic drugs (such as methotrexate), biologics, and small molecule targeted drugs. Although these drugs can relieve symptoms and control inflammation, they have significant limitations: First, most drugs focus on immunosuppression and cannot directly target and reverse the pathological metabolic phenotype of synovial lymphocytes (FLLSs), making it difficult to eradicate pathological synovial cells, and relapse is common after drug withdrawal. Second, long-term immunosuppression is accompanied by an increased risk of infection and tumors, and biologics are expensive with significant individual differences in response. Third, existing drugs have limited effectiveness in intervening in abnormal proliferation, invasion, and glycolytic metabolic reprogramming of synovial cells, and cannot fundamentally block the pathological process of RA synovitis.
[0004] Oridonin (ORI) is a natural enanthocarne diterpenoid extracted from *Rabdosia rubescens*, a plant in the Lamiaceae family. It possesses various pharmacological activities, including anti-inflammatory, antioxidant, immunomodulatory, and antitumor effects. Current research indicates that oridonin has a certain therapeutic effect on arthritis; however, its direct target, molecular binding mechanism, and downstream signaling pathways in combating RA synovitis remain unclear, limiting its further development and clinical translation as a targeted drug.
[0005] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key rate-limiting enzyme in the glycolysis pathway, catalyzing the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. It also participates in non-metabolic functions such as oxidative stress and inflammation regulation. Studies have found that GAPDH is highly expressed and abnormally active in RA synovial cells, making it a core target mediating glycolytic reprogramming and inflammatory activation in flaccid lesions (FLSs). Currently, there are no reports of natural products covalently targeting GAPDH and treating RA synovitis by regulating the GAPDH-KEAP1-NRF2 signaling axis, nor are there any drug compositions or application regimens based on this mechanism of action.
[0006] Related technology CN 108926525 A discloses a topical transdermal absorption preparation for arthritis, containing oridonin, its pharmaceutically acceptable derivatives, or a prodrug. This invention utilizes transdermal absorption technology, comprehensively considering factors such as the concentration of oridonin, the choice of matrix, and the selection of transdermal absorption enhancers, enabling oridonin to be absorbed into the body through the skin and directly reach the joints, overcoming the drawback of poor oral efficacy of oridonin. This technology clarifies that it belongs to the topical preparation type, is merely a dosage form improvement, has no clear molecular target, and only targets acute inflammation, not involving any core pathology related to synovitis in rheumatoid arthritis, and does not disclose the target mechanism, metabolic regulation, or covalent binding mechanism.
[0007] Related technology CN 111454916 A discloses a novel application of glyceraldehyde-3-phosphate dehydrogenase protein or its immune fragment. This invention provides the application of glyceraldehyde-3-phosphate dehydrogenase protein or its immune fragment in blood as a target in the development, screening, and / or preparation of drugs for the prevention and / or inhibition of tumor-related diseases. This technology is only applicable to the field of oncology and does not involve any core pathology related to synovitis in rheumatoid arthritis, nor does it disclose the target mechanism, metabolic regulation, or covalent binding mechanism.
[0008] Therefore, developing a natural drug that directly targets GAPDH, precisely blocks glycolytic metabolism in RA synovial cells, and simultaneously regulates inflammatory and oxidative stress signaling pathways, thus breaking through the current bottleneck in RA treatment, has significant clinical value and scientific research significance. Summary of the Invention
[0009] To address the technical problems of existing drugs for treating synovitis of rheumatoid arthritis, such as unclear targets, inability to reverse abnormal metabolism of synovial cells, and significant side effects, the purpose of this invention is to provide an application of oridonin in the preparation of drugs for the prevention and / or treatment of synovitis of rheumatoid arthritis. This invention clarifies the direct target, binding site, mechanism of action, dosage, and efficacy range of oridonin, providing an anti-RA synovitis drug with a clear target, well-defined mechanism, and high safety.
[0010] To achieve the above objectives, the present invention adopts the following technical solution: The application of oridonin in the preparation of a drug for the prevention and / or treatment of synovitis in rheumatoid arthritis, wherein the drug uses oridonin as the active ingredient, and inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase by covalently targeting and binding to the cysteine 152 site of glyceraldehyde-3-phosphate dehydrogenase, thereby blocking the glycolytic flux of synovial cells, activating the KEAP1-NRF2 / HO-1 antioxidant signaling pathway, inhibiting the NF-κB inflammatory pathway, reducing the expression of matrix metalloproteinases, inducing G2 / M phase arrest in synovial cells, thereby inhibiting abnormal proliferation, migration and invasion of synovial cells, and improving the pathological damage of joint tissue in AIA mice; The drug is an oral preparation or an injection; The purity of the oridonin A is greater than or equal to 98%.
[0011] The aforementioned oridonin, from a chemical structure perspective, belongs to the ent-kaurane type of enantio-kaurane diterpenoid, with the molecular formula C0. 20 H 28 O6. Its core structure consists of a tetracyclic hydroxyphenanthrene skeleton with 7,20-epoxy bonds and multiple oxygen-containing functional groups, including hydroxyl or acyloxy groups at C-1, C-6, C-7, and C-14, and a ketone carbonyl group at C-15. Its most characteristic chemically active center is the α,β-unsaturated ketone structure on ring A. This group, as a strong electrophile, can covalently bind to nucleophilic sites (such as cysteine thiol groups) in biomolecules via Michael addition reactions, and is a key pharmacophore for exerting various biological activities. In terms of physicochemical properties, oridonin is a white crystalline powder, poorly soluble in water, and highly lipid-soluble. This characteristic limits its oral absorption and bioavailability to some extent, and has become one of the main directions for its structural optimization.
[0012] Preferably, the oridonin A undergoes a specific Michael addition reaction with the cysteine residue at position 152 of the active site of glyceraldehyde-3-phosphate dehydrogenase via the α,β-unsaturated ketone group in its molecular structure, thereby achieving irreversible covalent binding. The equilibrium dissociation constant Kd for the covalent bond is 2.68 × 10⁻⁶. -10 M; The half-maximal inhibitory concentration (IC50) of oridonin in inhibiting glyceraldehyde-3-phosphate dehydrogenase activity 50 =2.277μM.
[0013] Preferably, the drug works by blocking the glycolytic flux of synovial cells, specifically by significantly accumulating upstream metabolites and significantly reducing downstream metabolites. The upstream metabolites include one or more of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate; The downstream metabolites include one or more of 3-phosphoglyceric acid, 2-phosphoglyceric acid, pyruvate, and lactate.
[0014] Preferably, the drug promotes the accumulation of intracellular methylglyoxal, induces KEAP1 protein dimerization, and thereby activates the KEAP1-NRF2 / HO-1 antioxidant signaling pathway.
[0015] Preferably, the drug improves the pathological damage of joint tissues in AIA mice by relieving paw swelling, reducing arthritis scores, alleviating synovial hyperplasia and inflammatory cell infiltration in the ankle joint, and reducing serum IL-1β, IL-6 and IL-8 levels in AIA mice.
[0016] Preferably, the drug inhibits the NF-κB inflammatory pathway by suppressing the phosphorylation level of NF-κB p65 and reducing the level of inflammatory factors; The inflammatory factors include IL-1β, IL-6, and IL-8.
[0017] Preferably, the drug reduces matrix metalloproteinase expression by inhibiting the expression of MMP1, MMP3, MMP9 and MMP13 in synovial tissue.
[0018] A pharmaceutical composition for treating rheumatoid arthritis synovitis by targeting glyceraldehyde-3-phosphate dehydrogenase, comprising an effective dose of oridonin A and pharmaceutically acceptable excipients.
[0019] Preferably, the effective dose used is: The optimal concentration for oridonin A administration to synovial cells is 0.5–2 μM. The dosage of oridonin A in AIA mice is 10-20 mg / kg.
[0020] Preferably, the pharmaceutical composition is a single formulation or a compound formulation; The single formulation is a formulation containing only oridonin as the active ingredient; The compound preparation is a combination of oridonin and methotrexate, or a combination of oridonin and tripterygium glycosides.
[0021] Compared with the prior art, the present invention has at least the following technical effects: This invention provides the application of oridonin in the preparation of drugs for the prevention and / or treatment of synovitis in rheumatoid arthritis, clarifying the direct target, binding site, mechanism of action, dosage, and efficacy range of oridonin, and providing an anti-RA synovitis drug with a clear target, clear mechanism, and high safety.
[0022] The core mechanism of oridonin A application is as follows: Oridonin A, through its α,β-unsaturated ketone group, specifically covalently binds to the Cys152 residue of the active site of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) via Michael addition, competitively occupying the NAD+ binding pocket of GAPDH and inhibiting GAPDH enzyme activity. This, in turn, blocks the abnormal glycolytic flux in RA synovial cells, leading to the accumulation of upstream glycolytic intermediates and promoting the accumulation of methylglyoxal (MGO). MGO induces KEAP1 dimerization, relieving its inhibition of NRF2, activating the NRF2 / HO-1 antioxidant and anti-inflammatory pathway, and simultaneously inhibiting the activation of the NF-κB inflammatory signaling pathway, thus blocking the pathological process of RA synovitis from multiple dimensions of metabolism, inflammation, and oxidative stress.
[0023] Based on the application mechanism, the following was achieved: (1) Precise target: For the first time, oridonin A is clearly identified as having GAPDH as its direct target and achieving specific inhibition through covalent binding of Cys152 residues. It has strong targeting and low off-target risk. (2) Multi-pathway regulation: Simultaneously blocking abnormal glycolysis of synovial cells, activating antioxidant and anti-inflammatory pathways, and inhibiting the activation of inflammatory signals, thereby intervening in the pathological process of RA synovitis in multiple dimensions; (3) Significant therapeutic effect: In vitro and in vivo experiments have confirmed that oridonin can significantly reduce paw swelling, synovial hyperplasia and inflammatory infiltration in RA model mice, and reduce the levels of inflammatory factors and matrix metalloproteinases. Its therapeutic effect is comparable to that of methotrexate. (4) High safety: At therapeutic doses, it has no obvious toxicity to major organs such as the heart, liver, spleen, lungs, and kidneys, and no significant adverse reactions; (5) Mechanism innovation: The GAPDH-MGO-KEAP1-NRF2 signal axis was discovered and verified for the first time, providing a new target and theoretical basis for RA treatment. Attached Figure Description
[0024] Figure 1 This is a schematic diagram illustrating the inhibitory effect of oridonin A on the viability of MH7A cells. Figure 2 Oridonin inhibits IL-1β-induced expression of inflammatory factors in MH7A cells; Figure 3 This is a schematic diagram illustrating the inhibition of IL-1β-induced oxidative stress in MH7A by oridonin A. Figure 4 This is a schematic diagram illustrating the inhibition of IL-1β-induced migration and invasion of MH7A cells by oridonin. Figure 5 A schematic diagram illustrating how oridonin promotes IL-1β-induced G2 / M phase arrest in MH7A cells; Figure 6 This is a schematic diagram showing that oridonin A has no significant effect on IL-1β-induced apoptosis in MH7A cells; Figure 7 A schematic diagram illustrating how oridonin improves the general condition and arthritis phenotype in AIA mice; Figure 8 A schematic diagram illustrating how oridonin reduces pathological damage to ankle joint tissue in AIA mice. Figure 9 Schematic diagram illustrating how oridonin reduces serum inflammatory factor levels in AIA mice; Figure 10 This is a schematic diagram illustrating how oridonin inhibits the expression of MMP1, MMP3, MMP9, and MMP13 in the ankle joint of AIA mice. Figure 11 Schematic diagram showing that no significant toxicity was observed in the major organs of mice at therapeutic doses of oridonin A and methotrexate. Figure 12 A schematic diagram illustrating the inhibition of glycolysis in MH7A cells induced by oridonin A treatment; Figure 13 A schematic diagram illustrating the accumulation of intermediate metabolites from glycolysis caused by the inhibition of GAPDH enzyme activity by oridonin. Figure 14 A schematic diagram illustrating how oridonin promotes the accumulation of methylglyoxal (MGO) in IL-1β-induced MH7A cells; Figure 15 This is a schematic diagram showing how oridonin induces KEAP1 dimerization and activates the NRF2 / HO-1 pathway via MGO. Figure 16 A schematic diagram of MH7A cells stimulated with IL-1β and treated with exogenous MGO; Figure 17 This is a schematic diagram illustrating the inhibition of IL-1β-induced NF-κB signaling pathway activation by oridonin. Figure 18 This is a schematic diagram illustrating how oridonin's regulation of the KEAP1-NRF2 / HO-1 and NF-κB pathways depends on GAPDH. Detailed Implementation
[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the accompanying drawings is 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. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.
[0026] One specific embodiment of the present invention is as follows: I. In vitro pharmacodynamic study of oridonin in the treatment of RA synovitis 1. Cell culture: MH7A cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution, and maintained in a constant temperature incubator at 37°C and 5% CO2.
[0027] 2. Experimental groups: control group, IL-1β (10 ng / mL) model group, low (0.5 μM), medium (1 μM), and high (2 μM) dose groups of oridonin A, and methotrexate positive control group.
[0028] 3. Detection indicators: (1) CCK-8 assay for cell viability: Oridonin A showed no cytotoxicity in the concentration range of 0.1-2 μM and could significantly inhibit IL-1β-induced proliferation of MH7A cells; (2) ELSA and RT-qPCR detection of inflammatory factors: Oridonin significantly downregulated the mRNA and protein expression of IL-6, IL-8, COX-2 and iNOS; (3) ROS and NO detection: Oridonin significantly reduced IL-1β-induced ROS and NO levels and alleviated oxidative stress; (4) Scratch and Transwell assays: Oridonin A significantly inhibited the migration and invasion of MH7A cells; (5) Cell cycle detection: Oridonin A induces G2 / M phase arrest in MH7A cells and regulates the CDK1 / Cyclin B1 signaling axis; (6) Apoptosis detection: Oridonin A has no significant effect on apoptosis and has good safety.
[0029] 4. Experimental Results: 4.1 Inhibitory effect of oridonin on MH7A cell viability: The toxic effect of oridonin (ORI) on human MH7A cells was detected by CCK-8 assay.
[0030] like Figure 1The diagram illustrates the inhibitory effect of oridonin A on MH7A cell viability. (A) The CCK-8 assay was used to detect the toxic effects of different concentrations of ORI (0.1, 0.5, 2, 10, and 50 μM) on MH7A cells (n=4). (B) The CCK-8 assay was used to assess the effect of ORI on MH7A cell viability (n=4), with MTX as a positive control. ns indicates no statistically significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0031] Results combined Figure 1 It can be seen that, after 24 hours of stimulation with oridonin, oridonin A showed no significant toxicity to MH7A cells in the range of 0.1–2 μM. Figure 1 (Middle A). Simultaneously, under stimulation with 10 ng / mL IL-1β, oridonin (0.5, 1, and 2 μM) inhibited the proliferation of MH7A cells ( Figure 1 (B). Therefore, in subsequent experiments, concentrations of 0.5, 1, and 2 μM were selected to treat low, medium, and high doses, respectively.
[0032] 4.2 Oridonin inhibits IL-1β-induced expression of inflammatory factors in MH7A cells. To evaluate the anti-inflammatory effects of oridonin A (ORI), this study used real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot to detect the expression of inflammatory factors at the gene and protein levels, respectively.
[0033] like Figure 2 The figures show the inhibition of IL-1β-induced inflammatory cytokine expression in MH7A cells by oridonin A. (A) RT-qPCR was used to detect the mRNA levels of inflammatory mediators (IL-6, IL-8, COX2, and iNOS) in MH7A cells after ORI treatment. (B) ELISA was used to determine the levels of IL-6 and IL-8 (n = 3). (C) Expression of iNOS and COX-2 proteins (n = 3) and (D) Western blot Image J quantification results. ns indicates no statistically significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0034] Results combined Figure 2It was found that oridonin A could downregulate the gene expression of IL-6, IL-8, COX2 and iNOS stimulated by IL-1β in MH7A cells (Figure A); oridonin A could downregulate the protein levels of IL-6 (left figure) and IL-8 (right figure) stimulated by IL-1β in MH7A cells (Figure B); ORI could significantly downregulate the expression of IL-6, IL-8, COX2 and iNOS at the mRNA and protein levels induced by IL-1β in MH7A cells (Figures C-D).
[0035] The above results indicate that ORI can effectively inhibit IL-1β-triggered inflammatory responses.
[0036] 4.3 Oridonin inhibits IL-1β-induced oxidative stress in MH7A cells. Oxidative stress further stimulates the inflammatory response, leading to cartilage and bone damage in rheumatoid arthritis (RA). Therefore, this study used enzyme-linked immunosorbent assay (ELISA) and DCFH-DA fluorescent probe method to detect nitric oxide (NO) and intracellular reactive oxygen species (ROS) levels, respectively.
[0037] like Figure 3 The diagram illustrates the inhibition of IL-1β-induced oxidative stress in MH7A by oridonin. (A) NO content was detected using ELISA (n=3); (B) ROS content was assessed using flow cytometry (n=3); (C) ROS quantification results; (D) ROS content was assessed using DCFH-DA staining combined with fluorescence imaging (n=3), and (E) quantification results were obtained. Scale bar = 100 μm. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0038] Results combined Figure 3 This indicates that oridonin can downregulate the level of NO stimulated by IL-1β in MH7A cells (Figure A); oridonin can reduce the accumulation of ROS stimulated by IL-1β in MH7A cells (Figures B and C); oridonin can reduce the accumulation of ROS stimulated by IL-1β in MH7A cells, thereby weakening the intensity of green fluorescence (Figures D and E).
[0039] The above results indicate that ORI can effectively inhibit IL-1β-induced oxidative stress.
[0040] 4.4 Oridonin inhibits IL-1β-induced migration and invasion of MH7A cells The pathological progression of rheumatoid arthritis (RA) is largely attributed to the migration and invasion of synovial cells. Therefore, the migration and invasion capabilities of MH7A cells were evaluated.
[0041] like Figure 4 The diagram illustrates the inhibition of IL-1β-induced migration and invasion of MH7A cells by oridonin. (A) Cell scratch assay to detect migration of MH7A cells at 0 h and 24 h after oridonin treatment (n=3), and (B) quantification results of cell migration. Scale bar = 100 μm; (C) Cell invasion assay to analyze the effect of oridonin on the invasive ability of MH7A cells (n=3), and (D) quantification results of cell invasion. Scale bar = 50 μm; (E) ELISA to detect the protein levels of CCL2 and CXCL10 in MH7A cells after oridonin treatment (n=3); (F) RT-qPCR to detect the mRNA expression levels of CCL2 and CXCL10 in MH7A cells after oridonin treatment (n=3). ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0042] Results combined Figure 4 The results (Figures A and B) show that oridonin can inhibit the migration of MH7A cells after IL-1β stimulation. (Figures C and D) show that oridonin can inhibit the invasion of MH7A cells after IL-1β stimulation. (Figure E) shows that oridonin can reduce the protein levels of IL-1β-stimulated CCL2 and CXCL10 in MH7A cells. (Figure F) shows that oridonin can downregulate the gene levels of IL-1β-stimulated CCL2 and CXCL10 in MH7A cells.
[0043] 4.5 Oridonin promotes IL-1β-induced G2 / M phase arrest in MH7A cells To investigate the mechanism by which oridonin A (ORI) inhibits the proliferation of MH7A cells, the effects of ORI treatment on cell cycle distribution and apoptosis were analyzed.
[0044] like Figure 5 The diagram shows the effect of oridonin promoting IL-1β-induced G2 / M phase arrest in MH7A cells. (A) Flow cytometry assessment of cell cycle distribution (n=3) in ORI-treated MH7A cells and its (B) quantitative results; (C) Western blot detection of protein expression levels (n=3) of CDK1, Cyclin B1, p-CDK (Thr 161), p-CDK1 (Tyr 15), CDC25C, and p-CDC25C (Ser 216) in ORI-treated MH7A cells and its (D) quantitative results. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0045] Results combined Figure 5 As shown in Figures A and B, treatment with oridonin arrested MH7A cells in the G2 / M phase and reduced the number of cells in the G0 / G1 phase. Figures C and D show that oridonin treatment downregulated the levels of CDK1, Cyclin B1, and CDC25C proteins, inhibited phosphorylation of CDK1 protein Thr 161 and CDC25C protein Ser 216, and promoted phosphorylation of CDK1 protein Tyr 15, indicating that oridonin treatment affected the levels of cell cycle-related proteins.
[0046] These results indicate that ORI inhibits the proliferation of MH7A cells by regulating the expression and phosphorylation status of G2 / M phase-related proteins.
[0047] 4.6 Oridonin A had no significant effect on IL-1β-induced apoptosis in MH7A cells. In addition, apoptosis was detected using Annexin V-FITC / PI double staining combined with flow cytometry.
[0048] like Figure 6 The diagram shows that oridonin had no significant effect on IL-1β-induced apoptosis in MH7A cells. (A) shows the flow cytometry results of MH7A cell apoptosis (n=3) after oridonin treatment, and (B) shows the quantitative results. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0049] Results combined Figure 6 The results (Figures A and B) show that treatment with oridonin A in IL-1β-stimulated MH7A cells did not significantly promote apoptosis.
[0050] This further demonstrates that ORI effectively inhibits IL-1β-induced abnormal proliferation of MH7A cells primarily by inducing cell cycle arrest rather than promoting apoptosis.
[0051] In summary, oridonin can significantly inhibit the abnormal proliferation, migration, and invasion of RA synovial cells in vitro, alleviate inflammation and oxidative stress, and has a clear anti-RA synovitis efficacy.
[0052] II. In vivo pharmacodynamic study of oridonin in the treatment of RA synovitis The adjuvant-induced arthritis (AIA) model is a classic experimental model that mimics the pathological characteristics of human rheumatoid arthritis (RA). It is characterized by progressive joint swelling, synovial inflammatory infiltration, cartilage destruction, and bone erosion, which highly coincide with the clinical pathological progression of RA. Therefore, it is widely used for the preclinical efficacy evaluation of anti-RA drugs. Based on this model, in this study, complete Freund's adjuvant (CFA) was subcutaneously injected into the plantar part of the right hind paw of C57BL / 6 mice to induce arthritis. During the 21-day continuous drug administration intervention period, the body weight changes, paw swelling degree, and arthritis score of the mice were dynamically monitored. Through pathological analysis of the ankle joint synovial tissue and combined with serum inflammatory factor detection, the in vivo therapeutic effect of oridonin on RA synovitis was systematically evaluated, providing in vivo pharmacodynamic basis for the subsequent study of its mechanism of action.
[0053] 1. Experimental animals: In this study, 30 SPF-grade male C57BL / 6 mice, 8 weeks old and weighing 20 ± 2 g, were purchased from Beijing Spey Foster Laboratory Animal Co., Ltd. [Production License No.: SCXK (Beijing) 2023-0105]. All animals were housed in a standard SPF environment, with free access to food and water. All experimental operations and animal management strictly complied with the relevant regulations of the "Regulations on the Administration of Laboratory Animals of the People's Republic of China". The experimental protocol of this study was reviewed and approved by the Laboratory Animal Ethics Committee of Chengdu University (Approval No.: 2024-013).
[0054] 2. Animal grouping, arthritis model construction, and drug administration protocol: 50 µL of complete Freund's adjuvant was subcutaneously injected once into the toes of the right hind paw of each mouse to induce the adjuvant-induced arthritis (AIA) model. Control group mice were injected with an equal volume of normal saline at the same site. On the 14th day after immunization, the AIA model mice were randomly divided into 4 groups (n = 6 in each group): AIA model group, low-dose oridonin group (10 mg / kg), high-dose oridonin group (20 mg / kg), and positive drug methotrexate group (1 mg / kg). Each group started drug administration on the 14th day and was orally gavaged once every 3 days. The blank control group and the AIA model group were given an equal volume of 0.5% sodium carboxymethylcellulose solution as a solvent control.
[0055] 3. Detection indicators: (1) Arthritis phenotype: The paw swelling degree, arthritis score, and body weight were regularly measured. Oridonin significantly alleviated swelling, reduced the score, and improved the body weight loss. (2) Histopathology: H&E staining showed that oridonin significantly reduced synovial hyperplasia, inflammatory infiltration, and joint cavity exudation. (3) Serum inflammatory factors: ELISA detection showed that oridonin significantly reduced the levels of IL-1β, IL-6 and IL-8; (4) Matrix metalloproteinases: Immunohistochemistry showed that oridonin significantly downregulated the expression of MMP1, MMP3, MMP9 and MMP13. (5) Organ toxicity: No significant damage was observed in the pathological tissues of the heart, liver, spleen, lungs and kidneys, indicating good safety.
[0056] 4. Conclusion: 4.1 Oridonin improves the general condition and arthritis phenotype in AIA mice In in vivo experiments, this study successfully constructed an adjuvant-induced arthritis model by injecting complete Freund's adjuvant to simulate the pathological process of synovitis in rheumatoid arthritis.
[0057] like Figure 7 The figure shows a schematic diagram illustrating the improvement of general condition and arthritis phenotype in AIA mice by oridonin. The changes in (A) paw swelling, (B) arthritis score, and (C) body weight (n = 6) of each group of mice are shown. No significant differences were found in the following data (ns). Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; compared with the AIA model group, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001.
[0058] Results combined Figure 7 The results showed that after complete Freund's adjuvant injection into the paws to establish the model, the model group (AIA group) mice exhibited significant paw swelling, a significant increase in arthritis scores, and a significant decrease in body weight. After gavage administration of oridonin (10 or 20 mg / kg) or methotrexate (1 mg / kg), the paw swelling was alleviated, the arthritis scores decreased, and the body weight increased. Data were analyzed using one-way ANOVA, followed by multiple t-tests, and p-values were corrected for multiple comparisons using Dunnett's post-hoc correction.
[0059] In conclusion, all dosage groups of oridonin and methotrexate group effectively relieved the above symptoms.
[0060] 4.2 Oridonin A reduces pathological damage to ankle joint tissue in AIA mice The therapeutic effect of oridonin A was further evaluated through histopathological examination of the ankle joint.
[0061] like Figure 8The image shown is a schematic diagram illustrating how oridonin reduces the pathological damage to the ankle joint tissue of AIA mice. It specifically displays representative images of the ankle joint and H&E stained tissue sections. Black arrows indicate joint cavity exudate, and red arrows indicate synovial hyperplasia.
[0062] Results combined Figure 8 This study observed the joint effusion, synovial hyperplasia, and inflammatory cells in the ankle joint of mice. Black arrows indicate joint effusion, and red arrows indicate synovial hyperplasia. Results showed that compared to other groups, the model group mice exhibited significant paw swelling, more joint effusion, more severe synovial hyperplasia, and more inflammatory cells. Treatment with oridonin (10 or 20 mg / kg) or methotrexate (1 mg / kg) alleviated paw swelling, reduced joint effusion, decreased synovial hyperplasia, and reduced inflammatory cells, thus alleviating synovitis.
[0063] 4.3 Oridonin A reduces serum inflammatory factor levels in AIA mice The development of RA synovitis is driven by a variety of pro-inflammatory cytokines and matrix metalloproteinases.
[0064] like Figure 9 The diagram illustrates how oridonin reduces serum inflammatory factor levels in AIA mice. Specifically, the levels of (A) IL-1β, (B) IL-6, and (C) IL-8 in the serum of each group of mice were detected using ELISA (n=6). ns indicates no statistically significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0065] Results combined Figure 9 It was found that the levels of IL-1β, IL-6 and IL-8 in the serum of model mice were significantly increased, while the levels of IL-1β, IL-6 and IL-8 were significantly reduced after treatment with oridonin (10 or 20 mg / kg) or methotrexate (1 mg / kg).
[0066] 4.4 Oridonin inhibits the expression of MMP1, MMP3, MMP9 and MMP13 in the ankle joint of AIA mice. like Figure 10The image shows a schematic diagram illustrating the inhibition of MMP1, MMP3, MMP9, and MMP13 expression in the ankle joint of AIA mice by oridonin. (A) Representative immunohistochemical (IHC) images of MMP-1, MMP-3, MMP-9, and MMP-13 proteins in joint tissues of each group (n=3), (B) Immunohistochemical quantification results of MMP-1, (C) MMP-3, (D) MMP-9, and (E) MMP-13 proteins. Scale bar = 100 μm. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Results combined Figure 10 As shown in Figures A-E, the results indicated that compared with other groups of mice, the levels of MMP-1, MMP-3, MMP-9, and MMP-13 proteins were significantly increased in the model group mice. Treatment with oridonin (10 or 20 mg / kg) or methotrexate (1 mg / kg) significantly reduced the levels of these proteins. ns indicates no statistically significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0067] The above results indicate that oridonin can effectively improve synovial inflammation and related pathological processes in AIA model mice.
[0068] 4.5 No significant toxicity was observed in the major organs of mice at therapeutic doses of oridonin and methotrexate. like Figure 11 The diagram shows that no significant toxicity was observed in the major organs of mice at therapeutic doses of oridonin and methotrexate. Specifically, it includes histopathological analysis of the heart, liver, spleen, lung, and kidney of each group of mice (n=6).
[0069] Results combined Figure 11 Histopathological evaluation of major organs showed that treatment with oridonin (10 or 20 mg / kg) or methotrexate (1 mg / kg) had no toxicity to the heart, liver, spleen, lungs, and kidneys of mice.
[0070] III. Effects of oridonin on the inhibition of GAPDH on glycolysis 1. Cell Culture and Drug Treatment: MH7A cells were cultured at a density of 1 × 10⁶ cells per well. 6 Inoculate at a density of [number] cells, incubate for 24 hours until fully adherent, then treat with the drug as described above. After treatment, collect approximately 5 × 10 [units]. 6 Cells were flash-frozen and preserved using liquid nitrogen.
[0071] 2. Sample preparation and detection for targeted energy metabolomics After thawing the sample on ice, 500 µL of pre-cooled (-20℃) 80% methanol aqueous solution was added, and the sample was vortexed at 2500 r / min for 2 minutes. The sample was then immersed in liquid nitrogen for 5 minutes, removed, and warmed on ice for 5 minutes, followed by another vortexing for 2 minutes. This freeze-thaw cycle was repeated three times. Next, the sample was centrifuged at 4℃ and 12000 r / min for 10 minutes. 300 μL of the supernatant was transferred to a new centrifuge tube and incubated at -20℃ for 30 minutes. This supernatant was then centrifuged again at 4℃ and 12000 r / min for 10 minutes. After centrifugation, 200 μL of the supernatant was filtered through a protein precipitation plate for subsequent LC-MS analysis.
[0072] 3. Metabolomics Data Processing and Analysis Principal component analysis (PCA) was used to perform pattern recognition on the metabolomics data. This analysis was performed using the `prcomp` function in the R software package. Before PCA, all data underwent unit variance scaling to eliminate dimensional differences. The absolute Log2FC value was used to determine the significant differences between groups of metabolites. Identified metabolites were annotated using the KEGG compound database, and the annotated metabolites were mapped to the KEGG pathway database. Subsequently, metabolite set enrichment analysis was performed on the pathways mapped to the significantly different metabolites, and statistical significance was determined using the p-value of the hypergeometric test.
[0073] 4. Conclusion: 4.1 Treatment with oridonin A induced inhibition of glycolysis in MH7A cells like Figure 12 The diagram shows the inhibition of glycolysis in MH7A cells induced by oridonin treatment. (A) Schematic diagram of the targeted metabolomics experimental workflow. (B) Principal component analysis of targeted energy metabolites in the control group, IL-1β model group, and ORI treatment group. (C) Heatmap of differentially identified metabolites in targeted metabolomics (screening criteria: fold change ≥ 1.5 or ≤ 0.67, n=4). (D) KEGG enrichment analysis of 65 energy metabolism-related metabolites and their regulation of metabolic pathways by oridonin compared to the model group.
[0074] Results combined Figure 12To investigate the role of oridonin (ORI) in inhibiting GAPDH enzyme activity during glycolysis, this study employed targeted metabolomics technology to analyze MH7A cells. Three groups were set up: a control group (untreated MH7A cells), an IL-1β group (IL-1β-treated MH7A cells), and an ORI group (IL-1β and ORI co-treated MH7A cells) (Figures A and B). Results showed a clear separation of metabolic profiles among the control group, the IL-1β model group, and the ORI treatment group, indicating significant differences in their energy metabolism status (Figure C). Quantitative analysis by LC-MS / MS identified 65 energy metabolism-related metabolites (Figure D). KEGG enrichment analysis of the oridonin-regulated metabolic pathway compared to the model group was also performed. The results showed that multiple metabolic pathways were significantly enriched in the oridonin A treatment group, including secondary metabolite biosynthesis, glycolysis / gluconeogenesis, carbon fixation in photosynthetic organisms, carbon metabolism, TCA cycle, other carbon fixation pathways, and pentose phosphate pathway.
[0075] These findings suggest that ORI blocks energy metabolism in MH7A cells by inhibiting GAPDH activity. This metabolic inhibition suppresses abnormally active glycolysis, thereby limiting the supply of biosynthetic precursors. Consistent with this metabolic disturbance, ORI arrests the IL-1β-stimulated MH7A cell cycle at the G2 / M phase, likely due to energy deprivation and insufficient biosynthetic support resulting from glycolytic inhibition.
[0076] 4.2 Oridonin inhibits GAPDH enzyme activity, leading to the accumulation of intermediate metabolites from glycolysis. like Figure 13 The figure shows a schematic diagram illustrating the accumulation of intermediate metabolites in glycolysis caused by oridonin inhibiting GAPDH enzyme activity. (A) is a volcano plot of differentially regulated metabolites compared to the model group. Metabolite changes are color-coded according to the following rules: downregulation (green), upregulation (red), and no significant change (gray). (B) A heatmap shows that oridonin inhibits glycolysis by targeting GAPDH. The figure displays differentially regulated metabolites identified in targeted metabolomics (fold change ≥1.5 or ≤0.67; n=4), with the level of change indicated by color: red indicates significant upregulation, and blue indicates significant downregulation. (C and D) Quantitative analysis of the relative content of intermediate metabolites in the glycolysis pathway was performed using LC-MS / MS-based targeted metabolomics technology. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0077] Among the detected energy metabolism-related metabolites, binding Figure 13As shown in Figure A, metabolite changes are indicated by color according to the following rules: downregulation (green), upregulation (red), and no significant change (gray). The results show that among the detected energy metabolism-related metabolites, compared to the IL-1β group, the oridonin group showed upregulation of 21 metabolites and downregulation of 13 metabolites. (Figure B) The results show that ORI treatment significantly altered the levels of glycolysis-related metabolites. (Figures C and D) The results show that after oridonin treatment, upstream GAPDH metabolites, including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), and dihydroxyacetone phosphate (DHAP), significantly accumulated; downstream GAPDH metabolites, such as 3-phosphoglycerate (3PG), 2-phosphoglycerate (2PG), pyruvic acid, and lactate, significantly decreased.
[0078] In summary, GAPDH upstream metabolites (G6P, F6P, FBP, DHAP) accumulated significantly, while downstream metabolites (3PG, 2PG, pyruvate, lactate) decreased significantly, confirming that oridonin blocks glycolysis flux.
[0079] IV. Oridonin inhibits the activation of the KEAP1-NRF2 / HO-1 and NF-κB signaling pathways by GAPDH. 1. Cell Culture and Drug Treatment: 50 nM GAPDH small interfering RNA was transfected into MH7A cells using the Lipo8000 transfection reagent. The transfection complex was prepared by mixing siRNA, transfection reagent, and Opti-MEM, and then added to cells cultured in complete medium. Scrambled siRNA was used as a negative control.
[0080] 2. Determination of intracellular methylglyoxal (MGO) content After treating MH7A cells according to the experimental design, intracellular metabolites were extracted. The culture medium was discarded, and 50 µL of pre-cooled extraction solvent was added per million cells to rapidly quench metabolism and stabilize the metabolome. The extraction solvent was acetonitrile / methanol (50:50, v / v) containing 100 µM DETAPAC. The sample was vortexed vigorously for 30 seconds, followed by three freeze-thaw cycles to fully disrupt the cells and release metabolites. The supernatant was collected after centrifugation. To detect methylglyoxal, the above extract was incubated with 100 µM 4-methoxy-o-phenylenediamine at room temperature in the dark for 4 hours for derivatization. After the reaction, the cells were centrifuged again, and the supernatant was used for high-performance liquid chromatography (HPLC). Chromatographic separation was performed on a Kromasil C18 column at 30 °C. The mobile phase was (A) an aqueous solution containing 0.1% formic acid and (B) methanol, with a flow rate of 1.0 mL / min. The elution gradient is as follows: 0-5 minutes, maintain 30% B; 5-10 minutes, linearly increase to 90% B; 10-15 minutes, maintain 90% B; 15-16 minutes, linearly decrease to 30% B; 16-20 minutes, maintain 30% B for column equilibration.
[0081] 3. Conclusion: 3.1 Oridonin promotes methylglyoxal (MGO) accumulation in IL-1β-induced MH7A cells. To further investigate the role of GAPDH in linking glycolysis and synovitis in rheumatoid arthritis, we focused on methylglyoxal (MGO), a non-enzymatic degradation product of G3P and DHAP. MGO is a highly reactive, electrophilic dicarbonyl compound capable of broadly modifying nucleophilic residues on proteins. Our research shows that MGO mediates the selective modification of the KEAP1 protein, forming methylimidazolium crosslinks, which induces KEAP1 dimerization. This process relieves the inhibition of KEAP1 on its substrate NRF2, promoting NRF2 accumulation and activation of its downstream target gene network.
[0082] Based on this, the hypothesis is proposed that ORI inhibition of GAPDH leads to MGO accumulation, which in turn promotes KEAP1 dimerization (forming high molecular weight KEAP1, HMM-KEAP1), and ultimately activates NRF2 and its downstream signaling pathways. To verify this hypothesis, changes in MGO levels, HMM-KEAP1, KEAP1 monomers, NRF2, and HO-1 protein expression in IL-1β-stimulated MH7A cells after ORI treatment were examined.
[0083] like Figure 14This diagram illustrates how oridonin promotes IL-1β-induced accumulation of methylglyoxal (MGO) in MH7A cells. (A) MGO content was determined by high-performance liquid chromatography (HPLC). (B) MGO content was determined using a methylglyoxal (MGO) assay kit. (C) MGO content was determined using a methylglyoxal assay kit. ns indicates no statistically significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0084] Results combined Figure 14 The results (Figures A and B) show that treatment with oridonin A in MH7A cells resulted in a dose-dependent increase in methylglyoxal content. (Figure C) shows that treatment with oridonin A in MH7A cells resulted in a dose-dependent increase in methylglyoxal content.
[0085] 3.2 Oridonin induces KEAP1 dimerization and activates the NRF2 / HO-1 pathway via MGO. like Figure 15 This diagram illustrates how oridonin induces KEAP1 dimerization and activates the NRF2 / HO-1 pathway via MGO. (A, C) Western blot analysis of the protein expression levels of HMM-KEAP1, KEAP1 monomers, NRF2, and HO-1 in ORI-treated IL-1β-induced MH7A cells, and their quantitative results (B, D). (E) Western blot analysis of NRF2 and HO-1 protein levels in nuclear extracts and their quantitative results (F). (G) Immunofluorescence assay for NRF2 nuclear translocation. Scale bar = 50 μm. ns indicates no statistically significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0086] Results combined Figure 15 As shown in Figures A and C, and Figures B and D, quantitative results indicate that, under IL-1β stimulation, oridonin treatment upregulated the protein expression of HMM-KEAP1, NRF2, and HO-1 in MH7A cells, while downregulating the protein expression of KEAP1 monomer. Quantitative results in Figures E and F show that oridonin treatment also upregulated the nuclear NRF2 protein level. Immunofluorescence results in Figure G show that oridonin treatment significantly enhanced the accumulation of NRF2 in the cell nucleus.
[0087] like Figure 16The figures show schematic diagrams of MH7A cells stimulated by IL-1β treated with exogenous MGO. Figures A and C show the Western blot analysis of the protein expression levels of HMM-KEAP1, KEAP1 monomers, NRF2, and HO-1 in IL-1β-induced MH7A cells treated with methylglyoxal (MGO) and the quantification results of HMM-KEAP1, KEAP1, NRF2, and HO-1 proteins in Figures B and D. Figures E and G show the Western blot analysis of the protein expression levels of HMM-KEAP1, KEAP1 monomers, NRF2, and HO-1 in IL-1β-induced MH7A cells treated with methylglyoxal or a glyoxalase 1 inhibitor and the quantification results of HMM-KEAP1, KEAP1, NRF2, and HO-1 proteins in Figures F and H. ns indicates no statistically significant difference; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0088] To further investigate the role of MGO in the KEAP1-NRF2 signaling pathway, IL-1β-stimulated MH7A cells were treated with exogenous MGO.
[0089] Results combined Figure 16 The results (Figures A and C) and (Figures B and D) show that methylglyoxal upregulated the protein levels of HMM-KEAP1, NRF2, and HO-1 in a dose-dependent manner, while downregulating the KEAP1 monomer level. The results (Figures E and G) and (Figures F and H) show that treatment with a GLO1 inhibitor further amplified the changes in KEAP1, NRF2, and HO-1 proteins.
[0090] 3.3 Oridonin inhibits IL-1β-induced NF-κB signaling pathway activation The NRF2 and NF-κB pathways are key regulators of cellular protective responses, primarily mediating antioxidant and anti-inflammatory processes. Evidence suggests that NRF2 can inhibit the expression of its downstream target gene IL-6 by interfering with the transcriptional activity of p65, a core component of the NF-κB pathway. To investigate the effects of ORI on the NF-κB signaling pathway, the phosphorylation status of its key subunit p65 was examined.
[0091] like Figure 17The diagram shows the inhibition of IL-1β-induced NF-κB signaling pathway activation by oridonin. (A) Western blot analysis of p-p65 and total p65 protein expression levels and their (B) quantitative results. (C) Western blot analysis of nuclear p-p65 protein content and its (D) quantitative results. (E) Immunofluorescence analysis of p65 nuclear translocation. Scale bar = 50 μm. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0092] Results combined Figure 17 The results (Figures A and B) show that, compared with the IL-1β stimulation group, oridonin treatment significantly inhibited p65 protein phosphorylation, but had no effect on p65 protein itself. (Figures C and D) show that oridonin treatment significantly reduced p65 protein phosphorylation in the cell nucleus. (Figure E) shows that oridonin effectively blocked IL-1β-induced p65 nuclear translocation.
[0093] In summary, these results demonstrate that ORI exerts its anti-synovitis effect in rheumatoid arthritis by inhibiting the activation of the NF-κB pathway.
[0094] 3.4 Oridonin's regulation of the KEAP1-NRF2 / HO-1 and NF-κB pathways depends on GAPDH. like Figure 18 The diagram shows the GAPDH-dependent regulation of the KEAP1-NRF2 / HO-1 and NF-κB pathways by oridonin. (A) Western blot analysis of GAPDH siRNA knockdown efficiency (n = 3) and quantification results (right side of Figure A). (B) RT-qPCR analysis of GAPDH siRNA knockdown efficiency (n = 3). (C) Western blot analysis of protein expression levels of HMM-KEAP1, KEAP1 monomer, NRF2, and HO-1 in MH7A cells after treatment with NC siRNA or GAPDH siRNA, and (D, E) quantification results of HMM-KEAP1, KEAP1, NRF2, and HO-1 proteins. (F) Western blot analysis of p-p65 protein expression level (n = 3) in MH7A cells after treatment with NC siRNA or GAPDH siRNA, and (G) quantification results. ns indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0095] To investigate the anti-inflammatory and antioxidant effects of GAPDH in MH7A cells, this study used siRNA and plasmid transfection technology to knock down GAPDH expression and evaluated its effects on the KEAP1-NRF2 / HO-1 and NF-κB signaling pathways.
[0096] Results combined Figure 18 As shown in Figure A, treatment with GAPDH siRNA reduced the protein level of GAPDH in MH7A cells. Figure B shows that treatment with GAPDH siRNA reduced the gene level of GAPDH in MH7A cells. Figures C, D, and E show that GAPDH knockdown significantly upregulated the expression of HMM-KEAP1, NRF2, and HO-1 proteins, while downregulating Keap1 monomeric expression. Figures F and G show that GAPDH knockdown inhibited p65 protein phosphorylation and suppressed NF-κB pathway activation.
[0097] 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 scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The use of oridonin A in the preparation of a medicament for the prevention and / or treatment of synovitis in rheumatoid arthritis, characterized in that, The drug uses oridonin as its active ingredient. It covalently targets and binds to the cysteine 152 site of glyceraldehyde-3-phosphate dehydrogenase, inhibiting the activity of glyceraldehyde-3-phosphate dehydrogenase, blocking the glycolytic flux of synovial cells, activating the KEAP1-NRF2 / HO-1 antioxidant signaling pathway, inhibiting the NF-κB inflammatory pathway, reducing the expression of matrix metalloproteinases, inducing G2 / M phase arrest in synovial cells, thereby inhibiting abnormal proliferation, migration and invasion of synovial cells, and improving the pathological damage of joint tissue in AIA mice. The drug is an oral preparation or an injection; The purity of the oridonin A is greater than or equal to 98%.
2. An application according to claim 1, characterized in that, The oridonin A undergoes a specific Michael addition reaction with the cysteine residue at position 152 of the active site of glyceraldehyde-3-phosphate dehydrogenase via the α,β-unsaturated ketone group in its molecular structure, thereby achieving irreversible covalent binding. The equilibrium dissociation constant Kd for the covalent bond is 2.68 × 10⁻⁶. -10 M; The half-maximal inhibitory concentration (IC50) of oridonin in inhibiting glyceraldehyde-3-phosphate dehydrogenase activity 50 =2.277μM.
3. An application according to claim 1, characterized in that, The drug works by blocking the glycolytic flux of synovial cells, specifically by significantly accumulating upstream metabolites and significantly reducing downstream metabolites. The upstream metabolites include one or more of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate, and dihydroxyacetone phosphate; The downstream metabolites include one or more of 3-phosphoglyceric acid, 2-phosphoglyceric acid, pyruvate, and lactate.
4. An application according to claim 1, characterized in that, The drug promotes the accumulation of intracellular methylglyoxal, induces KEAP1 protein dimerization, and thereby activates the KEAP1-NRF2 / HO-1 antioxidant signaling pathway.
5. An application according to claim 1, characterized in that, The drug improves joint tissue pathological damage in AIA mice by relieving paw swelling, reducing arthritis scores, alleviating synovial hyperplasia and inflammatory cell infiltration in the ankle joint, and reducing serum IL-1β, IL-6 and IL-8 levels in AIA mice.
6. An application according to claim 1, characterized in that, The drug inhibits the NF-κB inflammatory pathway by suppressing NF-κB p65 phosphorylation levels and reducing inflammatory factor levels. The inflammatory factors include IL-1β, IL-6, and IL-8.
7. An application according to claim 1, characterized in that, The drug reduces matrix metalloproteinase expression by inhibiting the expression of MMP1, MMP3, MMP9, and MMP13 in synovial tissue.
8. A drug composition of oridonin A targeting glyceraldehyde-3-phosphate dehydrogenase for the treatment of rheumatoid arthritis synovitis, characterized in that, It contains an effective dose of oridonin A, as well as pharmaceutically acceptable excipients.
9. The oridonin-based antirheumatoid arthritis synovitis pharmaceutical composition targeting glyceraldehyde-3-phosphate dehydrogenase according to claim 8, characterized in that, The effective dosage used is: The optimal concentration for oridonin A administration to synovial cells is 0.5–2 μM. The dosage of oridonin A in AIA mice is 10-20 mg / kg.
10. The oridonin-based antirheumatoid arthritis synovitis pharmaceutical composition targeting glyceraldehyde-3-phosphate dehydrogenase according to claim 8, characterized in that, The pharmaceutical composition is a single preparation or a compound preparation; The single formulation is a formulation containing only oridonin as the active ingredient; The compound preparation is a combination of oridonin and methotrexate, or a combination of oridonin and tripterygium glycosides.