Application of triptophenol terpene in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention relates to the field of medicines, and discloses application of triptophenol terpene in preparation of a medicine for treating rheumatoid arthritis. According to the application disclosed by the invention, the effect of treating rheumatoid arthritis of the triptophenol terpene is found for the first time, and obvious hepatorenal toxicity and reproductive toxicity are not observed under an effective in-vivo dosage, so that the triptophenol terpene has a higher clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of rapamycin in the preparation of rheumatoid arthritis medicines. Background Art

[0002] Rheumatoid arthritis is an autoimmune disease characterized by chronic, symmetrical polyarthritis. Its pathological hallmarks are abnormal proliferation of synovial tissue and infiltration of inflammatory cells, ultimately leading to progressive destruction of articular cartilage and bone. While the disease can occur at any age, it is most common in people over 40 years of age. Women are significantly more likely to develop the disease than men, with a prevalence approximately two to three times higher. In addition to joint involvement, rheumatoid arthritis can also affect a variety of extra-articular organs, including interstitial lung disease, the cardiovascular system, and the skin, severely impacting patients' quality of life. While the specific pathogenesis of rheumatoid arthritis remains incompletely elucidated, research suggests that abnormal activation of fibroblast-like synoviocytes plays a key role in disease progression. When the immune microenvironment is imbalanced, formerly quiescent fibroblast-like synoviocytes become highly active, proliferating abnormally and invading surrounding tissues. They also secrete large amounts of proinflammatory cytokines, further exacerbating synovial inflammation and joint destruction, and even causing the disease to spread to other joints or organs. Therefore, targeted inhibition of fibroblast-like synoviocyte activation may become an important direction for the future treatment of rheumatoid arthritis.

[0003] Currently, the clinical treatment of rheumatoid arthritis relies primarily on traditional disease-modifying antirheumatic drugs and biologics. While these drugs can effectively control clinical symptoms, they still have numerous limitations during long-term treatment, including an increased risk of opportunistic infections, adverse drug reactions such as hepatotoxicity and renal toxicity, and the high cost of treatment. In recent years, natural active ingredients have demonstrated significant application value and research potential in the treatment of rheumatoid arthritis. Studies using curcumin as an example have demonstrated that this class of natural compounds can intervene in the pathological process of rheumatoid arthritis through multi-target mechanisms, exerting therapeutic effects by regulating the balance of inflammatory cytokines and immune cell function. Importantly, natural active ingredients exhibit synergistic effects when combined with traditional antirheumatic drugs, enhancing efficacy while significantly reducing the incidence of adverse reactions. Natural products not only provide important candidate molecules for the development of novel, highly effective, and low-toxic drugs for the treatment of rheumatoid arthritis, but also lay the theoretical foundation for optimizing clinical treatment regimens and achieving personalized therapy. In-depth research on natural products has greatly expanded the therapeutic strategies for rheumatoid arthritis and provided new insights and directions for overcoming current treatment bottlenecks.

[0004] Tripterygium wilfordii has demonstrated unique clinical value in the treatment of autoimmune diseases such as rheumatoid arthritis. Modern research indicates that its pharmacological activity is primarily attributed to its diverse active ingredients, including diterpenes, triterpenes, and alkaloids. Among these, triptolide and celastrol, due to their high activity, have become important active ingredients in the treatment of rheumatoid arthritis (CN202311651871.5). However, its clinical application is severely limited by its significant dose-dependent toxicity, particularly hepatotoxicity and renal toxicity. Therefore, given the medicinal value of Tripterygium wilfordii, further research is needed to identify a natural active ingredient with significant therapeutic efficacy for rheumatoid arthritis while also exhibiting low hepatotoxicity, renal toxicity, and reproductive toxicity. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a method for preparing a medicament for treating rheumatoid arthritis. This invention, for the first time, demonstrates that terpenoid is effective in treating rheumatoid arthritis and exhibits no significant hepatotoxicity or renal toxicity at effective in vivo doses, thus demonstrating its high clinical application value.

[0006] The specific technical solutions of the present invention are: In a first aspect, the present invention provides the use of rapamycin in the preparation of a drug for rheumatoid arthritis.

[0007] Rapotrope is a diterpenoid component found in Tripterygium wilfordii. Currently, research on the pharmacological effects of rapotrope is relatively limited, with the only reports focusing on anti-tumor effects. Consequently, there have been no reports of rapotrope as a treatment for rheumatoid arthritis. This study, for the first time, discovered that rapotrope can effectively reduce joint swelling, bone erosion, and significantly lower the expression of proinflammatory cytokines in serum, thereby achieving therapeutic effects in rheumatoid arthritis. Furthermore, and crucially, compared to triptolide, which has a high biotoxicity, rapotrope showed no significant hepatotoxicity, renal toxicity, or reproductive toxicity at effective in vivo doses (0.35-0.70 mg / kg).

[0008] Specifically, this study systematically evaluated the therapeutic effects of rapamycin on rheumatoid arthritis by establishing a collagen-induced arthritis (CIA) rat model and an in vitro rheumatoid arthritis fibroblast-like synoviocyte (RA-FLS) model. This study demonstrates for the first time that rapamycin has significant activity in treating rheumatoid arthritis. Experimental results showed that rats in the rapamycin-treated group experienced significantly reduced clinical arthritis scores, significantly improved paw swelling, and effectively alleviated pathological changes such as bone erosion. At the molecular level, rapamycin significantly downregulated the expression levels of key proinflammatory factors in serum. Furthermore, serum biochemical testing and histopathological analysis (HE staining) demonstrated that rapamycin exhibited no significant hepatotoxicity, renal toxicity, or reproductive toxicity.

[0009] Preferably, the rheumatoid arthritis drug comprises: raphenol terpenes as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

[0010] More preferably, the carrier and / or excipient comprises one or more of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler and a disintegrant.

[0011] Preferably, the rheumatoid arthritis drug includes pharmaceutical preparations for gastrointestinal administration and parenteral administration.

[0012] More preferably, the gastrointestinal administration includes oral administration; the parenteral administration includes intravenous injection, intramuscular injection, subcutaneous injection, transdermal administration or mucosal administration.

[0013] More preferably, the gastrointestinal administration preparations include but are not limited to tablets, capsules, and granules; the parenteral administration preparations include but are not limited to injections, transdermal patches, and inhalation powders.

[0014] Preferably, the effective in vivo concentration of the rapaloglucosidone is 0.35-0.70 mg / kg.

[0015] Preferably, the drug is a drug for treating rheumatoid arthritis or a tool drug for scientific research on rheumatoid arthritis.

[0016] Based on the present invention's first discovery that rapamycin has a therapeutic effect on rheumatoid arthritis, rapamycin can not only be used to prepare drugs for treating rheumatoid arthritis, but can also be used as a tool drug for scientific research on rheumatoid arthritis, for example, it can be used for in vitro modeling and research on the pathogenesis or treatment mechanism of rheumatoid arthritis.

[0017] In a second aspect, the present invention provides a method for inhibiting the proliferation, invasion, metastasis or secretion of inflammatory factors of fibroblast-like synoviocytes in vitro, which comprises: adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro, thereby inhibiting the proliferation, invasion, metastasis and secretion of inflammatory factors of fibroblast-like synoviocytes.

[0018] In a third aspect, the present invention provides a method for inducing apoptosis of fibroblast-like synoviocytes in vitro, comprising: adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro, thereby inducing apoptosis of fibroblast-like synoviocytes.

[0019] In vitro experiments showed that rapamycin can effectively inhibit the proliferation and migration of RA-FLS, reduce the secretion of pro-inflammatory factors, and promote cell apoptosis.

[0020] In a fourth aspect, the present invention provides a method for constructing an in vitro model of fibroblast-like synoviocyte apoptosis, which comprises: adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro to induce apoptosis of fibroblast-like synoviocytes, thereby obtaining an in vitro model of fibroblast-like synoviocyte apoptosis.

[0021] Preferably, the effective in vitro concentration of the raphenol terpene is 20-80 μmol / L.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention discovered for the first time that raphenol terpenes can effectively reduce joint swelling, reduce bone erosion, and significantly lower the expression level of pro-inflammatory cytokines in serum, thereby achieving the effect of treating rheumatoid arthritis.

[0023] (2) Compared with triptolide, which has a higher biological toxicity, no obvious hepatotoxicity, renal toxicity and reproductive toxicity were observed at the effective in vivo dose (0.35-0.70 mg / kg). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figures are the morphological observation results of the paws of the experimental rats in each group; A is the gross appearance of the paws of the rats in each group; B is the X-ray examination results of the paws of the rats in each group.

[0025] Figure 2 The following are the results of pathological evaluation of arthritis in experimental rats in each group; A is the arthritis index score; B is the change in hind limb paw thickness; C is the trend of body weight change.

[0026] Figure 3 The results are the expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum of experimental rats in each group; ### P <0.001 vs normal control group;** P <0.01,*** P <0.001 vs CIA model group.

[0027] Figure 4 The results of the determination of serum liver function (ALT, AST) and renal function (BUN, CREA) in each group of experimental rats; ns means no significant difference ( P >0.05).

[0028] Figure 5 The following are the histopathological observation results of liver and kidney of experimental rats in each group (HE staining); A is the pathological morphology of liver tissue; B is the pathological morphology of kidney tissue; C is the pathological morphology of ovarian tissue.

[0029] Figure 6 Figure 2 shows the effect of rapamycin on the viability of MH7A cells (RA-FLS) stimulated or unstimulated by LPS; A shows the effect of rapamycin on the viability of RA-FLS cells in the basal state; B shows the effect of rapamycin on the viability of RA-FLS cells induced by LPS; *** P <0.001 vs untreated group; in panel B # P <0.05 vs untreated control group;*** P <0.001 vs LPS model group; ns indicates no statistical difference (P>0.05).

[0030] Figure 7 The effect of raphenol on the proliferation of MH7A cells (RA-FLS) stimulated by LPS; wherein: ## P <0.01 vs untreated control group;** P <0.01,*** P <0.001 vs LPS model group.

[0031] Figure 8 The effect of raphenol on the expression of apoptotic proteins (BAX / BCL-2) in LPS-stimulated MH7A cells (RA-FLS); ### P <0.001 vs untreated control group; *** P <0.001 vs LPS model group; ns indicates no statistical difference (P>0.05).

[0032] Figure 9 The effects of rapamycin on the migration and invasion of MH7A cells (RA-FLS) stimulated by LPS; A is a scratch test; B is a Transwell invasion test; ## P <0.01 vs untreated control group;* P <0.05,*** P <0.001 vs LPS model group; ns indicates no statistical difference ( P >0.05).

[0033] Figure 10 The effect of raphenol on the key inflammatory factors TNF-α and IL-1 in LPS-stimulated MH7A cells (RA-FLS) β and IL-6 expression levels; among which: # P <0.05 vs untreated control group;* P <0.05,***P <0.001 vs LPS model group. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the embodiments.

[0035] Overall embodiment In a first aspect, a rheumatoid arthritis drug comprises: rapamycin as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

[0036] More preferably, the carrier and / or excipient comprises one or more of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler and a disintegrant.

[0037] Preferably, the rheumatoid arthritis drug includes pharmaceutical preparations for gastrointestinal administration and parenteral administration.

[0038] More preferably, the gastrointestinal administration includes oral administration; the parenteral administration includes intravenous injection, intramuscular injection, subcutaneous injection, transdermal administration or mucosal administration.

[0039] More preferably, the gastrointestinal administration preparations include but are not limited to tablets, capsules, and granules; the parenteral administration preparations include but are not limited to injections, transdermal patches, and inhalation powders.

[0040] Preferably, the effective in vivo concentration of the rapaloglucosidone is 0.35-0.70 mg / kg.

[0041] Preferably, the drug is a drug for treating rheumatoid arthritis or a tool drug for scientific research on rheumatoid arthritis.

[0042] In the second aspect, a method for inhibiting the proliferation, invasion, metastasis or secretion of inflammatory factors of fibroblast-like synoviocytes in vitro comprises: adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro, thereby inhibiting the proliferation, invasion, metastasis and secretion of inflammatory factors of fibroblast-like synoviocytes.

[0043] In a third aspect, a method for inducing apoptosis of fibroblast-like synoviocytes in vitro comprises: adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro, thereby inducing apoptosis of the fibroblast-like synoviocytes.

[0044] In a fourth aspect, a method for constructing an in vitro model of fibroblast-like synoviocyte apoptosis comprises: adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro to induce apoptosis of fibroblast-like synoviocytes, thereby obtaining an in vitro model of fibroblast-like synoviocyte apoptosis.

[0045] Preferably, the effective in vitro concentration of the raphenol terpene is 20-80 μmol / L. Specific embodiments Example 1: Animal Experiment 1. Experimental animals: Wistar rats, female, weighing 180-190 g, 7-8 weeks old, provided by Shanghai Slake Company SPF.

[0047] 2. Animal Model Preparation (CIA Model): Equal volumes of type II collagen (purchased from Chondrex, USA) and incomplete Freund's adjuvant (purchased from Chondrex, USA) were emulsified and injected subcutaneously at the base of the tail (100 μg / animal). A second booster immunization with the same dose was performed 7 days later. Typical arthritis symptoms, such as joint swelling, were observed 14-21 days after model establishment.

[0048] 3. Animal Grouping: Experimental animals were randomly divided into four groups: a control group, a model group, a medium-dose rapamycin (purchased from Shanghai Yuanye) group (0.35 mg / kg), and a high-dose rapamycin group (0.7 mg / kg). Dosing began on day 15 after model establishment: the control and model groups were gavaged daily with 2 mL of normal saline; the medium-dose rapamycin group and the high-dose rapamycin group were gavaged daily with 2 mL of normal saline solution containing 0.35 mg / kg and 0.7 mg / kg, respectively. After 28 consecutive days of dosing, the animals were sacrificed in accordance with animal ethics standards.

[0049] 4. Pharmacodynamic testing plan for CIA animal model 4.1 Paw swelling measurement: The ankle diameter was measured with a vernier caliper every two days.

[0050] 4.2 Body weight: The body weight of Wistar rats was measured and recorded every 2 days.

[0051] 4.3 Arthritis Index Score: According to the scoring criteria, the clinical manifestations of arthritis are divided into the following scores: 0 points indicate normal, without redness or swelling; 1 point indicates erythema and mild swelling of the ankle joint; 2 points indicates erythema and mild swelling from the ankle joint to the metatarsal joint or metacarpal joint; 3 points indicates erythema and moderate swelling from the ankle joint to the metatarsophalangeal joint or metacarpal joint; 4 points indicates erythema and severe swelling from the ankle joint to the metatarsophalangeal joint.

[0052] 4.4 Imaging Examination: 28 days after administration, the rats' hind limbs were examined under anesthesia using a digital X-ray imaging system (VET1800B, Pride). The imaging parameters were set as follows: tube voltage 50 kV, tube current 160 mA, and exposure time 8 mAs. X-ray images focused on evaluating pathological changes such as joint swelling, joint space space, and bone invasion.

[0053] 4.5 Detection of inflammatory factors: Elisa kits were purchased from Hangzhou Fushen Biotechnology Co., Ltd. and operated according to the instructions.

[0054] (1) Dilution of standard: The initial standard solution is serially diluted: equal volumes of standard are mixed with diluent to obtain gradient dilutions of 1 / 2, 1 / 4, 1 / 8, etc.

[0055] (2) Sample addition and incubation: Add 50 μL of standard to the standard wells and 40 μL of diluent + 10 μL of sample (5-fold dilution) to the sample wells, and incubate at 37°C for 30 minutes.

[0056] (3) Washing and adding enzyme: Discard the reaction solution, fill each well with washing solution, let it stand for 30 seconds, then spin dry, repeat 5 times, and pat dry at the end. Add 50 μL of enzyme labeling reagent to each well, incubate at 37℃ for 30 minutes, and then wash the plate 5 times according to the previous method.

[0057] (4) Color development and detection: Add 50 μL of color developer A / B and develop the color at 37°C in the dark for 10 minutes. Add 50 μL of stop solution and measure the OD value at 450 nm within 15 minutes.

[0058] 5. Safety Assessment 5.1 Serum biochemical index detection: To systematically evaluate the effects of rapamycin on the liver and kidney function of rats, the animals were anesthetized and killed 28 days after administration, and heart blood samples were collected. The serum liver function indexes (alanine aminotransferase ALT, aspartate aminotransferase AST) and kidney function indexes (serum uric acid BUA, creatinine CREA) levels were detected using an automatic biochemical analyzer.

[0059] 5.2 HE staining (1) Sample preparation: Fresh tissue blocks were immediately fixed with 4% paraformaldehyde for 48 hours. After fixation, gradient dehydration was performed, using 70% ethanol for 2 hours, 80% ethanol for 2 hours, 90% ethanol for 1 hour, 95% ethanol for 1 hour, and anhydrous ethanol twice for 30 minutes each. The dehydrated tissue was transparentized with xylene twice for 15 minutes each, then immersed in paraffin at 60°C twice for 2 hours each, and finally placed in an embedding box for paraffin embedding and allowed to cool and solidify.

[0060] (2) Slice preparation: Place the embedded tissue block on a paraffin slicer and cut into 4-6 μm thick serial slices. Flatten the slices in 42°C warm water, pick them up with a glass slide, and dry them in a 60°C oven for 2 hours to ensure that the slices are firmly attached to the slide.

[0061] (3) HE staining: Before staining, the sections must be dewaxed and hydrated. The sections are sequentially treated with xylene twice for 10 minutes each, and then with anhydrous ethanol to 70% ethanol gradient for 2 minutes each. Finally, they are rinsed with distilled water for 5 minutes. Then, they are stained with Harris hematoxylin solution for 5 minutes and rinsed with running water for 15 minutes to fully turn the cell nuclei back to blue. After differentiation with 1% hydrochloric acid ethanol for 5 seconds, they are rinsed with running water again for 10 minutes. Then, they are stained with 0.5% eosin solution for 2 minutes. After a quick wash with distilled water, they are dehydrated with 70% to anhydrous ethanol gradient for 30 seconds each, and then transparentized with xylene twice for 2 minutes each. Finally, the sections are sealed with neutral gum, taking care to avoid bubbles.

[0062] (4) Microscopic examination and analysis: After staining, observe the sections under an optical microscope.

[0063] 6. Experimental results and analysis: After 28 days of treatment with raphenol, the paw swelling of rats was reduced compared to the model group. X-ray imaging analysis showed that the joint space of the model group rats was significantly narrowed, and the joint surface showed characteristic worm-eaten changes. Although the normal structure was not completely restored after the treatment, the joint space was partially reconstructed and the range of bone erosion was significantly reduced ( Figure 1 ).

[0064] Compared with the normal group, the rats in the model group continued to show severe paw swelling and high arthritis scores, accompanied by a continuous stagnation of weight gain. It is worth noting that after intervention with raphenol terpenes, these pathological changes were reversed to varying degrees ( Figure 2 ).

[0065] At the molecular level, the elevated pro-inflammatory cytokine profile (TNF- α , IL-1β and IL-6) showed an overall down-regulation trend after administration ( Figure 3 ).

[0066] In addition, according to the results of serum biochemical index detection, there was no statistically significant difference in liver function related indicators (ALT, AST) and kidney function related indicators (BUN, CREA) among the groups (blank control group, model group and drug treatment group), indicating that under the experimental conditions, raphenol terpenes had no significant effect on the liver and kidney functions of experimental animals (see Figure 4 ).

[0067] Histopathological evaluation showed that there was no significant difference in the HE staining results of the liver, kidney and ovarian tissues of the blank control group, model group and raphenol terpenoid-treated group. Specifically, the liver tissue structure was intact, the hepatic cords were arranged regularly, and no obvious degeneration, necrosis or inflammatory infiltration was observed (see Figure 5 A); The glomerular structure is clear, the renal tubular epithelial cells are normal in morphology, and no pathological changes such as cast formation or interstitial fibrosis are observed (see Figure 5B); Follicular cells at all levels are well developed, with distinct layers and regular cell morphology (see Figure 5 C).

[0068] In vivo experimental results showed that within the dose range of 0.35-0.70 mg / kg, raphenol terpenoid could improve the arthritis symptoms of CIA rats, and no obvious liver, kidney and reproductive toxicity was observed.

[0069] Example 2: Cell experiment 1. Cell Culture and Treatment: MH7A cells (purchased from Guangzhou Geneo Biotechnology) were routinely cultured in complete DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. Cells were passaged when they reached 80% confluency. The cells used in the experiment were within 15 passages to ensure cell stability. The experiment was divided into three groups: a blank control group (conventional culture), an LPS-stimulated group (1 μg / ml LPS-treated), and a combined LPS-administered group (1 μg / ml LPS-stimulated group plus 20, 40, and 80 μM rapamycin). All treatment groups, except the control group, received equal doses of LPS to establish an inflammatory model.

[0070] 2. CCK8 detection of cell viability (1) Cell plating steps: Adjust the cell concentration to 5 × 10³ cells / well (96-well plate) with complete culture medium, add 100 μL of cell suspension to each well, fill the edge wells with PBS to reduce the effect of evaporation, and set up the culture medium wells without cells as blank controls for background correction. Place the culture plate in a 37°C, 5% CO2 incubator and culture for 12-24 hours to allow the cells to fully adhere to the wall.

[0071] (2) Drug treatment: After the cells adhere to the wall, replace the culture medium with different concentrations of drugs and continue culturing for 24 or 48 hours.

[0072] (3) Add CCK-8 reagent: Mix CCK-8 reagent with fresh culture medium at a ratio of 1:10. After removing the original culture medium in the well, add 110 μL of diluted CCK-8 solution to each well, and then incubate the culture plate at 37°C in the dark for 1-4 hours. (4) Detection of absorbance: After the end of the application, the absorbance (OD value) at 450 nm was measured by a microplate reader. Calculation of absorbance: Cell viability (%) = (OD experimental group - OD blank group / OD control group - OD blank group) × 100%.

[0073] 3. Clone formation assay to detect cell proliferation (1) Cell inoculation and drug administration: Take cells in the logarithmic growth phase and digest them with trypsin to prepare a single-cell suspension. After counting, adjust the density to 500-1000 cells / well and inoculate them into 6-well plates. Add 2 mL of complete culture medium to each well, shake gently to mix, and place in a 37°C, 5% CO2 incubator for static culture. When the cells begin to clone, induce them with LPS and replace the drug-containing culture medium. During this period, replace the fresh drug-containing culture medium every 2-3 days. Continue to culture for 14 days until obvious clones are visible to the naked eye.

[0074] (2) Clone fixation and staining: After discarding the culture medium, gently wash twice with PBS, add 4% paraformaldehyde to fix for 20 minutes, rinse with PBS, and stain with 0.1% crystal violet for 15 minutes. Wash off the floating color with PBS and dry at room temperature.

[0075] (3) Photographing and counting clones: Collect images of each group of clones and calculate the number of clone groups.

[0076] 4. Western blot experiment (1) Sample preparation: After washing with PBS, the cell samples were lysed on ice for 30 minutes in RIPA lysis buffer containing protease inhibitors. The supernatant was then collected by centrifugation at 12,000 g for 15 minutes. The protein concentration was determined using the BCA assay. Finally, the cells were denatured by boiling at 95°C for 5 minutes in SDS loading buffer containing β-mercaptoethanol.

[0077] (2) SDS-PAGE electrophoresis: 12% separating gel and 5% stacking gel were used for SDS-PAGE electrophoresis. 20 μg of protein was loaded into each well, and a marker was used as a reference. Electrophoresis conditions: 80V for stacking gel, 120V for separating gel, and stop when bromophenol blue reached the bottom.

[0078] (3) Transfer: For protein transfer, use NC membrane and assemble the transfer sandwich from negative electrode to positive electrode. Transfer at a constant current of 260 mA for 70 minutes.

[0079] (4) Blocking: Block with 5% skim milk / TBST on a shaker at room temperature for 1 hour.

[0080] (5) Primary antibody incubation: The primary antibody was incubated with the antibody diluent at the dilution ratio recommended in the instructions (1:1000) and incubated at 4°C overnight, followed by washing three times with TBST buffer for 10 minutes each time.

[0081] (6) Secondary antibody incubation: Use HRP-labeled species-matched secondary antibody (1:2000 dilution) and incubate at room temperature for 1 hour, then wash three times with TBST buffer for 10 minutes each time.

[0082] (7) Development and detection: ECL development uses equal volumes of mixed A / B solution to react for 1 minute, and then the signal is detected using a chemiluminescence analyzer. The exposure time is optimized according to the signal intensity.

[0083] (8) Data analysis: The data were quantitatively analyzed using ImageJ software for grayscale value analysis, and β-actin / GAPDH was used as the internal reference protein for standardization and correction to calculate the relative expression of the target protein.

[0084] 5. Transwell invasion assay (1) Cell pretreatment: Before the experiment, cells were stimulated with 1 μg / mL LPS to enhance invasive activity.

[0085] (2) Matrigel plating: Place Matrigel (1:8 dilution) on the upper chamber of the Transwell (8 μm pore size) and solidify at 37°C for 4 hours.

[0086] (3) Cell inoculation: Digest LPS-treated cells and resuspend in serum-free medium (5×10 5 cells / mL), 200 μL of cell suspension was added to the upper chamber, and 500 μL of complete medium containing drugs was added to the lower chamber.

[0087] (4) Culture conditions: 37°C, 5% CO2 for 24 hours.

[0088] (5) Termination and staining: First, gently wipe the non-invaded cells in the upper chamber of the Transwell with a cotton swab. Then, fix the cells with 4% paraformaldehyde for 30 minutes and stain them with 0.1% crystal violet solution for 20 minutes. Finally, randomly select 5 fields under the microscope to count the number of invasive cells, and use ImageJ software to calculate the average value.

[0089] 6. Cell scratch assay to detect cell migration ability (1) Cell preparation: MH7A cells were seeded in 6-well plates or 35 mm culture dishes at a density of 90% to 100% confluence. They were cultured in a 37°C, 5% CO2 incubator until adherent. Except for the blank control group, all other groups were induced with LPS for 12 h.

[0090] (2) Scratch preparation: Aspirate the original culture medium and gently wash the cells twice with pre-cooled PBS to remove floating cells; then use the tip of a 200 μL pipette to vertically point the bottom of the culture dish and evenly scratch a straight line along the ruler or marking line (make sure the scratch width is consistent); finally, rinse with PBS to remove cell debris detached from the scratch, and replace with drug-containing culture medium and continue culturing for 24 hours.

[0091] (3) Image acquisition and analysis method: Microscopic images of the scratch area were collected at 0 hours (T0) and 24 hours (T24) after administration. The scratch area was quantitatively analyzed using Image J software, and the cell migration rate was calculated according to the following formula: Cell migration rate (%) = [(T0 scratch area - T24 scratch area) / T0 scratch area] × 100% 7. Detection of inflammatory factors: The Elisa kit was purchased from Hangzhou Fushen Biotechnology Co., Ltd. according to the instructions.

[0092] (1) Preparation of cell supernatant: After drug treatment, collect the cell culture medium, place it in a pre-cooled centrifuge tube, and centrifuge it at 2000×g for 20 minutes at 4°C. After centrifugation, carefully aspirate the supernatant to avoid inhaling the precipitate. After aliquoting, freeze it at -80°C for later use.

[0093] (2) The remaining steps are the same as the previous steps for detecting inflammatory factors in rat serum.

[0094] 8. Experimental results and analysis The results of CCK-8 experiments showed that the inhibitory effect of raphenol on MH7A cell viability showed significant dose-dependent and time-dependent characteristics (see Figure 6 After calculation, under normal culture conditions, the half inhibitory concentration (IC 50 ) were 91.75 μM and 79.27 μM, respectively. It is worth noting that after LPS induction, the inhibitory effect of raphenol terpenes was more significant, with its 24-hour and 48-hour IC 50 The values ​​dropped to 83.25 μM and 60.75 μM, respectively. Based on the experimental results, in order to optimize the experimental conditions and take into account the control of drug toxicity, the subsequent experiments selected 24 hours as the treatment time and used a concentration lower than IC 50 The drug concentrations were studied.

[0095] The present invention established an LPS-induced MH7A cell model to systematically study the multiple regulatory effects of rapamycin on RA-FLS. The experimental results showed that rapamycin could significantly inhibit the clone formation ability of RA-FLS cells, suggesting that it has the effect of inhibiting synovial hyperplasia (see Figure 7 Western blot analysis showed that raphenyl terpenes treatment could significantly upregulate the expression of pro-apoptotic protein BAX and downregulate the expression of anti-apoptotic protein BCL-2, indicating that it may induce cell apoptosis by regulating the expression of apoptosis-related proteins (see Figure 8 In terms of cell function, the results of cell scratch test and Transwell invasion test showed that terpenoid treatment could significantly weaken the migration and invasion ability of MH7A cells stimulated by LPS (see Figure 9 In addition, by detecting the levels of inflammatory factors in the cell culture supernatant, it was found that raphenol treatment could significantly reduce the secretion of key pro-inflammatory factors such as TNF-α, IL-1β and IL-6 (see Figure 10These findings clarify the potential mechanism of action of rapamycin in the treatment of rheumatoid arthritis from multiple aspects, including cell proliferation, apoptosis regulation, migration and invasion, and inflammatory response, providing important experimental basis for its further development and application.

[0096] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0097] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Application of rapamycin in the preparation of rheumatoid arthritis drugs.

2. The use according to claim 1, characterized in that: The rheumatoid arthritis drug comprises: raphenol terpenes as an active ingredient, and pharmaceutically acceptable solvents, excipients and / or carriers.

3. The use according to claim 1 or 2, characterized in that: The rheumatoid arthritis drugs include pharmaceutical preparations for gastrointestinal administration and parenteral administration.

4. The application according to claim 3, characterized in that: The gastrointestinal administration includes oral administration; the non-gastrointestinal administration includes intravenous injection, intramuscular injection, subcutaneous injection, transdermal administration or mucosal administration.

5. The use according to claim 1 or 2, characterized in that: The effective in vivo concentration of the raphenol terpene is 0.35-0.70 mg / kg.

6. The use according to claim 1 or 2, characterized in that: The medicine is a medicine for treating rheumatoid arthritis or a tool medicine for scientific research on rheumatoid arthritis.

7. A method for inhibiting the proliferation, invasion, metastasis, or secretion of inflammatory factors of fibroblast-like synoviocytes in vitro, characterized by: Adding drugs containing rapamycin to fibroblast-like synoviocytes cultured in vitro can inhibit the proliferation, invasion and metastasis of fibroblast-like synoviocytes and the secretion of inflammatory factors.

8. A method for inducing apoptosis of fibroblast-like synoviocytes in vitro, characterized by: Adding a drug containing rapamycin to fibroblast-like synoviocytes cultured in vitro can induce apoptosis of the fibroblast-like synoviocytes.

9. A method for constructing an in vitro model of fibroblast-like synoviocyte apoptosis, characterized by: A drug containing rapamycin is added to fibroblast-like synoviocytes cultured in vitro to induce apoptosis of the fibroblast-like synoviocytes, thereby obtaining an in vitro model of fibroblast-like synoviocyte apoptosis.

10. The method according to claim 7, 8 or 9, characterized in that: The effective in vitro drug concentration of the raphenol terpene is 20-80 μmol / L.

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