Application of tetrahydrojateorhizine in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of tetrahydrojateorhizine or pharmaceutically acceptable salt thereof in preparation of medicines for treating rheumatoid arthritis. Experimental results show that tetrahydrojateorhizine can effectively relieve the foot swelling level of CIA rats, reduce the arthritis score and inhibit the progress of rheumatoid arthritis. Blood routine analysis shows that the number of immune cells in blood is reduced and the immune level of an organism is weakened by administration of tetrahydrojateorhizine. In addition, experimental results also show that tetrahydrojateorhizine can inhibit synovial hyperplasia and relieve joint injury. Therefore, the tetrahydrojateorhizine has the prospect of being developed into the oral medicine for treating the rheumatoid arthritis, provides more treatment choices for clinically treating the rheumatoid arthritis, and has important social benefits and economic values.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to use of tetrahydrojatrorrhizine or a pharmaceutically acceptable salt thereof in preparing a medicine for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a common inflammatory autoimmune disease. Epidemiology shows that the incidence of RA is 0.5% to 1%, and the incidence in women is about three times that in men. RA is characterized by persistent synovitis, systemic inflammation, and continuous production of autoantibodies. It is a chronic autoimmune disease with a protracted course of disease. Most patients will experience repeated symmetrical pain, swelling, and dysfunction in the small joints of the hands and feet, accompanied by morning stiffness. Its pathological features are mainly inflammatory cell infiltration and synovial cell hyperplasia, involving multiple joints throughout the body, affecting the patient's normal life, and severe cases will cause joint deformity and disability.

[0003] Contrary to popular belief, RA and osteoarthritis are two diseases that differ greatly in both disease manifestations and treatment methods. Osteoarthritis is a non-infectious, chronic and progressive disease characterized by degenerative changes in the joints. The knee joint is the most commonly affected site, and the causative factors are related to age, weight, trauma, and labor intensity. Rheumatoid arthritis is a systemic autoimmune disease characterized by synovitis, which is mainly manifested as synovial hyperplasia and joint damage in the fingers, toes, wrists, and ankles. The causative factors are still unclear. Synovial cell hyperplasia is an important cell in the development of rheumatoid arthritis, which can erode cartilage and cause bone damage, while the degeneration of joints in osteoarthritis has little to do with synovial cells.

[0004] The treatment goal for RA patients is to control inflammation while preserving joint structure and function, preventing structural damage, and restoring normal human function to maximize the quality of life. At present, glucocorticoids, anti-inflammatory drugs, and disease-modifying antirheumatic drugs (DMARDs) are mainly used in clinical treatment of RA. In addition, in the context of precision medicine, biologics and targeted drugs will also be the future development direction for the treatment of RA. However, RA is a chronic disease that requires long-term medication to control the condition. Long-term high-dose administration can lead to gastrointestinal reactions and damage to the liver, kidneys, and blood system. Therefore, finding new drugs for the treatment of RA with higher efficacy and lower toxic side effects is still the future development direction for the prevention and treatment of RA.

[0005] Natural product monomer compounds are monomer components extracted and separated from natural products. They may have multiple pharmacological activities, such as antioxidant, anti-tumor, antibacterial, anti-inflammatory, etc. They have a single component and few side effects, and are valuable resources for new drug research and development. Tetrahydrochalcone (corypalmine) is an isoquinoline alkaloid. The compound is derived from traditional Chinese medicines such as summer nectar, coptis root, and yanhusuo. The molecular formula of the compound is C 20 H 23 NO 4 , its chemical structure is shown below:

[0006]

[0007] Chinese patent CN 111184719A discloses a dopamine receptor antagonist and its use, wherein the dopamine receptor antagonist is D-tetrahydrojatrorrhizine and its pharmaceutically acceptable salt or precursor compound, and the dopamine receptor antagonist can antagonize the calcium ion influx caused by dopamine receptor activation. Animal experimental results show that D-tetrahydrojatrorrhizine as a dopamine receptor antagonist has a good analgesic effect on acute pain, inflammatory pain and bone cancer pain; at the same time, D-tetrahydrojatrorrhizine also has anti-inflammatory, antiviral and anti-arrhythmic effects. In addition, Chinese patent CN117379379A also discloses the effect of nanoparticles containing tetrahydrojatrorrhizine in treating osteoarthritis.

[0008] At present, there is no report on the use of tetrahydrojatrorrhizine in the treatment of rheumatoid arthritis. Summary of the invention

[0009] The purpose of the present invention is to solve the problem of insufficient oral drugs of natural origin for treating rheumatoid arthritis that are currently highly effective, safe and inexpensive for clinical use, to achieve in-depth development and utilization of natural products using modern drug research methods, and to provide the use of tetrahydrojatrorrhizine in preparing drugs for treating rheumatoid arthritis in combination with a large number of pharmacodynamic experimental studies.

[0010] Specifically, the present invention is realized through the following technical solutions:

[0011] The present invention provides use of tetrahydrojatrorrhizine or a pharmaceutically acceptable salt thereof in preparing a medicine for treating rheumatoid arthritis.

[0012] As an optional mode, in the above use, the tetrahydrojatrorrhizine is L-tetrahydrojatrorrhizine or D-tetrahydrojatrorrhizine.

[0013] Preferably, the pharmaceutically acceptable salt is selected from hydrochloride.

[0014] As an optional mode, in the above use, the tetrahydrojatrorrhizine inhibits the progression of rheumatoid arthritis, reduces the number of immune cells in the blood, weakens the body's immune level, and tetrahydrojatrorrhizine inhibits synovial hyperplasia and relieves joint damage.

[0015] As an optional mode, in the above use, the medicament comprises a therapeutically effective amount of tetrahydrojatrorrhizine and a pharmaceutically acceptable carrier.

[0016] As an optional mode, in the above use, the medicine also comprises other natural products commonly used in clinical treatment of rheumatoid arthritis.

[0017] As an optional mode, in the above use, the active ingredients in the medicine are composed of tetrahydrojatrorrhizine and other natural products.

[0018] As an optional mode, in the above use, the other natural products are selected from one or more of the following: oridonin, triptolide, resveratrol, sclareol, curcumin, decarinine, borfenolin, punicalagin, gentisic acid or naringin.

[0019] Preferably, the other natural product is Sophora flavescens pine.

[0020] As an optional manner, in the above use, the weight ratio of the tetrahydrojatrorrhizine to the other natural products is 1:10 to 10:1.

[0021] Preferably, the weight ratio of the tetrahydrojatrorrhizine to the other natural products is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0022] As an optional mode, in the above-mentioned use, when tetrahydrojatrorrhizine is used in combination with other natural products, the dosage forms of the former and the latter may be the same or different. Furthermore, the former and the latter may be administered simultaneously or separately.

[0023] As an optional mode, in the above use, the drug is in oral dosage form.

[0024] As an optional mode, in the above-mentioned use, the oral dosage form is selected from oral liquid, tablet, powder, capsule or granule.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention combines my country's advantages in natural product research and discovers for the first time a new use of the natural product tetrahydrojatrorrhizine in the treatment of rheumatoid arthritis.

[0027] (2) The experimental results showed that tetrahydrojatrorrhizine can effectively alleviate the foot swelling level of CIA rats, reduce arthritis scores, and inhibit the progression of rheumatoid arthritis. Blood routine analysis showed that the administration of tetrahydrojatrorrhizine reduced the number of immune cells in the blood and weakened the body's immune level. In addition, the experimental results also showed that tetrahydrojatrorrhizine can inhibit synovial hyperplasia and relieve joint damage.

[0028] (3) Tetrahydrochalcone has the prospect of being developed into an oral drug for the treatment of rheumatoid arthritis, which provides more treatment options for the clinical treatment of rheumatoid arthritis and has important social benefits and economic value.

[0029] (4) In addition, the present invention also found that the combination of tetrahydrojatrorrhizine and certain natural products (especially, borfenolin) can produce significant synergistic effects in the treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 : Changes in the volume of the hind paws of rats during the drug administration period. *, p < 0.5; **, p < 0.01, compared with the CIA group.

[0032] Figure 2 : Changes in the thickness of the hind paws of rats during the drug administration period. *, p < 0.5; **, p < 0.01, compared with the CIA group.

[0033] Figure 3 : Changes in arthritis scores of rats during drug administration. *, p < 0.5; **, p < 0.01, compared with the CIA group.

[0034] Figure 4 : Micro-CT analysis of the ankle joints of rats after drug administration (bone injury evaluation).

[0035] Figure 5 : HE staining analysis of the ankle joints of rats after administration (evaluation of synovial hyperplasia).

[0036] Figure 6 : Safranin fast green staining analysis of the ankle joints of rats after administration (evaluation of cartilage destruction).

[0037] Figure 7: S100A4 immunohistochemical analysis (analysis of synovial fibroblast marker) in the ankle joints of rats after administration.

[0038] Figure 8 : Immunohistochemical analysis of Vimentin (analysis of synovial fibroblast marker) in the ankle joints of rats after administration.

[0039] Fig. 9 : Tetrahydrojatrorrhizine inhibits the migration of synovial fibroblasts.

[0040] Fig.10 : Statistical graph of tetrahydrojatrorrhizine inhibiting the migration of synovial fibroblasts. **, p<0.01, compared with the 0-dose group.

[0041] Fig.11 :MTT analysis of tetrahydrojatrorrhizine inhibiting proliferation of synovial fibroblasts. **, p<0.01, compared with the 0-dose group.

[0042] Fig.12 :Edu staining analysis of tetrahydrojatrorrhizine inhibiting proliferation of synovial fibroblasts.

[0043] Fig.13 : Statistics of edu staining analysis of tetrahydrojatrorrhizine inhibiting proliferation of synovial fibroblasts. *, p < 0.5; **, p < 0.01, compared with the 0-dose group. DETAILED DESCRIPTION

[0044] The inventors used modern drug research methods to deeply develop and utilize natural products, and through a large number of screenings, found for the first time that oral tetrahydrojatrorrhizine can effectively treat rheumatoid arthritis. On this basis, the present invention was completed.

[0045] The dosage form of the drug of the present invention is preferably an oral dosage form.

[0046] The oral dosage form is capsule, tablet, granule or oral solution, preferably tablet or capsule.

[0047] The pharmaceutically acceptable carrier refers to a conventional drug carrier in the field of pharmaceutical preparations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0048] The filler of the present invention includes but is not limited to starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricant includes but is not limited to magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binder includes but is not limited to water, ethanol, starch slurry, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, etc.; the disintegrant includes but is not limited to starch effervescent mixture, i.e. sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agent includes but is not limited to polysaccharides such as acacia gum, agar, alginic acid, cellulose ether and carboxymethyl chitosan, etc.; the solvent includes but is not limited to water, a balanced salt solution, etc.

[0049] The drug can be made into various solid oral preparations, liquid oral preparations, etc. Pharmaceutically acceptable oral solid preparations include: ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dripping pills, powders, etc., and oral liquid preparations include oral liquids, emulsions, etc.

[0050] The above-mentioned various dosage forms can be prepared according to conventional processes in the field of pharmaceutical preparations.

[0051] Tetrahydrojatrorrhizine can be extracted and separated from plants such as summer jasmine containing the active ingredient by conventional natural product extraction methods in the art, or can be purchased from commercial products.

[0052] In the medical use described herein, the administration time, number of administrations and frequency of administration of tetrahydrojatrorrhizine need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

[0053] The treatment plan for rats can be applied to humans, and the effective doses of all drugs for humans can be converted by the effective doses of the drugs for rats, which is also easy to achieve for ordinary technicians in this field.

[0054] In order to better understand the essence of the present invention, the pharmacodynamics experiment and its results are used in the following specific embodiments to further illustrate the new use of tetrahydrojatrorrhizine in the pharmaceutical field for treating rheumatoid arthritis.

[0055] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0056] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0057] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0058] In the following examples, for cost saving and other considerations, this application and another patent application for corydaline submitted by the inventor on the same day share a normal control group, a model group, a positive drug control group and a group of some natural product monomers for combined use.

[0059] Example 1: In vivo efficacy study of tetrahydrojatrorrhizine in a collagen-induced rheumatoid arthritis animal model

[0060] 1.1 Experimental methods:

[0061] Rheumatoid arthritis is an autoimmune disease characterized by synovitis, and its pathological manifestations include immune enhancement, synovial hyperplasia and destruction of bone tissue. The collagen-induced rat arthritis model (CIA model) can be used to simulate rheumatoid arthritis.

[0062] SD rats, 180-200 g, were obtained from the Department of Experimental Animal Science, Peking University Health Science Center. There were 75 male rats, 8 weeks old, SPF grade, and divided into 5 groups.

[0063] Animal groups: normal control group, 15 animals / group; CIA model group, 15 animals / group; positive drug group (methotrexate, 1 mg / kg / 2d), 15 animals / group; drug-treated group (L-tetrahydrojatrorrhizine, 5 mg / kg / d, 20 mg / kg / d), divided into 2 groups, 15 animals / group in each.

[0064] Administration: intragastric administration.

[0065] Procedure: The animals were first divided into 7 groups according to the grouping scheme and were adaptively fed for 6 days to adapt to the environment. The animal room maintained a 12-hour light and 12-hour dark cycle. During this period, the animals were free to eat and drink. The experiment used bovine type II collagen injection at the base of the tail to establish the model. The specific method is as follows: (1) Bovine type II collagen solution: bovine type II collagen was dissolved in an acetic acid aqueous solution with a pH of 3 at a concentration of 2 mg / mL and mixed on a shaker at 4°C for 2 hours. (2) Emulsion I: 2 mg / mL bovine type II collagen solution was mixed with complete Freund's adjuvant in an equal volume of 1:1 and vortexed. (3) Emulsion II: 2 mg / mL bovine type II collagen solution was mixed with incomplete Freund's adjuvant in an equal volume of 1:1 and vortexed. After the SD rats were anesthetized with an anesthesia machine, 0.1 mL of emulsion I was subcutaneously injected at the base of the tail of the rats in the model group and the treatment group. After 7 days, the living status of the rats was observed. If there was no abnormality, the SD rats were anesthetized using an anesthesia machine. 0.1 mL of emulsion II was subcutaneously injected at the base of the tail of the rats in the model group and the treatment group. After 7 days, the model was successful (signs of successful establishment of the arthritis model: obvious swelling of the toes of the rat's hind legs, inability to bend the hind leg joints, and creeping walking), and grouping and drug administration began.

[0066] Animals in the drug group were given oral administration, while the control group and the model group were only given an equal volume of 0.5% CMC-Na solution. The level of foot swelling, paw thickness and arthritis score were monitored during the drug administration. Four weeks after drug administration, the animals were anesthetized, micro-CT scans were performed on the hind limbs, blood was collected from the orbits and euthanized.

[0067] Animal treatment and subsequent analysis: After anesthesia, blood was collected from the orbits, serum was separated, and the animals were euthanized to obtain spleen, thymus, hind limbs and other organs. Three animals were randomly selected from each group for paraffin embedding, and ankle joints were taken for HE staining, safranin fast green staining, and immunohistochemical analysis of S100A4 and vimentin.

[0068] Experimental data presentation method: Foot swelling level (foot volume), sole thickness and arthritis score are recorded in Figures 1 to 3 , each time point is the mean ± standard deviation of each group of data. Anticoagulated blood was tested by routine blood analyzer, and the results are reported in Table 1. The experimental data were statistically analyzed using GraphPad-Prism 6.0 software, and the method was to compare whether there was a significant difference in the mean value of each column under one-way analysis of variance (ANOVA) using the t-test, and p<0.05 was considered to be significantly different.

[0069] 1.2 Experimental results:

[0070] The results showed that (see Figures 1 to 3), the foot volume, sole thickness and arthritis score of CIA rats were significantly higher than those of the normal group, and did not decrease during the administration period. Tetrahydrochalcone (CP) can effectively alleviate the foot swelling level of CIA rats, reduce the arthritis score, and inhibit the progression of rheumatoid arthritis. The high-dose group has better efficacy than the positive drug group.

[0071] Routine blood analysis showed that administration of tetrahydrojatrorrhizine reduced the number of immune cells (neutrophils, lymphocytes) in the blood (see Table 1), and weakened the body's immune level.

[0072] Table 1: Results of blood routine analysis of various experimental animals after administration of tetrahydrojatrorrhizine

[0073]

[0074]

[0075] CT analysis showed a reduction in bone damage (see Figure 4 ), HE staining showed that synovial proliferation was inhibited (see Figure 5 ), safranin fast green staining showed that it alleviated the cartilage destruction in CIA rats (see Figure 6 ), S100A4 and vimentin immunohistochemistry showed that the proliferation of synovial fibroblasts was inhibited (see Figures 7 and 8 ).

[0076] Example 2: In vitro effectiveness study of tetrahydrojatrorrhizine on primary synovial fibroblasts 2.1 Experimental methods:

[0077] The cells were obtained from primary synovial fibroblasts of rats with rheumatoid arthritis.

[0078] Primary rat synovial fibroblast culture: The synovial tissue of the hind foot of the rat was digested with 1 mg / mL type II collagenase at 37°C for 2 h, the supernatant was filtered with a filter, and the synovial fibroblasts were collected by centrifugation at 1000×g for 2 min. The cells were cultured in DMEM complete medium (DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixed solution) at 37°C and 5% CO. 2 and 95% humidity.

[0079] (1) MTT assay

[0080] Synovial fibroblasts were seeded in 48-well plates at a density of 2 × 10 4 / well, after growing in the incubator for 12 hours, a control group and a drug-treated group were randomly set up, and 4 wells were repeated. The control group was not treated, and the drug-treated group was treated with tetrahydrorhizine. The concentrations of tetrahydrorhizine were set to 1.5, 3, and 6 μM. After 24 hours of tetrahydrorhizine treatment, the original culture medium in the 48-well plate was carefully aspirated, and DMEM high-glucose medium containing 50 μg / mL MTT was added. Incubate at 37°C and 5% CO 2 The cells were incubated in a dark environment for 4 hours under 95% humidity. The culture medium in the 48-well plate was carefully aspirated, and 350 μL DMSO was added to dissolve the purple crystals. The sample absorbance was detected using a multifunctional microplate reader, and the detection wavelength was set to 570 nm. Cell survival rate = (absorbance of the drug group - blank) / (absorbance of the control group - blank) * 100%.

[0081] (2) Wound healing experiment

[0082] Synovial fibroblasts were seeded in 12-well plates at a density of 5 × 10 5 / well, after the cells are fully grown, a sterile 200μL pipette tip is used to make a uniform wound. After washing with PBS twice, a control group and a treatment group were randomly set up. The control group was not treated, and the treatment group was treated with tetrahydrorhachiridine. The concentration of tetrahydrorhachiridine was set to 1.5, 3 and 6μM. After 24h of tetrahydrorhachiridine treatment, the culture medium in the 12-well plate was discarded, and the plate was fixed with 4% paraformaldehyde solution for 30min. A Zeiss microscope was used to capture images, and Image J software was used to analyze relative mobility. Relative mobility = (initial wound area - wound area after healing) / initial wound area * 100%.

[0083] (3) Edu experiment

[0084] Synovial fibroblasts were seeded in 24-well plates at a density of 4 × 10 4 / well, after growing in the incubator for 12 hours, a control group and a drug-treated group were randomly set up, and repeated 3 wells. The control group was not treated, and the drug-treated group was treated with tetrahydrochalcone, and the concentrations of tetrahydrochalcone were set to 1.5, 3 and 6μM. After culturing for 4 hours, an EdU solution with a final concentration of 10μM preheated to 37°C was added. After incubation for 20 hours, the culture medium in the 24-well plate was carefully aspirated, and the cells were fixed with a 4% paraformaldehyde solution at room temperature for 40 minutes. After washing the cells twice with PBS, 0.5% Triton X-100PBS solution was used to punch holes at room temperature for 40 minutes. After washing the cells twice with PBS, 400μL / well Click reaction solution (430μL Click Reaction Buffer, 20μL CuSO 4, 1μL AlexaFluor 488 and 50μL Click Additive Solution mixed solution), react at room temperature in the dark for 30 minutes. Discard the Click reaction solution, wash with pre-cooled PBS three times, add 400μL Hoechst33342 diluted with PBS at a ratio of 1:1000 to each well, incubate at room temperature in the dark for 10 minutes, wash thoroughly with PBS, and take images with Zeiss fluorescence microscope. Image J software is used to analyze the number of EDU positive cells. The excitation / emission wavelengths of Alexa Fluor 488 are 495nm / 520nm, and the excitation / emission wavelengths of Hoechst33342 are 360nm / 450nm. EDU positive cell rate = number of 488 positive cells / number of cell nuclei * 100%

[0085] The experimental data were statistically analyzed using GraphPad-Prism 6.0 software. The method was to compare whether there was a significant difference in the mean value of each column using the t-test under one-way analysis of variance (ANOVA). A significant difference was considered when p < 0.05.

[0086] 2.2 Experimental results:

[0087] Wound healing experiments showed that Figures 9 and 10 ), the wound healing degree of the drug-treated group was lower than that of the non-drug-treated group, indicating that CP inhibited the migration of synovial fibroblasts. MTT experiment (see Fig.11 ), edu proliferation assay (see Figure 12 to Figure 13 ), the number of edu-positive cells in the drug-treated group was significantly lower than that in the non-drug-treated group, indicating that CP inhibits the proliferation of synovial fibroblasts. CP can alleviate rheumatoid arthritis by inhibiting the proliferation of synovial fibroblasts.

[0088] Example 3: In vitro efficacy study of tetrahydrojatrorrhizine combined with other natural products on primary synovial fibroblasts

[0089] In this embodiment, the inventors also preliminarily investigated the effect of using tetrahydrojatrorrhizine in combination with a variety of other natural product monomers that may have the effect of treating rheumatoid arthritis (oridinol, triptolide, resveratrol, sclareol, curcumin, decarinine, pedunculaginine, punicalagin, gentisic acid, naringin).

[0090] 3.1 Experimental methods:

[0091] The cells were obtained from primary synovial fibroblasts of rats with rheumatoid arthritis.

[0092] Primary rat synovial fibroblast culture: The synovial tissue of the hind foot of the rat was digested with 1 mg / mL type II collagenase at 37°C for 2 h, the supernatant was filtered with a filter, and the synovial fibroblasts were collected by centrifugation at 1000×g for 2 min. The cells were cultured in DMEM complete medium (DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin mixed solution) at 37°C and 5% CO. 2 and 95% humidity.

[0093] MTT assay

[0094] Synovial fibroblasts were seeded in 48-well plates at a density of 2 × 10 4 / well, after growing in the incubator for 12 hours, randomly set up a control group and a drug-treated group, and repeat 4 wells. The control group was not treated, and the drug-treated group was treated with the above-mentioned various natural product monomers to be tested, and the concentration was set to 3μM. After 24 hours of drug treatment, the original culture medium in the 48-well plate was carefully aspirated, and DMEM high-glucose medium containing 50μg / mL MTT was added, and incubated at 37°C, 5% CO 2 The cells were incubated in a dark environment for 4 hours under 95% humidity. The culture medium in the 48-well plate was carefully aspirated, and 350 μL DMSO was added to dissolve the purple crystals. The sample absorbance was detected using a multifunctional microplate reader, and the detection wavelength was set to 570 nm. Cell survival rate = (absorbance of the drug group - blank) / (absorbance of the control group - blank) * 100%.

[0095] The experimental data were statistically analyzed using GraphPad-Prism 6.0 software. The method was to compare whether there was a significant difference in the mean value of each column using the t-test under one-way analysis of variance (ANOVA). A significant difference was considered when p < 0.05.

[0096] The synergy index is determined by the Kim Jong-gwan q value method, and the q value is calculated by the following formula:

[0097] q=P A+B / (P A +P B -P A ×P B ). Where P A , P B and P A+B They are the treatment improvement rates of drug A group, drug B group and the combination of the two drugs. q<1 indicates that the two drugs produce antagonism after combination; q>1 indicates that the two drugs produce synergistic effect after combination; q=1 indicates that the two drugs produce additive effect after combination.

[0098] 3.2 Experimental results:

[0099] The experimental results are shown in Table 2 below.

[0100] Table 2 Survival rate of synovial fibroblasts in each drug group tested (%)

[0101]

[0102]

[0103] The experimental results show that although natural product monomer compounds such as oridonin and triptolide may have different degrees of inhibitory effects on the proliferation of rat synovial fibroblasts when used alone, these natural product monomer compounds may exhibit three types of results when used in combination with tetrahydrojatrorrhizine: antagonistic effects, additive effects, and synergistic effects. It can be seen that the effect of tetrahydrojatrorrhizine combined with other natural products on rat synovial fibroblasts is difficult to predict.

[0104] As shown in the results in Table 2, the combination of tetrahydrojatrorrhizine and borfenolin may have a synergistic effect in the treatment of rheumatoid arthritis.

[0105] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of tetrahydrojatrorrhizine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating rheumatoid arthritis.

2. The use according to claim 1, characterized in that: The tetrahydrojatrorrhizine is L-tetrahydrojatrorrhizine or D-tetrahydrojatrorrhizine.

3. The use according to claim 1, characterized in that: The tetrahydrojatrorrhizine inhibits the progression of rheumatoid arthritis, reduces the number of immune cells in the blood, weakens the body's immune level, inhibits synovial hyperplasia, and relieves joint damage.

4. The use according to claim 1, characterized in that: The medicament comprises a therapeutically effective amount of tetrahydrojatrorrhizine and a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The medicine also contains other natural products commonly used in clinical treatment of rheumatoid arthritis.

6. The use according to claim 5, characterized in that: The active ingredients in the medicine are composed of tetrahydrojatrorrhizine and other natural products.

7. The use according to claim 6, characterized in that: The other natural products are selected from one or more of the following: oridonin, triptolide, resveratrol, sclareol, curcumin, decarinine, borfenolin, punicalagin, gentisic acid or naringin.

8. The use according to claim 6, characterized in that: The weight ratio of the tetrahydrojatrorrhizine to the other natural products is 1:10 to 10:

1.

9. The use according to claim 1, characterized in that: The drug is in oral dosage form.

10. The use according to claim 9, characterized in that: The oral dosage form is selected from oral liquid, tablet, powder, capsule or granule.

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