| Rheumatoid arthritis(RA)is an autoimmune disease characterized by synovial hyperplasia and inflammation,bone tissue destruction and joint erosion.It has high morbidity,poor curability,and high disability rate.It is known as the"immortal cancer".Even under the dual treatment of drugs and surgery,modern medicine can only relieve the symptoms of RA patients but cannot cure it.Many medicines of RA have relatively large toxic and side effects for long-term using.RA is a heavy burden on both the patient and the society.Therefore,looking for high-efficiency and low-toxicity alternative drugs for RA has become a promising research direction in the treatment of RA.Natural compounds are always a treasure house of drug development.The pharmacodynamics and pharmacological effects of natural compounds have become research hotspots in various fields.According to statistics,about 64.4%of the drugs approved are natural compounds and their derivative by the US FDA from 1981 to 2019.The screening of RA alternative or complementary drugs from natural compounds is an important direction for the research.Tomatidine(Td)is a glycoalkaloid that exists in Solanaceae plants and tomato genus.It is rich in tomato roots,stems,leaves and fruits.It has immunomodulatory,anti-oxidant,anti-cancer,anti-inflammatory properties and anti-bone destruction.RA is a disorder of immune and oxidative stress,tumor-like hyperplasia occurs in synovial tissue,synovial inflammation and bone erosion are over-activated in RA patients.In view of the existing pharmacological reports of Td and the characteristics of RA,we speculate that Td is very likely to play a certain therapeutic effect on RA.In addition,through network pharmacology predictions and existing research reports,we found that Td can block the MAPK and NF-?B signaling pathways in many types of cells.These two signaling pathways are the most classic signals for the pathogenesis of RA.Based on this,we tested the inhibitory effect of Td on RA from different levels in vitro and in vivo,and analyzed the possible mechanism of action at the molecular level.Main experimental content and results:Part 1:In vitro experiment:Td significantly inhibited the over-activated phenotype of FLSs by blocking the NF-?B and MAPKs(ERK,JNK,p38)signaling pathways induced by TNF?.Fibroblast-like synoviocytes(FLSs)are important effector cells in the pathogenesis of RA.The cells are over-activated in RA,and tumor-like cell changes occur,including over-activated proliferation,migration,anti-apoptotic ability and secretion of inflammatory factors.Inhibiting the over-activated phenotypes of FLSs have become a potential therapeutic strategy RA immunotherapy.Here,we selected the FLSs from collagen induced arthritis rats as the in vitro study object to examine the effect of Td on migration,proliferation and inflammatory factor expression.We also analyzed the possible molecular mechanism.In vitro experiments included:the effect of Td on FLSs activity was tested by MTS assay;the effect of Td on FLSs migration ability was tested by wound-healing assay;the effect of Td on FLSs proliferation was tested by Ed U incorporation assay;the effect of Td on FLSs apoptosis was tested by flow cytometry.;the effect of Td on FLSs inflammatory factor expression was tested by real-time PCR;the possible target of Td on RA treatment was predicted by network pharmacology enrichment;the explore Td inhibits the possible mechanism of FLSs over-activated phenotype by Western blot and immunofluorescence assay.Results:The MTS assay showed that Td had no effect on the activity of FLSs at the concentrations of 0-20?M,and its half-inhibitory concentration IC50 was 34.31?M.Ed U incorporation assay showed that 5?M Td significantly inhibited proliferation of FLSs.Wound-healing assay showed that Td significantly inhibited TNF?-induced migration of FLSs at the concentrations of 2.5-10?M.Real-time PCR results showed that Td significantly down-regulated TNF?-induced expression of pro-inflammatory cytokines including TNF?,IL-1?,IL-6,matrix metalloproteinase MMP-9 and osteoclast differentiation factor RANKL.Further molecular mechanism studies demonstrated that Td inhibited the over-activation of FLSs by blocking the NF-?B and MAPKs(ERK,JNK,p38)signaling pathways.Part 2:In vivo experiment:the therapeutic effect of Td on CIA ratsPurchasing SPF female Wistar rats(160-70g,6-8 weeks).The rats were injected with chicken collagen type II emulsified in Freund's complete adjuvant twice intracutaneously and subcutaneously respectively to induce and successfully establish a CIA rat model.All the immunized rats developed CIA(clinical score?2).CIA rats were randomly divided into groups of 6:Negative control group(Control):Control rats were intraperitoneal injection of corn oil;positive control group(CIA+Vehicle):CIA rats were intraperitoneal injected with corn oil;positive drug treatment group(CIA+MTX 3 mg·kg-1):CIA rats were intraperitoneal injected with positive drug MTX every three days;Td low-dose treatment group(CIA+Td 5 mg·kg-1)and Td high-dose treatment group(CIA+Td 15 mg·kg-1):CIA rats were intraperitoneal injected with Td every day.CIA rats were treated with continuous administration for a 14-day period,then the materials were collected,and the therapeutic effect of Td on CIA rats was detected by various methods such as arthritis index score,paw volume measurement,X-ray photography,pathological section and score and inflammatory factor detection.The toxicology of Td in CIA rats was initially evaluated by detecting the weight of rats before and after administration,important organ indexes and biochemical indexes of liver function.Results:Experimental results showed that Td significantly inhibited the severity of the onset of CIA rats,manifested by decreased arthritis index score and paw volume.Imaging scores,histopathological scores and ELISA experiments showed that Td significantly inhibited synovial inflammation and bone tissue destruction,the inhibitory effect of Td low-dose group was similar to that of MTX,while Td high-dose group was significantly better than MTX group.Conclusion:We here demonstrated for the first time that Td could effectively improve the pathological symptoms such as synovial inflammation and bone destruction in CIA rats.The therapeutic effect of Td on CIA was mainly achieved by blocking the MAPKs and NF-?B signaling pathways,thereby inhibiting the over-activated phenotype of FLSs.Based on the above research results,Td has the potential to become a complementary or alternative medicine for RA therapy. |
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