Effects And Possible Mechanisms Of Triptolide On Cartilage Destruction Through Histone Methylation

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Effect and possible mechanisms of triptolide on bone destruction in collagen-induced arthritis ratsObjective:The morphological changes of rat joints and synovial membranes after treatment was investigated,cartilage and bone destruction was evaluated,and mRNA expression of matrix metalloproteinases(MMPs)and H3K27 were detected in order to evaluate the effects and possible mechanisms of Triptolide on cartilage destruction in collagen-induced arthritis rats.Methods:(1)Sixty SPF Wistar male rats were raised for one week to adapt to the environment Eight rats were randomly selected as the control group,the others were injected with type II collagen and complete Freund's adjuvant to establish CIA model.(2)Rats meet CIA criteria were divided into model(CIA),low dose of triptolide(TP L),and high dose of triptolide(TPH)and administered with equal volume of normal saline,3.15mg/kg·d triptolide and 5.36 mg/kg·d triptolide for another 28 days.(3)During the feeding period,the general condition,the hair color,appetite and mental state of the rats was observed.In addition,the joint claw diameter was measured every week and the body weight was recorded.(4)After 28 days gavage,peripheral blood was collected,hind paw and femur were fixed,synovial tissue was frozen.(5)The peripheral blood level of interleukin-6(IL-6)and interleukin-1?(IL-1?)were detected by ELISA.The posterior paw,femur bone mass and trabecular bone structure of rats were observed by Micro-CT.The pathological changes of synovial tissue and ankle joint were detected by HE.The cartilage damage was observed by safranin and toluidine blue Matrix metalloproteinase(MMP-3),gelatinase(MMP-9)and collagenase-3(MMP-13)were detected by histochemical methods.RT-PCR was used to evaluate the expression of H3K27 and corresponding histone methylase(EZH2),histone demethylase(JMJD3).Results:1.Compared with the control group,the spleen index of the model group was increased(p>0.05),while the spleen index in high-dose of triptolide group decreased significantly after gavage treatment(p<0.05),which showed a significant statistical difference 2.Micro-CT showed that the control group presented with intact joint structure and trabecular bone structure.However,the articular surface of the ankle joint in the CIA is blurred,cortical bone erosion occurred,especially in the proximal interphalangeal joint.Besides,some of the cortical bones appear worm-like changes;the trabecular bone becomes thinner with a decreased number and results in increased trabecular space.What worse,the trabecular structure is disordered,damaged,perforated or deformed into a rod shape,and even the structure disappears.After treatment with triptolide,bone destruction was alleviated,bone cortical defects were significantly reduced,and trabecular bone structure was improved.Compared with the control group,bone volume fraction(BV/TV),bone surface area to tissue volume ratio(BS/TV)decreased<0.05),and showed a significant statistical difference.Compared with the model group,treatment with TP could increase the ratio of BV/TV and BS/TV(p<0.05),and showed a significant statistical difference.3.Compared with the control group,IL-1? and IL-6 were significantly increased in the model group;compared with the model group,the expression of IL-1? and IL-6 was decreased by triptolide(p<0.05),it showed statistically significant differences.4.HE showed that compared with the control group,lamina cells increased in the CIA.Infiltrated inflammatory cells infiltrated,the narrower joint space in the ankle joint,and the blurry boundary between the synovial membrane?cartilage and bone tissue were detected in the CIA.Compared with the model group,TP could reduce the hyperplasia of synovial tissue,decreased the infiltration of inflammatory cell,and repair the bone structure.5.The isochromatic is obvious in control group while the red color was obviously weakened or even disappeared in the CIA.Besides,the vasospasm directly eroded the cartilage and the cartilage was destroyed.The restored heterogeneity of color showed improvement of cartilage.The toluidine blue results showed that cartilage and bone cells in control group stained by bright purple-red.The red purple-blue color in CIA decreased,which showed a structure damage in CIA.Compared with the model group,the cartilage condition was improved after TP treatment.6.Compared with the control group,the results of histochemistry showed that the expression of MMP-3,MMP-9 and MMP-13 increased in the model group increased,and treatment with triptolide could reduce the expression of MMP-3,MMP-9 and MMP-13.7.Compared with the control group,the mRNA expression of H3K27,EZH2 increased in the model group,which showed significant statistical differences.Compared with the model group,H3K27 and EZH2 decreased(p<0.05)in the triptolide group,which showed significant statistically differences.Compared with the control group,JMJD3 decreased in CIA(p>0.05),which showed no statistical differences.Compared with the model group,the triptolide group increased mRNA expression of JMJD3(p>0.05),which showed no significant statistical differences.Conclusion:Triptolide could decrease the expression of cytokines such as IL-1? and IL-6.The cartilage and bone lesions in CIA rats were improved parallel with the decreased expression of MMP-3,MMP-9 and MMP-13.Down-regulates the expression of EZH2,affecting the trimethylation of H3K27 and thus regulating matrix metalloproteinases might be the possible mechanisms of TP to reduce the cartilage destruction.Part ? Effect and possible mechanisms of Triptolide on synovial fibroblastObjective:Fibroblast synovial cells(FLS)play a pivotal role in cartilage destruction,and TP was considered effective in cartilage destruction in our previous studies.In this study,we further investigated the potential role of TP in the proliferation,apoptosis,and migration of FLS and explore the possible mechanisms from H3K27 methylation.Methods:(1)Synovial tissue of confirmed RA was obtained from Tongji Hospital Department of Orthopaedics,then primary fibroblast-like synoviocytes were extracted through enzymatic digestion The cells from third to seventh were used for subsequent experiments.(2)Flow cytometry was used to identification FLS.(3)MTT assay was used to detect the effect of triptolide on proliferation,Annexin V-FITC was used to detect the effect of triptolide on apoptosis,Transwell was used to examine the effect of triptolide on migration.(4)RT-PCR was used to detect the effect of TP on mRNA expression of matrix metalloproteinases,H3K27,EZH2,JMJD3.(5)Western Blot was used to evaluate the protein expression of matrix metalloproteinases and histone modifications.Results:Primary cells from synovial tissue cultured in vitro exhibit a typical uniform spindle morphology.Flow cytometry results showed that 92.7%of the cells were vimentin positive and CD55 negative.Pretreated with more than 31.625 nM TP for 24 showed significant inhibition of cell proliferation(p<0.05),it showed statistically significant differences.While pretreated FLS with TP for 48 hours,cell viability was inhibited by only 7.5 nM.The follow-up experiment was performed with 15nM,30nM and 60nM intervention for 24h.Compared with the control group,a high dose of triptolide could reduce the migration of FLS and induce apoptosis(p<0.05),showed a significant difference.TP could reduce the mRNA and protein expression of MMP-3 and MMP-9,which is parallel to the down-regulation of H3K27 and EZH2.Conclusion:TP could inhibit the viability of primary fibroblast-like synovial on time and doses dependent.TP could promote apoptosis,inhibit migration and invasion;decreased the expression of MMP-3 and MMP-9.The down-regulation of H3K27 and EZH2 might be a possible mechanism of TP to protect cartilage destruction.