| Rheumatoid arthritis(RA)is a chronic inflammatory disease characterized by symmetrical polyarthritis.The pathological features are inflammation and hyperplasia of synovium,accompanied by erosion of bone and cartilage.Its pathogenesis is not clear,and epigenetics may be the future research direction.RA fibroblast like synoviocytes(FLS),as the main constituent cells of synovitis,have the characteristics of invasion and antiapoptosis,as well as the ability to regulate immune response and chronic inflammation,which make FLS become the main participant of arthritis.Understanding the epigenetic characteristics of RA-FLS may help us to identify new diagnostic and prognostic markers and treatment targets for RA.In recent years,the research on the function of micro RNA in RA-FLS has become a hotspot in the research of RA mechanism.Our previous study found that mi R-146 a was down regulated in fibroblast like synoviocytes of RA patients,which aroused our interest.What role does mi R-146 a play in RA? Is it the proinflammatory gene or the anti-inflammatory gene.Bioinformatics prediction and double luciferase reporter genes confirmed that IL-1 receptor associated kinase 1(IRAK1)and tumor necrosis factor receptor-associated factor-6(TRAF6)were the target genes of mi R-146 a.TRAF6 is the upstream molecule of c-jun N-terminal kinase(JNK).JNK signaling pathway is closely related to cell proliferation,apoptosis and inflammatory response.In this study,we intend to study the effect of mi R-146 a on the biological behavior of RA-FLS and explore the molecular mechanism of mi R-146 a regulating RA-FLS.On this basis,we systematically studied whether the new immunomodulator,iguratimod,can inhibit the proliferation of RA synovium and inflammatory response by regulating mi R-146 a on the level of molecule,cell and animal model.Part 1 The regulatory mechanism of mi R-146 a targeting IRAK1 / TRAF6 / JNK1 signaling pathway on proliferation,invasion,migration,apoptosis and inflammatory response of RA-FLSObjective: To investigate the expression of mi R-146 a in RA-FLS,to study the effect of mi R-146 a on the proliferation,invasion,migration,apoptosis and inflammatory response of RA-FLS,and to explore the molecular mechanism of mi R-146 a regulating RA-FLS.Methods:(1)the synovium of RA and trauma patients was collected,and human primary fibroblast like synovium cells were extracted by enzyme digestion and cultured.(2)Real time quantitative PCR(RT-q PCR)was used to detect the expression of mi R-146 a in RA-FLS and Ctrl-FLS.(3)Mi R-146 a,mimic and inhibitor and their corresponding negative controls were transfected into RA-FLS.(4)The expression of mi R-146 a in RA-FLS was detected by RT-q PCR.(5)CCK-8 method was used to detect the cell activity and compare the proliferation of RA-FLS after transfection.(6)The invasiveness of RA-FLS after transfection was compared with that of Transwell.(7)The migration ability of RA-FLS after transfection was compared by scratch test.(8)The apoptosis rate of RA-FLS cells was detected by AV/PI flow cytometry.(9)The expression levels of TNF-?,IL-1?,IL-6 and IL-17 were detected by enzyme-linked immunosorbent assay(ELISA).(10)The total RNA and protein of FLS were extracted and the m RNA and protein levels of target genes and pathway molecules were detected by RT-q PCR and Western blot respectively.Results:(1)the results of RT-q PCR showed that mi R-146 a in RA-FLS group was significantly lower than that in Ctrl-FLS group.(2)Compared with mimic-NC group,over expression of mi R-146 a can significantly inhibit the proliferation of RA-FLS;compared with Inhibitor-NC group,inhibition of mi R-146 a can promote the proliferation of RA-FLS.(3)The results of Transwell invasion experiment showed that overexpression of mi R-146 a could significantly reduce the number of cells invaded by RA-FLS compared with mimic-NC group,and inhibition of mi R-146 a could increase the number of cells invaded by RA-FLS compared with Inhibitor-NC group.(4)Compared with mimic-NC group,overexpression of mi R-146 a can significantly reduce the mobility of RA-FLS;compared with Inhibitor-NC group,inhibition of mi R-146 a can promote the migration of RA-FLS.(5)The results of AV/PI flow cytometry showed that over expression of mi R-146 a could significantly increase the apoptosis rate of RA-FLS cells compared with mimic-NC group,and inhibition of mi R-146 a could reduce the apoptosis rate of RA-FLS cells compared with Inhibitor-NC group.(6)The results of ELISA showed that overexpression of mi R-146 a could inhibit the secretion of TNF-?,IL-1?,IL-6,IL-17 by RA-FLS,while inhibition of mi R-146 a could promote the secretion of TNF-?,IL-1?,IL-6,IL-17 by RA-FLS.(7)The results of RT-q PCR and Western blot indicated that overexpression of mi R-146 a could reduce the levels of IRAK1,TRAF6 m RNA and IRAK1,TRAF6,p-JNK1 protein.However,inhibition of mi R-146 a expression increased IRAK1,TRAF6 m RNA and IRAK1,TRAF6,p-JNK1 protein levels.Conclusion: In RA-FLS,the expression of mi R-146 a is down regulated.Over expression of mi R-146 a can inhibit the proliferation,invasion,migration and inflammatory response of RA-FLS,and promote the apoptosis of RA-FLS.This mechanism of mi R-146 a may be related to the inhibition of IRAK1/TRAF6/JNK1 signaling pathway.This study revealed the regulatory mechanism of mi R-146 a on the biological behavior of FLS,and provided a solid theoretical basis for the use of mi R-146 a as a therapeutic target of RA in the future.Part 2 The in vitro and in vivo study of T-614 inhibiting RA-FLS function by regulating mi R-146aObjective: To study the regulatory mechanism of T-614 on mi R-146 a and its target genes IRAK1 and TRAF6 in RA-FLS,and the effect of T-614 on the proliferation,invasion,migration,apoptosis and inflammatory response of RA-FLS after mi R-146 a inhibitor plasmid transfection.Then,the CIA rat model was established to elucidate the regulatory mechanism of mi R-146 a involved in the inhibition of RA synovium proliferation and inflammatory response.Methods:(1)CCK-8 method was used to detect the effect of eramod on the activity of RA-FLS.(2)The m RNA and protein levels of mi R-146 a and target gene pathway were detected by q PCR and Western blot after the intervention of RA-FLS.(3)The expression of mi R-146 a of RA-FLS was detected by RT-q PCR.(4)CCK-8 method was used to detect the cell survival rate and compare the proliferation of RA-FLS after plasmid transfection.(5)The Transwell invasion assay compared the invasivity of RA-FLS after plasmid transfection and T-614 intervention.(6)After T-614 intervention,the migration ability of RA-FLS was compared by Wound healing test.(7)The apoptosis rate of RA-FLS was detected by AV/PI flow cytometry.(8)The expression levels of TNF-?,IL-1 ?,IL-6 and IL-17 were detected by ELISA.(10)The total RNA and protein of FLS were extracted and the m RNA and protein levels of target genes and pathway molecules were detected by RT-q PCR and Western blot respectively.(9)The model of CIA rats was established.T-614 and mi R-146 a inhibitor were used to intervene.The arthritis severity of CIA rats was evaluated by arthritis score.(10)HE staining was used to observe the pathological changes of synovium.(11)TUNEL staining was used to detect the apoptosis of synovial cells.(12)The expression of TNF-?,IL-1?,IL-6 and IL-17 were detected by RT-q PCR and IHC respectively.(13)The expression of mi R-146 a and its target gene pathway in rat synovium was detected by RT-q PCR and IHC.Results:(1)20?g/ml,40?g/ml,80?g/ml,160?g/ml T-614 could significantly inhibit the activity of RA-FLS,significantly increase the level of mi R-146 a in RA-FLS,and decrease the level of IRAK1 and TRAF6.With the increase of T-614 concentration,the activity of RA-FLS was inhibited more obviously,the level of mi R-146 a was increased,and the levels of IRAK1 and TRAF6 were decreased.It was determined that 20?g/ml T-614 was used for subsequent experiments.(2)RT-q PCR results showed that the expression of mi R-146 a in the group of Inhibitor-mi R-146 a was significantly lower than that in the group of Inhibitor-NC;the expression of mi R-146 a in the group of T614 & inhibitor-NC was significantly higher than that in the group of Inhibitor-NC and T614 & inhibitor-mi R-146 a.(3)CCK-8 test results showed that the proliferation ability of RA-FLS cells in Iinhibitor-mi R-146 a group was higher than that in Inhibitor-NC group,and had significant difference at 72h;the proliferation ability of RA-FLS cells in T614 & inhibitor-NC group was lower than that in Inhibitor-NC group and T614 & inhibitor-mi R-146 a group,and had significant difference at 72 h.(4)The results of Transwell invasion experiment showed that the number of RA-FLS invasion cells in Inhibitor-mi R-146 a group was higher than that in Inhibitor-NC group;the number of RA-FLS invasion cells in T614 & inhibitor-NC group was significantly lower than that in Inhibitor-NC group and T614 & inhibitor-mi R-146 a group.(5)Compared with Inhibitor-NC group,the healing area of RA-FLS in Inhibitor-mi R-146 a group was significantly increased;the healing area of RA-FLS in T614 & inhibitor-NC group was significantly smaller than that in Inhibitor-NC group and T614 & inhibitor-mi R-146 a group.(6)The results of AV / PI flow cytometry showed that the apoptosis rate of RA-FLS in Inhibitor-mi R-146 a group was significantly lower than that in Inhibitor-NC group;the apoptosis rate of RA fibroblast like synoviocytes in T614 & inhibitor-NC group was significantly higher than that in Inhibitor-NC group and T614 & inhibitor-mi R-146 a group.(7)The levels of TNF-a,IL-1?,IL-6 and IL-17 in RA-FLS supernatant of Inhibitor-mi R-146 a group were higher than those of Inhibitor-NC group,and the levels of pro-inflammatory factors in T614 & inhibitor-NC group were lower than those of inhibitor-NC group and T614 & inhibitor-mi R-146 a group.(8)The results of RT-q PCR and Western blot showed that the expression levels of IRAK1,TRAF6 m RNA and IRAK1,TRAF6,p-JNK1 protein in RA-FLS of Inhibitor-mi R-146 a group were higher than that of Inhibitor-NC group;the expression levels of IRAK1 and TRAF6 m RNA and IRAK1,TRAF6,p-JNK1 protein in RA-FLS of T614 & inhibitor-NC group were lower than that of Inhibitor-NC group and T614 & inhibitor-mi R-146 a group.(9)The arthritis score of CIA rats in Inhibitor mi R-146 a group was higher than that of model group;the arthritis score of CIA rats in T-614 group was lower than that of model group and T-614 & inhibitor mi R-146 a group,while the arthritis score of CIA rats in T-614 & inhibitor mi R-146 a group was lower than that of Inhibitor mi R-146 a group.(10)HE staining of synovium showed that synovium epithelial hyperplasia and inflammatory cell infiltration were obvious in model group.The degree of synovium proliferation and inflammatory cell infiltration of Inhibitor-mi R-146 a group was higher than that of model group;the degree of synovium proliferation and inflammatory cell infiltration of T-614 group was lower than that of model group and T614&inhibitor-mi R-146 a group,and the degree of synovium proliferation and inflammatory cell infiltration of T614-inhibitor-mi R-146 a group was lower than that of Inhibitor-mi R-146 a group.(11)The results of TUNEL staining showed that the percentage of apoptotic cells in model group was lower than that in control group;the percentage of apoptotic cells in Inhibitor-mi R-146 a group was lower than that in model group;the percentage of apoptotic cells in T614 group was higher than that in model group and T614&inhibitor-mi R-146 a group;and the percentage of apoptotic cells in T614&inhibitor-mir-146 a group was higher than that in Inhibitor-mi R-146 a group.(12)The results of RT-q PCR and IHC showed that the expression of TNF-?,IL-1?,IL-6 and IL-17 in model group was higher than that in control group;the expression of TNF-?,IL-1 ?,IL-6 and IL-17 in inhibitor mi R-146 a group was higher than that in model group;the expression of TNF-?,IL-1?,IL-6 in T-614 group was higher than that in model group.The expression of TNF-?,IL-1 ?,IL-6 and IL-17 in T614&inhibitor-mi R-146 a group was lower than that in model group and T614&inhibitor-mi R-146 group.(13)The results of RT-q PCR and IHC showed that the expression of mi R-146 a decreased,while that of IRAK1,TRAF6 and p-JNK1 increased in the model group compared with the control group;the expression of mi R-146 a decreased and that of IRAK1,TRAF6 and p-JNK1 increased in the Inhibitor-mi R-146 a group compared with the model group;Compared with the model group and T614 & inhibitor-mi R-146 group,the expression of mi R-146 a in T-614 group increased,while that of IRAK1,TRAF6 and p-JNK1 decreased;and compared with the Inhibitor-mi R-146 a group,the expression of mi R-146 a in T614 & inhibitor-mi R-146 a group increased,while that of IRAK1,TRAF6 and p-JNK1 decreased.Conclusion:Iguratimod can inhibit the proliferation,migration,invasion and inflammation of RA-FLS cells,and promote its apoptosis;at the same time,it can increase the expression of mi R-146 a in RA-FLS,and reduce the levels of IRAK1,TRAF6 and p-JNK1;Inhibition of mi R-146 a can weaken the effect of Iguratimod on the proliferation,migration,invasion and inflammation of RA-FLS cells,and promote its apoptosis.When the expression of mi R-146 a was inhibited in CIA rats,The inhibitory effect of Iguratimod on synovium proliferation and inflammation of RA was weakened,while the expression of IRAK1,TRAF6 and p-JNK1 increased.In vitro and in vivo,Iguratimod can inhibit the proliferation and inflammatory response of RA-FLS and promote its apoptosis by upregulating mi R-146 a and downregulating its target genes IRAK1 and TRAF6.The protective effect of Iguratimod on RA was partly realized by up regulating mi R-146 a and down regulating IRAK1 / TRAF6 / JNK1 signal pathway. |
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