| Rheumatoid arthritis(RA)is an autoimmune disease characterized by synovial inflammation,synovial hyperplasia and cartilage degradation.Patients with RA experience early inflammation,synovial proliferation,and eventually cartilage degradation,with joint involvement and immobility.Early inflammation and synovial hyperplasia should be given more attention.Rheumatoid arthritis synovial fibroblasts(RASF)are an important component of synovium and play an important role in synovial proliferation.Inflammatory factor Tumor necrosis factors(TNF-α)is widely detected in the early and active stages of RA and plays a key role in the development of Ra.In addition,it should be noted that a decrease in p H has been observed in both synovial and synovial fluids of RA,and previous studies have shown that acid-sensing ion channel 1A(ASIC1a)is highly expressed in RA;ASIC1a can be activated by extracellular H~+and plays an important role in the pathogenesis of RA.Acid-sensing ion channels(ASICs)have been shown to be regulated by a variety of cytokines.This study revealed that TNF-αcould up-regulate the expression of ASIC1a in human RASFs,and the ASIC1a channel was opened under the action of extracellular H~+,which aggravated the process of acid-induced RASF migration and invasion.We conducted the following studies:1.Human rheumatoid arthritis synovial fibroblasts were isolated and cultured.Cell surface marker CD55 was detected by Flow cytometry to identify RASF.The results showed that RASF was successfully extracted.2.Expression of TNF-αand ASIC1a in synovium of human and CIA model mice.The expression of TNF-αand ASIC1a in the synovial tissues of human and CIA model mice was detected by immunohistochemistry.The results showed that TNF-αand ASIC1a were highly expressed in the synovium of human and CIA model mice.3.Expression of ASIC1a in synovial fibroblasts stimulated by TNF-α.Western Blot,q-PCR and immunofluorescence were used to detect the expression of ASIC1a in synovial fibroblasts stimulated by TNF-α.TNF-αexpression gradient of ASIC1a total protein was increased by stimulation with TNF-α(0 ng/ml,6.25 ng/ml,12.5ng/ml,25 ng/ml,50 ng/ml,100 ng/ml)and 25 ng/ml of TNF-α(0 h,3 h,6 h,12 h,24h,48 h),respectively.TNF-αstimulated the expression of ASIC1a membrane protein and m RNA.The fluorescence intensity of ASIC1a increased.The results showed that TNF-αstimulation could increase the expression of ASIC1a in synovial fibroblasts.4.The effect of TNF-αon acid-induced RASF activation by up-regulating ASIC1a.Edu proliferation test and PCNA were used to examine the proliferation.Cell Scratch Test,cell chamber test and EMT-related proteins were used to determine the migration and invasion ability of the cells.The results showed that TNF-αhad little effect on the proliferation of RASF induced by acid,and TNF-αpromoted the migration and invasion of RASF induced by acid.5.TNF-αup-regulates the possible pathway of ASIC1a.Second-generation gene sequencing and JASPAR predicted a possible pathway by which TNF-αup-regulates ASIC1a.The related pathway proteins were detected by Western Blot,and the up-regulation was reversed when the pathway inhibitors were added.The results showed that TNF-αup-regulated ASIC1a by activating NF-κB pathway.6.Effects of NF-κB blocker PDTC on TNF-αup-regulation of acid-induced RASF migration and invasion.Cell Scratch Test,cell chamber test and EMT-related proteins were used to determine the migration and invasion ability of the cells.The results showed that PDTC decreased the RASF migration and invasion induced by TNF-αupregulation.Taken together,our results suggest that TNF-αupregulates ASIC1a through regulation of NF-κB pathway,which in turn enhances ASIC1a-mediated RASF migration invasion. |
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