lncRNA-H19/miR-93-5p/KLF2信号轴调控痛风性关节炎所致Th17/Treg细胞焦亡机制研究OA

Objective:To explore specific mechanism by which lncRNA-H19/miR-93-5p/Krüppel-like factor 2(KLF2)signal-ing axis regulates pyroptosis of Th17/Treg cells induced by gouty arthritis(GA).Methods:Primary Th17 and Treg cells from rats were cultured in vitro,and RNA fluorescence in situ hybridization(FISH)was performed to analyze subcellular localization of lncRNA-H19 in Th17 and Treg cells.Dual luciferasereporter assays and qRT-PCR were conducted to analyze interaction between lncRNA-H19 and miR-93-5p,as well as between miR-93-5p and KLF2.An in vitro GA model was established using Th17 and Treg cells.CCK-8 was used to detect cell viability;biochemical assay was used to detect lactate dehydrogenase(LDH)release rate;ELISA was used to detect TNF-α,IL-1β levels;flow cytometry was performed to assess cell apoptosis rate.Western blot was conducted to detect expres-sions of pyroptosis-related proteins.A rat model of GA was established.qRT-PCR was used to detect changes in molecular levels of lncRNA-H19/miR-93-5p/KLF2 signaling axis in synovial tissue.ELISA and Western blot were used to assess expressions of TNF-α,IL-1β and pyroptosis-related proteins in serum and synovial tissue.Results:lncRNA-H19 was mainly localized in cytoplasm of Th17 and Treg cells.There were obvious binding sites between lncRNA-H19 and miR-93-5p,as well as between miR-93-5p and KLF2.Compared with NC group of Th17 and Treg cells,lncRNA-H19 and KLF2 mRNA expressions were increased(P<0.05),while miR-93-5p expression was decreased in H19 group(P<0.05).Compared with sh-NC group,lncRNA-H19 and KLF2 mRNA expressions were decreased(P<0.05),while miR-93-5p expression was increased in sh-H19 group(P<0.05).Compared with control group,lncRNA-H19 and KLF2 mRNA expressions were increased(P<0.05),while miR-93-5p expression was decreased in GA group(P<0.05).Compared with Scramble group,sh-H19 group exhibited increased cell viability(P<0.05)and decreased LDH release rate,TNF-α,IL-1β,apoptosis rate,Caspase-3,NLRP1,NLRP3,Gasdermin-D(GSDMD)and IL-18 proteins expressions(P<0.05).In comparison to sh-H19 group,sh-H19+miR-93-5p inhibitor group showed decreased cell viability(P<0.05),and increased LDH release rate,TNF-α,IL-1β,apoptosis rate,Caspase-3,NLRP1,NLRP3,GSDMD and IL-18 proteins expressions(P<0.05).Com-pared with GA group in rats,there were no significant differences in lncRNA-H19,miR-93-5p,KLF2 mRNA,Caspase-3,NLRP1,NLRP3,GSDMD,IL-18 protein levels in synovial tissue,and TNF-α,IL-1β in serum between GA+sh-NC group(P>0.05).However,in GA+sh-H19 group,lncRNA-H19,KLF2 mRNA,Caspase-3,NLRP1,NLRP3,GSDMD,IL-18 protein levels in synovial tissue,and TNF-α,IL-1β in serum were decreased(P<0.05),while miR-93-5p expression in synovial tissue was increased(P<0.05).Con-clusion:lncRNA-H19/miR-93-5p/KLF2 signaling axis is involved in regulating pyroptosis process of Th17/Treg cells induced by GA.