Mechanism study of microRNA-146a alleviating ventilator induced lung injury by inhibiting nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 expression
Objective To investigate the regulatory effect of microRNA-146a(miR-146a)on nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3(NLRP3)and its role in the ventilator-induced lung injury(VILI)and its mechanism.Methods ① MLE-12 cells were divided into 3 groups(n=3)according to the random number table method:mechanical stretch(CS)0 h group,CS 2 h group,and CS 4 h group.CS with an amplitude of 20％and a frequency of 0.5 Hz was performed for 0,2 h,and 4 h,respectively.② MLE-12 cells were divided into 4 groups(n=3)according to the random number table method:control group(NC+CS 0 h group),miR-146a overexpression group(Mimic+CS 0 h group),CS 4 h group(NC+CS 4 h group),miR-146a overexpres-sion+CS 4 h group(Mimic+CS 4 h group).After transfecting Negative control(NC)/miR-146a-5p Mimic,CS was administered for 0 or 4 h.③ MLE-12 cells were divided into 4 groups(n=3)according to the random number table method:control group(N.C.+CS 0 h),miR-146a inhibition group(Inhibitor+CS 0 h group),CS 4 h group(N.C.+CS 4 h group),miR-146a inhibition+CS 4 h group(Inhibitor+CS 4 h group),CS 0 or 4 h after transfection with mircoRNA Inhibitor N.C.(N.C.)/miR-146a-5p Inhibitor,respectively.④ MLE-12 cells were divided into 8 groups(n=3)according to the random number table method:control group(NC group),miR-146a overexpres-sion group(Mimic group),autophagy lysosomal pathway inhibition agent group(NC+CQ group),miR-146a overexpression+autophagy lysosomal pathway inhibitor group(Mimic+CQ group),ubiquitin proteasome inhibitor group(NC+MG132 group),miR-146a overexpres-sion+ubiquitin protease body inhibitor group(Mimic+MG132 group),protein synthase inhibitor group(NC+CHX group),and miR-146a overexpression+protein synthase inhibitor group(Mimic+CHX group).After transfecting sequence NC/miR-146a-5p Mimic,autophagic lysosomal pathway inhibitor chloroquine(CQ),ubiquitin proteasome inhibitor MG 132 or protein synthetase inhibitor cycloheximide(CHX)were added respectively.The protein expression levels of E-cadherin,Occludin and NLRP3 were detected by Western blot,and the expression of miR-146a was detected by real-time fluorescence quantitative polymerase chain reaction.Results ① Compared with CS 0 h group,the levels of miR-146a,E-cadherin and Occludin were low while the NLRP3 protein levels were higher in CS 2 h and CS 4 h groups(all P＜0.05).Compared with CS 2 h group,the levels of miR-146a,E-cadherin and Occludin were significantly low-er,while the NLRP3 protein level was higher in the CS 4 h group(all P＜0.05).② Compared with NC+CS 0 h group,the NLRP3 protein level was significantly higher,while the E-cadherin and Occludin levels were lower in NC+CS 4 h group(all P＜0.05);the NLRP3 pro-tein level of Mimic+CS 0 h group was lower than that of NC+CS 0 h group(P＜0.05).Compared with the NC+CS 4 h group,the NLRP3 protein level was lower,and the E-cadherin and Occludin protein levels were higher in the Mimic+CS 4 h group(all P＜0.05).③ Com-pared with N.C.+CS 0 h group,NLRP3 protein level was higher,while the E-cadherin and Occludin protein levels were lower in N.C.+CS 4 h group and Inhibitor+CS 0 h group(all P＜0.05).Compared with the N.C.+CS 4 h group,NLRP3 protein level was higher,E-cadherin and Occludin protein level was lower in the Inhibitor+CS 4 h group(all P＜0.05).④ Compared with the NC group,the NLRP3 protein levels of Mimic group and NC+CHX group were lower(all P＜0.05),and there was no significant difference between NC+CQ group and NC+MG132 group(all P＞0.05).Compared with the Mimic group,the NLRP3 protein level of the Mimic+CHX group was higher(P＜0.05),while the NLRP3 protein level of Mimic+CQ group and Mimic+MG132 group had no statistical significance(all P＞0.05).Conclusions After CS,the expression of miR-146a decreased and the expression of NLRP3 increased,which activated the excessive activation of inflammatory response and led to the degradation of epithelial barrier-related connexins E-cadherin and Occlu-din to form VILI.MiR-146a may inhibit the expression of NLRP3 through the protein synthesis pathway to alleviate inflammation and thus alleviate VILI.
万方数据
