The Mechanism Of MiR-146a Regulating NLRP3 In Ventilator-induced Lung Injury

Background:Mechanical ventilation(MV)is a frequently used life-supportive technology for acute respiratory distress syndrome(ARDS)patients.The patients can safely go through the treatment period by controlling or assisting the patients’ spontaneous breathing with the help of MV.Ventilator-induced lung injury(VILI)is a common complication during MV,which can aggravate or cause lung damage in patients and severely impair patients’ quality of life.Currently,protective mechanical ventilation is advocated for preventing VILI,such as low tidal volume ventilation,prone position ventilation,high-frequency oscillatory ventilation,positive end-expiratory pressure ventilation,and extracorporeal membrane oxygenation.Although some progress has been made in preventing VILI,the occurrence of VILI cannot be avoided entirely due to the complexity of the pathogenesis.Therefore,further exploring the mechanisms of VILI and finding new therapeutic targets is essential for reducing medical costs and hospitalization of patients.The pathogenesis of VILI is complex.Barotrauma,atelectrauma,volutrauma and biotrauma are involved in VILI.Among them,biotrauma has attracted increasing attention.The recruitment of inflammatory cells,the generation of inflammatory mediators and the activation of complement impair the function of the alveolar air-blood barrier,eventually leading to lung injury.Among them,Nod-like receptor protein 3(NLRP3)inflammasome plays a vital role in VILI-induced inflammatory lung injury.MV induces the activation of NLRP3 inflammasome and the release of inflammatory factors to cause the degradation of cell junction proteins.The degradation of cell junction proteins increases the permeability of alveoli.A large amount of edema fluid flows into the alveolar septum and alveolar cavity,which reduces the gas exchange efficiency,seriously affecting the patients’ breathing and oxygenation.Reducing the activation of the NLRP3 inflammasome during MV is beneficial for VILI.Therefore,it is very important to explore the negative regulatory mechanism of NLRP3 inflammasome to provide new theoretical support for the prevention and treatment of VILI.Recently,as the research on microRNA(miRNA)continues,there is plenty of experimental evidence indicating that miRNA could participate in the occurrence and progression of multiple diseases.miR-146a was the first finding of miRNA associated with immune function.Studies have shown that miR-146a can regulate the secretion of various inflammatory factors and has an inflammatory inhibitory effect on multiple cells and tissues.Inflammatory responses play a central role in regulating the pathogenesis of VILI.However,it has not yet been reported whether miR-146a is involved in VILI development.Therefore,this study mainly detected the expression of miR-146a and deeply explored the specific roles and molecular mechanisms in VILI,offering a promising target for the prevention of VILI.Objective:The mouse alveolar epithelial cells(Mouse lung epithelial cells,MLE-12)models of VILI were constructed by cyclic stretch.To clarify(1)the expression of NLRP3 and cell junction proteins at different time points of cyclic stretch;(2)the expression of miR-146a after cyclic stretch;(3)The expression of NLRP3 and cell junction proteins in VILI model after high expression of miR-146a;(4)The expression of NLRP3 and cell junction proteins in VILI model after inhibiting the activity of miR-146a;(5)The mechanism of miR-146a regulating the expression of NLRP3 in VILI.Methods:(1)MLE-12 cells were treated with the FX-5000T Flexercell Tension Plus system at a frequency of 0.5 Hz with 20%elongation for 0,2,or 4 h,respectively.WB was used to detect the expression of NLRP3,E-cadherin and Occludin.(2)MLE-12 cells were treated with the FX-5000T Flexercell Tension Plus system at a frequency of 0.5 Hz with 20%elongation for 0,2,or 4 h,respectively.The expression of miR146a after cyclic stretch was detected by qRT-PCR.(3)MLE-12 cells were transfected with miR-146a mimic for 48 h,then cyclic stretch was performed.WB was used to detect the expression of NLRP3,E-cadherin and Occludin.(4)MLE-12 cells were transfected with miR-146a inhibitor for 48 h,then cyclic stretch was performed.WB was used to detect the expression of NLRP3,E-cadherin and Occludin.(5)MLE-12 cells were transfected with miR-146a mimic for 48 h,then the cells were treated with protein degradation pathway inhibitor or protein synthesis pathway inhibitor,respectively.Then the expression of NLRP3 was detected by WB.Results:(1)The expression of NLRP3 was increased,while the expression of E-cadherin,Occludin was decreased after cyclic stretch.(2)The expression of miR-146a was decreased after cyclic stretch.(3)High expression of miR-146a can inhibit the expression of NLRP3.In VILI model,compared with the cyclic stretch group,the expression of NLRP3 was decreased,and the degradation of cell junction proteins was alleviated after high expression of miR-146a.(4)Inhibiting the activity of miR-146a can promote the expression of NLRP3.In VILI model,compared with the cyclic stretch group,inhibiting the activity of miR-146a increased the expression of NLRP3 and the degradation of cell junction proteins.(5)miR-146a negatively regulates NLRP3 through the protein synthesis pathway.Conclusion:After cyclic stretch,the expression of miR-146a was decreased,and the expression of NLRP3 was increased,inducing inflammatory activation and the degradation of E-cadherin and Occludin,which leads to the VILI.miR-146a may negatively regulate the expression of NLRP3 through the protein synthesis pathway,then reduce the inflammatory response,alleviate the degradation of cell junction proteins and eventually improve VILI.