MiR-139-5p Improves Sepsis-induced Lung Injury By Targeting ROCK1

Background and Object:Sepsis is a kind of multiple organ dysfunction caused by host's maladjusted response to infection,which is a serious threat to human health.The lung is vulnerable to multiple organ failure in sepsis.Sepsis-induced acute lung injury(ALI)is an inflammatory process that involves both inflammatory cytokines production and apoptosis.In this study,we investigated the regulatory role of miR-139-5p in sepsis-induced ALI using a murine model of cecal ligation puncture(CLP)and an in vitro model induced by normal human bronchial epithelial(NHBE)cells.Methods:1?Pathological changes of lungs from sepsis-induced ALI mice were detected using Hematoxylin and eosin staining.Lung water content was determined and the expression of proinflammatory cytokines in the bronchoalveolar lavage fluid(BALF)and serum of sepsis-induced ALI mice was quantified using ELISA.Levels of oxidative stress in lung tissues were determined using two commercial kits.TUNEL assay was used to detect the level of apoptosis.miR-139-5p and Rho kinase 1(ROCK1)were determined using qRT-PCR and Western Blotting.2?ROCK1 was predicted and confirmed to be a direct target of miR-139-5p using a dual-luciferase reporter assay.3?NHBEs were cotransfected with lentiviral vector expressing ROCK1(or empty vector)and miR-139-5p mimics or control mimics prior to lipopolysaccharide(LPS)treatment.The transcriptional activity of caspase-3,the apoptosis ratio,the levels of MUC5AC,MUC1,TNF-?,IL-1?,IL-6,NLRP3,ASC,and caspasel were assessed.The apoptosis of NHBE cells was detected by flow cytometry.Results:1?Compared to the normal group,mice after CLP showed pulmonary morphological abnormalities,elevated production of TNF-?,IL-1?,and IL-6,increased ROS,MDA,LDH levels and apoptotic ratios and ROCK1,and decreased SOD,GSH-px levels and miR-139-5p in the lung.Overexpression of miR-139-5p in septic mice alleviated sepsis-induced pulmonary injury,apoptosis,and inflammation.2?ROCK 1 was confirmed to be a direct target of miR-139-5p using a dual-luciferase reporter assay.3?In NHBEs,LPS stimulation resulted in a significant induction of inflammatory cytokine levels and apoptosis compared to untreated cells.MiR-139-5p also exhibited anti-apoptotic and anti-inflammatory effects in LPS-treated NHBEs.The overexpression of ROCK1 in NHBEs recovered miR-139-5p-suppressed ROS activity and proinflammatory cytokine production.Conclusion:In conclusion,miR-139-5p alleviated sepsis-induced ALI via suppressing its downstream target ROCK1,suggesting a therapeutic potential of miR-139-5p for the management of sepsis-induced ALI.