Protective Effect Of Oxycodone And MiRNA-140 On Acute Lung Injury

Part 1 Effect of oxycodone pretreatment on sespsis-induced acute lung injury in ratsObjective The aim of the present study was to investigate the effect of oxycodone pretreatment on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in rats,and to explore its possible mechanism.Methods Thrity-six adult male Sprague-Dawley rats were randomly divided into three groups(n= 12 each):sham group(Group S),ALI group(Group A)and oxycodone pretreatment group(Group O).The ALI model was established by intravenous injection of LPS 8 mg/kg in Group A.Group O was intravenously injected with oxycodone of 2 mg/kg 10 min before LPS injection.Six hous after LPS injection,blood samples were taken from the femoral vein for determination of TNF-α、IL-1β plasma levels.The serum and bronchoalveolar lavage fluid were collected to examine lung permeability index(LPI).Pulmonary specimens were harvested for microscopic examination of the pathological changes,detection of apoptotic index(by TUNEL staining),determination of wet/dry lung weight(W/D)ratio,analysis of expression level of Toll-like receptor 4(TLR4),NF-κB,AQP-1,AQP-5 in lung tissues(by Western blottingting).Results Compared with group S,LPI,W/D ratio,TNF-α,IL-1β plasma level,the expression level of TLR4 and NF-κB were increased,while PaO2,PaO2/FiO2 and the expression level of AQP-1,AQP-5 were decreased in group A;Compared with group A,LPI,W/D ratio,TNF-α,IL-1β plasma levels,the expression level of TLR4 and NF-κB were decreased,while PaO2,PaO2/FiO2,and the expression level of AQP-1,AQP-5 were increased in group O.LPS induced significantly pathologic injuries in lung tissues which were significantly attenuated by oxycodone.Oxycodone exerted beneficial effects on the inhibition of proinflammatory cytokine production.Conclusions Oycodone might reduce pulmonary microvascular permeability in sepsis-induced acute lung injury model by upregulating expression of AQP-1 and AQP-5,and inhibit the inflammatory response via suppressing TLR4/NF-κB signaling pathway.Part 2 Differential expression of microRNA-140 in sepsis-induced acute lung injury and the mechanistic investigation in inflammationObjective This study aimed to investigate the differential expression of miR-140 in sepsis-induced acute lung injury(ALI)and its correlation with inflammatory cytokines in vivo and in vitro.Methods Thrity adult male Sprague-Dawley rats were randomly divided into five groups(n=6 each):sham group,ALI 3h,6h,12h and 24h group.The ALI model was established by intravenous injection of LPS 8 mg/kg in ALI group.We cultured human pulmonary Ⅱ epithelial cells(A549),which were divided into five groups:negative control group(NC group),LPS 3h,6h,12h and 24h group.A549 cells were added with LPS(2 μg/ml)to establish the ALI model in vitro.At different time point,we observed the pathological changes of lung tissue in ALI rats,detected the expression level of miR-140 in plasma and lung tissue(by RT-qPCR),TNF-a and IL-1β plasma concentration(by ELISA),the expression level of TNF-α and IL-1β in lung tissues by RT-qPCR.We used software TargetScan and RNAhybrid database to predict the target gene of miR-140.The recombinant Luciferase vector was constructed,miR-140 mimic and recombinant plasmid were co-transfected into A549 cells.The effect of miR-140 on the activity of TLR4 was detected by the dual-luciferase reporter assay.In restore experiment,TLR4 overexpression vector was constructed,and the expression of TLR4 and NF-κB were detected(by Western blotting).Results(1)Compared with the control group,the plasma level of TNF-a and IL-1β in ALI rats were significantly increased,while plasma miR-140 level was significantly downregulated at different time point.(2)TLR4 expression in lung tissue of ALI rats was increased,while miR-140 expression in lung tissue was down-regulated,also showing a negative correlation.(3)In LPS-stimulated A549 cells,the trendency of miR-140,TNF-α and IL-1β level was consistent with that of ALI rats,which showed a negative correlation between miR-140 and inflammatory cytokines.(4)Dual luciferase reporter assay and restore experiment indicated that TLR4 was a target gene of miR-140.Conclusions(1)The expression level of miR-140 was significantly down-regulated in sepsis-induced acute lung injury;(2)TLR4 is a target gene of miR-140;(3)miR-140 reduced the inflammatory response in acute lung injury via down-regulating the expression of TLR4.Part 3 The mechanism of miR-140 regulating Toll-like receptor 4 signaling pathway on lipopolysaccharide-induced epithelial cell injuryObjective This study aimed to investigate the regulatory mechanism of miR-140 on TLR4-TRAF 6-NF-κB signaling pathway,and observed its effect on apoptosis in the cell model of sepsis-induced acute lung injury in vitro.Methods We cultured human pulmonary Ⅱ epithelial cells(A549),and transfected the A549 cells with miR-140 mimic or miR-140 inhibitor for 48h respectively.A final concentration of 2 ug/ml lipopolysaccharide(LPS)was used to stimulate the A549.The epithelial cells were divided into four groups:control group(CON),LPS group,miR-140 mimic group(MI),negative control group(NC),miR-140 inhibior group(IN).In LPS group,cells were added with LPS.In MI group,cells were transfected with miR-140 mimic,and then added with LPS.In NC group,cells were transfected with mimic-negative control and then added with LPS.In IN group,cells were transfected with miR-140 inhibitor,and then added with LPS.Then cells were cultured for 24h.At the end of the experiments,cells and supernatants were collected for determination.The mRNA expression level of target gene were detected by RT-qPCR.The expression of target protein was detected the by Western blotting.Cell apoptosis was measured by flow cytometry and TUNEL staining.Results Compared with control group,miR-140 expression was down-regulated and the apoptosis rate was significantly increased in LPS group(P<0.05);Compared with LPS group,miR-140 mimic significantly upregulated the expression of miR-140,increased the expression of TLR4,NF-κB,TRAF6,RIP,Caspase-3,decreased the the expression of XIAP,SCOS-3 and the apoptosis index in MI group(P<0.05);miR-140 inhibitor significantly down-regulated the expression of miR-140,decreased the expression of TLR4,NF-κB,TRAF6,RIP,Caspase-3 in 140-mimic group,increased the the expression of XIAP,SCOS-3 and the apoptosis index in IN group(P<0.05).The mRNA expression of TLR4,TRAF6,RIP,XIAP showed a similar trend.There was no difference between LPS group and NC group.Conclusions(1)miR-140 might inhibit cell apoptosis by down-regulating TLR4-TRAF6-NF-κB signaling pathway and thus played a protective role in LPS-induced epithelial cell injury.(2)Upregulating miR-140 or inhibiting activation of TLR4-TRAF6-NF-κB signaling pathway is expected to become an effective target in the prevention and treatment of acute lung injury.