The Mechanism Research Of MiR-9-5p On Ventilator-Induced Lung Injury By Targeting GSK-3β Regulating The Wnt/β-catenin Pathway

Objective:In mechanical ventilation,the improper use of ventilator can cause ventilator-induced lung injury(VILI).VILI main pathophysiological changes include inflammation,apoptosis,ect.At present,the pathogenesis of VILI remains unclear.Micro RNAs(miRs)are potential targets for disease therapy.Studies found that miR-9-5p mediates inflammation and apoptosis and affects the progression of diseases.Wnt/β-catenin pathway plays an important role in lung development,regeneration and repair.The purpose of this study was to explore the role of miR-9-5p in VILI and the potential regulatory mechanisms among miR-9-5p,GSK-3β and Wnt/β-catenin pathways.Methods:This study is divided into animal experiment and cell experiment.1.In animal experiments,Sprague--Dawley male rats were randomly divided into the following sections:(1)Control group(C): kept breathing autonomously,without mechanical ventilation(MV);(2)Low tidal volume group(L): 7ml/kg;(3)High tidal volume group(H): 40ml/kg;(4)H+miR-9-5p overexpression group(agomir);(5)H+agomir negative control(NC);(6)H+miR-9-5p silent expression group(antagomir);(7)H+antagomir NC;(8)H+ dimethyl sulfoxide(DMSO);(9)H+DMSO+GSK-3β inhibitor(SB216763);(10)H+antagomir+DMSO+SB216763;(11)11 H+DMSO+SB216763+β-catenin inhibitor(MSAB);n=6/group.miR-9-5p agomir,agomir NC,antagomir and antagomir NC were injected via caudal vein;SB216763,MSAB and DMSO were intraperitoneally injected.The rats were anesthetized,trachea cannula and then given MV with an animal ventilator for 4h.After MV,the rats were euthanized,and the changes of lung general appearance were observed.Bronchoalveolar lavage fluid(BALF)was collected by ligation of the whole lobe of the right lung and lavage of the whole lobe of the left lung,and the total protein level of BALF was determined.Enzyme linked immunosorbent assay(ELISA)was used to detect the expression levels of Interleukin-6(IL-6),IL-1β and Tumor necrosis factor-α(TNF-α)in BALF.The upper lobe of the right lung was weighed as wet weight(W),and was then placed in an oven at 65℃ for 72 h.The dry weight(D)was weighed and the W/D ratio was calculated.The middle lobe of the right lung was used for pathological experiments,while the lower and posterior lobes of the right lung were used for molecular experiments.The expression levels of miR-9-5p,GSK-3β,β-catenin,c-Myc,Bax and Bcl2 were measured by real-time quantitative polymerase chain reaction(qRT-PCR),western blot and immunofluorescence analysis.Hematoxylin and Eosin(HE)staining was used to evaluate pulmonary lesions.mediated dUTP nick-end labeling(TUNEL)was used to measure the apoptosis rate of lung tissue.The relationship between GSK-3β and miR-9-5p was identified by dual luciferase reporter analysis.2.In cell experiment,rat Type Ⅱ alveolar epithelial cell(AEC Ⅱ)was randomly divided into:(1)Control group(CMS): no mechanical stress stretching;(2)Low mechanical stress cyclic tensile group(LMS): 5%;(3)High mechanical stress cyclic tensile group(HMS): 18%;(4)HMS+miR-9-5p mimic;(5)HMS+mimic NC;(6)HMS+miR-9-5p inhibitor;(7)HMS+inhibitor NC;(8)HMS+DMSO;(9)HMS+DMSO+SB216763;(10)HMS+inhibitor+DMSO+SB216763.Before loading,miR-9-5p mimic/inhibitor/NC,SB216763 and DMSO were transfected correspondingly.After transfection,when the cell density reached 90%,the cells were subjected to mechanical stress and cyclic stretching for 4h to simulate MV.After loading,the expression of IL-6,IL-1β and TNF-α were detected by ELISA.AEC Ⅱ was collected and the expression of miR-9-5p,GSK-3β,β-catenin,c-Myc,Bax and Bcl2 were detected by qRT-PCR.The apoptosis rate was detected by flow cytometry.Results:1.The VILI rat model was successfully replicated by MV 4h with high tidal volume(40ml/kg).Compared with C group and L group,the gross appearance of lung in group H showed obvious congestion and bleeding.BALF total protein level and W/D ratio were significantly increased(p < 0.05).After HE staining,alveolar,interstitial and perivascular exudation increased significantly,and inflammatory cell infiltration was observed.ELISA results showed that the expression levels of IL-6,IL-1β and TNF-α in BALF increased significantly(p < 0.05).TUNEL staining showed that TUNEL(+)/DAPI(+)apoptotic cells increased significantly in lung tissue(p<0.05).Western blot analysis showed that the expression of Bax was up-regulated and that of Bcl2 was down-regulated(p<0.05).It can be seen that the VILI model was successfully replicated in this study.2.The expression of miR-9-5p was decreased and GSK-3β was increased in the lung tissues of VILI rat models(p < 0.05).Compared with group H,after overexpression of miR-9-5p,the congestion and bleeding in the lung were significantly reduced.Total protein level of BALF and W/D ratio were significantly decreased(p<0.05).After HE staining,alveolar,interstitial,perivascular exudation and alveolar bleeding were significantly reduced under the microscope.ELISA results showed that the expression levels of IL-6,IL-1β and TNF-α in BALF were significantly decreased(p < 0.05).TUNEL staining result showed that TUNEL(+)/DAPI(+)apoptotic cells in lung tissue were significantly reduced(p <0.05).Western blotting analysis showed that the expression of Bax was significantly decreased and that of Bcl2 was significantly increased(p<0.05).However,silencing of miR-9-5p aggravated lung injury.3.Double luciferase reporter gene analysis identified GSK-3β as the target gene of miR-9-5p.Overexpression of miR-9-5p down-regulated the expression of GSK-3βand up-regulated the expression of β-catenin and c-Myc in lung tissue of group H(p< 0.05).The opposite was true for silent miR-9-5p.Inhibition of GSK-3β by SB216763 upregulated the expression levels of β-catenin and c-Myc,and reversed the low expression of β-catenin and c-Myc in lung tissues that silenced miR-9-5p(p<0.05).Meanwhile,after SB216763 intervention,the appearance of lung congestion and bleeding were significantly reduced.BALF total protein level and W/D ratio were significantly decreased(p<0.05).Lung injury was significantly alleviated;the release of proinflammatory factors decreased significantly(p < 0.05);TUNEL(+)/DAPI(+)apoptotic cells decreased significantly(p<0.05);The expression of Bax decreased significantly,while the expression of Bcl2 increased significantly(p<0.05).However,GSK-3β was simultaneously inhibited by SB216763 in miR-9-5p-silenced lung tissue.The results showed that the lung injury effect of silenced miR-9-5p was partially reversed by the inhibition of GSK-3β expression(p<0.05).However,the inhibition of β-catenin expression by MSAB attenuated the anti-inflammatory and antiapoptotic effects of the inhibition of GSK-3β in VILI.4.After cyclic stretching AEC Ⅱ at 18% mechanical stress,the expressions of pro-inflammatory factors were significantly increased in HMS group compared with CMS and LMS.Flow cytometry showed that apoptosis of AEC Ⅱ increased significantly(p<0.05).qRT-PCR results showed that the expression of Bax m RNA increased and that of Bcl2 m RNA decreased(p<0.05).The AEC Ⅱ model of VILI was successfully replicated.In this model,miR-9-5p expression decreased and GSK-3β increased(p<0.05),which was consistent with animal experiments results.After overexpression of miR-9-5p,the pro-inflammatory factors and apoptosis of AEC Ⅱ was significantly reduced(p<0.05),GSK-3β expression was down-regulated and β-catenin and c-Myc expression was up-regulated(p<0.05).The results were reversed when silencing miR-9-5p.5.SB216763 inhibited GSK-3β to up-regulate the expression of β-catenin and c-Myc,and reversed the low expression of β-catenin and c-Myc in silencing miR-9-5p(p < 0.05).After transfection with SB216763,the pro-inflammatory factors and apoptosis of AEC Ⅱ was significantly reduced.The pro-inflammatory and pro-apoptotic effects of silencing miR-9-5p were partially reversed by SB216763(p<0.05).Conclusions:1.The expression of miR-9-5p was decreased and GSK-3β was increased in VILI.miR-9-5p can reduce inflammation and apoptosis of lung tissue and AEC Ⅱ.2.GSK-3β is a target gene of miR-9-5p.Down-regulation of GSK-3β by miR-9-5p can activate the Wnt/β-catenin pathway,which play a lung protective role by reducing inflammatory response and apoptosis of AEC Ⅱ,thereby improving lung barrier structure and function,and alleviating lung injury.3.miR-9-5p may be a potential therapeutic target for VILI,and AEC Ⅱ may be an important effector cell for the lung protective effect of miR-9-5p in VILI.