Study On The Effect And Mechanism Of Syndecan-4-Mediated Mechanical Stretch-Biological Signal In Ventilator-Induced Lung Injury

Ventilator-induced lung injury(VILI)refers to the process of an excessive alveolar expansion or excessive pulmonary pressure leading to the disruption of alveolar epithelium and/or vascular endothelial barrier,which leads to lung injury.Mechanobiological signal transduction is the process in which alveolar epithelium and vascular endothelial cells sense mechanical stress and convert the stimulus into biochemical signal transduction,causing intracellular downstream signaling cascades.It is considered as the initial stage of VILI.Syndecans are a family of transmembrane proteoglycans prevalent in mammals.Similar to integrins,syndecans are important transmembrane protein receptors for cellextracellular matrix interactions,getting more and more attention.Syndecan-4 is one of the most active subtypes,and its extracellular segment and integrin are co-located to the focal adhesion complex(FAC),which is regarded as an important recognition site for mechanical force transduction and perception.The intracellular segment of syndecan-4contains a unique cytoplasmic V region,Endowing syndecan-4 with the function of regulating various intracellular kinases and cytoskeletal interactions.Thus,syndecan-4 has been proposed as a mechanical stretch-sensitive protein,which may play an important role in the cellular mechanosensory and signaling pathway transduction system.This study aims to explore the role and possible mechanism of syndecan-4 in the regulation of VILI by establishing vivo VILI and in vitro cellular mechanical cyclic stretch models,applying gene transfection and Rho A/ROCK pathway inhibitors.The research results would provide a further experimental basis for a new molecular target for the clinical prevention and treatment of VILI.Part one: Effects of vivo VILI and vitro cellular excessive mechanical stretch on syndecan-4 protein expressionObjective: To clarify the effect of vivo VILI and the cellular excessive mechanical stretch in vitro on syndecan-4 protein expression.Methods:(1)Establishing VILI rat models(VT 25ml/kg),Sixty clean grade wild-type male Wistar rats aged 7-8 weeks were randomly divided into sham operation group and high tidal volume ventilation 2h,4h,6h,and 8h groups,with 12 rats in each group.Arterial blood gas analysis,lung histopathology,pulmonary edema,total protein concentration,and inflammatory factor levels in BALF were detected to verify the successful establishment of VILI.Meanwhile,syndecan-4protein concentration in lung tissues was detected by immunofluorescence(IF)and Weston blotting(WB),and the shedding levels of syndecan-4 in plasma and BALF were detected by EnzymeLinked Immune Sorbent Assay(ELISA).(2)Establishing in vitro 20%cyclic stretch(CS)models of A549 and human pulmonary microvascular cell(HPMEC).According to the CS time,each cell-line was divided into control(0h),2h,4h,6h,and 8h groups.WB was used to detect the cellular syndecan-4 protein level,ELISA was used to detect the syndecan-4 protein level in the cell culture medium,and flow cytometry was used to detect the apoptosis rates.Results:(1)In the HVMV group,severity of lung injury and the level of inflammatory factors were all aggravated in time-dependent manner of mechanical ventilation.IF staining showed that syndecan-4protein expression in lung tissues of the HVMV 8h group decreased significantly compared with the sham operation group.WB further showed that the syndecan-4 protein level in lung tissues decreased gradually with the time of HVMV.The shedding level of syndecan-4 in serum and BALF increased in time-dependent manner of HVMV.(2)After 20% cyclic stretch for 0,2,4,6,and 8 h,the relative expression level of syndecan-4 protein in A549 and HPMEC decreased significantly,and the shedding level of syndecan-4 in the culture medium of the two cell lines increased obviously in a stretch time-dependent manner.Flow cytometry showed that the apoptosis rates of A549 and HPMEC increased gradually with the time of 20% CS.The change ranges of the above indicators were all more obvious in A549 than that in HPMEC,thus A549 was selected as the target cell for further study.Conclusions: In-Vivo VILI model and In-Vitro cell mechanical cyclic stretch model all confirmed that excessive mechanical stretch would induce significant shedding of syndecan-4 from the cell surface,resulting in a remarkable decrease in syndecan-4 protein level in lung tissue or cell.Part two: Molecular mechanism of syndecan-4 in regulating excessive mechanical stretch-biological signal transductionObjective: To investigate the molecular mechanism of syndecan-4in regulating excessive mechanical stretch-biological signal transduction.Methods:(1)A549 cells underwent 20%CS,and were divided into control group(20%CS 0h)、 20%CS 2h 、 4h 、 6h 、 8h groups.(2)A549 were transfected with si RNA,and were divided into si Scr+5%CS group,si Scr+20%CS group,si Syndecan-4 +20%CS group,si-Syndecan-4 +5%CS group.(3)A549 were transfected with overexpressed plasmids,dividing into Vector+5%CS group,Vector+20%CS group,syndecan-4 c DNA +20%CS group,syndecan-4 c DNA+5%CS group.(4)Before CS,A549 was pretreated with Y27632 or DMSO,and were divided into Vehicle +5%CS group,Vehicle+20%CS group,Y27632+20%CS group,Y27632+5%CSgroup.The activity of Rho A was detected by Pull down method,and the expressions of syndecan-4 protein,total Rho A protein,and downstream ROCK1,as well as ZO-1 protein were detected by WB.The shedding levels of syndecan-4 in the cell culture medium were detected by ELISA.IF staining was used to observe the expression of syndecan-4 protein in A549 cells and its co-localization with ROCK1,and the structural changes of ZO-1 an F-actin in cytoskeletal after transfection with si Syndecan-4 or syndecan-4 c DNA.Results:(1)With the extension of CS time(2,4,6,8h),the syndecan-4 protein level of the A549 cell lysate decreased gradually compared with the control group(CS 0h).GTP-Rho A,ROCK1 protein level was up-regulated,and the expression of tight junction protein ZO-1was decreased gradually compared with the control group.IF showed that syndecan-4 protein was expressed in A549,and syndecan-4 protein and ROCK1 protein were co-labeled in A549.(2)si Syndecan-4 transfection before 20%CS could further enhance the expression of GTP-Rho A and ROCK1 and reduce the level of ZO-1 protein and further aggravated the structural damage of ZO-1 and F-actin cytoskeleton under 20%CS.After transfection with Syndecan-4 c DNA plasmid before 20%CS,compared with the Vector +20%CS group,the increasement of GTP-Rho A and ROCK1,as well as the decreasment of ZO-1 level were all ameliorated.IF staining showed that it effectively improved the structure destruction of ZO-1 and the disorganization of F-actin cytoskeleton under 20%CS.(3)The expression of syndecan-4 both decreased significantly with or without Y27632 pretreatment before 20%CS in A549,and the change of syndecan-4 level was not associated with the application of DMSO or Y27632 pretreatment.Pretreatment with Y27632 significantly reduced the activation of ROCK1 and ameliorated the reduction of ZO-1 after20%CS.Conclusion:(1)Excessive mechanical stretch resulted in decreased syndecan-4 protein level,activation of Rho A/ROCK1 pathway,and decreased expression of ZO-1.(2)syndecan-4 may serve as an upstream factor for suppression of the activation of Rho A/ROCK1 signaling pathway in the pathological CS model,and then ameliorate the structural damage of ZO-1 and cytoskeleton.Part Three: Further verifying the effect of pulmonary syndecan-4-protein on VILI in the rat modelObjective: Verify the effect of syndecan-4 protein in rat lung tissue on VILI.Methods: Sixty-four 7-8 weeks old clean-grade wild-type Wistar male rats were selected and randomly divided into 4 groups with 16 rats in each group,as Vehicle+LVMV(VT 6ml/kg)group,Vehicle+HVMV(VT 25ml/kg)group,R-syndecan-4+HVMV group,R-syndecan-4+LVMV group.The rats in R-syndecan-4 pre-treatment groups were atomized with 5ug/day recombinant syndecan-4 protein for 7 consecutive days before MV,the rats were exposed to HVMV or LVMV for 6 hours.Artery blood gas analysis,lung histopathology,lung dry-wet weight ratio,total protein concentration in BALF,the level of inflammatory factors,pulmonary syndecan-4 protein level,and MPO activity were also detected.The survival rates of the rest 10 rats in each group within 7 days after HVMV was recorded.Results: Pretreatment with recombinant syndecan-4 can significantly improve the protein level of syndecan-4 in the lung tissue after HVMV,and improve oxygenation,alleviate lung histopathological injury,reduce W/D ratio and total protein concentration in BALF,as well as inflammatory factor level and MPO activity in lung tissue,and improve the survival rate of the VILI rats within 7 days after HVMV.Conclusions: Pre-treatment of recombinant syndecan-4 to VILI rats could enhance pulmonary syndecan-4 protein level significantly,which would alleviate lung injury and improve the survival rate of VILI rats,and further verify the positive regulatory effect of syndecan-4 protein in lung tissue on VILI.