The Role Of Integrin β1-Mediated Mechanochemical Pathway In Pulmonary Fibrosis Induced By Paraquat

In the late stage of paraquat(PQ)poisoning,the proliferation of alveolar and interstitial fibroblasts and abnormal deposition of extracellular matrix(ECM)can be seen,resulting in irreversible pulmonary fibrosis,which eventually leads to respiratory failure,which is the main cause of death after paraquat poisoning.Studies have shown that a large number of abnormal accumulation of ECM is not only a characteristic pathological result of pulmonary fibrosis,on the contrary,abnormal ECM will also aggravate the process of pulmonary fibrosis,the two are mutual cause and effect.Integrin β1,as a cell adhesion molecule,is widely distributed on ECM and cell surface.It is a bridge between intracellular and extracellular structures.Integrin β1 has a two-way channel for mechanical and biochemical signal transmission and can respond to extracellular and extracellular stimuli.Through the formation of ECM-integrin-cytoskeleton transmembrane signal system,integrin β1mediated FAK-Src-p130Cas mechanochemical pathway is activated,and then several downstream signal transduction pathways are activated,among which mitogen-activated protein kinase(FAK-Ras-MAPK)pathway is considered to be closely related to fibrotic diseases.However,the role and mechanism of integrin β1(Itgb1)-mediated mechanochemical pathway in paraquat-induced pulmonary fibrosis is still unclear.From the point of view of the biological effects of ECM in pulmonary fibrosis,this study was to clarify the activation of fibroblasts by changes in ECM microenvironment induced by paraquat,and to clarify the role and mechanism of integrin β1-mediated mechanochemical pathway in paraquat-induced pulmonary fibrosis.Part One:Differential Small Molecule Proteomics on Paraquat-induced Pulmonary Fibrosis Decellularized ScaffoldObjective:To investigate the changes of extracellular matrix small molecular proteins in pulmonary fibrosis induced by paraquat poisoning.Method:Fifteen healthy adult male SD rats were randomly divided into three groups:group A(n=5),group B(n=5)and group C(n=5).Group A was normal control group,group B and C were exposed to paraquat for 2 weeks and 4 weeks respectively.Acellular scaffolds of normal and fibrotic lungs were prepared by acellular technique.The expression of differentially expressed small molecular protein(DESMPs)in different groups and subcellular sites was analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry(LC-ESI-MS-MS).Result:1.The model of pulmonary fibrosis was established,and the acellular scaffold of normal and fibrotic lungs were prepared.2.Through differential small molecule proteomic analysis,a large number of paraquatinduced differential DESMPs were found in acellular scaffold of rat fibrotic lungs.The DESMPs of normal control group and paraquat group showed diversity in molecular function,biological process and biological pathway.The DESMPs proteins with the highest interaction correlation were albumin(Alb),integrin β1,apolipoprotein(Apoal),prolyl-4hydroxylase β(P4hb)and fibrinogen gene(Fgg).Compared with normal acellular scaffolds,the expression of integrin β1 protein in acellular scaffold of pulmonary fibrosis was significantly increased.Conclusion:There are a large number of differentially expressed small molecular proteins in acellular scaffold(fibrotic ECM)induced by paraquat poisoning.Integrin β1,as the main DESMPs,may play an important role in the pathogenesis of paraquat-induced pulmonary fibrosis.Part Two:Role and Mechanism of Integrin β1 in Paraquat-induced Pulmonary FibrosisObjective:To investigate the role and possible mechanism of integrin β1-mediated mechanochemical pathway in paraquat-induced pulmonary fibrosis in rats.Method:Eighteen healthy adult male SD rats were randomly divided into three groups:normal control group(Control group),paraquat intoxication group(PQ group)and integrinβ1 inhibitor group(ATN group).The degree of pulmonary fibrosis was evaluated by hematoxylin-eosin(HE),Masson,Sirius red staining and hydroxyproline content,and the protein gene expression and protein phosphorylation of integrinβ1-mediated mechanochemical pathways(FAK,Src,p130Cas)and downstream mitogen-activated protein kinase(FAK-Ras-MAPK)pathways(MEK1/2,ERK1/2,p38MAPK)were detected by Western blotting and RT-PCR,respectively.Result:1.The rat model of pulmonary fibrosis induced by paraquat was successfully established;2.Western blot showed that the protein expression of t-FAK,p-FAK,t-Src,p-Src,tp130Cas,p-p130Cas,p-MEK1/2,p-ERK1/2 and p-p38 MAPK in the PQ group was significantly higher than that in the normal control group(P<0.05),and the expression of tp38 MAPK protein in the paraquat group was higher than that in the normal control group,but the difference was not statistically significant(P>0.05).Compared with PQ group,the expression of t-FAK,p-FAK,t-Src,p-Src,t-p130Cas,p-p130Cas,p-MEK1/2,p-ERK1/2 and p-p38 MAPK protein in ATN group decreased significantly(P<0.05),while the expression of t-p38 MAPK protein showed a downward trend,but the difference was not statistically significant(P>0.05).3.RT-PCR showed that the mRNA expression of FAK,Src,p130Cas,MEK1 and MEK2 in PQ group was higher than that in normal control group((P<0.05)),and the mRNA expression of p38 MAPK in ATN group was higher than that in normal control group,but the difference was not statistically significant(P>0.05),Compared with PQ group,the mRNA expression of FAK,Src,p130Cas,MEK1 and MEK2 in ATN group decreased significantly((P<0.05)),and the mRNA expression of p38 MAPK decreased,but there was no significant difference((P>0.05)).Conclusion:1.The activation of mechanochemical pathway and its downstream FAK-Ras-MAPK signal pathway mediated by integrin β1 may be one of the important mechanisms of paraquat-induced pulmonary fibrosis in rats.2.Integrin β1 inhibitor ATN-161 can alleviate paraquat-induced pulmonary fibrosis in rats by inhibiting mechanochemical pathway,down-regulating downstream ERK1/2 signal pathway,or inhibiting the phosphorylation level of downstream ERK1/2 and p38 MAPK pathway.3.ATN-161,an inhibitor of integrin β1,is expected to become a new target for clinical intervention of paraquat in the treatment of pulmonary fibrosis.ATN-161 is a potential antipulmonary fibrosis drug.Part Three:Role and Mechanism of Pulmonary Fibrosis Decellularized Scaffold in Promoting Fibroblast ActivationObjective:To investigate the role and mechanism of integrin β1 in paraquat-induced pulmonary fibrosis acellular scaffold in promoting fibroblast activation.Methods:We constructed co-culture system of fibroblasts and lung acellular scaffold in vitro,the cell experiments were divided into 5 groups:group A without scaffold,group B with normal scaffold,group C with normal scaffold+integrin β1 inhibitor,group D with pulmonary fibrosis scaffold and group E with pulmonary fibrosis scaffold+integrin β1 inhibitor.The protein and gene expression of Vimentin,α-SMA and extracellular signalregulated kinase 1/2(ERK1/2)were detected by immunofluorescence staining and RT-PCR,respectively.Result:1.Immunofluorescence staining showed that the fluorescence brightness of Vimentin,α-SMA and p-ERK1/2 protein in the normal and pulmonary fibrosis scaffold group were brighter than that in the non-scaffold group,and the fluorescence brightness in the pulmonary fibrosis scaffold group was stronger than that in the normal scaffolds group,which was more obvious than that in the normal scaffold group(positive).The fluorescence brightness in the normal and pulmonary fibrosis scaffold+integrin β1 inhibitor group were weak expression.2.RT-PCR showed that the mRNA expressions of Vimentin,α-SMA,ERK1 and ERK2 in the normal and pulmonary fibrotic scaffold group were higher than those in the nonscaffold group(P<0.05),and the mRNA expressions of Vimentin,α-SMA,ERK1 and ERK2 in the pulmonary fibrotic scaffold group were significantly higher than those in the normal scaffold group(P<0.05),while the mRNA expressions of Vimentin,α-SMA,ERK1 and ERK2 in the pulmonary fibrosis scaffold+integrin β1 inhibitor group were significantly lower than those in the pulmonary fibrosis scaffold group(P<0.05).Conclusion:1.The acellular scaffold of pulmonary fibrosis induced by paraquat can promote the activation of fibroblasts;2.Integrin β1 may be involved in the activation of fibroblasts induced by ECM in pulmonary fibrosis by up-regulating the expression of downstream ERK1/2 signal pathway or inhibiting its protein phosphorylation.