The Changes Of TRPCs Expressed In PMVECs Play A Role In BLM-induced Pulmonary Fibrosis Of Rats

Background:Idiopathic pulmonary fibrosis is closely related to myofibroblasts transdifferentedfrom fibroblasts. Besides the resident fibroblasts, bone marrow stem cells, alveolarepithelial cells and microvascular endothelial cells may also be a source ofMFb.However,related research has been accepted that CTGF and TGF-β are criticalcytokines in causing MFb phenotype. Our previous study found that CTGF and TGF-βexpressed continuously high in rats’serum of BLM induced pulmonary fibrosis.,andpulmonary microvascular endothelial cells may be a source of the fibrotic cytokinementioned above.The study confirmed that endothelial cells proliferation and differentiation areregulated by cytokine related signaling pathway, such as vascular endothelial growthfactor(VEGF), VEGF/Flk-1are the main signal pathway in regulating ECs proliferation.Transient receptor potential channels (TRPCs) can mediate increased,calcium ion influx,and can increase the activation of VEGF/Flk-1.The research of recent year found thatTRPC1and TRPC3/6/7mediated ECs proliferation induced by VEGF,meanwhile,VEGF can induce the high expression of CTGF, however, whether the highexpression of CTGF is related to abnormal expression of TRPC1and TRPC3/6/7is not reported.Method and Purpose:This experiment make PF model by injecting BLM in trachea in rats, VG staining isused to examine the expression of collagen fiber in the lung of rats at day7,14after BLMinjection. Contrasting with Normal rats’lung, the result proves that we make successfulPF model. Primary culture of rats’PMVECs in Vitro is identified by light microscope,transmission electron microscope and immunohistochemistry staining, the result ofwhich confirm PMVECs cultured successfully. Immunohistochemistry andimmunocytochemistry are used to detect the expression of CTGF, α-SMA andproliferating cell nuclear antigen(PCNA).We observed [Ca2+]i in PMVECs while adding the inhibitor of TRPC channels andFLK-1receptor by laser conflcal microscopy. Immunocytochemistry and Westernblot(WB) are used to detect TRPC1, TRPC3/6/7, FLK-1expresssion in PMVECs, andwe dectect CTGF, α-SMA and PCNA in PMVECs after treating with TRPC inhibitor2-APB by WB. Flow Cytometry ia used to detect the cell cycle of PMVECs in all groups.The research observe the PMVECs’function in PF and the role of [Ca2+]i regulation inalteration of cell function, meanwhile investigate the relationship between TRPC and PFinduced by BLM, in order to elucidate the mechanism of PF and provide experimentalbasis for further studying.Result:1.Result under light microscope, we saw that PMVECs grow into typical appearance ofpolygon or triangle; Transmission electron microscope show that95%of primarycultured PMVECs express WPB, which is the marker of EC, and Immunofluorescentstaining prove that VIII is highly expressed in PMVECs.; all experiments aboveillustrate that we successfully culture primary PMVECs.2. Flow cytometry is performed for cell cycle analysis, which proved that PMVECsproliferate more excessively in BLM7d group than in normal group，whose proliferation index(PI) is0.599higher than nomal PMVECs’PI.3.Immunohistochemistry and immunofluorochemistry show that CTGF is expressedhigher in the group of BLM treated rats’PMVECs than in normal group,α-SMA andPCNA is also expressed higher in PMVECs of BLM group than that of normal groupshowed by immunofluorochemistry.4.Laser confocal microscopy show that [Ca2+]i descend obviously in PMVECs of BLMgroup comparing with normal group, after treating with TRPC inhabitor and FLK-1inhabitor individually.5. Immunofluorochemistry and WB show that TRPC1、TRPC3/6/7and FLK-1are allexpressed higher in BLM group than in normal group.6.WB show that the expression of CTGF,α-SMA and PCNA decline markedly inPMVECs of BLM group after treated with TRPC inhabitor.Conclusion:1.The proliferation phynotype and excessive secretion of CTGF in PMVECs are one ofthe important reason in BLM induced pulmonary fibrosis2.TRPCs dysfunction play a key role in BLM induced PMVECs’proliferation,differention, intracellular calcium overloaded and VEGF/Flk-1signal activation. Theresult in vitro suggest that pulmonary fibrosis injuried by BLM may be an functionaldisorder.