Effect Of TRPC On Proliferation And Apoptosis Of Pulmonary Artery Smooth Muscle Cells In Ascites Syndrome Broilers

Pulmonary hypertension syndrome?PHS?is a metabolic condition of broilers also known as ascites syndrome?AS?.The pulmonary vasoconstriction and vascular remodeling are the two primary pathological findings.The pathological changes of pulmonary artery are closely related to the abnormal proliferation of pulmonary artery smooth muscle cells.Canonical transient receptor potential channel?TRPC?is the main channel for calcium influxes in the cell membrane,which is closely related to the programmed cell death,transcription factor activation and cell proliferation.Although TRPC is considered to be closely related to abnormal proliferation of pulmonary artery smooth muscle cells in mammals,its role in the pathological changes of pulmonary arteries in PHS broilers has not yet been reported.Intravenous injection of cellulose particles were used to induce PHS in broilers.This study was conducted to evaluate changes of TRPC,proliferation and apoptosis of smooth muscle cells in pulmonary arterioles as for investigation of the pathogenesis of PHS.At 20 d of age,a total number of 120 chickens were divided into a control group?n=30?and a treatment group?n=90?.Ascites syndrome was induced in treatment group chickens by intravenous injection of a cation exchange cellulose suspension.A single dose of 0.3 ml?0.02 g/ml cellulose and 150 U/ml heparin saline?was used per chicken as previously described.Control group chickens were injected intravenously with the same volume of normal saline as controls.Chickens were euthanized at day 42 by cervical dislocation.The tension of pulmonary artery rings was measured by the wire-myograph.And crdiomyocyte intracellular Ca²⁺levels were evaluated by flow cytometry using fluo-4/AM probes.At the same time,we detected mRNA relative expression level of TRPC1,TRPC4 and TRPC6 by real-time quantitative PCR and the localization of TRPC6 in lung tissue was investigated by immunohischemisty assays.The results showed that compared with the control group,the ratio between right ventricle and total ventricle?RV/TV?,the packed cell volume?PCV?,the serum aspartate aminotransferase?AST?and the creatine kinase?CK?were significantly increased?P<0.01?,but pulmonary vascular elasticity and sensitivity to the calcium channel blockers were decreased?P<0.01?.We also observed obvious pathological changes in myocardium and lung tissue of experimental group in pathological sections.Intracellular calcium levels in cardiomyocytes were significantly increased in cardiomyocytes from AS chickens?P<0.01?.The relative levels of TRPC1 was significantly decreased?P<0.05?,but the relative levels of TRPC4 and TRPC6 were significantly increased in the pulmonary artery?P<0.05?.In the lung tissue,the expression of TRPC1 was significantly decreased?P<0.05?,but the expression of TRPC1 was significantly increased?P<0.05?.By immunohistochemistry,the expression of TRPC6 in PASMCs and bronchial epithelial cells was increased in PHS broilers.The primary pulmonary artery smooth muscle cells?PASMCs?of the broiler were cultured with the collagenase digestion method.The cell viability was tasted with CCK-8assay,the effects on cell cycle distribution and cell apoptosis were detected by flow cytometry,and the expression levels of TRPC1,TRPC4 and TRPC6 mRNA were determined with dd-PCR.And we treated PASMCs by TRPC6 selective inhibitor SKF96365 and activator Fluofenamic acid?FFA?respectively.The results showed that the cell viability,cell cycle distribution,intracellular Ca²⁺levels and expression levels of TRPC1,TRPC4 and TRPC6 mRNA were not increased in hypoxia for 24 hours?P>0.05?.But in hypoxia for 48 hours,the cell viability,expression levels of TRPC1,TRPC4 and TRPC6 mRNA were observably increased?P<0.05?.And the distribution of the cell in phase G1,total and early apoptosis were significantly decreased?P<0.05?,but the distribution of cell in phase S was increased?P<0.05?.And the results showed SKF96365significantly inhibited cell viability and intracellular Ca²⁺levels?P<0.01?,induced cell arrest in the G1 phase?P<0.05?,and enhanced cell apoptosis in 24h.The TRPC6 activator FFA stimulated cell growth,decreased intracellular Ca²⁺levels?P<0.05?.And the expression of TRPC6 mRNA was significantly increased?P<0.05?,the proportion of cells in S phase and G2 phase were increased significantly?P<0.01?and the proportion of apoptotic cells was increased?P<0.01?.However,the cell cycle and apoptosis did not change after treatment with SKF96365 or FFA after anoxic treatment for 48 hours.In this study we found that the vascular elasticity of PHS broilers decreased,and TRPC participated in the pathological changes of lung tissue and pulmonary artery of PHS broilers.The abnormal proliferation and decrease of apoptosis of hypoxic PASMCs are related to TRPC.