The Role Of TGF-β1/smad3 Pathway On Phenotype Transformation And Collagen Synthesis Of Rat Cardiac Fibroblasts Induced By High Sodium

Objective: To investigate the effect of high sodium on the phenotype transformation of rat cardiac fibroblasts(CFs) and the mechanisms.Methods: Rat cardiac fibroblasts were isolated from neonatal rat and cultured using tissue block culture method. The CCK8 assays were used to determine optimal concentration and action time of high sodium, smad3 inhibitor(SIS3) and TGF-β1 receptor inhibitor(SB431542)on CFs.This experiment were divided into control group(Na+122.27mmol/L),high-sodium group(Na+149mmol/L,152mmol/L, 155mmol/L,158mmol/L,161mmol/L,164mmol/L,167mmol/L,170mmol/L,173mmol/L,176mmol/L), control+SIS3 group( Na+ 122.27mmol/Ll+SIS3 0.5μM,1μM,2μM,3μM,4μM,5μM, high- sodium +SIS3 group(Na+ 161mmol/L+SIS3 0.5μM,1μM,2μM,3μM,4μM,5μM), control +SB group(Na+ 122.27mmol/l +SB 1μM,5μM,10μM,15μM,20μM,25μM), high- sodium +SB group(Na+ 161mmol/L+SB 1μM,5μM,10μM,15μM,20μM,25μM), mannitol group(osmotic pressure corresponding to the high- sodium group).After synchronization for serum-free medium 24 h prior, CFs were divided into control group(Na+ 122.27mmol/L),highsodium group(161mmol/L),control +SIS3 group(Na+ 122.27mmol/l+SIS3 1μM),high- sodium +SIS3 group(Na+ 161mmol/L+SIS3 1μM), mannitol group(osmotic pressure corresponding to the Na+ 161mmol/L group),then continued to culture for 48 h.The m RNA levels of α- smooth muscle actin(α-SMA), fibronectin extra domain-A(Fn ED-A), Collagen I, Collagen III, transforming growth factor-β1(TGF-β1),smad3 were analyzed by quantitative real-time polymerase chain reaction(RT-PCR).The protein levels of α-SMA,Fn ED-A,Collagen I, Collagen III, TGF-β1,phosphorylate-smad3(p-smad3) were further investigated using Western-blot and immunoflurescence. The TGF-β1 content in culture supernatant was assessed by ELISA method. The migration of rat CFs induced by high sodium was observed through wound healing.Results: CCK8 assay showed high- sodium(Na+ 161mmol/L) promoted rat CFs proliferation for 48 h markedly(P<0.05), and excluding the effect of osmotic pressure on CFs. The proliferation of rat CFs was inhibited significantly in a dose-dependent manner by smad3 inhibitor and TGF-β1 receptor inhibitor. Compared with the control group, the m RNA and protein levels of α-SMA, Fn ED-A, Collagen I, Collagen III,TGF-β1,p-smad3 were increased in high- sodium group(P<0.05).The secretion of TGF-β1 by rat CFs was elevated in culture media(P<0.05).Highsodium can enhance mobility of rat CFs and excluded the effect of osmotic pressure.Meanwhile, the protein levels of α-SMA, Fn ED-A, Collagen I, Collagen III were upregulated by SB431542 and SIS3 compare to the high- sodium group(P<0.05).Conclusion: I. High sodium can induce rat CFs phenotype transformation toward to myofibroblasts which enhance collagen synthesis and proliferation ability.II. The collagen synthesis and phenotype transformation of rat CFs induced by high sodium may be regulated via TGF-β1/ smad3 pathway.