A7793 The role of TGF-β1/smad3 pathway on phenotype transformation and collagen synthesis of rat cardiac fibroblasts induced by high sodium

Abstract

Objectives: In order to research the effects of TGF-β1/smad3 signaling pathway on the phenotype transformation and collagen synthesis of rat cardiac fibroblasts (CFs) induced by high sodium in vitro. Methods: The CCK8 assays were used to determine optimal concentration and action time of smad3 inhibitor (SIS3) and TGF-β1 receptor inhibitor (SB431542) on CFs from Wistar rats. CFs were divided into control group (Na+139 mmol/L), high-sodium group (Na+149 mmol/L- Na+176 mmol/L), high-sodium+SIS3 group (Na+161 mmol/L+SIS3 0.5 μM- 5 μM), high-sodium+SB group (Na+161 mmol/L+SB431542 1 μM- 25 μM), mannitol group (osmotic pressure corresponding to the Na+ 161 mmol/L group), and cultured for 48 h. The mRNA and protein levels of α-SMA, fibronectin extra domain-A (Fn ED-A), Collagen I, Collagen III, TGF-β1, smad3 were detected by real time-PCR, Western blot and immunofluorescence. The content of TGF-β1 in culture supernatant was determined by ELISA. The CFs migration was observed through the cell scratch test. Results: High sodium promoted the proliferation and migration of rat CFs under the exclusion of osmotic pressure. The proliferation of CFs was inhibited in a dose-dependent manner by SIS3 and SB431542. The mRNA and protein levels of α-SMA, Fn ED-A, Collagen I, Collagen III, TGF-β1, p-smad3 were increased in high-sodium group and excluded the influence of osmotic pressure. The secretion of TGF-β1 in culture medium was elevated as well. Meanwhile, the protein levels of α-SMA, Fn ED-A, Collagen I, Collagen III were downregulated by SB431542 and SIS3. Conclusion: High salt can cause the proliferation and phenotype transformation of CFs toward to myofibroblasts and promote collagen synthesis via TGF-β1/ smad3 pathway. TGF-β1/ smad3 may be the key intervention targets for preventing high-salt induced myocardial fibrosis.