Effect Of TGF-β1 On Expression Of Collagen And Alpha-Actinin-2 On Rat Atrial And Ventricular Fibroblasts

Objective To observe the effect of TGF-β1 inducing different expression of collagen and a-actinin-2 between rat atrial and ventricular fibroblasts, and to investigate the specific molecular mechanisms.Methods Tissue block method is used to culture rat atrial and ventricular fibroblasts, which is identified by SABC immunocytochemical staining,and then the following experiments are carried out.(1) investigating the impact of TGF-β1 on content of hydroxyproline in rat atrial and ventricular fibroblasts at different concentration(0.5\10ng/ml,respectively) and induction time (6h、12h、 24h、48h,respectively).(2) Rat atrial and ventricular fibroblasts are stimulated by TGF-β1 with optimum concentration and action time, The content of hydroxyproline in the two cells was measured by hydroproline kit.The mRNA expression of a-actinin-2、type Ⅰ、Ⅲ collagen are evaluated by reverse-transcription PCR.Western blot was used to measured the protein expression of a-actinin-2、Smad2/3、p-Smad2/3 and Smad7.Result (1) The TGF-β1 stimulation of 5ng/ml,24h was proved optimized in induce the expression of collagen in rat atrial and ventricular fibroblasts to the greatest extent.(2)After the optimized stimulating of TGF-β1, compared with control group,the increased mRNA and protein expression of type I and III collagen in ventricular fibroblasts stimulation intervention group were no significant difference; the increased mRNA and protein expression of type I and III collagen in atrial fibroblasts stimulation intervention group were significant (P<0.01); compared with ventricular fibroblasts stimulation intervention group, the type I and III collagen mRNA and protein expression increased more obviously in atrial fibroblasts stimulation intervention group,the difference were significant (P < 0.01).(3)The mRNA,protein level of a-actinin-2 were significantly up-regulated (P<0.05) in both ventricular and atrial fibroblasts TGF-β1 stimulation intervention group as compared to control groups; compared with ventricular fibroblasts TGF-β1 stimulation intervention group, the a-actinin-2 mRNA and protein expression increased more obviously in atrial fibroblasts TGF-β1 stimulation intervention group,the difference was significant (P<0.01).(4)compared with control group,the expression of Smad2/3、p-Smad2/3 were up-regulated and Smad7 was down-regulated in ventricular fibroblasts TGF-β1 stimulation intervention group,there were no significant difference; the increased protein expression of Smad2/3 in atrial fibroblasts TGF-β1 stimulation intervention group were no significant difference,and the increased expression of p-Smad2/3 and reduced expression of Smad7 were significantly (P< 0.01);compared with ventricular fibroblasts TGF-β1 stimulation intervention group, the increased expression of p-Smad2/3 and reduced expression of Smad7 in atrial fibroblasts TGF-β1 stimulation intervention group were more obvious,the difference were significant (P<0.01)Conclusion (1)TGF-β1 may not only cause cardiac fibroblasts collagen metabolic abnormalities, but also cause the abnormal expression of cytoskeletal protein.(2)The abnormal collagen metabolic and expression of cytoskeletal protein caused by TGF-β1 in atrial fibroblasts performed more obvious than ventricular fibroblasts,suggested that atrial fibroblasts may exerts greater susceptibility than ventricular fibroblasts. (3)Through detected the TGF-β1 downstream signaling molecules Smad2/3、p-Smad2/3 and Smad7, suggested that TGF-β1 has different effects on atrial and ventricular fibroblasts, and smads pathway may interpret the phenomenon.