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目的探讨阿司匹林(aspirin,aspi)对醛固酮(ald)诱导的新生大鼠心肌成纤维细胞(cardiac fibroblasts,CF)增殖的影响及可能机制。方法 0.08%胰酶消化1~3 d SD新生大鼠,差速贴壁法纯化SD新生大鼠原代CF。正式实验采用培养3~5代CF,分为4组,空白对照组:无小牛血清的高糖DMEM;醛固酮组(ald):无小牛血清的高糖DMEM+1×10-8mol/L ald;阿司匹林+醛固酮组(aspi+ald):无小牛血清的高糖DMEM+1×10-8mol/L ald+1.11×10-6mol/L aspi;螺内酯+醛固酮组(spiro+ald):无小牛血清的高糖DMEM+1×10-8mol/L ald+1×10-6mol/L spiro。HE染色法观察各组CF形态学变化;四氮唑盐(MTT)比色法检测细胞增殖;Western印迹分析TGF-β-Smad 2、3、4的蛋白表达。结果 HE染色结果显示,与对照组相比,ald可使细胞增生活跃,分裂相细胞显著增多(P<0.01);与ald相比,Aspi+ald组及spiro+ald组均可使细胞分裂显著减少(P<0.01)。MTT法检测细胞增殖结果提示,与对照组相比,ald能够明显增加CF对MTT的代谢率,MTT值明显升高,与对照组相比差异显著(P<0.01);与ald组相比,aspi+ald组、spiro+ald组均可使CF对MTT代谢率减少,MTT值显著降低(P<0.01)。Western印迹检测结果显示,以ald刺激CF后,TGF-β-Smad 2、Smad 3及Smad 4蛋白水平明显增高,以aspi及spiro干预后,CF中TGF-β-Smad 2、Smad 3及Smad 4蛋白水平表达较ald组明显降低,均有显著性差异(P<0.05)。结论 aspir可抑制ald诱导的CF增殖,其作用机制可能与下调TGF-β-Smad 2、3、4的蛋白水平相关。
Objective To investigate the effect of aspirin (aspi) on aldosterone-induced proliferation of cardiac fibroblasts (CF) in neonatal rats and its possible mechanism. Methods SD neonatal SD rats were digested with 0.08% trypsin for 1 ~ 3 days and primary CF of SD neonatal rats were purified by differential adherence. Formal experiments were conducted on 3 to 5-generation CFs and divided into 4 groups. Blank control group: high glucose-free DMEM without calf serum; ald: high glucose-free DMEM + 1 × 10-8mol / L without calf serum ald; aspirin + aldosterone group (aspi + ald): high glucose-free DMEM + 1 × 10-8 mol / L ald + 1.11 × 10-6 mol / L aspi without splenic serum; spiro + ald Bovine serum high glucose DMEM + 1 × 10-8mol / L ald + 1 × 10-6mol / L spiro. The morphological changes of CF were observed by HE staining. Cell proliferation was detected by MTT colorimetric assay. The protein expression of TGF-β-Smad 2,3,4 was analyzed by Western blotting. Results The results of HE staining showed that compared with the control group, ald increased cell proliferation and increased the numbers of dividing cells (P <0.01). Compared with the control group, the cell division was significantly inhibited by Aspi + ald and spiro + ald groups Decrease (P <0.01). MTT assay showed that ald significantly increased the metabolic rate of MTT and increased the MTT of CF compared with the control group (P <0.01). Compared with the control group, Aspi + ald group and spiro + ald group decreased the metabolic rate of MTT, and the MTT decreased significantly (P <0.01). The results of Western blotting showed that the levels of TGF-β-Smad 2, Smad 3 and Smad 4 were significantly increased after stimulation with CF with ald. The levels of TGF-β-Smad 2, Smad 3 and Smad 4 Protein expression was significantly lower than that of ald group, with significant difference (P <0.05). Conclusion aspir can inhibit ald-induced CF proliferation, the mechanism may be related to the down-regulation of TGF-β-Smad 2,3,4 protein levels.