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目的:研究丹酚酸B(salvianolic acid B,Sal-B)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的新生SD大鼠心肌成纤维细胞(cardiac fibroblasts,CFs)增殖与分化的影响,探讨Sal-B是否具有对抗心肌纤维化的作用。方法:胰酶消化,差速贴壁分离纯化CFs,抗波形蛋白免疫细胞化学法鉴定CFs。建立AngⅡ诱导的CFs增殖分化模型。实验分为空白组(无血清DMEM),模型组(1×10~(-6)mol·L~(-1)AngⅡ),Sal-B低剂量组(2.5×10~(-5)mol·L~(-1)Sal-B+1×10~(-6)mol·L~(-1)AngⅡ)及Sal-B高剂量组(5×10~(-5)mol·L~(-1)Sal-B+1×10-6mol·L~(-1)AngⅡ)共4组。Sal-B预保护1 h,加入AngⅡ共同作用24 h后,采用噻唑蓝(MTT)法分析Sal-B对AngⅡ诱导CFs增殖的细胞存活率,采用试剂盒(消化法)测定羟脯氨酸含量,蛋白质免疫印迹(Western blot)分析α-平滑肌肌动蛋白(α-SMA),Ⅰ型胶原(collagenⅠ,ColⅠ)的表达情况。结果:MTT结果显示,与空白组比较,模型组AngⅡ显著诱导CFs异常增殖(P<0.01),与模型组比较,Sal-B低、高剂量显著抑制AngⅡ诱导的CFs增殖(P<0.01);羟脯氨酸含量测定结果显示,与空白组比较,模型组羟脯氨酸含量显著升高(P<0.01),与模型组比较,Sal-B低、高剂量组含量均显著降低(P<0.01);Western blot结果显示,与空白组比较,模型组α-SMA,ColⅠ蛋白表达显著上调(P<0.01),与模型组比较,Sal-B低、高剂量组α-SMA,ColⅠ蛋白表达明显下调(P<0.05,P<0.01)。结论:Sal-B可抑制AngⅡ诱导的CFs增殖,减少α-SMA,ColⅠ蛋白的表达,对AngⅡ体外诱导心肌纤维化进程具有抑制作用。
Objective: To investigate the effect of Salvianolic acid B (Sal-B) on the proliferation and differentiation of cardiac fibroblasts (CFs) induced by angiotensin Ⅱ (AngⅡ) -B has the effect against myocardial fibrosis. Methods: Trypsin digestion, differential adherence purification CFs, anti-vimentin immunocytochemistry to identify CFs. Establishment of Ang Ⅱ-induced proliferation and differentiation of CFs model. The experiment was divided into blank group (serum-free DMEM), model group (1 × 10 -6 mol·L -1 AngⅡ) and Sal-B low dose group (2.5 × 10 -5 mol·L -1) (-1) Sal-B + 1 × 10 -6 mol·L -1 AngⅡ) and Sal-B high dose group (5 × 10 -5 mol·L -1) 1) Sal-B + 1 × 10-6mol·L -1 AngⅡ). Sal-B was pre-protected for 1 h, AngⅡ was added for 24 h, and the cell viability of Sal-B-induced CFs proliferation was analyzed by MTT assay. The hydroxyproline content Western blot was used to analyze the expression of α-SMA and collagenⅠ (ColⅠ). Results: Compared with the blank group, Ang Ⅱ significantly induced the abnormal proliferation of CFs in the model group (P <0.01). Compared with the model group, low and high doses of Sal-B significantly inhibited the proliferation of CFs induced by Ang Ⅱ (P <0.01). The content of hydroxyproline in the model group was significantly higher than that in the blank group (P <0.01). Compared with the model group, the content of Sal-B in the low and high dose groups was significantly decreased (P < 0.01). Western blot results showed that compared with the blank group, the expression of α-SMA and ColⅠ in model group was significantly increased (P <0.01). Compared with the model group, the expressions of α-SMA and ColⅠ in Sal-B low and high dose group Significantly decreased (P <0.05, P <0.01). Conclusion: Sal-B can inhibit the proliferation of CFs induced by AngⅡ, decrease the expression of α-SMA and ColⅠ, and inhibit the progression of myocardial fibrosis induced by AngⅡ.