丹酚酸B抑制血管紧张素Ⅱ诱导心肌成纤维细胞增殖与分化的作用

Effect of Salvianolic Acid B in Inhibiting Angiotensin Ⅱ-induced Proliferation and Differentiation of Rat Cardiac Fibroblast

摘要:

目的：研究丹酚酸B（salvianolic acid B，Sal-B）对血管紧张素Ⅱ（angiotensin Ⅱ，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^-6 mol·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。

Abstract:

Objective: To investigate the inhibitory effect of salvianolic acid B (Sal-B) on proliferation and differentiation of cardiac fibroblasts (CFs) induced by angiotensin Ⅱ(Ang Ⅱ) of neonatal Sprague Dawley rats, and clarify the anti-fibrotic mechanism of Sal-B in vitro. Method: Primary CFs were harvested from 1-3-day-old neonatal rats by 0.08% trypsin digesting, and then separated and purified by differential attachment. The CFs were identified by anti-vimentin immunocytochemistry. Proliferation and differentiation of the CFs model were reproduced by Ang Ⅱ. The CFs was randomly divided into 4 groups as follows: normal group (serum-free DMEM), model group (1×10^-6 mol·L^-1 Ang Ⅱ), Sal-B low-dose group (2.5×10^-5 mol·L^-1 Sal-B+1×10^-6 mol·L^-1 Ang Ⅱ) and high-dose group (5×10^-5 mol·L^-1 Sal-B + 1×10^-6 mol·L^-1 Ang Ⅱ). The CFs were pretreated with Sal-B for 1 h, and then co-cultured with Ang Ⅱ for 24 hours. The inhibition of Sal-B on CFs proliferation was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The content of hydroxyproline was detected by commercial kit. The expressions of alpha-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were detected by Western blot. Result: The results of MTT assay suggested that compared with the normal group, Ang Ⅱ in model group significantly induced abnormal CF proliferation (P<0.01); Compared with model group, both low and high-dose Sal-B significantly inhibited Ang Ⅱ-induced CFs proliferation (P<0.01). The content of hydroxyproline increased in model group (P<0.01) compared with the normal group, but decreased (P<0.01) after being co-cultured with Sal-B compared with model group. Western blot revealed that the expressions of α-SMA and Col Ⅰ significantly increased in the model group (P<0.01), low-dose and high-dose Sal-B suppressed Ang Ⅱ-induced up-regulation of α-SMA and Col Ⅰ(P<0.05,P<0.01). Conclusion: Sal-B could significantly inhibit Ang Ⅱ-induced cardiac fibroblasts proliferation and expressions of α-SMA and Col Ⅰ, and plays an important role in the process of anti-fibrosis induced by Ang Ⅱ in vitro.