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

HUANG Hai-feng; WANG Chun-hua; ZHAO Ling-lu; LUO Hong; XU Yi-ni; TAO Ling; ZHANG Min; SHEN Xiang-chun

doi:10.13422/j.cnki.syfjx.2017130128

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

更新时间：2024-01-04

- Effect of Salvianolic Acid B in Inhibiting Angiotensin Ⅱ-induced Proliferation and Differentiation of Rat Cardiac Fibroblast
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 23, Issue 13, Pages: 128-132(2017)
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- DOI：10.13422/j.cnki.syfjx.2017130128
  CLC： R285
- Published：2017
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HUANG Hai-feng, WANG Chun-hua, ZHAO Ling-lu, et al. Effect of Salvianolic Acid B in Inhibiting Angiotensin Ⅱ-induced Proliferation and Differentiation of Rat Cardiac Fibroblast[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(13): 128-132.
DOI：

HUANG Hai-feng, WANG Chun-hua, ZHAO Ling-lu, et al. Effect of Salvianolic Acid B in Inhibiting Angiotensin Ⅱ-induced Proliferation and Differentiation of Rat Cardiac Fibroblast[J]. Chinese journal of experimental traditional medical formulae, 2017, 23(13): 128-132. DOI： 10.13422/j.cnki.syfjx.2017130128.

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.

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