吴茱萸总碱对大鼠原代心肌细胞肥大的抑制作用

Evodia total alkaloids protect against rat cardiomyocyte hypertrophy in vitro

目的研究吴茱萸总碱对血管紧张素Ⅱ(AngⅡ)诱导大鼠心肌细胞肥大的抑制作用,探讨其可能的作用机制。方法应用胰酶和Ⅱ型胶原酶混合消化法制备新生大鼠原代心肌细胞。实验随机分为正常对照组,模型组(AngⅡ0.1μmol/L),Eta 1、10、100 mg/L组和Eta 100 mg/L+L-NAME(1.5×10^(-2)mol/L)组。心肌细胞加入不同浓度Eta 30 min后再加入AngⅡ0.1μmol/L作用24 h。采用Image-Pro Plus 6.0图像分析系统测量心肌细胞的表面积,BCA法测定总蛋白含量,比色法测定细胞培养上清液中一氧化氮(NO)含量和一氧化氮合酶(NOS)活性,RT-PCR检测心房利钠肽(ANP)、丝裂素活化蛋白激酶磷酸酶-1(MKP-1)和内皮型一氧化氮合酶(e NOS)基因的表达。结果与正常对照组比较,AngⅡ0.1μmol/L明显诱导心肌细胞肥大,增加心肌细胞表面积、细胞蛋白质含量和ANP mRNA的表达;降低NOS活性、NO含量和e NOS mRNA表达。与模型组比较,Eta 10和100 mg/L明显抑制AngⅡ诱导的心肌细胞肥大,心肌细胞表面积、蛋白质含量及ANP mRNA的增加;明显增加e NOS和MKP-1 mRNA表达、NOS活性和NO的产生。L-NAME能取消Eta抗心肌细胞肥大效应。结论 Eta可抑制AngⅡ诱导的心肌细胞肥大,作用机制可能与Eta增加心肌细胞NO的生成,促进MKP-1表达,增加MAPK去磷酸化有关。

Objective This study was designed to investigate the inhibition of evodia total alkaloids（ Eta） on rat cardiomyocyte hypertrophy induced by angiotensinⅡ（ Ang II） in vitro and to explore the underlying mechanisms.Methods The primary cardiomyocytes were isolated from neonatal Sprague Dawley rats by using trypsin and typeⅡ collagenase,and stained with anti-α-smooth-muscle-actin antibody by an immunohistochemistry method for identification. The cultured cardiomyocytes were randomly divided into 6 groups and marked as normal control group,model（ AngⅡ0. 1 μmol/l） group,Eta（ 1,10,100 mg/l） group,and Eta（ 100 g/l） ＋ L-NAME（ 1. 5 × 10-2mol/l） group. The cardiomyocytes were pretreated with Eta for 30 min and subsequently stimulated with Ang II 0. 1 μmol/l. The cardiomyocyte surface area was measured with Image-Pro Plus 6. 0 imaging analytic system,and the content of cell protein was determined by BCA assay. The activity of nitric oxide synthetase（ NOS） and the content of nitric oxide（ NO） were detected,respectively. The mRNA levels of atrial natriuretic peptide（ ANP）,mitogen-activated protein kinase phosphatase-1（ MKP-1） and endothelial nitric oxide synthase（ e NOS） in the cardiomyocytes were analyzed by RT-PCR. Results Compared with the normal control group,Ang II（ 0. 1 μmol/l） significantly induced cardiomyocytes hypertrophy which includes larger cell surface area,higher protein content,as well as the re-expression of ANP. The results further revealed that Eta（ 10,100 mg/l） could obviously protect against Ang II-induced cardiomyocyte hypertrophy,which was associated with enhancing the activity of NOS and the content of NO,and up regulating the gene expressions of e NOS and MKP-1. The data also demonstrated that L-NAME could abolish the effect of Eta on anti-cardiomyocyte hypertrophy. Conclusion The present study suggests that Eta（ 10,100 mg/l） could attenuate Ang II-induced cardiomyocyte hypertrophy by increasing the production of NO,promoting the gene expression of MKP-1,and improving the phosphorylation of MAPK.