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目的探讨血管紧张素(-1-7)[Angiotensin(-1-7),Ang(-1-7)]对血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)在大鼠心脏成纤维细胞(Cardiac fibroblasts,CFs)中信号转导的影响及其机制。方法原代分离培养并鉴定新生SD大鼠的CFs,将细胞分为8组:空白对照组、AngⅡ组、Ang(-1-7)组、AngⅡ+Ang(-1-7)组、AngⅡ+Ang(-1-7)+A-779组、AngⅡ+Ang(-1-7)+PAO组、Ang(-1-7)+PAO组和AngⅡ+PAO组,Western blot法检测细胞外信号调节激酶1/2(Extracellular signal regulated kinase 1/2,ERK1/2)和磷酸化的ERK1/2(p-ERK1/2)的表达;免疫沉淀捕捉分析法检测蛋白酪氨酸磷酸酶-1(Src-homology domain 2 containing protein tyrosine phosphatase-1,SHP-1)的酶活性;实时荧光定量PCR检测转化生长因子-β1(Transforming growth factor-β1,TGF-β1)、I型胶原蛋白(CollagenⅠ,ColⅠ)和Ⅲ型胶原蛋白(CollagenⅢ,ColⅢ)基因mRNA的转录水平。结果 AngⅡ可增加细胞内p-ERK1/2的表达水平和p-ERK/ERK值(磷酸化的p-ERK1/2与总的ERK1/2的比值),Ang(-1-7)通过与Mas受体结合可拮抗AngⅡ引起的上述效应;Ang(-1-7)通过与Mas受体结合而激活胞内SHP-1的活性,并可拮抗AngⅡ所致的SHP-1活性降低;抑制SHP-1的活性后,Ang(-1-7)拮抗AngⅡ诱导的p-ERK1/2表达水平和p-ERK/ERK值增高的效应被抑制。Ang(-1-7)对TGF-β1、ColⅠ和ColⅢ基因mRNA的转录水平无显著影响,但可抑制AngⅡ所诱导的上述基因mRNA转录水平的增加。结论 Ang(-1-7)通过与Mas受体结合而激活SHP-1,该效应与Ang(-1-7)拮抗AngⅡ诱导的p-ERK1/2的表达水平和p-ERK/ERK值增高密切相关;Ang(-1-7)可抑制AngⅡ所诱导的TGF-β1、ColⅠ和ColⅢ基因表达上调,这些现象可能体现了一种Ang(-1-7)拮抗AngⅡ所致的不良效应的保护性机制。
Objective To investigate the effects of Angiotensin (-1-7), Ang (-1-7) on angiotensin Ⅱ (AngⅡ) in cardiomyocyte fibroblasts (CFs) ) And its mechanism of signal transduction. Methods CFs of neonatal SD rats were isolated and cultured in primary culture. The cells were divided into 8 groups: blank control group, AngⅡgroup, Ang (-1-7) group, AngⅡ + Ang (-1-7) Ang (-1-7) + A-779 group, AngⅡ + Ang (-1-7) + PAO group, Ang (-1-7) + PAO group and AngⅡ + PAO group The expression of extracellular signal-regulated kinase 1/2 (ERK1 / 2) and phosphorylated ERK1 / 2 (p-ERK1 / 2) was detected by immunoprecipitation assay. Protein tyrosine phosphatase-1 (TGF-β1), Collagen Ⅰ (ColⅠ) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) And type Ⅲ collagen (Collagen Ⅲ, Col Ⅲ) gene mRNA transcription level. Results AngⅡ increased the expression of p-ERK1 / 2 and the ratio of p-ERK / ERK (the ratio of phosphorylated p-ERK1 / 2 and total ERK1 / 2) Receptor binding can antagonize the above effects induced by AngⅡ. Ang (-1-7) activates intracellular SHP-1 through binding to Mas receptor and inhibits the decrease of SHP-1 activity induced by AngⅡ. Inhibition of SHP- 1, the effect of Ang (-1-7) antagonizing AngⅡ-induced increase of p-ERK1 / 2 and p-ERK / ERK was inhibited. Ang (-1-7) had no significant effect on mRNA transcription of TGF-β1, ColⅠand ColⅢ, but inhibited AngⅡ-induced mRNA transcription. Conclusions Ang (-1-7) activates SHP-1 by binding to Mas receptor. This effect and Ang (-1-7) antagonize AngⅡ-induced increase of p-ERK1 / 2 and p-ERK / ERK Ang (-1-7) could inhibit AngⅡ-induced TGF-β1, ColⅠand ColⅢ gene expression, these phenomena may reflect a Ang (-1-7) protection against Ang Ⅱ-induced adverse effects of protection Sexual mechanisms.