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目的:研究氯沙坦通过影响高血压大鼠心肌成纤维细胞间质成分抑制心室重构的药理机制。方法:培养心肌成纤维细胞,免疫细胞化学法鉴定细胞,MTT法测氯沙坦和AngⅡ对细胞增殖活性影响的量效关系,Western Blotting测AngⅡ刺激成纤维细胞心肌JNK1/2、磷酸化JNK1/2表达的时效关系。根据实验所得AngⅡ刺激心肌成纤维细胞增殖活性作用的EC50值、氯沙坦抑制心肌成纤维细胞活性作用的IC50值和AngⅡ刺激JNK1/2表达的最佳时间,心肌成纤维细胞分为无血清DMEM组、AngⅡ100 nmol/L组、AngⅡ100 nmol/L+losartan 100 nmol/L组、losartan 100 nmol/L组,药物干预后分别收集各组蛋白质、培养上清液,Western blotting检测各组MMP-2、JNK1/2、磷酸化JNK1/2蛋白表达,ELISA检测分泌至培养上清液中MMP-2的量。结果:AngⅡ刺激心肌成纤维细胞增殖活性作用的EC50为53 nmol/L,氯沙坦抑制心肌成纤维细胞增殖活性作用的EC50为56.3 nmol/L。JNK1/2蛋白表达在AngⅡ刺激2 min达高峰,随后表达逐渐降低。AngⅡ增加JNK1/2表达,氯沙坦降低AngⅡ刺激导致的JNK1/2表达。AngⅡ组MMP-2蛋白表达较无血清DMEM组明显增高,氯沙坦降低AngⅡ刺激增高的MMP-2表达。AngⅡ组MMP-2分泌较无血清DMEM组增高,氯沙坦减少AngⅡ刺激所致MMP-2分泌增高。结论:氯沙坦防治高血压引起的心室重构,可能与其对抗AngⅡ刺激心肌成纤维细胞增殖活性、MMP-2合成、分泌有关,信号通路涉及JNK1/2。
OBJECTIVE: To study the pharmacological mechanism of losartan in inhibiting ventricular remodeling induced by interstitial components of cardiac fibroblasts in hypertensive rats. METHODS: Myocardial fibroblasts were cultured and identified by immunocytochemistry. MTT assay was used to determine the dose-response relationship of losartan and AngⅡ on cell proliferation. Western Blotting was used to measure the expression of JNK1 / 2 and JNK1 / 2 expression of the aging relationship. According to the experimental results of the EC50 value of stimulating cardiac fibroblast proliferation by AngⅡ, the IC50 value of losartan inhibiting the activity of cardiac fibroblasts and the optimal time of AngⅡ-stimulating JNK1 / 2 expression, the fibroblasts were divided into serum-free DMEM The Ang Ⅱ 100 nmol / L group, Ang Ⅱ 100 nmol / L + losartan 100 nmol / L group and losartan 100 nmol / L group were collected. The protein and the supernatant were collected after the intervention. The expressions of MMP- JNK1 / 2, phosphorylated JNK1 / 2 protein expression, the amount of MMP-2 secreted to the culture supernatant was detected by ELISA. Results: The EC50 of AngⅡ on the proliferation of cardiac fibroblasts was 53 nmol / L. The EC50 of Losartan on the proliferation of cardiac fibroblasts was 56.3 nmol / L. The expression of JNK1 / 2 peaked at 2 min after AngⅡ stimulation, and then decreased gradually. Ang Ⅱ increased JNK1 / 2 expression, losartan reduced Ang Ⅱ stimulation induced JNK1 / 2 expression. The expression of MMP-2 in AngⅡgroup was significantly higher than that in serum-free DMEM group. Losartan decreased the expression of MMP-2 in AngⅡgroup. The secretion of MMP-2 in AngⅡ group was higher than that in serum-free DMEM group, but losartan decreased the secretion of MMP-2 by AngⅡ stimulation. CONCLUSION: The prevention and treatment of ventricular remodeling induced by losartan by losartan may be related to the anti-angiotensin Ⅱ-induced cardiac fibroblast proliferation activity, MMP-2 synthesis and secretion, signaling pathway involves JNK1 / 2.