基于不同Caspase凋亡通路的玄参环烯醚萜总苷抑制心肌梗死模型大鼠心肌细胞凋亡的作用机制研究
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篇名: 基于不同Caspase凋亡通路的玄参环烯醚萜总苷抑制心肌梗死模型大鼠心肌细胞凋亡的作用机制研究
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摘要: 目的:研究玄参环烯醚萜总苷抑制心肌梗死模型大鼠心肌细胞凋亡的作用机制。方法:将雄性Wistar大鼠随机分为假手术组、模型组和玄参环烯醚萜总苷低、中、高剂量给药组,每组10只。采用结扎大鼠左冠状动脉前降支的方法建立心肌梗死模型,假手术组只穿线不结扎。各剂量药物组大鼠在造模成功后分别灌胃玄参环烯醚萜总苷混悬液,每次剂量为50、100、200 mg/kg(以总苷提取物质量计),灌胃体积为10 mL/次,每天2次,连续给药7 d;假手术组和模型组大鼠同法灌胃等体积生理盐水。记录大鼠术前、术后及给药7 d过程中的Ⅱ导联心电图S-T段变化情况;检查大鼠心功能指标;采用比色法、免疫抑制法或酶联免疫吸附(ELISA)法检测各组大鼠血清中乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白Ⅰ(cTnⅠ)、N末端钠尿肽原(NT-pro BNP)、肿瘤坏死因子α(TNF-α)水平;采用TUNEL法观察心肌细胞凋亡情况;采用比色法检测心肌细胞中超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量;采用ELISA法、酶解比色法或酶解荧光法检测心肌细胞中Bcl-2、Bax、细胞色素C(Cyt C)、胱天蛋白酶8(Caspase-8)、Caspase-9、Caspase-12、Caspase-3、Calpain的蛋白表达水平。结果:与假手术组比较,模型组大鼠心电图S-T段显著抬高,左室舒张末期内径、左室收缩末期内径均显著增加,左室射血分数、短轴缩短率均显著降低;血清中LDH、CK-MB、cTnⅠ、NT-pro BNP、TNF-α水平均显著升高;心肌组织中有大量黄褐色凋亡细胞;心肌组织中SOD活性显著降低,MDA含量显著升高;心肌细胞中Bcl-2蛋白表达水平及Bcl-2/Bax比值均显著降低,Bax、Cyt C、Caspase-3、Caspase-8、Caspase-9、Caspase-12、Calpain的蛋白表达水平均显著升高(P<0.05或P<0.01)。与模型组比较,各剂量药物组大鼠的上述指标及心肌组织病理学变化均有显著改善;心肌细胞中Bcl-2蛋白表达水平及Bcl-2/Bax比值均显著升高,Bax、Cyt C、Caspase-3、Caspase-8、Caspase-9、Caspase-12、Calpain的蛋白表达水平均显著降低(P<0.05或P<0.01)。结论:玄参环烯醚萜苷可同时通过抑制Caspase-8、Caspase-9、Caspase-12相关的3条凋亡通路,继而抑制Caspase-3的激活,从而起到抗心肌细胞凋亡作用。
ABSTRACT: OBJECTIVE: To study the effect mechanism of iridoid glycosides extracted from Scrophularia ningpoensis inhibiting cardiomyocytes apoptosis in myocardial infarction model rats. METHODS: The male Wistar rats were randomly divided into sham operation group, model group and S. ningpoensis iridoid glycosides low-dose, medium-dose and high-dose groups, with 10 rats in each group. Myocardial infarction models were established by ligating the left anterior descending coronary artery of the rats, and sham operation group was only threaded without ligation. After the model was established, each administration group was given S. ningpoensis iridoid glycosides suspension intragastrically at three different doses of 50,100,200 mg/kg (by the amount of total glycosides extract) with 10 mL/time, twice a day, for consecutive 7 days. Sham operation group and model group were given constant volume of normal saline intragastrically with same method. The changes of S-T segment of lead ECG Ⅱ were recorded before, after and during 7 days of administration. Cardiac function of rats was examined. The serum levels of LDH, CK-MB, cTnⅠ, NT-pro BNP and TNF-α were determined by colorimetry, immunosuppression or ELISA. The apoptosis of myocardial cells was observed by TUNEL method. SOD activity and MDA content in cardiac myocytes were detected by colorimetry. The expressions of Bcl-2, Bax, Cyt C, Caspase-8, Caspase-9, Caspase-12, Caspase-3 and Calpain in cardiac myocytes were detected by ELISA, enzymolysis colorimetry or enzymatic fluorescence assay. RESULTS: Compared with sham operation, electrocardiogram S-T segment was significantly elevated and the left ventricular end-diastolic diameter and left ventricular end-systolic diameter were significantly increased in the model group; left ventricular ejection fraction and short axis shortening rate decreased significantly; serum levels of LDH, CK-MB, cTnⅠ, NT-pro BNP and TNF-α were increased significantly; there were a large number of yellow-brown apoptotic cells in myocardial tissue; the activity of SOD in myocardial tissue was significantly decreased while the content of MDA was significantly increased; the protein expression level of Bcl-2 and Bcl-2/Bax were significantly decreased, while the levels of Bax, Cyt C, Caspase-3, Caspase-8, Caspase-9, Caspase-12 and Calpain were significantly increased (P<0.05 or P<0.01). Compared with model group, above indexes and pathological changes of myocardial tissue were improved significantly in administration group; the level of Bcl-2 and Bcl-2/Bax in cardiomyocytes increased significantly, while the levels of Bax, Cyt C, Caspase-3, Caspase-8, Caspase-9, Caspase-12 and Calpain decreased significantly (P<0.05 or P<0.01). CONCLUSIONS: S. ningpoensis iridoid glycosides can inhibit the activation of Caspase-3 by inhibiting three apoptotic pathways related to Caspase-8, Caspase-9 and Caspase-12, and then inhibit the apoptosis of cardiomyocytes.
期刊: 2019年第30卷第6期
作者: 梁俭,骆杰炉,蔡庆群,许良葵,李脉,黄海潮
AUTHORS: LIANG Jian,LUO Jielu,CAI Qingqun,XU Liangkui,LI Mai,HUANG Haichao
关键字: 玄参;环烯醚萜总苷;心肌梗死;半胱天冬蛋白酶;凋亡途径;机制;大鼠
KEYWORDS: Scrophularia ningpoensis; Iridoid glycosides; Myocardial infarction; Caspase;Apoptosis pathway; Mechanism; Rat
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