基于ERK-Calpain2通路探讨黄连素对冠心病大鼠心肌细胞凋亡的影响
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摘要
[目的]研究黄连素(BBR)对冠心病大鼠心肌细胞凋亡的影响及ERK-Calpain2信号通路在其中的作用。[方法]以高脂饮食喂养并注射脑垂体后叶素建立冠心病大鼠模型。大鼠分为实验组(模型大鼠)、对照组(模型大鼠)和空白组(正常大鼠),实验组大鼠予以BBR 80 mg/(kg·d)灌胃,对照组和空白组予以等量双蒸水灌胃,治疗周期为12周。采用TUNEL免疫荧光检测心肌细胞凋亡情况。采用实时荧光定量聚合酶链式反应(RT-qPCR)检测心肌组织葡萄糖调节蛋白78(GRP78)、增强子结合蛋白同源蛋白(CHOP)及钙蛋白酶2(Calpain2)m RNA水平。采用蛋白质印迹法检测心肌组织GRP78、CHOP、磷酸化细胞外调节蛋白激酶(p-ERK)、细胞外调节蛋白激酶(ERK)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase-3)、Caspase-12及Calpain2蛋白表达水平。[结果]心肌TUNEL染色后发现,与对照组比较,实验组细胞凋亡显著减少(P<0.01)。与对照组比较,实验组大鼠Caspase-12、Caspase-3、GRP78和CHOP蛋白均有不同程度下调(P<0.01),p-ERK和Calpain2蛋白表达下调,Capastatin蛋白表达升高(P<0.01)。与对照组比较,实验组大鼠GRP78、CHOP、Calpain2 mRNA表达下降(P<0.01)。[结论] BBR能抑制冠心病大鼠心肌细胞发生内质网应激凋亡,该作用可能与抑制ERK-Calpain2信号通路有关。
[Objective] To study the effects of berberine(BBR) on cardiomyocyte apoptosis in coronary heart disease rats and the role of ERK-Calpain2 signaling pathway. [Methods] A rat model of coronary heart disease was established by feeding on a high-fat diet and injection of pituitrin. The model animals were divided into test group and control group. Blank groups were established with normal SD rats. The test group was given intragastric administration of BBR 80 mg/(kg·d). The control group and blank group were given double distilled water. The treatment cycle is 12 weeks. TUNEL immunofluorescence was used to detect cardiomyocyte apoptosis. RT-q PCR was used to detect GRP78, CHOP and Calpain2 mRNA levels. Western blot method was used to detect GRP78, CHOP, p-ERK, ERK,Caspase-3, Caspase-12 and Calpain2 protein expression in myocardial tissue. [Results] After myocardial TUNEL staining, compared with the control group, the apoptosis of the test group significantly reduced(P <0.01). Compared with the control group, the expression of Caspase-12, Caspase-3, GRP78 and CHOP proteins were down-regulated in different degrees(P<0.01). The expression of p-ERK and Calpain2 protein was down-regulated and the expression of Capastatin Protein increased(P<0.01) in the test group. Compared with the control group, the expression of GRP78, CHOP and Calpain2 m RNA in the experimental group decreased(P<0.01). [Conclusion] BBR can inhibit endoplasmic reticulum stress-induced apoptosis of myocardial cells in CHD rats. This effect may be related to the inhibition of ERK-Calpain2 signaling pathway.
[Objective] To study the effects of berberine(BBR) on cardiomyocyte apoptosis in coronary heart disease rats and the role of ERK-Calpain2 signaling pathway. [Methods] A rat model of coronary heart disease was established by feeding on a high-fat diet and injection of pituitrin. The model animals were divided into test group and control group. Blank groups were established with normal SD rats. The test group was given intragastric administration of BBR 80 mg/(kg·d). The control group and blank group were given double distilled water. The treatment cycle is 12 weeks. TUNEL immunofluorescence was used to detect cardiomyocyte apoptosis. RT-q PCR was used to detect GRP78, CHOP and Calpain2 mRNA levels. Western blot method was used to detect GRP78, CHOP, p-ERK, ERK,Caspase-3, Caspase-12 and Calpain2 protein expression in myocardial tissue. [Results] After myocardial TUNEL staining, compared with the control group, the apoptosis of the test group significantly reduced(P <0.01). Compared with the control group, the expression of Caspase-12, Caspase-3, GRP78 and CHOP proteins were down-regulated in different degrees(P<0.01). The expression of p-ERK and Calpain2 protein was down-regulated and the expression of Capastatin Protein increased(P<0.01) in the test group. Compared with the control group, the expression of GRP78, CHOP and Calpain2 m RNA in the experimental group decreased(P<0.01). [Conclusion] BBR can inhibit endoplasmic reticulum stress-induced apoptosis of myocardial cells in CHD rats. This effect may be related to the inhibition of ERK-Calpain2 signaling pathway.
引文
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[9]He Y,Zhou L,Fan Z,et al.Palmitic acid,but not high-glucose,induced myocardial apoptosis is alleviated by Nacetylcysteine due to attenuated mitochondrial-derived ROS accumulation-induced endoplasmic reticulum stress[J].Cell Death Dis,2018,9(5):568.
[10]Martinez JA,Zhang Z,Svetlov SI,et al.Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells[J].Apoptosis,2010,15(12):1480-1493.
[11]Banerjee C,Goswami R,Datta S,et al.Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus[J].Toxicol Appl Pharmacol,2011,6(1):44-51.
[12]Chen Y,Li Z,He Y,et al.Estrogen and pure antiestrogen fulvestrant(ICI 182 780)augment cell-matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor(GPR30)-to-calpain signaling axis[J].Toxicology&Applied Pharmacology,2014,275(2):176-181.
[2]杨文.冠心病与心肌细胞凋亡的关系[J].转化医学杂志,2002,15(1):41-42.
[3]王松海,车奕宏,陈捷.黄连素对缺氧复氧诱导大鼠心肌细胞内Nrf2/Keap1的影响[J].中国临床解剖学杂志,2017,35(3):282-285.
[4]万强,杨玉萍,刘中勇.黄连素抑制ERK1/2途径减轻大气细颗粒物对EA.hy926内皮细胞损伤的研究[J].中药材,2016,39(7):1623-1627.
[5]窦清理,于湘友,顾亚楠,等.重组血红蛋白对冠心病大鼠氧供需平衡的影响[J].中华危重病急救医学,2016,28(12):1118-1122.
[6]滕海麟,吴福根,杨晓卫,等.过敏性紫癜患儿血清中血管内皮生长因子与一氧化氮水平变化及意义[J].中国全科医学,2010,13(2):150-152.
[7]李然,刘立萍,王哲,等.丹栀逍遥散对人乳腺癌MCF-7细胞株裸鼠移植瘤的影响[J].中国实验方剂学杂志,2016,22(2):78-81.
[8]梁晓飞,王铭,肖永红.粉防己碱对小鼠肺成纤维细胞的凋亡及Caspase-3蛋白的影响[J].中国临床药理学杂志,2016,32(6):531-533.
[9]He Y,Zhou L,Fan Z,et al.Palmitic acid,but not high-glucose,induced myocardial apoptosis is alleviated by Nacetylcysteine due to attenuated mitochondrial-derived ROS accumulation-induced endoplasmic reticulum stress[J].Cell Death Dis,2018,9(5):568.
[10]Martinez JA,Zhang Z,Svetlov SI,et al.Calpain and caspase processing of caspase-12 contribute to the ER stress-induced cell death pathway in differentiated PC12 cells[J].Apoptosis,2010,15(12):1480-1493.
[11]Banerjee C,Goswami R,Datta S,et al.Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus[J].Toxicol Appl Pharmacol,2011,6(1):44-51.
[12]Chen Y,Li Z,He Y,et al.Estrogen and pure antiestrogen fulvestrant(ICI 182 780)augment cell-matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor(GPR30)-to-calpain signaling axis[J].Toxicology&Applied Pharmacology,2014,275(2):176-181.