摘要
目的探究CaMK Ⅱ在20-HETE诱导的乳鼠心肌细胞凋亡和肥大中的作用。方法原代培养乳鼠心肌细胞,随机分为正常对照组(Con组),20-HETE组,20-HETE+KN-93组以及KN-93组;采用CCK-8法检测细胞活性、TUNEL法检测凋亡、HE染色后检测心肌细胞表面积、BAC法检测细胞内蛋白浓度;RT-q PCR检测心肌肥大特征性基因心钠肽(ANP)的表达;Western Blot检测CaMK Ⅱ及其磷酸化蛋白表达。结果与对照组相比,20-HETE组心肌细胞活性显著下降,细胞凋亡率显著升高(P<0.05);同时,20-HETE明显促进心肌细胞表面积增加、蛋白浓度升高以及肥大基因ANP的表达上调(P<0.05);使用CaMK Ⅱ抑制剂KN-93共孵育后,阻断了20-HETE诱导的细胞凋亡和肥大的作用(P<0.05);20-HETE促进心肌细胞CaMK Ⅱ蛋白以及磷酸化CaMK Ⅱ蛋白表达(P<0.05),具有激活CaMK Ⅱ作用。结论 20-HETE激活CaMK Ⅱ信号通路诱导心肌细胞肥大和凋亡。
Objective To investigate the role of CaMKII on 20 - HETE - induced cardiomyocyte apoptosis and hypertrophy in neonatal rats. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into normal control group （ Con group）, 20 - HETE group, 20 - HETE ＋ KN -93 group and KN -93 group. CCK - 8 method was used to detect the cell activity and TUNEL assay was performed to analyze the cell apoptosis. The surface area of cardiomyocytes was measured after HE staining and total intracellular protein levels were detected by commercial BCA protein kit. RT - qPCR was used to measure the expression of hypertrophic biomarkers ANP. Western blot assay was performed to measure the expression of CaMKII and phospho - CaMKII. Results Com- pared with Con group, the cell viability was obviously decreased and the apoptotic rate was significantly increased in 20 - HETE group （ P 〈 0.05 ）. Meanwhile, the cell surface area and total protein levels were significantly increased and the mRNA expression of hypertrophic markers ANF was up - regulated by 20 - HETE treatment （P 〈0.05） ,whereas cotreatment with KN-93 （3 μmol/L） significantly attenuated the effects of 20 -HETE - induced apoptosis and hypertrophy （ P 〈 0.05 ）. Finally, treatment with 20 - HETE significantly increased the protein expression of CaMKII and phospho - CaMKII in cardiomyocytes （ P 〈 0.05 ）, indicating 20 - HETE acti- vated the CaMKII signaling pathway. Conclusion 20 - HETE activates the CaMKII signaling pathway, which is involved in 20 - HETE - induced apoptosis and hypertrophy in neonatal rat cardiomyocytes.
出处
《遵义医学院学报》
2017年第5期475-481,共7页
Journal of Zunyi Medical University
基金
国家自然科学基金资助项目(NO:81460040)
贵州省科学技术基金资助项目(NO:黔科合LH字[2014]7544)
遵义医学院博士启动基金项目(NO:F-659)
