PHB对高糖环境下的心肌细胞中活性氧及细胞凋亡的影响及机制研究

Study effects and mechanism of PHB on reactive oxygen species and apoptosis in cardiomyocytes under high glucose

目的:探讨抗增殖蛋白(PHB)对高糖诱导的H9C2心肌细胞凋亡的影响以及机制研究。方法:选取p CDNA3-PHB、p CDNA3-NC重组质粒转染和高糖干预心肌细胞,实验分为4组:正常对照组(Control组)、高糖刺激组(HG组)、高糖+空载体组(HG+p CDNA3-NC组)、高糖加p CDNA3-PHB重组质粒组(HG+p CDNA3-PHB);Western blot检测细胞转染后各组细胞中PHB蛋白、B细胞淋巴瘤/白血病-2相关X蛋白(Bax)/B细胞淋巴瘤/白血病-2(Bcl-2)、剪切的半胱氨酸的天冬氨酸蛋白水解酶-3(c-caspase3)、蛋白激酶B(AKt)、磷酸化蛋白激酶B(p-AKt)蛋白的表达量,二氢乙啶(DHE)染色法检测高糖环境下心肌细胞内的活性氧(ROS)水平,实时荧光定量PCR(qRT-PCR)检测细胞中TNF-αmRNA和IL-6 mRNA含量的测定,流式细胞仪检测细胞的凋亡率。结果:p CDNA3-PHB重组质粒可使PHB表达量增加;与Control组相比,HG组、HG+p CDNA3-NC组、HG+p CDNA3-PHB组细胞凋亡率显著增高(P<0.05);与HG组相比,HG+p CDNA3-NC组细胞凋亡率变化不显著(P>0.05);HG+p CDNA3-PHB组细胞凋亡率明显降低(P<0.05)。与Control组相比,HG组、HG+p CDNA3-NC组、HG+p CDNA3-PHB组细胞内ROS的水平、TNF-αmRNA、IL-6 mRNA、Bax/Bcl-2的相对表达量、c-caspase3(P<0.05)蛋白相对表达量显著增高;与HG组相比,HG+p CDNA3-NC组细胞内ROS的水平(P>0.05)、TNF-αmRNA(P<0.05)、IL-6 mRNA(P<0.05)、Bax/Bcl-2的相对表达量(P>0.05)、c-caspase3蛋白相对表达量(P>0.05)无明显差异;但p CDNA3-PHB组细胞中ROS的水平(P<0.05)、TNF-αmRNA(P<0.05)、IL-6 mRNA(P<0.05)、Bax/Bcl-2的相对表达量(P<0.05)、c-caspase3(P<0.05)蛋白相对表达量明显降低。经统计分析显示,各组细胞中AKt蛋白的相对表达量无显著差异(P>0.05)。经比较后,HG组、HG+p CDNA3-NC组、HG+p CDNA3-PHB组心肌细胞中p-AKt蛋白的相对表达量显著低于Control组(P<0.05);HG+p CDNA3-NC组心肌细胞中p-AKt蛋白的相对表达量与HG组间无显著差异(P>0.05);但HG+p CDNA3-PHB组心肌细胞中p-AKt蛋白的相对表达量明显高于HG组(P<0.05)。结论:PHB过表达可抑制高糖环境下H9C2心肌细胞的凋亡和炎症反应,主要是通过调控PI3K/Akt信号通路、ROS、Bax/Bcl-2、c-caspase3蛋白的水平,为糖尿病心肌病的治疗提供新的思路与方法。

Objective：To investigate the effect and mechanism of PHB on the apoptosis of H 9C2 cardiomyocytes under high glucose.Methods：Select pCDNA3-PHB and pCDNA3-NC recombinant plasmid transfect into cardiomyocytes and effected by high glu-cose.The experiment were divided into four groups：control group,high glucose group（HG group）,high glucose＋empty vector group （HG＋pCDNA3-NC group）,high glucose＋pCDNA3-PHB plasmid group（HG＋pCDNA3-PHB group）.The expression of PHB,Bax/Bcl-2,c-caspase3,AKt and p-AKt protein after transfected were detected by Western blot .ROS were detected under high glucose by DHE staining.The TNF-αmRNA and IL-6 mRNA were detected by Real-time quantitative PCR（qRT-PCR）.Flow cytometry was used to detect the apoptotic rate .Results：The recombinant plasmid pCDNA 3-PHB could increase the expression of PHB .The apoptotic rate of HG group,HG＋pCDNA3-NC group and HG＋pCDNA3-PHB group were significantly higher than control group （P〈0.05）.Compared with HG group,the apoptotic rate of HG＋pCDNA3-NC group was not significant（P〉0.05）,but that of HG＋pCDNA3-PHB group was significantly lower（P〈0.05）.The levels of ROS,TNF-αmRNA,IL-6 mRNA,Bax/Bcl-2 and c-caspase3（P〈0.05） protein in the HG group,HG＋pCDNA3-NC group,HG＋pCDNA3-PHB group were significantly higher than the control group .Compared with HG group , the level of ROS,TNF-αmRNA,IL-6 mRNA,Bax/Bcl-2 and c-caspase3 （P〉0.05）protein in the HG＋pCDNA3-NC group were no＆nbsp;significant difference,but the ROS,TNF-αmRNA,IL-6 mRNA、Bax/Bcl-2 and c-caspase3 （P〈0.05）protein in the HG＋pCDNA3-PHB group were significantly decreased .Statistical analysis showed that there was no significant difference of AKt protein in each group （P〉0.05）.The relative expression of p-AKt protein in HG group,HG＋pCDNA3-NC group and HG＋pCDNA3-PHB group were signif-icantly lower than that in control group （ P〈0.05 ） ,HG＋pCDNA3-NC group was no significantly different than HG group （ P〉0.05 ） , However ,HG＋pCDNA3-PHB group was significantly higher than HG group （ P〈0.05 ） .Conclusion： PHB overexpression can inhibit the apoptosis of H9C2 cardiomyocytes in high glucose environment ,andreduce the inflammatory response by effected the regulation of PI3K/Akt signaling pathway,reduced ROS,Bax/Bcl-2,c-caspase3 protein levels,that give us a new ideas and methods of DCM .