摘要
探究参芎葡萄糖注射液拮抗H2O2诱导H9c2细胞凋亡的机制。体外培养H9c2细胞,预先给予体积分数1. 7%,3. 4%,6. 8%的参芎葡萄糖注射液处理H9c2细胞,再给予H2O2诱导细胞凋亡。MTS法检测细胞存活率,AO/EB荧光染色观察细胞凋亡的形态学变化,Annexin/PI法检测细胞凋亡率,基因芯片技术检测细胞表达谱的变化,荧光定量PCR技术(qRTPCR)检测PIK3CA,Bcl-2,Bax,caspase-3,GAPDH mRNA的表达,Western blot法检测PIK3CA,AKT,P-AKT,Bcl-2,Bax,caspase-3蛋白的表达水平,用试剂盒检测细胞乳酸脱氢酶(LDH)漏出量和丙二醛(MDA)含量。结果显示,不同浓度的参芎葡萄糖注射液可显著提高H2O2诱导的H9c2细胞存活率(P<0. 01),明显逆转H2O2诱导的细胞凋亡情况(P<0. 01)。芯片实验发现,参芎葡萄糖注射液处理后,H9c2细胞有138个基因表达发生改变,其中与PI3K/AKT通路相关的PIK3CA差异表达倍数较高,为3. 59倍。qRT-PCR和Western blot结果表明,参芎葡萄糖注射液可下调H2O2处理后H9c2细胞中caspase-3的mRNA和蛋白表达水平(P<0. 01),上调PIK3CA和Bcl-2的mRNA和蛋白的表达水平(P<0. 01),并上调AKT蛋白的磷酸化水平(P<0. 01)。在PI3K/AKT通路抑制剂LY294002作用下,参芎葡萄糖注射液拮抗H2O2细胞损伤作用显著降低。结果表明,参芎葡萄糖注射液可能是通过调控PI3K/AKT信号通路来抑制H2O2诱导的H9c2细胞凋亡。
The aim of this paper was to explore the mechanism of Shenxiong Glucose Injection antagonizing apoptosis of H9 c2 cells induced by H2O2. H9 c2 cells were pretreated with 1. 7%,3. 4% and 6. 8% Shenxiong Glucose Injection,and then H2O2 was introduced to induce apoptosis in vitro. Cell viability was detected by MTS assay,morphological changes of apoptosis were observed by AO/EB fluorescence staining,apoptosis rate was detected by Annexin/PI method,cell expression profile was detected by gene chip technology,the mRNA of PIK3 CA,Bcl-2,Bax,caspase-3 and GAPDH were detected by qRT-PCR,the protein expression levels of PIK3 CA,AKT,P-AKT,Bcl-2,Bax and caspase-3 were detected by Western blot,and the contents of LDH and MDA were detected by kit. The results showed that Shenxiong Glucose Injection of different concentrations significantly increased the viability of H9 c2 cells treated with H2O2( P<0. 01),and reversed H2O2-induced apoptosis( P< 0. 01). The microarray experiments showed that 138 genes were altered in H9 c2 cells after treatment with Shenxiong Glucose Injection. The differential expression fold of PIK3 CA associated with PI3 K/AKT pathway was 3. 59. The results of qRT-PCR and Western blot showed that Shenxiong Glucose Injection could down-regulate the mRNA and protein expression levels of caspase-3( P<0. 01),up-regulate the mRNA and protein expression level of PIK3 CA and Bcl-2( P<0. 01),and up-regulate the phosphorylation levels of AKT( P<0. 01) in H2O2-treated H9 c2 cells. The protective effect of Shenxiong Glucose Injection on H2O2 cells injury was significantly inhibited by LY294002,a PI3 K/AKT pathway inhibitor. The results suggested that Shenxiong Glucose Injection may inhibit H2O2-induced H9 c2 cells apoptosis by regulating PI3 K/AKT signaling pathway.
作者
陆定艳
李靖
孙佳
薛维娜
何彬
李勇军
王永林
林昌虎
刘亭
LU Ding-yan;LI Jing;SUN Jia;XUE Wei-na;HE Bin;LI Yong-jun;WANG Yong-lin;LIN Chang-hu;LIU Ting(Guizhou Provincial Key Laboratory of Pharmaceutics/State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,China;Engineering Research Center for the Development and Application of Ethnic Medicine and Traditional Chinese Medicine(Ministry of Education),Guizhou Medical University,Guiyang 550004,China;School of Medicine and Health Management,Guizhou Medical University,Guiyang 550025,China;School of Pharmacy,Guizhou Medical University,Guiyang 550025,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2019年第17期3773-3779,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81760699,81803827)
贵州省科技计划项目(黔科合基础[2019]1280号)
贵州省科学技术厅人才团队项目(黔科合平台人才[2016]5613\5677)
中央引导地方科技专项项目(黔科中引地[2018]4006)
