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目的探讨通心络(Tongxinluo,TXL)对同型半胱氨酸(homocysteine,Hcy)诱导大鼠心肌微血管内皮细胞内质网应激(endoplasmic reticulum stress,ERS)致细胞凋亡的影响及其机制。方法采用植块法原代培养大鼠心肌微血管内皮细胞,倒置显微镜观察细胞形态并通过CD31免疫荧光法对培养的大鼠心肌微血管内皮细胞进行辨别、鉴定。取2~4代生长良好的大鼠心肌微血管内皮细胞,采用免疫荧光法检测葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)表达以确定Hcy最佳作用时间。后续实验分为5组:空白对照组、Hcy诱导组(Hcy 10 mmol/L,10 h)、Hcy+TXL组(Hcy 10 mmol/L+TXL 400μg/m L)、Hcy+LY294002组(Hcy 10 mmol/L+LY294002 5μmol/L,LY294002为PI3K抑制剂)及Hcy+LY294002+TXL组(Hcy 10 mmol/L+LY294002 5μmol/L+TXL 400μg/m L)。采用流式细胞仪检测大鼠心肌微血管内皮细胞凋亡率,实时荧光定量反转录PCR(Real-Time Reverse Transcription PCR,RT-PCR)及Western blot法检测GRP78、C/EBP同源蛋白(C/EBP homologous protein,CHOP)及天冬氨酸特异性半胱氨酸蛋白酶-12(cysteinyl aspartate specific proteinase-12,Caspase-12)mRNA及蛋白表达,Western blot法检测磷酸化磷脂酰肌醇3-激酶(phosphorylation of phosphatidylinositol 3-kinase,P-PI3K)、总磷脂酰肌醇3-激酶(total phosphatidylinositol 3-kinase,T-PI3K)、磷酸化蛋白激酶B(phosphorylation of kinase B,P-Akt)及总的蛋白激酶B(total kinase B,T-Akt)蛋白表达。结果确定Hcy作用时间为10 h。与空白对照组比较,Hcy诱导组细胞凋亡率(22.77%)升高,GRP78、CHOP和Caspase-12 mRNA及蛋白表达升高,P-PI3K和P-Akt蛋白表达降低,PPI3K/T-PI3K及P-Akt/T-Akt降低(P<0.05,P<0.01);与Hcy诱导组比较,Hcy+TXL组细胞凋亡率(10.17%)降低,GRP78、CHOP和Caspase-12 mRNA及蛋白表达降低,P-PI3K和P-Akt蛋白表达升高,PPI3K/T-PI3K及P-Akt/T-Akt升高(P<0.05,P<0.01);与Hcy+TXL组比较,Hcy+TXL+LY294002组细胞凋亡率(17.9%)升高,GRP78、CHOP和Caspase-12 mRNA及蛋白表达升高,P-PI3K和P-Akt蛋白表达降低,P-PI3K/T-PI3K及P-Akt/T-Akt降低(P<0.05,P<0.01)。结论通心络可抑制Hcy诱导的ERS致大鼠心肌微血管内皮细胞凋亡,其机制可能与激活PI3K/Akt信号通路有关。
Objective To investigate the effect and mechanism of Tongxinluo (TXL) on apoptosis induced by homocysteine (Hcy) in endoplasmic reticulum stress (ERS) of rat cardiac microvascular endothelial cells. Methods Primary cultured rat cardiac microvascular endothelial cells were cultured by explant method. The morphology of the cells was observed with inverted microscope. The cultured rat myocardial microvascular endothelial cells were identified by CD31 immunofluorescence. Take 2 to 4 generations of well-developed rat myocardial microvascular endothelial cells, the expression of glucose regulated protein 78 (GRP78) was detected by immunofluorescence to determine the best Hcy time. The follow-up experiments were divided into five groups: blank control group, Hcy induction group (Hcy 10 mmol / L, 10 h), Hcy + TXL group (Hcy 10 mmol / L + TXL 400 μg / m L), Hcy + LY294002 group LY294002 5μmol / L, LY294002 as PI3K inhibitor) and Hcy + LY294002 + TXL group (Hcy 10 mmol / L + LY294002 5μmol / L + TXL 400μg / ml). The apoptosis rate of myocardial microvascular endothelial cells was detected by flow cytometry. The expression of GRP78, C / EBP homologous protein (C) was detected by Real-Time Reverse Transcription PCR (RT-PCR) and Western blot / EBP homologous protein (CHOP) and caspase-12 (Caspase-12) mRNA and protein expression were detected by Western blot. Phosphatidylinositol 3- Phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T-PI3K), phosphorylation of kinase B (P-Akt) Total protein kinase B (T-Akt) protein expression. The results confirmed that the Hcy action time was 10 h. Compared with the blank control group, the apoptosis rate (22.77%) of Hcy induced group increased, the mRNA and protein expression of GRP78, CHOP and Caspase-12 increased, the expressions of P-PI3K and P-Akt decreased, while PPI3K / T-PI3K And P-Akt / T-Akt (P <0.05, P <0.01). Compared with Hcy induction group, the apoptosis rate of Hcy + TXL group (10.17%) and the expression of GRP78, CHOP and Caspase-12 mRNA and protein (P <0.05, P <0.01). Compared with Hcy + TXL group, the expression of Hcy + TXL + P-Akt increased significantly The apoptosis rate of LY294002 group (17.9%) was increased, the mRNA and protein expressions of GRP78, CHOP and Caspase-12 increased, the expressions of P-PI3K and P-Akt decreased, T-Akt decreased (P <0.05, P <0.01). Conclusion Tongxinluo can inhibit Hcy-induced ERS-induced myocardial microvascular endothelial cell apoptosis, which may be related to the activation of PI3K / Akt signaling pathway.