尼古丁促进高糖/高脂培养的大鼠心肌细胞系H9C2凋亡

目的研究在不同浓度尼古丁(Nic)条件下高糖/高脂(HGHL)对大鼠心肌细胞系H9C2凋亡的影响。方法体外培养H9C2细胞,给予不同浓度Nic(6×10^-8、6×10^-7、6×10^-6mol/L)2 h后,HGHL(33 mmol/L葡萄糖和500μmol/L棕榈酸酯)培养24 h,CCK8法检测细胞增殖;乳酸脱氢酶(LDH)试剂盒测定细胞活力;Western blot检测Bax、Bcl-2、cleaved-caspase3及caspase3的表达及ERK1/2、JNK和p38MAPK磷酸化水平;流式细胞计量术测定细胞凋亡和上清液ROS水平。结果与对照组相比,HGHL和尼古丁联合HGHL均显著抑制细胞活力,增加上清液中LDH水平(P<0.001);HGHL及Nic+HGHL细胞凋亡率显著增加(P<0.001)及cleaved-caspase3蛋白表达明显增加(P<0.01);Bcl-2/Bax蛋白表达显著下降(P<0.01);在Nic+HGHL组,ROS水平表达最显著(P<0.001);HGHL能激活MAPK磷酸化,Nic+HGHL组ERK1/2磷酸化水平显著升高(P<0.01);ROS清除剂(NAC)能显著减少caspase3蛋白表达水平(P<0.001)。结论尼古丁、HGHL能导致H9C2细胞损伤并增加其凋亡,其发生与ROS及MAPK信号通路密切相关。

Nicotine promotes apoptosis of rat cardiomyocyte cell line H9C2 cultured with high glucose/high fat

Objective To study the apoptosis and mechanism of nicotine(Nic) in H9 C2 cells under high glucose and high fat(HGHL) conditions. Methods H9 C2 cells were cultured with different concentrations of nicotine(6×10^-8 mol/L Nic, 6×10^-7 mol/L Nic, 6×10^-6 mol/L Nic) for 2 hours, and HGHL(33 mmol/L glucose and 500 μmol/L palmitate) was added for 24 hours, cell proliferation was detected by CCK8 assag, cell viability was measured by lactate dehydrogenase(LDH) kit.The expression of Bax, Bcl-2, cleaved-caspase3 and caspase3 and the phosphorylation levels of ERK1/2, JNK and p38 MAPK were detected by Western blot. The supernatant ROS level was determined by flow cytometry. Results Compared with normal controls, high glucose, high fat and nicotine combined with high glucose and high fat significantly inhibited cell viability and increased LDH level in the supernatant(P<0.001). The apoptosis rate of HGHL and Nic+HGHL cells was significantly increased(P<0.001), and the expression of cleaved-caspase3 protein was significantly increased(P<0.01). The expression of Bcl-2/Bax protein was significantly decreased(P<0.01). ROS level was most significantly expressed in the Nic+HGHL group(P<0.001). At the same time, HGHL activated MAPK phosphorylation level. ERK1/2 phosphorylation level was significantly increased in Nic+HGHL group(P<0.01). ROS scavenger(NAC) significantly reduced caspase3 protein expression(P<0.001). Conclusions Nicotine, high glucose and high fat can cause H9 C2 cell damage and increase apoptosis, which is closely related to the regulation of ROS and MAPK signaling pathway.