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目的:探讨乳酸堆积和二氯乙酸钠(DCA)对肝癌细胞(HepG2)凋亡和bax、bcl-2表达及caspase-3活性的影响。方法:通过体外培养HepG2,建立稳定的体外培养模型,配制成终浓度分别为0 mmol/L、1.0 mmol/L、2.0 mmol/L、4.0 mmol/L、8.0 mmol/L的乳酸培养液以及在不同浓度乳酸组中加入终浓度为10-3mmol/L DCA培养液与HepG2共同培养,其中以0 mmol/L乳酸组为对照组。采用MTT法检测乳酸对HepG2的抑制率,流式细胞仪检测乳酸和DCA对HepG2的凋亡百分率,用Real-time PCR法测定bax及bcl-2 mRNA的表达,用免疫荧光法检测caspase-3的活性。结果:乳酸对HepG2的IC50值为13.6 mol/L,与对照组比较,随着乳酸浓度的增加,HepG2凋亡率增加,bax mRNA表达升高,bcl-2 mRNA的表达降低,caspase-3活性增加,其中1.0 mmol/L乳酸组与对照组比较(P>0.05),2.0 mmol/L,4.0 mmol/L和8.0 mmol/L乳酸组与对照组比较差异有统计学意义(P<0.05)。加入DCA后,HepG2凋亡减少,2.0 mmol/L乳酸+DCA组、4.0 mmol/L乳酸+DCA组、8.0 mmol/L乳酸+DCA组与同浓度的乳酸组比较,bax mRNA表达减少(P<0.05),bcl-2 mRNA表达增加(P<0.05),caspase-3活性减低(P<0.05)。结论:乳酸可诱导HepG2凋亡,且随着乳酸浓度的增高,HepG2的凋亡率增加,其机制可能是通过对bcl-2及bax mRNA表达的改变以及激活caspase-3活性而实现,DCA可以降低HepG2凋亡,对乳酸堆积造成的HepG2凋亡有抑制作用。
Objective: To investigate the effects of lactic acid accumulation and sodium dichloroacetate (DCA) on apoptosis and the expression of bax, bcl-2 and caspase-3 in HepG2 cells. Methods: HepG2 cells were cultured in vitro and stably cultured in vitro. The final concentration of HepG2 was 0 mmol / L, 1.0 mmol / L, 2.0 mmol / L, 4.0 mmol / L and 8.0 mmol / L of lactic acid medium, Different concentrations of lactate group were added to a final concentration of 10-3mmol / L DCA culture medium and HepG2 co-culture, with 0 mmol / L lactic acid group as the control group. The inhibitory rate of lactic acid on HepG2 was detected by MTT assay. The percentage of apoptosis of HepG2 by lactic acid and DCA was determined by flow cytometry. The expression of bax and bcl-2 mRNA was detected by Real-time PCR. The expressions of caspase-3 Activity. Results: The IC50 of lactic acid to HepG2 was 13.6 mol / L. Compared with the control group, the apoptotic rate of HepG2 increased, the expression of bax mRNA increased, the expression of bcl-2 mRNA decreased, the activity of caspase-3 (P <0.05). Compared with the control group, the concentration of 1.0 mmol / L lactic acid group was significantly higher than that of the control group (P> 0.05). The difference was statistically significant (P <0.05) between the 2.0 mmol / L, 4.0 mmol / L and 8.0 mmol / L lactic acid groups. After adding DCA, the apoptosis of HepG2 decreased, the expression of bax mRNA decreased in 2.0 mmol / L lactate + DCA group, 4.0 mmol / L lactic acid + DCA group and 8.0 mmol / L lactic acid + DCA group compared with the same concentration of lactic acid group (P < 0.05). The bcl-2 mRNA expression increased (P <0.05) and caspase-3 activity decreased (P <0.05). CONCLUSION: Lactic acid can induce the apoptosis of HepG2 cells. With the increase of lactate concentration, the apoptosis rate of HepG2 increases. The mechanism may be through the change of bcl-2 and bax mRNA expression and the activation of caspase-3 activity. DCA can Reduce HepG2 apoptosis, and inhibit HepG2 apoptosis caused by lactic acid accumulation.