甲酸诱导细胞凋亡与自噬的实验研究

Experimental Study on Apoptosis and Autophagy Induced by Formic Acid

目的通过实验研究凋亡及自噬相关因子表达变化,探讨甲酸对细胞凋亡及自噬的影响,为甲酸诱导凋亡及自噬的发生机制提供实验依据。方法取对数生长期的PC12细胞,采用CCK-8法检测染毒不同浓度(0、8.32、10.40、12.48、14.56、16.64、18.72 mmol·L^-1)、不同时间(0、6、12、24、36、48 h)对细胞存活率的影响,确定后续实验染毒浓度为0、8.32、12.48、16.64 mmol·L^-1,染毒时间为24 h。采用吖啶橙溴乙锭(AO/EB)染色初步观察PC12细胞凋亡情况;采用Annexin V-FITC/PI双染定量检测不同浓度的甲酸染毒PC12细胞的凋亡率;采用MDC染色法观察甲酸诱导PC12细胞自噬的情况;采用Western blot技术检测自噬相关蛋白P62、Beclin1、LC3的表达水平。结果确定甲酸的染毒浓度0、8.32、12.48、16.64 mmol·L^-1为后续实验浓度,染毒时间为24 h。细胞经AO/EB双染后,对照组绝大部分着绿色,有极少量凋亡的细胞;随着甲酸浓度的增加,晚期凋亡细胞均增多,细胞染色呈桔红色,细胞的体积逐渐变小;不同浓度的甲酸染毒PC12细胞,凋亡率随着甲酸浓度的增加而增加(P均<0.01)。MDC染色后对照组可见极少量的蓝色荧光,随着甲酸浓度的增高,蓝色荧光增多(P均<0.05)。自噬相关蛋白LC3Ⅱ/LC3Ⅰ、Beclin-1相对表达量呈浓度依赖性,而P62蛋白相对表达量逐渐降低(P均<0.01)。结论甲酸能够诱导PC12细胞死亡,可能与干扰自噬与凋亡过程有关。

Objective To explore the effect of formic acid on apoptosis and autophagy by studying the changes of apoptosis and autophagy related factors,so as to provide experimental basis for the mechanism of apoptosis and autophagy induced by formic acid.Methods PC12 cells in logarithmic growth phase were collected and the effects of different concentrations(0,8.32,10.40,12.48,14.56,16.64,18.72 mmol·L^-1)and different time(0,6,12,24,36,48 h)on cell survival rate were detected by CCK8 method.The following experimental exposure concentrations were 0(control group),8.32,12.48,16.64 mmol·L^-1,and the exposure time was 24 h.The apoptosis of PC12 cells was observed by AO/EB staining;the apoptosis rate of PC12 cells exposed to different concentrations of formic acid was detected by annexin V-FITC/PI double staining;the autophagy of PC12 cells induced by formic acid was observed by MDC staining;the expression levels of autophagy related proteins P62,Beclin1 and LC3 were detected by Western blot.Results The concentrations of formic acid 0,8.32,12.48 and 16.64 mmol·L^-1 were determined as the follow-up experimental concentrations,and the exposure time was 24 hours.After AO/EB double staining,most of the cells in the control group were green with a very small number of apoptotic cells,with the increase of formic acid concentration,the late apoptotic cells increased,the cell staining was orange,and the cell volume gradually decreased,and the apoptosis rate of PC12 cells exposed to different concentrations of formic acid increased with the increase of formic acid concentration(P all<0.01).After MDC staining,a very small amount of blue fluorescence was observed in the control group,and the difference was statistically significant with the increase of formic acid concentration(P all<0.05).The relative expression of autophagy-related proteins LC3ⅡPC3Ⅰand Beclin-1 increased in a concentration-dependent manner,while the relative expression of P62 protein decreased gradually,the difference was statistically significant.Conclusion Formic acid can induce the death of PC12 cells,and the mechanism may be by interfering with the process of autophagy and apoptosis.