摘要
目的探讨甲酸(FA)染毒对PC12细胞的损伤效应,阐明细胞自噬在其中的关键作用及机制。方法采用CCK-8法检测并确定自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3-MA)、自噬溶酶体抑制剂巴弗洛霉素A1(Bafilomycin A1,BA)染毒PC12细胞的浓度。将PC12细胞分为对照组(0 mmol/L)、3-MA组(1.25 mmol/L)、BA组(1 nmol/L)、FA组(16.64mmol/L)、FA+3-MA组(16.64 mmol/L+1.25 mmol/L)、FA+BA组(16.64 mmol/L+1 nmol/L),染毒24 h。采用3-MA、BA调控自噬水平后,用透射电镜观察各组凋亡及自噬情况;采用单丹磺酰尸胺(MDC)染色检测PC12细胞自噬情况;采用Western blotting法检测自噬相关蛋白LC3Ⅱ/LC3Ⅰ、Beclin1、P62的表达;采用流式细胞仪检测各组PC12细胞的凋亡情况。结果透射电镜观察结果显示,对照组、3-MA组、BA组可见大量的正常线粒体,核膜光滑,染色质均匀;FA组线粒体嵴溶解、断裂、消失,部分存在空泡,自噬现象明显;FA+3-MA组大量线粒体空泡,线粒体呈局灶性,染色质聚集,溶酶体增多,细胞核固缩明显,少见自噬小体形成;FA+BA组大量线粒体空泡,线粒体嵴溶解、断裂甚至消失,染色质边集明显,溶酶体增多,但未形成自噬溶酶体。MDC染色结果显示,对照组、3-MA组、BA组可见极少量的蓝色荧光,FA组、FA+3-MA组、FA+BA组可见大量的蓝色荧光。Western blot检测法结果显示,与对照组相比,FA组LC3Ⅱ/LC3Ⅰ、Beclin1蛋白表达量增加,P62蛋白表达量降低,差异有统计学意义(P<0.05);FA+3-MA组LC3Ⅱ/LC3Ⅰ、Beclin1蛋白表达量与FA组相比明显降低,P62蛋白表达量明显增加,差异有统计学意义(P<0.01);FA+BA组的LC3Ⅱ/LC3Ⅰ、P62蛋白表达量高于FA组,Beclin1蛋白表达量明显降低,差异有统计学意义(P<0.01)。流式细胞仪检测结果显示,BA组、3-MA组的细胞早期凋亡率、晚期凋亡率及总凋亡率与对照组均无明显差异(P>0.05);FA组3种类型的凋亡率均高于对照组,差异有统计学意义(P<0.05);与FA�
Objective Reveal the damage effect of formic acid on PC12 cells,and clarify the key role and mechanism of cell autophagy in it.Methods CCK-8 method was used to detect the concentration of 3-MA and BA-exposed PC12 cells.Relevant indicators were detected after 3-MA and BA were used to control autophagy levels.Transmission electron microscope was used to observe the apoptosis and autophagy of each group.The autophagy of PC12 cells was detected by MDC staining.Western blotting was used to detect the expression of LC3Ⅰ,Beclin1 and P62.Flow cytometry was used to detect the apoptosis level of PC12 cells in each group.Results Transmission electron microscopy observations showed that a large number of normal mitochondria were seen in the control,3-MA,and BA groups,with a smooth nuclear membrane and chromatin.Uniform;FA group mitochondrial crest dissolves,breaks,disappears,some vacuoles exist,and autophagy is obvious;FA+3-MA group has a large number of mitochondrial vacuoles,mitochondria are focal,chromatin aggregates,lysosomes increase,and cell nuclei shrink Obviously,the formation of autophagosomes was rare;there were a large number of mitochondrial vacuoles in the FA+BA group,and the mitochondria were dissolved and even disappeared.The chromatin edge set was obvious and the lysosomes increased,but no autophagolysosomes were formed.MDC staining results showed that a small amount of blue fluorescence was seen in the control group,3-MA group,and BA group,while FA group(16.64 mmol/L),FA+3-MA group(16.64 mmol/L+1.25 mmol/L),and FA+BA group(16.64 mmol/L+1 nmol/L)can see a lot of blue fluorescence.Western blot results showed that compared with the control group,the expression levels of LC3Ⅱ/LC3Ⅰand Beclin1 proteins in the FA group were increased,while the expression levels of P62 protein were significantly reduced.The ratio was significantly reduced,and the expression of P62 protein was significantly increased.The expression of LC3Ⅱ/LC3Ⅰ,P62 protein in the FA+BA group was significantly higher than that in the FA grou
作者
刘贺荣
郝乐乐
严姣
李亚妮
陈楠
LIU He-rong;HAO Le-le;YAN Jiao;LI Ya-ni;CHEN Nan(School of Public Health,Ningxia Medical University,Yinchuan,Ningxia 750004,China;不详)
出处
《环境与健康杂志》
CAS
2024年第2期95-99,92,共6页
Journal of Environment and Health
基金
国家自然科学基金(81560522)
关键词
甲酸
PC12细胞
凋亡
自噬
Formic acid
PC12 cells
Apoptosis
Autophagy
