硫化氢对Aβ25-35诱导的小鼠Neuro-2a细胞自噬的影响

目的 探讨硫化氢（H2S）对Aβ25-35诱导的小鼠成神经细胞瘤细胞Neuro-2a自噬的影响及其潜在机制。方法 将Neuro-2a细胞随机分为空白对照组、Aβ25-35处理组、Aβ25-35+NaHS组、Aβ25-35+3-甲基腺嘌呤（3-MA）组、NaHS组及Aβ25-35+NaHS+LY294002组。Aβ25-35+NaHS组和Aβ25-35+3-MA组细胞分别用NaHS和3-MA预处理2 h后均加用Aβ25-35继续处理24 h；Aβ25-35+NaHS+LY294002组除加入NaHS预处理外需在Aβ25-35处理前0.5 h加入特异性PI3K/Akt通路抑制剂LY294002。采用MTT法观察各组细胞存活率，蛋白质印迹法检测自噬蛋白Beclin-1、微管相关蛋白（LC3）及P62的表达，免疫荧光法检测LC3的表达及分布，透射电镜法观察自噬体的形成。结果 （1）与空白对照组相比，Aβ25-35作用细胞后细胞存活率降低（P<0.05），Beclin-1及LC3-Ⅱ表达增加，P62表达降低（P<0.05），荧光显微镜及电镜下可见细胞自噬体明显增多。（2）与Aβ25-35组相比，Aβ25-35+3-MA组和Aβ25-35+NaHS组细胞存活率均增高（P<0.05），Beclin-1及LC3-Ⅱ表达降低，P62表达增高（P<0.05），自噬体减少。（3）Aβ25-35+NaHS组p-Akt和p-mTOR的表达高于Aβ25-35组（P<0.05），而加入LY294002后，Aβ25-35+NaHS+LY294002组细胞p-Akt和p-mTOR表达与Aβ25-35+NaHS组相比降低（P<0.05）。结论 外源性H2S能对抗Aβ25-35的细胞毒性，可能与活化PI3K/Akt/mTOR途径抑制Aβ25-35引起的细胞自噬相关。
Objective To investigate the effect of exogenous hydrogen sulfide on Aβ25-35-induced autophagy in mouse Neuro-2a cells and the underlying mechanism. Methods The Neuro-2a cells were randomly divided into control group, Aβ25-35 treatment group, Aβ25-35+NaHS group, Aβ25-35+3-MA (3-methyl adenine) group, NaHS group and Aβ25-35+NaHS+LY294002 group. In Aβ25-35 + NaHS group and Aβ25-35+3-MA group, NaHS and 3-MA were used for pretreatment for 2 h, which was followed by 24 h co-incubation with Aβ25-35; in Aβ25-35+NaHS+LY294002 group, after pretreatment with NaHS, LY294002 was administered for 0.5 h before Aβ25-35 was given. MTT assay was used to detect the viability of Neuro-2a cells. The expression of the autophagy related proteins including Beclin-1, LC3 and P62 was measured by Western blotting analysis. Immunofluorescence was used to detect the expression and distribution of LC3. The formation of autophagosomes was determined by transmission electron microscopy (TEM). Results (1) Compared with control group, Aβ25-35 group had significantly declined cell viability (P<0.05), significantly increased expression of Beclin-1 and LC3-Ⅱ and decreased P62 expression (P<0.05), accompanied by increased autophagsomes under fluorescence microscope and electron microscope. (2) Compared with Aβ25-35 group, the cell viability was significantly increased in Aβ25-35+3-MA and Aβ25-35+NaHS groups (P<0.05), and expression of Beclin-1 and LC3-Ⅱ was decreased and expression of P62 was increased (P<0.05), accompanied by reduced autophagosomes. (3) The expression of p-Akt and p-mTOR in Aβ25-35+NaHS group was significantly higher than that in Aβ25-35 group (P<0.05); however, the expression of p-Akt and p-mTOR was significantly reduced after given the specific PI3K/Akt pathway