自噬在皮质酮致HT-22细胞损伤中的作用

Role of Autophagy in Corticosterone-Induced Neurotoxicity in HT-22 Cells

主要探讨自噬在皮质酮(CORT)诱导小鼠海马神经元HT-22细胞损伤中的作用。以不同浓度CORT(0,50,100,200μmol·L^-1)处理HT-22细胞24 h或200μmol·L^-1 CORT处理HT-22细胞不同时间(0,4,8,12,24,48 h)后,镜下观察细胞形态,MTS法检测细胞活力,Western blot检测自噬标志物LC3及p62蛋白表达;同时进一步考察自噬诱导剂Rapamycin和自噬抑制剂3-Methyladenine(3-MA)对CORT神经毒性的影响。结果发现CORT可呈剂量和时间依赖性地降低HT-22细胞活力并诱导细胞发生明显的形态学变化。与对照组相比,随着CORT浓度的增加,自噬体形成标志蛋白LC3Ⅱ/LC3Ⅰ的比值显著升高,自噬底物蛋白p62的表达并无明显变化。然而,200μmol·L^-1 CORT作用于HT-22细胞不同时间后,LC3Ⅱ/LC3Ⅰ比值均升高,在48 h时最为显著(P<0.01),同时p62的表达呈现先增加后逐步减少趋势。此外,与CORT组比,诱导自噬加重了CORT对HT-22细胞的损伤,而抑制自噬则减轻了CORT的细胞毒性。研究表明皮质酮增加了HT-22细胞的自噬水平,自噬介导了皮质酮诱导的HT-22细胞损伤,抑制自噬可以减轻皮质酮的神经毒性。

This article was prompted to evaluate whether autophagy was involved in corticosterone(CORT)-induced HT-22 cells injury.HT-22 cells were treated with different concentrations of CORT(0,50,100,200μmol·L^-1)for 24 h or 200μmol·L^-1 CORT for different time(0,4,8,12,24,48 h)respectively.The cell morphologies were observed by inverted microscope,the cell viabilities were detected by MTS assay and the expressions of autophagy markers LC3 and p62 were detected by Western blot.The effects of autophagy on CORT-induced neurotoxicity were also determined after 3-Methyladenine(3-MA)and Rapamycin treatment.CORT had a dose and time dependent effect on cell viabilities and induced obvious morphological changes in cells.Compared with the control group,the ratio of LC3II/LC3I was significantly increased with increasing concentrations of CORT,while p62 expression did not show any significant changes.Additionally,with increasing exposure time of 200μmol·L^-1 CORT,the ratio of LC3II/LC3I was also significantly increased with the highest level at 48 h after treatment(P<0.01),while p62 expression was increased at 4,8,12 h and then gradually decreased at 24 and 48 h.Moreover,co-treatment with autophagy inducer enhanced the neurotoxicity of CORT and co-treatment with autophagy inhibitor allievated the neurotoxicity of CORT.These results indicated that CORT increased the level of autophagy in HT-22 cells and autophagy mediated the CORT-induced cytotoxicity.Inhibition of autophagy could reduce the neurotoxicity of CORT.