摘要
目的探讨lncRNA H19(H19)/Sirt3介导自噬在脑出血(ICH)后损伤中的作用机制。方法通过胶原酶注射建立ICH小鼠模型。将经氧合血红蛋白(OxyHb)处理的SH⁃SY5Y细胞用作体外ICH模型。含有sh⁃H19的慢病毒载体用于抑制ICH小鼠和培养的SH⁃SY5Y细胞中的H19表达。在ICH模型建立后48 h,对小鼠神经功能缺损进行mNSS评分,并对病灶体积进行分析,同时分别采用RT⁃qPCR法、Tunel染色和免疫荧光分析损伤组织中H19表达、神经元细胞凋亡和LC3表达。分别采用MTT法测量细胞活力、Tunel染色检测细胞凋亡以及Western blot分析自噬相关蛋白和Sirt3/AMPK/mTOR信号通路蛋白表达情况。结果H19表达在ICH后和OxyHb处理细胞中以时间依赖性方式增强(P<0.05)。H19敲低降低了ICH模型建立后第2天病灶体积和mNSS评分(P<0.01),减少神经元细胞凋亡(P<0.01),并增强了ICH损伤期间自噬的调节作用。H19敲低增强了暴露于OxyHb的SH⁃SY5Y细胞活力,减少了细胞凋亡,促进细胞自噬。H19主要通过调节Sirt3表达和激活AMPK⁃mTOR信号通路促进自噬。结论H19在体内外ICH模型中以时间依赖性方式增强。H19敲低可能通过Sirt3/AMPK/mTOR信号通路诱导自噬减轻ICH损伤。
Objective To explore the mechanism of action of lncRNA H19(H19)/Sirt3 mediated autoph⁃agy in injury after intracerebral hemorrhage(ICH).Methods Amouse model of ICH was established by injections with collagenase.SH⁃SY5Y cells treated with oxyhemoglobin(OxyHb)were used as an in vitro ICH model.Alenti⁃viral vector containing sh⁃H19 was used to inhibit H19 expressions in ICH mice and cultured SH⁃SY5Y cells.48 hours after establishment of the ICH model,the mouse neurologic deficits were analyzed by mNSS score and lesion volume.RT⁃qPCR,Tunel staining and immunofluorescence were used to analyze the expression of H19,neuronal apoptosis and LC3 express in the injured tissues respectively.Cell viability was measured by MTT assay,apoptosis was detected by Tunel staining,and expressions of autophagy⁃related proteins and Sirt3/AMPK/mTOR signaling pathway were analyzed by Western blot.Results The expression of H19 was increased after ICH and in OxyHb⁃treated cells in a time⁃dependent manner(P<0.05).H19 knockdown reduced the lesion volume and mNSS score on the second day after establishment of the ICH model(P<0.01),decreased neuronal apoptosis(P<0.01),and enhanced regulation of autophagy during ICH injury.H19 knockdown enhanced the viability of SH⁃SY5Y cells exposed to OxyHb,reduced apoptosis,and accelerated autophagy.H19 accelerated autophagy mainly through regu⁃lating Sirt3 expression and activating AMPK⁃mTOR pathway.Conclusions H19 is enhanced in a time⁃dependent manner in ICH models in vivo and in vitro.H19 knockdown relieves ICH damage via autophagy induced by Sirt3/AMPK/mTOR signaling pathway.
作者
袁敏
王素洁
李茜
张志月
YUAN Min;WANG Sujie;LI Qian;ZHANG Zhiyue(Tangshan Workers Hospital(Tangshan Clinical Medical College Affiliated to Hebei Medical University),Tangshan 063000,China)
出处
《实用医学杂志》
CAS
北大核心
2022年第10期1213-1219,共7页
The Journal of Practical Medicine
基金
河北省卫生健康委2019年度医学科研立项项目(编号:20190052)。