长链非编码RNAZEB1-AS1通过HMGB1/TLR-4信号轴加剧大鼠脑缺血/再灌注损伤

Long noncoding RNA ZEB1-AS1 aggravates cerebral ischemia/reperfusion injury in rats through the HMGB1/TLR-4 signaling axis

目的探讨长链非编码RNA ZEB1-AS1在脑缺血/再灌注损伤(CI/RI)中的作用及其机制。方法通过脑中动脉闭塞构建雄性SD大鼠局灶性CI/RI模型,实时定量PCR和Western blot分别检测ZEB1-AS1和HMGB1的时序表达并确定了CI/RI最佳时间(2 h/24 h)。将大鼠分为Sham组、CI/RI组、CI/RI+si-NC组、CI/RI+si-ZEB1-AS1组、CI/RI+OE-NC组、CI/RI+OE-ZEB1-AS1组。向CI/RI大鼠侧脑室注射ZEB1-AS1沉默/过表达载体及对照载体后,通过神经功能缺损评分和转盘实验分析认知功能;干湿重法分析脑水含量;Western blot检测白蛋白水平以评价血脑屏障完整性;ELISA法分析脑脊液和血清IL-1β和TNF-α水平。分别用FJC和TUNEL法检测CI/RI大鼠皮层中神经元丢失和细胞凋亡情况,Western blot分析HMGB1和TLR-4水平。此外,体外建立SH-SY5Y细胞氧-糖剥夺/复氧模型来模拟缺血/再灌注微环境,TUNEL法、Hoechst 33258染色、流式细胞术检测ZEB1-AS1沉默对神经元凋亡的影响;Western blot分析HMGB1和TLR-4水平。结果qPCR和Western blot结果显示,ZEB1-AS1和HMGB1在CI/RI大鼠脑组织中的表达随着再灌注时间的延长升高(24 h达到峰值),而后逐渐降低。神经功能缺损评分和转盘试验结果显示,与OE-NC组相比,ZEB1-AS1过表达加重了CI/RI大鼠认知功能受损(P<0.05);而si-ZEB1-AS1组大鼠的认知功能则好于si-NC组(P<0.01)。OE-ZEB1-AS1组大鼠的脑水含量高于OE-NC组(P<0.05);而与si-NC组相比,si-ZEB1-AS1组大鼠的脑水含量减少(P<0.01)。血脑屏障渗漏检测数据提示,si-ZEB1-AS1组大鼠脑脊液中白蛋白的含量低于si-NC组(P<0.01);OE-ZEB1-AS1组大鼠脑脊液中的白蛋白含量则高于OE-NC组(P<0.05)。ELISA结果显示,si-ZEB1-AS1组大鼠脑脊液和血清IL-1β和TNF-α水平低于si-NC组(P<0.01);而OE-ZEB1-AS1组大鼠脑脊液和血清IL-1β和TNF-α含量高于OE-NC组(P<0.01)。OE-ZEB1-AS1组大鼠皮层FJC阳性神经元数量高于OE-NC组(P<0.01);与此相反,si-NC组相比,ZEB1-AS1敲减则降低了FJC阳性神经元数量(P<0.01)。Western blot结果显示,与OE-NC组相比,ZEB1-AS1过表达促进了HMGB1和TLR-4水平(P均<0.01);而ZEB1-AS1敲减则产生相反的结果(P<0.01)。细胞水平上,与si-NC组相比,ZEB1-AS1敲减降低了TUNEL阳性细胞数(P<0.01);流式细胞术检测结果进一步证实该现象。此外,Western blot结果进一步证实,ZEB1-AS1敲减下调了HMGB1和TLR-4水平(P均<0.01)。结论长链非编码RNA ZEB1-AS1通过HMGB1/TLR-4信号轴加剧脑缺血/再灌注损伤。

Objective To investigate the role of long non-coding RNA ZEB1-AS1 in cerebral ischemia/reperfusion injury(CI/RI).Methods We detected the temporal changes of ZEB1-AS1 and HMGB1 expression using qPCR and Western blotting in SD rats following CI/RI induced by middle cerebral artery occlusion(MCAO).The rat models of CI/RI were subjected to injections of vectors for ZEB1-AS1 overexpression or knockdown into the lateral ventricle,and the changes in cognitive function,brain water content,blood-brain barrier integrity,and IL-1βand TNF-αlevels in the cerebrospinal fluid(CSF)and serum were observed.Neuronal loss and cell apoptosis in the cortex of the rat models were detected by FJC and TUNEL methods,and HMGB1 and TLR-4 expressions were analyzed with Western blotting.We also examined the effects of ZEB1-AS1 knockdown on apoptosis and expressions of HMGB1 and TLR-4 in SH-SY5Y cells with oxygen-glucose deprivation/reoxygenation(OGD/R).Results In CI/RI rats,the expressions of ZEB1-AS1 and HMGB1 in the brain tissue increased progressively with the extension of reperfusion time,reaching the peak levels at 24 h followed by a gradual decline.ZEB1-AS1 overexpression significantly aggravated icognitive impairment and increased brain water content,albumin content in the CSF,and IL-1βand TNF-αlevels in the CSF and serum in CI/RI rats(P<0.05),while ZEB1-AS1 knockdown produced the opposite effects(P<0.05 or0.01).ZEB1-AS1 overexpression obviously increased the number of FJC-positive neurons in the cortex and enhanced the expressions of HMGB1 and TLR-4 in the rat models(P<0.01);ZEB1-AS1 knockdown significantly reduced the number of FJC-positive neurons and lowered HMGB1 and TLR-4expressions(P<0.01).In SH-SY5Y cells with OGD/R,ZEB1-AS1 knockdown significantly suppressed cell apoptosis and lowered the expressions of HMGB1 and TLR-4(P<0.01).Conclusion ZEB1-AS1 overexpression aggravates CI/RI in rats through the HMGB1/TLR-4 signaling axis.