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-1β and TNF-α levels 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-1β and TNF-α levels in the CSF and serum in CI/RI rats (P < 0.05), while ZEB1-AS1 knockdown produced the opposite effects (P < 0.05 or 0.01). 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-4 expressions (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.
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。
Keywords: HMGB1; Toll-like receptor-4; cerebral ischemia/reperfusion injury; lncRNA ZEB1-AS1.