lncRNA C2dat2调控小鼠大脑中动脉栓塞再灌注损伤及神经元自噬和凋亡

The lncRNA C2dat2 regulates reperfusion injury and neuronal autophagy and apoptosis in middle cerebral artery embolized mice

摘要:

目的分析lncRNA C2dat2调控小鼠大脑中动脉栓塞再灌注损伤(MCAO/R)及神经元自噬、凋亡的作用机制。 方法构建小鼠MCAO/R模型,实验分为假手术组、模型组、negative shRNA组和C2dat2 shRNA组。HE染色检测大脑组织病理学变化,改良神经功能缺陷评分评估小鼠神经功能,TUNEL染色检测大脑组织细胞凋亡水平,荧光定量PCR检测脑组织半胱氨酸蛋白酶-3(Caspase-3)、磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)mRNA表达。Western blotting检测脑组织自噬蛋白水平。 结果模型组小鼠脑组织病理损伤明显,降低C2dat2表达后病理损伤明显改善。除假手术组外,神经功能缺陷评分其他组均随时间依次下降,且C2dat2 shRNA组低于negative shRNA组(P＜0.05)。细胞凋亡率模型组和negative shRNA组高于假手术组,C2dat2 shRNA组低于negative shRNA组(P＜0.05)。LC3 Ⅱ/LC3 Ⅰ、Belin 1、Caspase-3和p-p38MAPK表达水平模型组和negative shRNA组高于假手术组,C2dat2 shRNA组低于negative shRNA组(P＜0.05)。 结论C2dat2表达下调可减轻小鼠MCAO/R损伤,抑制细胞自噬和凋亡,其机制可能与MAPK信号通路有关。

Abstract:

To analyze the mechanisms of CAMK2D-related transcript 1 (lncRNA C2dat2) on regulating reperfusion of middle cerebral artery occlusion (MCAO/R) injury, neurons autophagy and apoptosis in mice. MethodsThe MCAO/R models were constructed. A total of 60 mice were divided into sham operation group, model group, negative shRNA group and C2dat2 shRNA group. The histopathological changes in the brain tissues were detected by HE staining. The changes of neurologicalfunction of the mice were evaluated by scores of modified neurological deficits. The apoptosis in the brain tissues was observed by TUNEL staining. The expressions of cysteine protease protein-3 (Caspase-3) and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) mRNA in the brain tissues were detected by real-time fluorescence quantitative PCR. The autophagy proteins in the brain tissues were detected by Western blotting. ResultsThe pathological damage of the brain tissue of the mice in the model group was obvious, and the pathological damage was significantly improved after reducing the expression of C2dat2. Except for the sham operation group, the neurological deficit scores of other groups decreased sequentially with time, and the C2dat2 shRNA group was lower than the negative shRNA group (P＜0.05). The number of apoptosis in the model group and the negative shRNA group was higher than that in the sham operation group, and the C2dat2 shRNA group was lower than that in the negative shRNA group (P＜0.05). The expression levels of LC3 Ⅱ/LC3 I, Belin 1, Caspase-3 and p-p38MAPK in the model group and the negative shRNA group were higher than those in the sham operation group, and the C2dat2 shRNA group was lower than the negative shRNA group (P＜0.05). ConclusionThe down-regulation of C2dat2 can alleviate MCAO/R injury, inhibit cells autophagy and apoptosis in mice, which may be related to MAPK signaling pathway.