G蛋白γ2亚基沉默通过激活PI3K/AKT信号通路减轻异氟醚诱导的大鼠认知损害

Silencing of G Proteinγ2 Subunit Reduces Isoflurane-induced Cognitive Impairment by Activating PI3K/AKT Signaling Pathway in Rat

目的研究G蛋白γ2亚基(GNG2)沉默对异氟醚诱导认知损害的影响及其机制。方法健康SD大鼠(1周龄)行异氟醚麻醉,分别用GDC0941(PI3K抑制剂pictilisib)或Sh-GNG2进行处理,并将其分为6组(各组n=15):空白对照组(仅腹腔注射0.9%氯化钠溶液20μL),异氟醚组(异氟醚处理后,腹腔注射0.9%氯化钠溶液20μL),Sh GNG2组(异氟醚处理后,腹腔注射转染GNG2沉默基因腺病毒0.9%氯化钠溶液20μL),阴性转染对照组(异氟醚处理后,腹腔注射含阴性ShRNA沉默基因腺病毒的0.9%氯化钠溶液20μL),pictilisib组(异氟醚处理后,腹腔注射pictilisib 16 mg·kg^-1)和Sh+PCB组(异氟醚处理后,腹腔注射pictilisib 16 mg·kg^-1+含转染GNG2沉默基因腺病毒0.9%氯化钠溶液20μL)。采用莫里斯水迷宫和被动回避测试评估大鼠认知功能,应用TUNEL染色及免疫组化法分别检测海马CA1区神经元凋亡及GNG2蛋白的表达,PCR和蛋白印迹方法分别检测相关基因mRNA及蛋白表达水平。结果与对照组比较,异氟醚或pictilisib处理可增加大鼠水迷宫逃逸潜伏时间、反应时间和错误次数,诱导神经元凋亡;GNG2沉默可以缩短逃逸潜伏期、反应时间和错误次数,减少神经元凋亡,差异有统计学意义(P<0.05)。与对照组比较,异氟醚或pictilisib处理后大鼠海马组织中ChAT、PI3K、Akt等蛋白表达以及p-PI3K和p-Akt蛋白水平均显著降低(均P<0.05);GNG2沉默可以上调ChAT、PI3K、Akt蛋白表达及p-PI3K、p-Akt水平(均P<0.05)。结论GNG2沉默可通过激活PI3K/Akt信号通路减轻异氟醚引起的大鼠认知损害。

Aim To study the effect and mechanism of GNG2 silencing on isoflurane-induced cognitive dysfunction.Methods Healthy SD rats(1 week old)were anesthetized with isoflurane and treated with GDC0941(PI3K inhibitor pictilisib)or ShGNG2,respectively.They were divided into 6 groups(n=15 in each group):a control group(no treatment),a fluoroether group(isoflurane treatment),a ShGNG2 group(isoflurane treatment,a intraperitoneal injection of GNG2 silent gene adenovirus 20μL),a negative transfection control group(intraperitoneal injection of 20μL of adenovirus containing negative transfected GNG2 silencing gene after isoflurane treatment),a pictilisib group(intraperitoneal injection of pictilisib after isoflurane treatment,a 16 mg·kg^-1)and Sh+PCB group(treated with isoflurane followed by intraperitoneal injection of pictilisib and transfection with 20μL of GNG2 silencing adenovirus).Morris water maze and passive avoidance tests were used to evaluate the cognitive function of rats.TUNEL staining and immunohistochemistry were used to detect neuronal apoptosis and GNG2 protein expression in hippocampal CA1 area.PCR and Western blot methods were used to detect mRNA of the related genes and protein expression level.Results Compared with the control group,isoflurane or pictilisib treatment can increase the escape latency,reaction time,error times,and induce neuronal cell apoptosis,while GNG2 silencing can shorten the escape latency,reaction time,error times,and reduce nerves(P<0.05).Western blot analysis showed that compared with the control group,the expression of ChAT,PI3K,Akt and other proteins in the hippocampus of rats treated with isoflurane or pictilisib and the phosphorylation levels of p-PI3K and p-Akt were significantly reduced(P<0.05),while GNG2 silencing can up-regulate ChAT,PI3K,Akt protein expression and p-PI3K,p-Akt phosphorylation level(P<0.05).Conclusion GNG2 silencing can alleviate cognitive dysfunction caused by isoflurane by activating P13 K/Akt signaling pathway.