腺病毒介导的RNAi对VaD大鼠脑内NgR/RhoA/ROCK2信号通路的影响

The effect of adenovirus-mediated RNAi on NgR/RhoA/ROCK2 signal pathway in VaD rat brain

目的探讨基于腺相关病毒为载体构建的Nogo受体(NgR)抑制剂通过NgR/Ras基因家族成员(Rho)A/Rho相关激酶(ROCK)2信号通路改善血管性痴呆(VaD)大鼠学习记忆能力及轴突再生分子机制。方法将40只SD大鼠随机分为假手术组、模型组、NgR干扰剂组(腺相关病毒)、阴性对照组(NgR空载体病毒),每组10只。采用双侧颈总动脉永久结扎术法制作VaD大鼠模型,模型成功后,将AAV9-NgR-shRNA通过脑立体定位术注射至大鼠海马组织,阴性对照组注入等量空载体腺相关病毒。4 w后,采用Morris水迷宫测定学习、记忆能力,采用实时荧光定量-聚合酶链反应(PCR)、Western印迹检测各组NgR/RhoA/ROCK2通路NgR、RhoA、ROCK2 mRNA及蛋白表达水平,电镜观察大鼠海马突触超微结构变化。结果术后1 w,模型组、阴性对照组潜伏期时间与跨越平台次数与NgR干扰组比较,差异无统计学意义(P>0.05),与假手术组比较有显著性差异(均P<0.05)。术后4 w,与模型组及阴性对照组比较,NgR干扰组潜伏期显著缩短,跨越平台次数显著增加,海马中NgR、RhoA、ROCK2 mRNA及其蛋白表达显著减少(均P<0.05),且海马组织内突触前后囊泡正常、细胞器更加完整;阴性对照组上述指标与模型组比较差异无统计学意义(均P>0.05)。结论NgR抑制剂可能通过抑制NgR/RhoA/ROCK2信号通路起到促进神经轴突再生,改善海马突触结构,减轻VaD大鼠认知功能障碍的作用。

Objective To explore the molecular mechanism of Nogo receptor(NgR)inhibitor constructed based on adeno-associated virus as a vector through NgR/Ras family members(Rho)A/Rho-associated kinase(ROCK)2 signaling pathway to improve the learning and memory ability and axon regeneration of vascular dementia(VaD)rats.Methods 40 SD rats were randomly divided into sham operation group,model group,NgR interference group(Adeno-associated virus),negative control group(NgR empty vector virus),with 10 rats in each group.The VaD rats model was prepared by permanent ligation of bilateral common carotid arteries.After confirming the model successfully,AAV9-NgR-shRNA was injected into the hippocampus tissue of the rat by stereotaxic technique,the negative control group was injected with the same amount of empty vector adeno-associated virus.After 4 w,Morris water maze was used to measure the ability of learning and memory.Real-time polymerase chain reaction(PCR)and Western blot were used to detect the mRNA and protein expression levels of NgR,RhoA,ROCK2 in the NgR/RhoA/ROCK2 pathway of rats.The ultrastructural changes of hippocampal synapses in rats were observed by electron microscopy.Results 1 w after the operation,there was no significant difference between the incubation period and the number of crossing platforms in the model group and the negative control group compared with the NgR interference group(P>0.05),and there was a significant difference compared with the sham operation group(all P<0.05).4 w after operation,compared with the model group and the negative control group,the incubation period of NgR interference group was significantly shortened,the number of crossing platforms was significantly increased,the mRNA and protein expressions of NgR,RhoA and ROCK2 in hippocampus were significantly decreased(all P<0.05),and the pre-synaptic and post-synaptic vesicles were normal and the organelles were more complete.There was no significant difference in the above indexes between the negative control group and the model group(all P>0.05).Conclusions NgR inhibitors might promote axon regeneration,improve hippocampal synaptic structure and reduce cognitive dysfunction in VaD rats by inhibiting the NgR/RhoA/ROCK2 signaling pathway.