摘要
目的基于ROCK2-shRNA转染技术,探讨温肺降浊方对血管性痴呆大鼠学习、记忆能力及海马组织病理形态的影响。方法将60只SD大鼠随机分为假手术组、模型组、中药组、ROCK2干扰组、阴性对照组、中药+ROCK2干扰组,每组10只。除假手术组外,其余组均采用双侧颈总动脉永久性结扎法制备血管性痴呆模型。造模成功后,ROCK2干扰组及中药+ROCK2干扰组将AAV9-ROCK2-shRNA注射至大鼠海马组织,ROCK2干扰组同时给予生理盐水灌胃4周,中药+ROCK2干扰组同时给予温肺降浊方2 g/(kg·d)灌胃4周;阴性对照组同法注射空载体腺相关病毒,同时给予生理盐水灌胃4周;中药组给予温肺降浊方2 g/(kg·d)灌胃4周,假手术组、模型组给予生理盐水灌胃4周。分别于术后1周及4周,采用Morris水迷宫测试大鼠学习、记忆能力;术后4周处死各组大鼠,电镜观察海马神经元突触超微结构,尼氏染色法观察海马组织中神经元的分布、形态及尼氏小体数量,Tunel染色检测海马神经元凋亡情况。结果术后1周,模型组与各干预组大鼠逃避潜伏期、跨越平台次数比较差异均无统计学意义(P均>0.05);术后4周,与假手术组比较,模型组大鼠逃避潜伏期延长(P<0.05),穿越平台次数明显减少(P<0.05);与模型组比较,中药组、ROCK2干扰组及中药+ROCK2干扰组大鼠逃避潜伏期均明显缩短(P均<0.05),跨越平台次数均明显增加(P均<0.05);与ROCK2组及中药组比较,中药+ROCK2干扰组大鼠逃避潜伏期更短(P均<0.05),穿越平台次数更多(P均<0.05)。与假手术组比较,模型组大鼠神经元分布散乱,突触形态紊乱,细胞器破裂消失,尼氏小体数目明显减少(P<0.05),海马神经元细胞凋亡数明显增加(P<0.05);与模型组比较,中药组、ROCK2干扰组及中药+ROCK2干扰组大鼠神经元损伤明显改善,海马神经元突触形态及细胞器结构较完整规则,尼氏小体数量明显增加(P均<0.05),海马神经元细胞凋亡数明显减少(P均<0.05);与ROCK2组及中药组比较,中药+ROCK2干扰组大鼠神经元突触形态更规则、细胞器更完整,且尼氏小体数目明显增多(P均<0.05),海马神经元细胞凋亡数明显减少(P均<0.05);中药组与ROCK2干扰组各观察指标比较差异均无统计学意义(P均>0.05)。结论温肺降浊方能明显改善血管性痴呆大鼠的学习与记忆能力,机制可能与其能降低ROCK2表达,进而抑制RhoA/ROCK2信号通路的传导,改善海马神经元结构损伤,减少神经细胞凋亡有关。
Objective It is to explore the effect of Wenfei Jiangzhuo decoction on learning,memory and hippocampal histopathological morphology in vascular dementia rats based on ROCK2-shRNA transfection technology.Methods Sixty SD rats were randomly divided into sham operation group,model group,Chinese medicine group,ROCK2 interference group,negative control group,Chinese medicine+ROCK2 interference group,10 rats in each group.Except for the sham operation group,the other groups were treated with permanent ligation of bilateral common carotid arteries to establish vascular dementia models.After successful modeling,AAV9-ROCK2-shRNA was injected into hippocampus of the rats in ROCK2 interference group and Chinese medicine+ROCK2 interference group,at the same time,the ROCK2 interference group was given normal saline by gavage for 4 weeks,the traditional Chinese medicine+ROCK2 interference group was given Wenfei Jiangzhuo decoction 2 g/(kg·d)by gavage for 4 weeks;the negative control group was injected with adeno-associated virus with empty vector in the same way,and was given normal saline by gavage at the same time for 4 weeks;the traditional Chinese medicine group was given Wenfei Jiangzhuo decoction 2 g/(kg·d)by gavage for 4 weeks,and the sham operation group and the model group were given normal saline by gavage for 4 weeks.At 1 week and 4 weeks after operation,the learning and memory abilities of the rats were tested by Morris water maze;the rats in each group were sacrificed at 4 weeks after operation,and the synaptic ultrastructure of hippocampal neurons was observed by electron microscope,and the distribution,morphology and number of Nissl bodies in hippocampal tissue was observed by Nissl staining,and the apoptosis of hippocampal neurons was detected by Tunel staining.Results At 1 week after operation,there was no significant difference in the escape latency and the number of crossing platforms between the model group and each intervention group(all P>0.05).At 4 weeks after operation,compared with the sham operation group,the escape latency of rats in the model group was prolonged(P<0.05),and the number of crossing platforms was significantly reduced(P<0.05);compared with the model group,the escape latency of the Chinese medicine group,the ROCK2 interference group and the Chinese medicine+ROCK2 interference group were significantly shortened(all P<0.05),and the number of crossing platforms was significantly increased(all P<0.05);compared with ROCK2 group and Chinese medicine group,the escape latency of Chinese medicine+ROCK2 interference group was shortened(P<0.05),and the number of crossing platforms was increased(P<0.05).Compared with the sham operation group,the distribution of neurons was scattered,the synaptic morphology was disordered,the organelles were ruptured and disappeared,the number of Nissl bodies was significantly reduced(P<0.05),and the number of hippocampal neurons apoptotic cells was significantly increased in the model group(P<0.05);compared with the model group,the damage of neurons in the Chinese medicine group,ROCK2 interference group and Chinese medicine+ROCK2 interference group was significantly improved,the synaptic morphology and organelle structure of hippocampal neurons were more complete and regular,and the number of Nissl bodies was significantly increased(all P<0.05),the apoptotic number of hippocampal neurons was significantly decreased(P<0.05);compared with the ROCK2 group and the Chinese medicine group,the synaptic morphology of the neurons in the Chinese medicine+ROCK2 interference group was more regular,the organelles were more complete,and the number of Nissl bodies was significantly increased(all P<0.05),apoptotic number of hippocampal neurons was significantly decreased(all P<0.05),there was no significant difference in each observation index between the traditional Chinese medicine group and the ROCK2 interference group(all P>0.05).Conclusion Wenfei Jiangzhuo decoction can significantly improve the learning and memory ability of vascular dementia rats,and the mechanism may be related to its ability to reduce the expression of ROCK2,thereby inhibiting the conduction of RhoA/ROCK2 signaling pathway,improving the structural damage of hippocampal neurons and reducing neuronal apoptosis.
作者
向军军
莫雪妮
杨璧璘
李晓琼
林荣清
黎军宏
胡跃强
XIANG Junjun;MO Xueni;YANG Bilin;LI Xiaoqiong;LIN Rongqing;LI Junhong;HU Yueqiang(The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine,Nanning 530023,Guangxi,China;Guangxi University of Traditional Chinese Medicine,Nanning 530200,Guangxi,China)
出处
《现代中西医结合杂志》
CAS
2022年第13期1753-1758,共6页
Modern Journal of Integrated Traditional Chinese and Western Medicine
基金
国家自然科学基金项目(81860842)
广西高等学校高水平创新团队及卓越学者计划(桂教人才202006)
广西中医脑病临床研究中心项目(桂科AD20238028)。
作者简介
向军军,男,博士,研究方向为缺血性脑血管病的中医药防治;通信作者:胡跃强,E-mail:137463195@qq.com。
