基于PERK/ATF4/CHOP通路探讨游泳运动干预血管性痴呆大鼠的机制

Mechanism of Swimming Exercise Intervention on Rats with Vascular Dementia Based on PERK/ATF4/CHOP Pathway

目的:基于PERK/ATF4/CHOP通路探讨游泳运动对血管性痴呆(VD)大鼠的脑保护作用。方法:选择48只SPF级雄性SD大鼠,按随机数字表法分为假手术组和实验组,分别为12、36只。实验组采用永久性结扎双侧颈总动脉法进行模型制备,按随机数字表法分为模型组、游泳运动组和药物组,每组12只。游泳运动组给予无负重游泳训练30 min/(次·d),连续4周;药物组腹腔注射单唾液酸四己糖神经节苷脂钠0.33 mg/(kg·d),连续4周;其余2组自由活动4周。干预4周后,采用Morris水迷宫实验观察大鼠的行为学变化;采用透射电镜观察大鼠海马CA1区神经元超微结构;采用实时荧光定量PCR(RT-qPCR)、Western blot法分别检测蛋白激酶R样内质网激酶(PERK)、活化转录因子4(ATF4)、C/EBP同源蛋白(CHOP)mRNA水平及蛋白的相对表达量。结果:①行为学变化:与假手术组比较,模型组平均逃避潜伏期明显延长,穿越平台次数明显减少,差异具有统计学意义(P<0.05)。与模型组比较,游泳运动组、药物组平均逃避潜伏期明显缩短,穿越平台次数明显增加,差异具有统计学意义(P<0.05)。②神经元超微结构:模型组胞浆内细胞器数量减少,胞浆基质溶解空泡化,线粒体嵴断裂,结构不清晰,神经纤维可见坏死、溶解;与模型组比较,游泳运动组胞浆内各细胞器结构、空泡样变程度明显改善。③脑组织PERK、ATF4、CHOP mRNA水平及蛋白含量:与假手术组比较,模型组海马CA1区PERK、ATF4、CHOP mRNA水平均明显升高(P<0.05),PERK、ATF4、CHOP蛋白含量均明显升高(P<0.05);与模型组比较,游泳运动组、药物组海马CA1区PERK、ATF4、CHOP mRNA水平及蛋白含量均明显降低(P<0.05)。结论:游泳运动可保护VD大鼠脑组织,改善其认知功能;其作用机制可能与调控PERK/ATF4/CHOP通路,抑制VD大鼠海马区ERS相关蛋白PERK、ATF4、CHOP mRNA及蛋白表达有关。

Objective:To explore the neuroprotective effects of swimming exercise on rats with vascular dementia(VD)based on the PERK/ATF4/CHOP pathway.Methods:A total of 48 male SPF SD rats were randomly divided into sham operation group and experimental group,with 12 and 36 cases respectively.The permanent bilateral common carotid artery occlusion method was used to establish the model in the experimental group,which was randomly divided to model group,swimming exercise group and drug group,with 12 cases in each group.The swimming exercise group underwent load-free swimming training for 30 min/(time·day),lasting for four weeks;the drug group received intraperitoneal injections of monosialotetrahexose ganglioside sodium,0.33 mg/(kg·day),lasting for four weeks;the other two groups were allowed free activity for four weeks.After four weeks of intervention,Morris water maze experiment was used to observe behavioral changes of rats;transmission electron microscopy was used to observe the ultrastructure of neurons in the hippocampal CA1 region of rats;real-time fluorescent quantitative PCR(RT-qPCR)and Western blot were used to detect the relative expression levels of protein kinase R-like endoplasmic reticulum kinase(PERK),activating transcription factor 4(ATF4),and C/EBP homologous protein(CHOP)mRNA and protein.Results:(1)Behavioral changes:compared with the sham operation group,the average escape latency in the model group was significantly prolonged,and the number of platform crossing significantly reduced,and the differences were statistically significant(P<0.05).Compared with the model group,the average escape latency of the swimming exercise group and the drug group were significantly shortened,and the number of platform crossing significantly increased(P<0.05).(2)Neuron ultrastructure:in the model group,the number of organelles in the cytoplasm decreased,cytoplasmic matrix dissolution and vacuolization occurred,mitochondria cristae were broken and unclear,and neural fiber necrosis and dissolution were visible;compared with the model group,the swimming exercise group showed significant improvement in the structure of various organelles in the cytoplasm and the degree of vacuolar changes.(3)Expression levers of brain tissue PERK,ATF4,CHOP mRNA and protein:compared with the sham operation group,the expression levels of PERK,ATF4,CHOP mRNA,and protein in the hippocampal CA1 region of the model group significantly increased(P<0.05);compared with the model group,the expression levels of PERK,ATF4,CHOP mRNA and protein in the hippocampal CA1 region of the swimming exercise group significantly decreased(P<0.05).Conclusion:Swimming exercise can protect brain tissue in rats with VD and improve their cognitive functions.The mechanism may be related to the regulation of the PERK/ATF4/CHOP pathway,suppressing the expression of ERS-related proteins PERK,ATF4,CHOP mRNA and protein in the hippocampal region of rats with VD.