益肾养心安神片对应激模型大鼠学习记忆的改善作用研究

Pharmacological mechanism of Yishen Yangxin Anshen Tablets on learning and memory in stress rats

目的探究益肾养心安神片(YYA)通过对应激大鼠下丘脑-垂体-肾上腺(HPA)轴调控进而改善学习记忆能力的作用。方法SPF级SD大鼠随机分别为对照组、模型组、艾司唑仑片(ES,阳性对照,0.6 mg·kg^(-1))、安神补脑液(AS,阳性对照,2 mL·kg^(-1))和YYA高、中、低剂量(7.2、3.6、1.8 g·kg^(-1))组,采用咖啡因和环磷酰胺ip同时联合水环境法制作大鼠应激模型,造模成功后ig给药,每天1次,连续给药28 d。实验结束后采用Morris水迷宫分析学习记忆能力的变化;HE染色观察海马区病理形态学改变;实时荧光定量PCR(qRT-PCR)法和免疫组化法检测海马区血管活性肠肽(VIP)和神经生长因子(NGF)的表达;ELISA法检测脑组织超氧化物歧化酶(SOD)活力的变化、血清促肾上腺皮质激素(ACTH)、肾上腺皮质激素释放激素(CRH)和肾上腺皮质激素(ACH)以及脑组织中5-羟色胺(5-HT)含量。结果与模型组比较,各给药组大鼠穿越平台和在目标象限探索的时间明显增加,其中ES组和YYA中、高剂量组尤为显著(P<0.05);海马区神经元病变程度明显改善;海马区神经元细胞胞浆内NGF和VIP染色明显增强,NGF和VIP mRNA表达水平显著升高(P<0.05、0.01);ES组、YYA中、高剂量组SOD水平显著升高(P<0.01);各给药组血清中ACTH和CRH水平均显著降低(P<0.05、0.01),AS组、YYA高剂量组ACH水平显著降低(P<0.05);AS组、ES组、YYA高剂量组5-HT水平显著升高(P<0.05、0.01)。结论YYA可以通过抑制HPA通路,降低脑组织氧化应激水平,从而改善海马组织损伤,营养保护神经,进而增强应激大鼠的学习记忆能力。

Objective To explore the pharmacological mechanism of Yishen Yangxin Anshen(YYA) Tablets by axial regulation of the hypothalamic-pituitary-adrenal pathway on the model of stress rats.Methods SPF grade SD rats were randomly divided into control group,model group,Eszolam tablets(ES,positive control,0.6 mg·kg^(-1)),Anshen Bu Nao Ye(AS,positive control,2 mL·kg^(-1)),and YYA high,medium,and low dose(YYA 7.2,3.6,1.8 g·kg^(-1)) groups.The model of stress rats was made by intraperitoneal injecting cyclophosphamide and caffeine and water environment.After successful modeling,ig administration was administered once a day for 28 consecutive days.Morris Water Maze was used to detect learning memory ability.HE staining was used to observe the pathological changes in the hippocampus.Immunohistochemistry and RT-PCR were applied to detect the expression of nerve growth factor(NGF) and vasoactive intestinal peptide(VIP) in the hippocampus.ELISA was used to detect the level of serum adrenocorticotropic hormone(ACTH),adrenocorticoid-releasing hormone(CRH),adrenal corticoid hormone(ACH),and surperoxide dismutase(SOD),5-hydroxytryptamine(5-HT) content in the brain tissue.Results Compared with the model group,the time for rats to cross the platform and explore in the target quadrant significantly increased in each treatment group,with the ES group and YYA medium and high dose groups being particularly significant(P < 0.05).The degree of neuronal damage in the hippocampus has significantly improved.The staining of NGF and VIP in the cytoplasm of hippocampal neurons was significantly enhanced,and the expression levels of NGF and VIP mRNA were significantly increased(P < 0.05,0.01).The SOD levels in the ES group,YYA medium and high-dose groups significantly increased(P < 0.01).The levels of ACTH and CRH in the serum of each treatment group were significantly reduced(P < 0.05,0.01),while the levels of ACH in the AS group and YYA high-dose group were significantly reduced(P < 0.05).The levels of 5-HT were significantly increased in the AS group,ES group,and YYA highdose group(P < 0.05,0.01).Conclusion YYA can reduce the oxidative stress level of the brain tissue,reduce the hippocampal tissue damage,protect nerves,and enhance the learning and memory ability of the stressed rats by inhibiting the HPA pathway.