基于PI3K/AKT信号通路及自噬探讨恒清Ⅰ号方治疗血管性痴呆的作用机制

Discussion on the mechanism of HengqingⅠDecoction in the treatment of vascular dementia based on PI3K/AKT signaling pathway and autophagy

目的基于PI3K/AKT信号通路及自噬研究恒清Ⅰ号方治疗血管性痴呆的作用机制。方法将60只健康雄性SD大鼠随机分为假手术组、模型组、恒清Ⅰ号方低剂量组、恒清Ⅰ号方中剂量组、恒清Ⅰ号方高剂量组和欧来宁组,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建模。术后7 d,恒清Ⅰ号方低、中、高剂量组分别给予0.268 g/mL、0.535 g/mL、1.070 g/mL的恒清Ⅰ号方灌胃,欧来宁组给予欧来宁14.8 g/kg灌胃,假手术组、模型组给予等量生理盐水灌胃,均2次/d,持续灌胃30 d。灌胃结束后采用Morris水迷宫实验测试各组大鼠的逃避潜伏期、穿越平台次数,以ELISA法检测海马组织中肿瘤坏死因子-α(TNF-α)、核因子-κB(NF-κB)含量,以Western blot法检测海马组织中PI3K、AKT、p-AKT、LC3-Ⅰ及LC3-Ⅱ蛋白表达情况。结果水迷宫实验第1~4天,模型组大鼠逃避潜伏期均明显长于假手术组(P均<0.05);恒清Ⅰ号方中、高剂量组和欧来宁组大鼠逃避潜伏期均明显短于模型组和恒清Ⅰ号方低剂量组(P均<0.05),且恒清Ⅰ号方高剂量组明显短于恒清Ⅰ号方中剂量组和欧来宁组(P均<0.05);模型组大鼠穿越平台次数明显少于假手术组(P<0.05),恒清Ⅰ号方中、高剂量组和欧来宁组大鼠穿越平台次数均明显多于模型组和恒清Ⅰ号方低剂量组(P均<0.05),且恒清Ⅰ号方高剂量组明显多于恒清Ⅰ号方中剂量组和欧来宁组(P均<0.05)。与假手术组比较,模型组大鼠海马组织中TNF-α、NF-κB含量和LC3-Ⅱ/LC3-Ⅰ比值均明显增高(P均<0.05),PI3K、AKT、p-AKT蛋白表达量均明显降低(P均<0.05);与模型组和恒清Ⅰ号方低剂量组比较,恒清Ⅰ号方中、高剂量组和欧来宁组大鼠海马组织中TNF-α、NF-κB含量和LC3-Ⅱ/LC3-Ⅰ比值均明显降低(P均<0.05),PI3K、AKT、p-AKT蛋白表达量均明显升高(P均<0.05);与恒清Ⅰ号方中剂量组和欧来宁组比较,恒清Ⅰ号方高剂量组大鼠海马组织中TNF-α、NF-κB含量和LC3-Ⅱ/LC3-Ⅰ比值均明显降低(P均<0.05),PI3K、AKT、p-AKT蛋白表达量均明显升高(P均<0.05)。结论恒清Ⅰ号方可以呈量效关系改善血管性痴呆大鼠的学习记忆能力,且高剂量恒清Ⅰ号方的作用优于欧来宁,可能与其抑制炎症反应、阻滞细胞自噬和调控PI3K/AKT信号通路有关。

Objective It is to study the mechanism of HengqingⅠDecoction(HQ)in the treatment of vascular dementia(VD)based on PI3K/AKT signaling pathway and autophagy.Methods Sixty healthy male SD rats were randomly divided into sham operation group,model model group,HQ low dose(HQ-L)group,HQ medium dose(HQ-M)group,HQ high dose(HQ-H)group and oxiracetam group.Except for the sham operation group,the other groups were modeled by permanent ligation of bilateral common carotid arteries.On the 7th postoperative day,the low,medium and high dose groups of HengqingⅠDecoction were respectively given 0.268 g/mL,0.535 g/mL,and 1.070 g/mL HengqingⅠDecoction by gavage,the Oulaining group was given Oulaining 14.8.g/kg by gavage,the sham operation group and the model group were given the same amount of normal saline by gavage,all twice/d,continuously treated for 30 days.At the end of the gavage,the Morris water maze test was used to test the escape latency and the number of crossing platforms of the rats in each group,and the content of tumor necrosis factor-α(TNF-α)and nuclear factor-κB(NF-κB)in the hippocampus were detected by ELISA,the expression of PI3K,AKT,p-AKT,LC3-Ⅰand LC3-Ⅱprotein in hippocampus was detected by Western blot.Results From the 1st day to the 4th day of the water maze experiment,the escape latency of rats in the model group was significantly longer than that of the sham operation group(all P<0.05);the escape latency of rats in the HQ-M group,HQ-H group and Oulaining group were significantly shorter than those in the model group and the HQ-L group(P<0.05),and the escape latency of the HQ-H group was significantly shorter than those of the HQ-M group and Oulaining group(all P<0.05).The number of crossing platforms of the rats in the model group was significantly less than that in the sham operation group(P<0.05),the numbers of crossing the platform of the rats in the HQ-M group,HQ-H group and Oulaining group were significantly more than those in the model group and HQ-L group(P<0.05),and the HQ-H group were significantly more than the HQ-M group and Oulaining group(all P<0.05).Compared with the sham operation group,the contents of TNF-α,NF-κB and in the hippocampus and the ratio of LC3-Ⅱ/LC3-Ⅰof rats in the model group were significantly increased(all P<0.05),and the expression of PI3K,AKT,and p-AKT proteins were all significantly decreased(all P<0.05);compared with the model group and the HQ-L group,the content of TNF-αand NF-κB in the hippocampus and LC3-Ⅱ/LC3-Ⅰratios of rats in the HQ-M group,HQ-H group and Oulaining group were significantly reduced(P<0.05),the expression levels of PI3K,AKT,p-AKT protein were significantly increased(P<0.05);compared with the HQ-M group and Oulaining group,the contents of TNF-α,NF-κB in the hippocampus and the ratio of LC3-Ⅱ/LC3-Ⅰof the HQ-H group were significantly reduced(all P<0.05),the expression levels of PI3K,AKT,p-AKT protein was significantly increased(all P<0.05).Conclusion HQ can improve the learning and memory ability of rats with vascular dementia in a dose-effect relationship,and the effect of high-dose HQ is better than that of Oulaining,the mechanism may be related to inhibiting inflammation,blocking autophagy and regulating PI3K/AKT signal pathway.