基于PI3K/Akt/mTOR通路探讨补肾抗衰片介导自噬调控动脉粥样硬化的机制

Mechanism of Bushen Kangshuai Tablet Interfere Autophagy of Atherosclerosis Based on PI3K/Akt/mTOR Signaling Pathway

目的观察补肾抗衰片对动脉粥样硬化(AS)和巨噬细胞自噬的影响并探讨其分子机制。方法采用单纯高脂饮食喂养法建立AS兔模型。于4周末,24只兔随机分为对照组、模型组、补肾抗衰片[1g/(kg·d)]组及阿托伐他汀[5mg/(kg·d)]组,每组6只,干预8周。检测血清TC、TG、LDL-C和HDL-C的水平;油红O染色观察动脉粥样硬化斑块形成情况,HE染色观察血管内膜病理变化,计算内膜增生面积与中膜面积的比值;检测LC3Ⅱ蛋白、Beclin-1蛋白表达水平。建立氧化低密度脂蛋白(100μg/mL)诱导的巨噬细胞株RAW264.7自噬模型,分为模型组、补肾抗衰片组(10%含药血清)和雷帕霉素(10nmol/L)组,另设正常对照组。Western blot技术检测各组细胞LC3Ⅱ、p62、PI3K、p-Akt、p-mTOR的蛋白表达水平。结果在体研究结果显示,与模型组比较,阿托伐他汀组血清TC、TG、LDL-C水平降低(P<0.01),HDL-C水平升高(P<0.01);阿托伐他汀组和补肾抗衰片组主动脉内中膜面积比值降低(P<0.05,P<0.01),主动脉LC3Ⅱ阳性面积百分比、Beclin-1蛋白的表达升高(P<0.05,P<0.01)。体外研究结果显示,与模型组比较,补肾抗衰片组和雷帕霉素组细胞LC3Ⅱ/Ⅰ表达水平升高(P<0.01),p62、PI3K、p-Akt、p-mTOR表达降低(P<0.01)。结论补肾抗衰片能减轻AS病变程度,上调自噬水平,其机制可能通过抑制PI3K/Akt/mTOR信号通路,促进巨噬细胞自噬相关。

Objective To observe the effects of Bushen Kangshuai Tablet（BSKST） on atherosclerosis（ AS） and macrophage autophagy,and to explore its possible molecular mechanism. Methods AS rabbit model was established by feeding the pure high-fat diet for 4 weeks,and the successful model rabbits were randomly divided into 4 groups,including normal control group,model group,BSKST [1 g/（ kg·d）]group and atorvastatin calcium tablet（ACT, 5 mg/kg·d） group,6 in each group. After 8 weeks intervention,levels of serum TC,TG,LDL-C and HDL-C were detected; the aortic plaque general formation of the aortic tissue was observed by Oil red O staining,and the pathological morphological changes were observed by H E staining,then to calculate the ratio of intima to media area. The protein of LC3 Ⅱ and Beclin-1 expression were detected. The macrophage autophagy model was induced by oxidized low density lipoprotein（ox-LDL,100μg/mL） in macrophage cell lines RAW2 64. 7. The cells were divided into 4 groups,including normal control group, model group,BSKST group and rapamycin（10 nmol/L） group. The autophagy related proteins expression of LC3-Ⅱ,p62,PI3 K,phospho-mAkt（p-Akt） and phospho-mTOR（pTOR） were detected by Western blot. Results In vivo research,compared with model group, serum TC,TG and LDL-C level reduced in ACT group（ P 〈0.01）,and HDL-C level was increased（P〈0.01）. The ratio of intima to media area decreased in ACT group and BSKST group（P〈0.05,P〈0.01）. Aortic a utophagy related protein LC 3 Ⅱ and Beclin 1 expression increased（ P〈0.05,P〈0.01）. Compared with model group,the expression of the autophagy-related protein LC3 Ⅱ was increased and the expression of p6 2 PI3 K,p-Akt,and p-mTOR were decreased in the BSKST intervention and rapamycin group（P〈0.01）. Conclusion BSKST can reduce the degree of atherosclerosis.It promotes macrophage autophagy by increasing the level of autophagy protein and inhibiting the PI3 K/Akt/mTOR signaling pathway.