解毒活血方调控PI3K/Akt/mTOR信号通路对ApoE^(-/-)动脉粥样硬化小鼠斑块稳定性的影响

Jiedu Huoxue Prescription Affects Plaque Stability in ApoE^(-/-) Atherosclerosis Mice by Modulating PI3K/Akt/mTOR Signaling Pathway

目的:研究解毒活血方是否可通过调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路诱导巨噬细胞自噬抑制炎症反应稳定AS易损斑块。方法:30只高脂饲料喂养Apo E^(-/-)小鼠随机分为模型组、解毒活血方(中药)低、中、高剂量组、雷帕霉素组,6只普通饲料喂养的Apo E^(-/-)小鼠为正常组,6只普通饲料喂养的C57BL/6J小鼠为空白组,造模7周后开始灌胃给药,中药组予解毒活血方溶液(5.35、10.7、21.4 g·kg^(-1)·d^(-1)),雷帕霉素组予自噬诱导剂雷帕霉素(2 mg·kg^(-1)·d^(-1)),连续给药4周,余各组予等容积生理盐水。检测血清血脂及炎症因子单核细胞趋化蛋白-1(MCP-1)、白细胞介素-6(IL-6)水平,苏木素-伊红(HE)染色观察主动脉根部血管壁病理变化,免疫组化法测定巨噬细胞/单核细胞单克隆抗体(MOMA-2)、α-平滑肌肌动蛋白(α-SMA)表达水平,透射电镜观测主动脉自噬体数量,蛋白免疫印迹法(Western blot)检测巨噬效应蛋白(Beclin-1)、自噬相关蛋白1轻链3(LC3)蛋白及PI3K、Akt、mTOR通路蛋白水平。结果:与正常组比较,模型组血清血脂和炎症因子MCP-1、IL-6水平升高(P<0.05),MOMA-2、α-SMA蛋白表达减少(P<0.05,P<0.01),自噬蛋白Beclin-1表达增加(P<0.05),通路蛋白PI3K、Akt、mTOR表达减少(P<0.05,P<0.01);主动脉内壁可见明显粥样斑块,斑块内可见炎性细胞浸润,斑块附着处血管内膜明显增厚且结构紊乱;自噬体和线粒体数量更少,且线粒体结构不完整。与模型组比较,中药组及雷帕霉素组总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)水平和MCP-1、IL-6水平降低(P<0.05),高密度脂蛋白(HDL)水平升高(P<0.05),MOMA-2、α-SMA蛋白表达减少(P<0.05,P<0.01),Beclin-1、LC3Ⅱ表达增加(P<0.05,P<0.01),PI3K、Akt、mTOR表达减少(P<0.05,P<0.01);主动脉内壁可见少量粥样斑块,炎性细胞浸润程度及血管壁厚度减轻;自噬体和自噬溶酶体数量增多。结论:解毒活血方改善AS小鼠脂质代谢,增强巨噬细胞自噬活性,减轻AS炎症反应,提高易损斑块稳定性,其机制可能与抑制PI3K/Akt/mTOR信号通路活化有关。

Objective:To investigate whether Jiedu Huoxue prescription can induce macrophage autophagy and inhibit inflammatory response to stabilize vulnerable plaques of atherosclerosis (AS) by regulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin(m TOR) signaling pathway.Method:Thirty Apo E^(-/-)mice fed with high-fat diet were randomly assigned into model,low-,medium-,and high-dose (5.35,10.7,and 21.4 g·kg^(-1)·d^(-1),respectively) Jiedu Huoxue prescription (Chinese medicine),and rapamycin (2 mg·kg^(-1)·d^(-1)) groups.Six Apo E^(-/-)mice fed with common diet were used as the control group,and 6 C57BL/6J mice fed with common diet as the blank group.The drugs or equal volume of normal saline were administrated by gavage after 7 weeks of modeling,and the treatment lasted for 4 weeks.The serum levels of lipids and inflammatory cytokines[monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6)]were measured.Hematoxylin-eosin (HE) staining was employed to observe the pathological changes of the vascular wall of the aortic root.Immunohistochemistry was employed to detect the expression of macrophages/monocytes monoclonal antibody (MOMA-2) and α-smooth muscle actin (α-SMA).Transmission electron microscopy was employed to count the autophagosomes in the aorta,and Western blot to determine the protein levels of Beclin-1,LC3,PI3K,Akt,and m TOR.Result:Compared with the control group,the model group showed elevated serum levels of lipids,MCP-1,and IL-6 (P<0.05),inhibited expression of MOMA-2 and α-SMA (P<0.05,P<0.01),up-regulated protein level of Beclin-1 (P<0.05),and down-regulated protein levels of PI3K,Akt,and m TOR (P<0.05,P<0.01).The model group presented obvious atherosclerotic plaques on the inner wall of the aorta,infiltration of inflammatory cells in the plaque,thickened and disarranged vascular intima where the plaque was attached,decreased autophagosomes and mitochondria,and destroyed mitochondrial structure.Chinese medicine and rapamycin groups showed lower levels of total cholesterol,triglycerides,low-density lipoprotein cholesterol,MCP-1,and IL-6 (P<0.05),higher level of high-density lipoprotein cholesterol (P<0.05),inhibited expression of MOMA-2 and α-SMA (P<0.05,P<0.01),higher protein levels of Beclin-1 and LC3Ⅱ(P<0.05,P<0.01),and lower protein levels of PI3K,Akt,and m TOR (P<0.05,P<0.01) than the model group.Moreover,Chinese medicine and rapamycin groups showed only a small number of atherosclerotic plaques on the inner wall of the aorta,reduced infiltration of inflammatory cells and thickness of the blood vessel wall,and increased autophagosomes and autophagic lysosomes.Conclusion:Jiedu Huoxue prescription can improve lipid metabolism,enhance macrophage autophagy,and reduce AS-induced inflammation to improve the stability of vulnerable plaques in AS mice by inhibiting the PI3K/Akt/m TOR signaling pathway.