小檗碱激活巨噬细胞自噬并改善动脉粥样硬化斑块脆弱性

摘要/Abstract

摘要： 目的探索小檗碱(BBR)对高糖及高脂引起的动脉粥样硬化斑块脆弱性的影响,并进行初步的机制研究。方法将雄性载脂蛋白E基因敲除(ApoE^-/-)小鼠随机分为对照组、高糖组[腹腔注射链脲佐菌素(STZ)45 mg/(kg·d),连续5 d]和高脂组[高脂饮食(HFD)];造模2周后,高糖组随机取10只小鼠组成药物预防组[灌胃,BBR 50 mg/(kg·d)];造模4周后,高糖组随机分为模型(STZ)组(灌胃,溶剂)、BBR 25、50、100(STZ)组[灌胃,BBR分别25、50、100 mg/(kg·d)]、阿托伐他汀组[灌胃,2.5 mg/(kg·d)]、恩格列净组[灌胃,1.25 mg/(kg·d)],高脂组随机分为模型(HFD)组(灌胃,溶剂)、BBR50(HFD)组[灌胃,BBR 50 mg/(kg·d)];6周后,取小鼠主动脉进行病理切片染色及免疫组织学检测,评估动脉粥样硬化病变情况及斑块脆弱性指数。以佛波酯(PMA)诱导人单核细胞系Thp1分化为巨噬细胞,研究小檗碱对自噬和炎性反应信号通路的影响;采用Western blot检测小鼠主动脉及细胞中MIF、NLRP3、AMPK/mTOR信号通路及自噬相关蛋白表达;ELISA检测血清及细胞上清液中IL-1β的水平。结果与对照组相比,高糖及高脂组动物有典型的动脉粥样硬化病变,且斑块脆弱性指数明显升高,给予小檗碱治疗后,斑块面积减小,脆弱性指数显著下降(P＜0.05);小檗碱下调小鼠主动脉组织和Thp1细胞中MIF蛋白的表达,促进AMPK的活化,抑制mTOR磷酸化,促进自噬相关蛋白LC3Ⅱ/LC3Ⅰ表达比例升高、SQSTM1/P62表达降低,下调炎性体NLRP3及IL-1β的表达。结论小檗碱能够减轻高糖及高脂引起的ApoE^-/-小鼠动脉粥样硬化斑块沉积并改善斑块的脆弱性,其机制可能与下调MIF的蛋白表达,以及通过AMPK/mTOR信号通路促进巨噬细胞自噬进而抑制炎性反应有关。

关键词: 小檗碱, 动脉粥样硬化斑块脆弱性, 自噬, NLRP3, MIF

Abstract: Objective To explore the effect of berberine (BBR) on the vulnerability of atherosclerotic plaques caused by hyperglycemia and hyperlipidemia, and to discover the potential mechanism. Methods Male ApoE^-/-mice were randomly divided into the control group, hyperglycemia group (intraperitoneal injection of streptozotocin(STZ), a dose of 45 mg/(kg·d) for 5 consecutive days), and hyperlipidemia group (high-fat diet, HFD). After two weeks, 10 mice were randomly selected from the hyperglycemia group to form a drug prevention group[gavage, BBR 50 mg/(kg·d)].Four weeks after modeling,the animals in the hyperglycemia model group were randomly divided into the model (STZ) group (gavage, solvent), BBR25, 50, and 100 (STZ) group (gavage, different doses of berberine [25, 50, and 100 mg/(kg·d)], the atorvastatin group [gavage, 2.5 mg/(kg·d)], the englitazone group [gavage, 1.25 mg/(kg·d)], and the hyperlipidemia model group were randomly divided into the model (HFD) group (gavage, solvent), BBR50 (HFD) group [gavage, berberine 50 mg/(kg·d)]. After 6 weeks of continuous treatment, the aorta of the mouse was taken for pathological and immunohistological examination to evaluate the atherosclerotic plaque lesions and their vulnerability index; Thp1 cells were stimulated to differentiate into macrophages with phorbol ester (PMA) and were used to investigate the effect of berberine on autophagy and inflammatory signaling pathways; Western blot was conducted to detect the expression of MIF, as well as NLRP3, AMPK/mTOR signal pathway and autophagy-related protein level in mouse aorta and cells.ELISA was used to detect the level of IL-1β in serum and cell supernatant. Results Compared with the control group, the atherosclerotic lesions in the hyperglycemia and hyperlipidemia groups were worsened, and the plaque vulnerability index was significantly increased. After the treatment with berberine, the plaque lesions were significantly improved, and the plaque vulnerability index decreased significantly(P＜0.05).Berberine down-regulated MIF protein expression in mouse aortic tissue homogenated and Thp1 cells, promoted AMPK activation, inhibited mTOR phosphorylation, regulated autophagy-related protein expression (LC3Ⅱ/LC3Ⅰ ratio increased, SQSTM1/P62 decreased), and down-regulated NLRP3 and IL-1β expression. Conclusions Berberine can effectively inhibit formation of atherosclerotic plaque and its vulnerability in ApoE^-/-mice caused by hyperglycemia and hyperlipidemia. The mechanism may be attributed to the down-regulation of MIF protein expression and the promotion of autophagy of macrophages through the AMPK/mTOR signaling pathway to inhibit the inflammatory response.

Key words: berberine, atherosclerotic plaque vulnerability, autophagy, NLRP3, MIF

中图分类号:

刘琴, 杨黎星, 石婧, 黄雨晴, 高洪婷, 鞠瑞, 郭磊. 小檗碱激活巨噬细胞自噬并改善动脉粥样硬化斑块脆弱性[J]. 基础医学与临床, 2020, 40(5): 668-677.

LIU Qin, YANG Li-xing, SHI Jing, HUANG Yu-qing, GAO Hong-ting, JU Rui, GUO Lei. Berberine activates macrophage autophagy and improves atherosclerotic plaque vulnerability[J]. Basic & Clinical Medicine, 2020, 40(5): 668-677.

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