小檗碱靶向Wnt5a/NPC1信号通路调节脂自噬抑制动脉粥样硬化形成  

Berberine Inhibits Atherosclerosis by Regulating Lipophagy Via Targeting Wnt5a/NPC1 Signaling Pathway

作　　者：杨彩云 鲁启文 罗桑 涂梦婷 赵瞳 郑翠翠 万强[3] YANG Caiyun;LU Qiwen;LUO Sang;TU Mengting;ZHAO Tong;ZHENG Cuicui;WAN Qiang(Affiliated Hongdu Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China;Jiangxi University of Chinese Medicine,Nanchang 330004,China;The Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China)

机构地区：[1]江西中医药大学附属洪都中医院,南昌330006 [2]江西中医药大学,南昌330004 [3]江西中医药大学附属医院,南昌330006

出　　处：《中国实验方剂学杂志》2023年第18期62-68,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：江西省自然科学基金项目(20232BAB206144);江西省卫生健康委科技计划项目(202310664);江西中医药大学科技创新团队项目(CXTD22011)。

摘　　要：目的:探讨小檗碱(BBR)在防治小鼠动脉粥样硬化(AS)病变中对脂自噬的调节作用及分子机制。方法:采用载脂蛋白E基因敲除(ApoE-/-)小鼠50只随机分为AS模型组、阿托伐他汀组(5 mg·kg-1)、BBR低、中、高剂量组(2.5、5、10 mg·kg-1);另设10只C57BL/6J小鼠为空白组。12周后苏木素-伊红(HE)及油红O染色检测主动脉AS斑块病理学改变;生化检测血清脂质水平;酶联免疫吸附测定法(ELISA)检测血清炎症因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平、氧化应激标记物活性氧(ROS)含量及血清脂自噬标记物Beclin1、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)水平;黄嘌呤氧化酶法检测血清超氧化物歧化酶(SOD)活力;免疫组化(IHC)检测主动脉无翅型MMTV整合位点家族成员5a(Wnt5a)、C1型尼曼-匹克蛋白(NPC1)分布;蛋白免疫印迹法(Western blot)检测主动脉Wnt5a、NPC1蛋白表达。结果:与空白组比较,AS模型组小鼠可见显著的AS斑块形成,血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、IL-6、TNF-α、ROS、主动脉Wnt5a分布及蛋白表达均显著升高(P<0.01),血清高密度脂蛋白胆固醇(HDL-C)、SOD、Beclin1、LC3Ⅱ、主动脉NPC1分布及蛋白表达均显著降低(P<0.01);与AS模型组比较,阿托伐他汀组、BBR中剂量组及BBR高剂量小鼠AS斑块面积明显减少(P<0.05,P<0.01),血清TC、TG、LDL-C、IL-6、TNF-α、ROS、主动脉Wnt5a分布及蛋白表达均明显降低(P<0.05,P<0.01),血清HDL-C、SOD、Beclin1、LC3Ⅱ、主动脉NPC1分布及蛋白表达均明显升高(P<0.05,P<0.01);与阿托伐他汀组比较,BBR中剂量组小鼠以上检测指标差异无统计学意义。结论:BBR可通过竞争性结合Wnt5a激活NPC1表达,上调脂自噬水平降低血脂,抑制炎症介质的释放及氧化应激损伤从而发挥防治AS功效。Objective:To investigate the regulatory effect and molecular mechanism of berberine(BBR)on lipophagy in the prevention and treatment of atherosclerotic(AS)lesions in mice.Method:Fifty apolipoprotein E-knockout(ApoE−/−)mice were randomly divided into an AS model group,an atorvastatin group(5 mg·kg-1),and low-,medium-,and high-dose BBR groups(2.5,5,10 mg·kg-1).Ten C57BL/6J mice were assigned to the control group.After 12 weeks,hematoxylin-eosin(HE)and oil red O staining were performed to assess the histopathological changes of AS plaques in the aorta.Biochemical analysis was used to measure serum lipid levels,and enzyme-linked immunosorbent assay(ELISA)was employed to measure the levels of inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α),oxidative stress marker reactive oxygen species(ROS),and serum lipophagy marker Beclin1 and microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ).The xanthine oxidase method was used to measure serum superoxide dismutase(SOD)activity.Immunohistochemistry(IHC)was used to detect the distribution of wingless-type MMTV integration site family member 5a(Wnt5a)and Nieman Pick type C1(NPC1)in the aorta,and Western blot was used to determine the protein expression of Wnt5a and NPC1 in the aorta.Result:Compared with the control group,the AS model group showed significant AS plaque formation,significantly elevated levels of serum total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),IL-6,TNF-α,and ROS,aortic Wnt5a distribution and protein expression(P<0.01),and significantly reduced levels of serum high-density lipoprotein cholesterol(HDL-C),SOD,Beclin1,LC3Ⅱ,and aortic NPC1 distribution and protein expression(P<0.01).Compared with the AS model group,the atorvastatin group,and high-and medium-dose BBR groups showed a significant reduction in AS plaque area(P<0.05,P<0.01),significantly decreased levels of serum TC,TG,LDL-C,IL-6,TNF-α,ROS,and aortic Wnt5a distribution and protein expression(P<0.05,P<0.01),and significantly

关 键 词：小檗碱 脂自噬 载脂蛋白E基因敲除小鼠 无翅型MMTV整合位点家族成员5a(Wnt5a)/C1型尼曼-匹克蛋白(NPC1)信号通路 

分 类 号：R2-0[医药卫生—中医学] R33R972.6R289