双氢杨梅素通过诱导自噬减轻慢性帕金森病小鼠的细胞焦亡和坏死性凋亡  

Dihydromyricetin alleviates pyroptosis and necroptosis in mice with MPTP-induced chronic Parkinson's disease by inducing autophagy

作　　者：张蒙 张源源 牛梦竹 朱悦 童诗逸 寇现娟[1,2] ZHANG Meng;ZHANG Yuanyuan;NIU Mengzhu;ZHU Yue;TONG Shiyi;KOU Xianjuan(College of Sports Medicine,Wuhan Sports University,Wuhan 430079,China;Hubei Key Laboratory of Exercise Training and Monitoring,Wuhan 430079,China)

机构地区：[1]武汉体育学院运动医学院,湖北武汉430079 [2]运动训练监控湖北省重点实验室,湖北武汉430079

出　　处：《南方医科大学学报》2023年第8期1268-1278,共11页Journal of Southern Medical University

基　　金：国家自然科学基金(81601228);教育部人文社会科学研究规划基金(21YJA890014)。

摘　　要：目的探讨8周的双氢杨梅素(DHM)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)/丙磺舒(probenecid)(MPTP/p)诱导慢性帕金森病(PD)小鼠运动功能障碍的影响及其分子机制。方法C57BL/6小鼠腹腔注射MPTP/p构建PD模型,将60只健康雄性小鼠随机分为4组(n=15):对照组(Control)、模型组(PD)、DHM干预组(PD+DHM)、阳性对照NEC-1抑制剂组(PD+NEC-1)。除对照组外,其余小鼠(n=45)腹腔注射MPTP(25 mg·kg^(-1)·d^(-1))联合Probenecid(250 mg·kg^(-1)·d^(-1)),每周2次,干预5周构建慢性PD模型。造模结束后,DHM干预组进行8周DHM灌胃处理(100 mg·kg^(-1)·d^(-1),5 d/周),阳性对照组进行5周NEC-1腹腔注射(6.25 mg·kg^(-1)·d^(-1),2 d/周)。随机选取小鼠进行运动协调性测试(n=10);免疫荧光染色观察黑质内TH、GFAP、Iba-1的表达(n=3);ELISA检测小鼠血清中IL-1β;试剂盒测定纹状体内LDH酶活性;RT-PCR测定TNF-α及IL-6的m RNA水平;Western blot检测小鼠纹状体中TH和病理蛋白α-syn、神经炎症、细胞焦亡、坏死性凋亡及自噬相关蛋白的表达。1-甲基-4-苯基吡啶离子(MPP+)激活Bv-2小胶质细胞,采用不同浓度的DHM(3.12、6.25、12.5μg/m L)及自噬抑制剂3-MA进行干预;Western blot检测干预后Bv-2细胞自噬及NLRP3炎症小体相关蛋白的表达水平;PI染色检测细胞的死亡情况。结果与对照组相比,PD组小鼠存在运动功能障碍,且TH的阳性细胞数及蛋白水平显著下降(P<0.001),而DHM干预显著改善了小鼠的运动协调能力,增加TH(P=0.0023),降低α-syn的表达(P<0.001)。8周的DHM干预降低了PD小鼠纹状体中胶质细胞异常活化,下调GFAP(P=0.045)和Iba-1(P<0.001)的表达;降低TNF-α(P=0.0015)、IL-6(P<0.001)的m RNA和蛋白水平,上调IL-4(P<0.001),减轻神经炎症反应;DHM通过下调Caspase1的活性、NLRP3炎症小体、GSDMD-N、IL-1β、IL-18的表达水平(P<0.001)抑制细胞焦亡;降低LDH、p-RIPK1、RIPK1、p-RIPK3、RIPK3、MLKL的水平(P<0.001),上调Caspase8(P=0.8944),改善Objective To investigate the effect of 8-week dihydromyricetin(DHM)treatment on motor ability of mice with MPTP/probenecid-induced Parkinson's disease(PD)and explore the molecular mechanism.Methods Sixty C57BL/6 mice were randomized into the control group,PD model group,PD+DHM group and PD+NEC-1 group(n=15).In the latter 3 groups,the mice were treated with 25 mg·kg^(-1)·d^(-1)MPTP and 250 mg·kg^(-1)·d^(-1)probenecid twice a week for 5 weeks to establish PD models;DHM(100 mg·kg^(-1)·d^(-1))was administered 5 times a week via gavage for 8 weeks and NEC-1(6.25 mg·kg^(-1)·d^(-1),twice a week)via intraperitoneal injection for 5 weeks.The changes in motor function of the mice were assessed,and the expressions of TH,GFAP and Iba-1 in the substantia nigra were detected with immunofluorescence assay;serum levels of IL-1βand LDH were detected using ELISA.The mRNA expressions of TNF-αand IL-6 were determined with RT-PCR,and the expressions of TH and proteins associated with pyroptosis,neuroinflammation,necroptosis and autophagy in the striatum were detected using Western blotting.MPP+-activated Bv-2 cells were treated with different concentrations of DHM or 3-MA,and the expressions of proteins associated with autophagy and NLRP3 were detected using Western blotting;PI staining was used to detect cell necroptosis.Results The PD mouse models showed significantly reduced TH-positive cells and TH protein expression(P<0.001).DHM obviously ameliorated motor deficits and TH loss in PD mice,increased TH expression(P=0.0023),decreasedα-syn levels(P<0.001),lowered the protein expressions of GFAP(P=0.045)and Iba-1(P<0.001)and the mRNA and protein levels of TNF-α(P=0.0015)and IL-6(P<0.001),and increased IL-4 level(P<0.001).The 8-week DHM treatment significantly suppressed pyroptosis and necroptosis and activated autophagy in the striatum of the PD mice.In MPP+-induced Bv-2 cells,DHM treatment effectively reversed autophagy impairment and inhibited NLRP3 and TNF-α,IL-6 and IL-1βrelease,and the anti--inflammatory effects of

关 键 词：双氢杨梅素 MPTP 丙磺舒 细胞焦亡 坏死性凋亡 自噬 神经炎症 

分 类 号：R285.5[医药卫生—中药学]