基于RIP1/RIP3/MLKL介导程序性坏死通路探讨人参皂苷Rb1拮抗LPS诱导的黑质多巴胺神经元损伤机制  

Ginsenoside Rb1 antagonizes LPS-induced nigrostriatal dopamine neuron damage via RIP1/RIP3/MLKL-mediated necroptosis pathway

作　　者：姜明春 孙丽霞[2] 李大伟[3] 孙宪昌 JIANG Mingchun;SUN Lixia;LI Dawei;SUN Xianchang(Clinical and Basic Medical College,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250017,Shandong,China;Tai’an Central Hospital,Tai'an 2710003,Shandong,China;Xintai People’s Hospital,Xintai 271200,Shandong,China)

机构地区：[1]山东第一医科大学(山东省医学科学院)临床与基础医学院,山东济南250017 [2]泰安市中心医院,山东泰安271000 [3]新泰市人民医院,山东新泰271200

出　　处：《现代中西医结合杂志》2024年第16期2191-2200,共10页Modern Journal of Integrated Traditional Chinese and Western Medicine

基　　金：山东省自然科学基金项目(ZR2020MH151);山东省医药卫生科技发展计划项目(202002010298)。

摘　　要：目的观察受体相互作用蛋白1(RIP1)/RIP3/混合激酶样蛋白(MLKL)介导的程序性坏死在脂多糖(LPS)诱导的大鼠黑质多巴胺能神经元损伤中的作用,探讨人参皂苷Rb1保护多巴胺能神经元的作用机制。方法将48只SD大鼠随机分为对照组、帕金森组、Rb1+帕金森组和Nec-1+帕金森组,每组12只。对照组大鼠黑质内注射生理盐水2μL,然后连续14 d腹腔内注射生理盐水(2 mL/kg);帕金森组大鼠黑质内注射LPS 5μg(2μL),然后连续14 d腹腔内注射生理盐水(2 mL/kg);Rb1+帕金森组大鼠腹腔注射人参皂苷Rb1(20 mg/kg)3 d,第4天黑质内注射5μg LPS后,再连续14 d腹腔内注射人参皂苷Rb1(20 mg/kg);Nec-1+帕金森组大鼠黑质内注射LPS 5μg,然后连续14 d腹腔内注射Nec-1(2 mg/kg)。LPS注射后14 d,皮下注射阿扑吗啡(APO)观察大鼠行为学变化;免疫组化法观察黑质内多巴胺能神经元数量及小胶质细胞形态;高效液相色谱法测定纹状体中多巴胺(DA)及其代谢物高香草酸(HVA)、二羟苯乙酸(DOPAC)的含量;RT-PCR法检测黑质中白细胞介素-1β(IL-1β)及肿瘤坏死因子-α(TNF-α)mRNA表达情况;化学试剂盒检测黑质中超氧化物歧化酶(SOD)活性与丙二醛(MDA)含量;蛋白免疫印迹法检测黑质中酪氨酸羟化酶(TH)、离子钙结合适配器分子1(Iba-1)及程序性细胞坏死相关蛋白(RIP1、RIP3、MLKL)表达情况。结果皮下注射APO后,帕金森组大鼠出现明显的旋转行为,Rb1+帕金森组和Nec-1+帕金森组大鼠旋转行为均较帕金森组明显改善。与对照组比较,帕金森组大鼠注射侧黑质内多巴胺能神经元大量丢失和小胶质细胞过度激活;注射侧纹状体中DA、DOPAC及HVA含量均明显降低(P均<0.05);注射侧黑质中SOD活性和TH蛋白相对表达量均明显降低(P均<0.05),MDA含量和IL-1β、TNF-αmRNA相对表达量及Iba-1、RIP1、RIP3、MLKL蛋白相对表达量均明显升高(P均<0.05)。与帕金森组比较,Rb1+帕金森组和Nec-1+帕金森组Objective It is observe the role of receptor-interacting protein 1(RIP1)/RIP3/mixed lineage kinase domain-like(MLKL)-mediated necroptosis in lipopolysaccharide(LPS)-induced damage in substantia nigra dopaminergic neurons of rats,and to explore the mechanism of action of ginsenoside Rb1 in protecting dopaminergic neurons.Methods Forty-eight SD rats were randomly divided into control group,Parkinson group,Rb1+Parkinson group and Nec-1+Parkinson group,with 12 rats in each group.The rats of the control group were injected with 2μL of saline in substantia nigra,followed by 14 consecutive days of intraperitoneal injection of saline(2 mL/kg);the rats of the Parkinson were injected with 5μg(2μL)of LPS in substantia nigra,followed by 14 consecutive days of intraperitoneal injection of saline(2 mL/kg);the rats of the Rb1+Parkinson group were treated with ginsenoside Rb1(20 mg/kg)by intraperitoneal injection for 3 days,and then injected with 5μg of LPS in substantia nigra on the 4th day,then continuously treated with ginsenoside Rb1(20 mg/kg)by intraperitoneal injection for 14 days;the rats of the Nec-1+Parkinson group were injected with 5μg of LPS in substantia nigra for 3 days,followed by intraperitoneal injection of ginsenoside Rb1(20 mg/kg)for 14 days.14 days after LPS injection,the rats were given apomorphine(APO)by subcutaneous injection to observe their behavioral changes;the number of dopaminergic neurons in substantia nigra and the morphology of microglia were observed by immunohistochemistry;the contents of dopamine(DA)and its metabolites homovanillic acid(HVA)and dihydroxyphenylacetic acid(DOPAC)in striatum were dettected by HPLC;the mRNA expressions of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in substantia nigra were detected by RT-PCR;the activity of superoxide dismutase(SOD)and content of malondialdehyde(MDA)in substantia nigra were detected by chemical assay kit;the expressions of tyrosine hydroxylase(TH),ionic calcium-binding adapter molecule 1(Iba-1)and necroptosis-associated proteins

关 键 词：帕金森病 人参皂苷RB1 脂多糖 程序性坏死 小胶质细胞 多巴胺能神经元 

分 类 号：R-332[医药卫生]