基于Keap1/Nrf2与TLR4/NF-κB p65信号通路研究藏族药二十五味松石丸改善对乙酰氨基酚致小鼠肝损伤的作用机制  

Mechanism of Tibetan medicine Ershiwuwei Songshi Pills against liver injury induced by acetaminophen in mice based on Keap1/Nrf2 and TLR4/NF-κB p65 signaling pathways

作　　者：沙玉茹 罗晓敏 丁翼 杨斌 简程芳 龚普阳 顾健 谭睿[2] SHA Yu-ru;LUO Xiao-min;DING Yi;YANG Bin;JIAN Cheng-fang;GONG Pu-yang;GU Jian;TAN Rui(College of Pharmacy,Southwest Minzu University,Chengdu 610041,China;College of Life Science and Engineering,Southwest Jiaotong University,Chengdu 610031,China)

机构地区：[1]西南民族大学药学院,四川成都610041 [2]西南交通大学生命科学与工程学院,四川成都610031

出　　处：《中国中药杂志》2022年第8期2049-2055,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82174083);四川省重点研发项目(20ZDYF3291);四川省科技厅项目(2019YFS0157);教育部重点实验室开放课题(KF2020009);西南民族大学中央高校基本科研业务费专项(2020NZD06)。

摘　　要：该研究基于Keap1/Nrf2与TLR4/NF-κB p65信号通路研究藏族药二十五味松石丸(Ershiwuwei Songshi Pills, ESP)改善对乙酰氨基酚(acetaminophen, APAP)致小鼠肝损伤的作用机制。将昆明小鼠随机分为空白组,模型组,二十五味松石丸高(400 mg·kg^(-1))、中(200 mg·kg^(-1))、低(100 mg·kg^(-1))剂量组和乙酰半胱氨酸(N-acetyl-L-cysteine, NAC)组。各给药组小鼠均连续给药14 d后,除空白组外,其余各组小鼠腹腔注射200 mg·kg^(-1)APAP,12 h后收集小鼠血清与肝脏组织。通过肝脏病理切片苏木精-伊红染色(hematoxylin-eosin staining, HE),并检测小鼠血清中天冬氨酸氨基转移酶(aspartate aminotransferase, AST)、丙氨酸氨基转移酶(alanine aminotransferase, ALT)水平以及肝组织匀浆中谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)、超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、髓过氧化物酶(myeloperoxidase, MPO)、总抗氧化能力(total antioxidant capacity, T-AOC)水平,观察与分析二十五味松石丸对APAP致小鼠肝损伤的保护作用。通过ELISA法测定小鼠血清中肿瘤坏死因子(tumor necrosis factor-alpha, TNF-α)、白细胞介素(interleukin-1 beta, IL-1β)与(interleukin-6,IL-6)水平,Western blot法测定小鼠肝细胞核内核转录因子E2相关因子2(nuclear transcription factor E2 related factor 2,Nrf2)、细胞质kelch样环氧氯丙烷相关蛋白1(kelch-like ECH-associated protein 1,Keap1)、肝脏内Toll样受体4(Toll-like receptor 4,TLR4)以及细胞核内核因子-κB p65(nuclear factor-kappa B,NF-κB p65)蛋白表达,实时荧光定量PCR测定小鼠肝脏中谷氨酸半胱氨酸连接酶催化亚基(glutamate-cysteine ligase catalytic subunit, GCLC)、谷氨酸半胱氨酸连接酶调节亚基(glutamate-cysteine ligase regulatory subunit, GCLM)、血红素氧合酶1(heme oxygenase-1,HO-1)、还原型辅酶/醌氧化还原酶[NAD(P)H dehydrogenase quinone 1,NQO-1]基因mRNA相对表达水平,初步探究�The present study investigated the mechanism of the Tibetan medicine Ershiwuwei Songshi Pills(ESP) against the liver injury induced by acetaminophen(APAP) in mice based on the kelch-like ECH-associated protein 1(Keap1)/nuclear transcription factor E2 related factor 2(Nrf2) and Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) p65 signaling pathways. Kunming mice were randomly divided into a blank control group, a model group, an N-acetyl-L-cysteine(NAC) group, and high-(400 mg·kg^(-1)), medium-(200 mg·kg^(-1)), and low-dose(100 mg·kg^(-1)) ESP groups. After 14 days of continuous administration, except for those in the control group, the mice were intraperitoneally injected with 200 mg·kg^(-1)APAP. After 12 h, the serum and liver tissues of mice were collected. Hematoxylin-eosin(HE) staining was performed on pathological sections of the liver, and the levels of aspartate aminotransferase(AST) and alanine aminotransferase(ALT) in the serum and the levels of glutathione(GSH), malondialdehyde(MDA), superoxide dismutase(SOD), catalase(CAT), myeloperoxidase(MPO), and total antioxidant capacity(T-AOC) in liver tissue homogenate were detected to observe and analyze the protective effect of ESP on APAP-induced liver injury in mice. The serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β), and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay(ELISA). The protein expression of Nrf2, Keap1, TLR4, and NF-κB p65 in the liver was determined by Western blot. Quantitative real-time was used to determine the mRNA expression of glutamate-cysteine ligase catalytic subunit(GCLC), glutamate-cysteine ligase regulatory subunit(GCLM), heme oxygenase-1(HO-1), and NAD(P)H dehydrogenase quinone 1(NQO-1) in the liver to explore the mechanism of ESP in improving APAP-induced liver damage in mice. As revealed by results, compared with the model group, the ESP groups showed improved liver pathological damage, decreased ALT and AST levels in the serum and MDA and MPO content in the l

关 键 词：藏族药 二十五味松石丸 对乙酰氨基酚 信号通路 核转录因子E2相关因子2 核因子-κB p65 

分 类 号：R29[医药卫生—民族医学]