芳樟醇调控Nrf2/HO-1抑制黄曲霉毒素B_(1)所致肝损伤的实验  

Experimental Study on Regulation of Nrf2/HO-1 by Linalool to Inhibit Hepatic Injury Induced by Aflatoxin B_(1)

作　　者：王萌 薛春苗[1] 黄鑫 刘文卉 高若瑜 白雪慧 华国栋 朱宝琛 WANG Meng;XUE Chunmiao;HUANG Xin;LIU Wenhui;GAO Ruoyu;BAI Xuehui;HUA Guodong;ZHU Baochen(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]北京中医药大学中药学院,北京102488

出　　处：《中国实验方剂学杂志》2024年第14期89-96,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医药管理局高水平中医药重点学科建设项目临床中药学(zyyzdxk-2023257)。

摘　　要：目的:探讨芳樟醇对黄曲霉毒素B(1 AFB1)诱导的大鼠急性肝损伤的保护作用,并初步探讨其保护机制。方法:20只雄性SPF级SD大鼠,采用随机数字表法分为3组:正常组(n=6),AFB1组(n=7),芳樟醇组(n=7)。预防性给予芳樟醇溶液(200 mg·kg^(-1))持续14 d,正常组和AFB1组给予等体积双蒸水灌胃。芳樟醇预防性给药结束后,连续2 d腹腔注射AFB1(1 mg·kg^(-1),溶于生理盐水)溶液,以建立大鼠急性肝损伤模型。模型建立14 d后进行样本采集和处理。苏木素-伊红(HE)染色和马松(Masson)染色观察大鼠肝组织病理学改变;生化检测各组大鼠的丙氨酸氨基转氨酶(ALT)、天冬氨酸转氨酶(AST)、γ-谷氨酰转移酶(GGT)、乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)、总胆红素(TBil)、直接胆红素(DBil)和间接胆红素(IBil)、丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽(GSH)和肝组织中的Fe^(3+)和Fe^(2+)含量;蛋白免疫印迹法(Wester blot)检测核转录因子红系2相关因子2(Nrf2)和血红素氧合酶-1(HO-1)蛋白的表达水平;分子对接技术验证芳樟醇与Nrf2/HO-1信号通路关键蛋白之间的结合性能;分子动力学技术验证芳樟醇与Nrf2/HO-1信号通路关键蛋白结合能的稳定性和亲和力。结果:病理结果表明,与AFB1组比较,芳樟醇组肝脏结构趋向正常,蓝色胶原纤维显著减少;芳樟醇组的ALT、AST、GGT、LDH含量及ALP、TBil、DBil、IBil水平均显著降低(P<0.01),肝组织中的Fe^(3+)和Fe^(2+)含量及氧化应激产物MDA的水平显著降低(P<0.01);抗氧化物质SOD水平、CAT含量和GSH含量则显著增加(P<0.01);分子对接结果显示芳樟醇与Nrf2、HO-1靶点结合的分子对接能分别为-5.4956、-5.1994 kcal·mol^(-1)(1 cal≈4.186 J);分子动力学结果显示芳樟醇与Nrf2、HO-1结合能与亲和力较强。Western blot结果显示芳樟醇组中Nrf2蛋白的表达水平明显升高(P<0.05),而HO-1蛋白的表达水平显著降低(P<0.01)。结Objective:To investigate the effect of linalool against acute liver injury induced by aflatoxin B1(AFB1)in rats and explore its protective mechanism.Method:Twenty male SPF SD rats were randomly divided into three groups:Control(n=6),AFB1(n=7),and linalool(n=7)groups.Linalool solution(200 mg·kg^(-1))was administered preventatively for 14 days,while the control and AFB1 groups intragastrically received an equivalent volume of double distilled water.After preventative administration of linalool,AFB1 solution(1 mg·kg^(-1),dissolved in saline)was intraperitoneally injected for two consecutive days to induce acute liver injury in rats.Samples were collected and processed 14 days after model establishment.Pathological changes in liver tissue of rats were observed using Hematoxylin-eosin(HE)staining and Masson staining.Biochemical detection was performed to measure the levels of alanine transaminase(ALT),aspartate transaminase(AST),γ-glutamyl transferase(GGT),lactate dehydrogenase(LDH),alkaline phosphatase(ALP),total bilirubin(TBil),direct bilirubin(DBil),indirect bilirubin(IBil),malondialdehyde(MDA),superoxidedismutase(SOD),catalase(CAT),glutathione(GSH),Fe^(3+),and Fe^(2+)in the liver tissue.Western blot was adopted to assess protein expression levels of nuclear factor-erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1).Molecular docking was performed to verify the binding between linalool and key proteins of the Nrf2/HO-1 signaling pathway.Molecular dynamics techniques were used to confirm the stability and affinity of linalool binding with key proteins of the Nrf2/HO-1 signaling pathway.Result:Pathological results showed that compared to that in the AFB1 group,the liver structure in the linalool group tended to be normal,with a significant decrease in blue collagen fibers.The linalool group exhibited significantly reduced levels of ALT,AST,GGT,LDH,ALP,TBil,DBil,and IBil(P<0.01),Fe^(3+)and Fe^(2+)content,and oxidative stress marker MDA(P<0.01).The levels of antioxidants SOD,CAT,and GSH significantly increa

关 键 词：黄曲霉毒素B1 芳樟醇 急性肝损伤 核转录因子红系2相关因子2(Nrf2)/血红素氧合酶-1(HO-1) 铁死亡 氧化应激 

分 类 号：R284.2[医药卫生—中药学] R285[医药卫生—中医学] R289R287R22R2-031R33R24