原儿茶醛对环磷酰胺所致急性肝损伤的保护作用及机制研究  

Prophylactic effect and mechanism of protocatechuic aldehyde on cyclophosphamide-induced acute liver injury

作　　者：杨世瑜 高思齐 肖雨 何继江 邱昌龙 李沙 齐亚娟 李继安 储金秀 YANG Shiyu;GAO Siqi;XIAO Yu;HE Jijiang;QIU Changlong;LI Sha;QI Yajuan;LI Ji’an;CHU Jinxiu(Hebei Key Laboratory for Chronic Diseases,School of Basic Medical Sciences,North China University of Science and Technology,Tangshan 063210,China;Hebei Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Diabetes Mellitus and Its Complications,School of Traditional Chinese Medicine,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]华北理工大学基础医学院,河北省慢性疾病基础医学重点实验室,河北唐山063210 [2]华北理工大学中医学院,河北省中西医结合防治糖尿病及其并发症重点实验室,河北唐山063210

出　　处：《中草药》2024年第22期7714-7723,共10页Chinese Traditional and Herbal Drugs

基　　金：河北省自然科学基金中医药联合基金重点项目(H2023209038);河北省自然科学基金中医药联合基金重点项目(H2022209087);河北省高等学校科学技术研究项目(ZD2022141)。

摘　　要：目的 探讨原儿茶醛(protocatechuic aldehyde,PCA)对环磷酰胺(cyclophosphamide,CPA)所致急性肝损伤的预防作用及其机制。方法 分别以PCA(15、30 mg/kg)和甘草酸二铵(diammonium glycyrrhizinate,DG,67.5 mg/kg)ig小鼠,1次/d,连续14 d后,ip CPA(200 mg/kg)建立急性肝损伤小鼠模型,第2天收集小鼠血清和肝脏组织;采用生化法检测小鼠血清中丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartate aminotransferase,AST)水平以及肝脏组织匀浆中超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和丙二醛(malondialdehyde,MDA)水平;采用苏木素-伊红(hematoxylin eosin,HE)染色观察各组小鼠肝组织病理变化;TUNEL染色检测小鼠肝脏细胞凋亡情况;采用超氧阴离子荧光探针检测小鼠肝细胞内活性氧(reactive oxygen species,ROS)的表达情况;采用免疫组织化学染色检测小鼠肝细胞中核因子-E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)的易位情况;采用蛋白质免疫印迹法检测小鼠肝组织凋亡淋巴细胞瘤-2(B cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)和半胱氨酸天冬氨酸蛋白酶-3(cysteine containing cysteinyl aspartate-3,Caspase-3)的表达,及氧化应激相关蛋白Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein1,Keap1)、Nrf2、血红素加氧酶(heme oxygenase,HO-1)、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 modifier subunit,GCLM)、醌氧化还原酶1 [NAD(P)H quinone oxidoreductase 1,NQO1]的表达情况。结果 PCA可以增加肝脏指数,降低血清ALT和AST水平,减少肝组织的细胞间隙和细胞肿胀,呈剂量相关性地减轻CPA诱导的肝损伤(P<0.05、0.01、0.001);PCA能明显降低肝组织TUNEL荧光染色强度(P<0.005)。此外,蛋白质免疫Objective To explore the prophylactic effect and mechanism of protocatechuic aldehyde(PCA)against cyclophosphamide(CPA)-induced acute liver injury.Methods Mice were orally treated with PCA at dosages of 15,30 mg/kg or diammonium glycyrrhizinate(DG)at 67.5 mg/kg once daily for 14 days,followed by a single intraperitoneal injection of CPA at 200 mg/kg to establish an acute liver injury model.The serum and liver tissue were collected on the next day.The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in mice serum and superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)in liver tissue homogenate were detected by biochemical kit.Hematoxylin eosin(HE)staining was used to observe the pathological changes of liver tissue.The apoptosis of liver cells was detected by dUTP nickel end-labeled fluorescent staining(TUNEL)mediated by terminal deoxynucleotide transferase.The expression of reactive oxygen species(ROS)in hepatocytes was detected by superoxide anion fluorescence staining.Immunohistochemical staining was used to detect the translocation of nuclear factor erythroid 2-related factor 2(Nrf2)in mouse hepatocytes.Western blotting was adopted to detect the protein expression of B cell lymphocyte 2(Bcl-2),Bcl-2-associated X protein(Bax),cysteine containing cysteinyl aspartate-3(Caspase-3),Kelch-like ECH-associated protein 1(Keap1),Nrf2,heme oxygenase-1(HO-1),glutamate-cysteine ligase catalytic subunit(GCLC),glutamate-cysteine ligase modifier subunit(GCLM)and NAD(P)H quinone oxidoreductase(NQO1).Results PCA could increase liver index,decrease serum ALT and AST levels,reduce cell space and cell swelling in liver tissue,and alleviate CPA-induced liver injury in a dose-dependent manner(P<0.05,0.01,0.001).PCA significantly decreased the intensity of TUNEL fluorescence staining(P<0.001).In addition,western blotting results showed that PCA could increase the expression of Bcl-2(P<0.001)and decrease the expression of cleaved Caspase-3 and Bax(P<0.05,0.001),indica

关 键 词：原儿茶醛 肝损伤 环磷酰胺 氧化应激 细胞凋亡 Keap1/Nrf2/HO-1信号通路 

分 类 号：R285.5[医药卫生—中药学]