连翘提取物对对乙酰氨基酚诱导小鼠肝损伤的保护作用

doi:10.16431/j.cnki.1671-7236.2021.10.037

摘要/Abstract

摘要： 试验旨在探究连翘提取物（FSE）预防性干预对对乙酰氨基酚（acetaminophen，APAP）诱导小鼠肝损伤的保护作用及其潜在作用机制。采用半仿生-生物酶法提取连翘有效成分，使用APAP构建小鼠肝损伤模型。随机将60只雌性昆明小鼠分入正常对照（NC）组、APAP肝损伤模型（LD）组、连翘提取物（FSE）对照组、连翘提取物高剂量（HFSE＋LD）组、连翘提取物中剂量（MFSE＋LD）组和连翘提取物低剂量（LFSE＋LD）组，每组10只。HFSE＋LD组、MFSE＋LD组、LFSE＋LD组分别按每天200、100、50 μg/g灌胃给予连翘提取物，NC组和LD组分别灌胃等量生理盐水，每天2次，连续给药6 d。预防性给药3 d后，腹腔注射APAP，每天1次。末次给药12 h后，试验小鼠眼球采血并快速取出肝脏，检测血清中丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）活性，以评价肝损伤程度；检测肝脏匀浆液中还原型谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、丙二醛（MDA）和过氧化氢（H₂O₂）水平，以评价肝脏氧化应激程度；制作肝脏病理切片，经苏木精伊红（HE）染色后观察肝脏病理变化；采用探针药物法测定肝脏线粒体细胞色素P4502E1（CYP2E1）活性；采用实时荧光定量PCR检测肝脏线粒体CYP2E1 mRNA表达水平；采用Western blotting法检测肝脏CYP2E1蛋白表达情况。结果表明：与NC组相比，LD组小鼠血清中ALT、AST活性均显著增加（P<0.05）；肝脏SOD活性、GSH含量均显著降低（P<0.05），MDA、H₂O₂含量，CYP2E1 mRNA及蛋白表达水平均显著升高（P<0.05），成功构建小鼠肝损伤模型。200、100和50 μg/g的连翘提取物可显著降低肝损伤小鼠血清ALT和AST活性（P<0.05），显著提高肝脏中SOD活性（P<0.05）；200、100 μg/g连翘提取物可显著提高肝脏中GSH水平（P<0.05），显著降低肝脏中MDA、H₂O₂水平及CYP2E1的mRNA和蛋白表达量（P<0.05）。以上结果表明，连翘提取物可减轻APAP诱导的小鼠肝损伤程度且呈剂量依赖性，其潜在作用机制可能与所含活性物质的抗氧化作用以及对CYP2E1酶活性和表达的抑制有关。

关键词: 连翘提取物; 对乙酰氨基酚; 细胞色素P4502E1(CYP2E1); 肝损伤

Abstract: This study was aimed to explore the protective effect and potential mechanism of Forsythiae suspensa extract (FSE) preventive intervention on acetaminophen (APAP)-induced liver injury in mice. The semi-bionic enzyme method was used to extract the active components of Forsythia suspense, and the mouse liver injury model was constructed by APAP in this experiment. 60 female Kunming mice were randomly divided into 6 groups:normal control(NC) group, APAP liver injury model (LD) group, Forsythia suspense extract (FSE) group, high-dose Forsythia suspense extract (HFSE+LD) group, medium-dose Forsythia suspense extract (MFSE+LD) group and low-dose Forsythia suspense extract (LFSE+LD) group. 10 mice for each group. The HFSE+LD group, the MFSE+LD group, and the LFSE+LD group were intragastrically administrated with Forsythia suspense extract 200, 100, and 50 μg/g, respectively, twice a day, for 6 consecutive days. The NC group and the LD group were given the same amount of normal saline. After preventive administration with FSE for 3 days, APAP was injected intraperitoneally once a day. Blood samples were collected from the eyeball, and the liver was quickly taken out 12 h after the last administration. Serum aminotransferase (ALT) and aspartate aminotransferase (AST) were detected to evaluate the degree of liver injury. The homogenates of liver glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were detected to evaluate the degree of liver oxidative stress of liver. Liver samples were visualized by hematoxylin and eosin staining. Furthermore, liver mitochondrial cytochrome P4502E1 (CYP2E1) activity were measured by probe drug method. The expression level of liver mitochondrial CYP2E1 mRNA was detected by Real-time quantitative PCR and the liver CYP2E1 protein expression was detected by Western blotting. The results showed that:Compared with NC group, the ALT and AST activities in serum were increased significantly (P<0.05), the SOD activity and GSH content in liver were significantly reduced (P<0.05), the MDA and H₂O₂ contents, and the expression of CYP2E1 mRNA and protein in liver were significantly increased (P<0.05) in LD group. The mouse liver injury model was successfully established. 200, 100, and 50 μg/g FSE treatment significantly reduced the serum ALT and AST activities in mice with liver injury (P<0.05), and significantly increased the liver SOD avtivity (P<0.05). 200 and 100 μg/g FSE significantly increased the GSH level in liver (P<0.05), and significantly reduced the level of MDA, H₂O₂ and the expression of CYP2E1 mRNA and CYP2E1 protein in liver (P<0.05). The results showed that FSE reduced APAP-induced mice liver injury in a manner of dose-dependent, and its potential mechanism was related to the antioxidant effect and the inhibition of CYP2E1 enzyme activity and expression of the active substances.

Key words: Forsythiae suspensa extract; acetaminophen; cytochrome P4502E1 (CYP2E1); liver injury

中图分类号:

S853.74

赵晨栋, 王萌, 张昊, 代国年, 安志霞, 沈雅丽, 王桂荣. 连翘提取物对对乙酰氨基酚诱导小鼠肝损伤的保护作用[J]. 中国畜牧兽医, 2021, 48(10): 3845-3854.

ZHAO Chendong, WANG Meng, ZHANG Hao, DAI Guonian, AN Zhixia, SHEN Yali, WANG Guirong. Protective Effect of Forsythiae suspensa Extract on Acetaminophen-induced Liver Injury in Mice[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3845-3854.

参考文献

[1] 侯健, 孙娥, 宋捷, 等.肝脏药物代谢酶CYP450与中药肝毒性的关系[J]. 中国中药杂志, 2016, 41(15):2774-2780. HOU J, SUN E, SONG J, et al.Relationship between hepatic drug-metabolizing enzymes CYP450 and traditional Chinese medicine-induced hepatotoxicity[J]. China Journal of Chinese Materia Medica, 2016, 41(15):2774-2780.(in Chinese)
[2] 王玉琳, 柳雨亭, 陶艳艳, 等.以线粒体为靶点筛选中药抗肝毒性的活性成分研究思路与方法[J]. 中国中药杂志, 2021, 46(2):306-311. WANG Y L, LIU Y T, TAO Y Y, et al.Research ideas and method on screening active components of traditional Chinese medicine against hepatotoxicity with mitochondria as target[J]. China Journal of Chinese Materia Medica, 2021, 46(2):306-311.(in Chinese)
[3] LEE W M.Acetaminophen (APAP) hepatotoxicity-Isn't it time for APAP to go away?[J]. Journal of Hepatology, 2017, 67:1324-1331.
[4] 李会英, 潘家琪, 汤治元.扑热息痛肝毒性中炎症反应的研究进展[J]. 中国现代应用药学, 2014, 31(6):763-767. LI H Y, PAN J Q, TANG Z Y.Progress of inflammation in liver toxicity caused by acetaminophen[J]. Chinese Journal of Modern Applied Pharmacy, 2014, 31(6):763-767.(in Chinese)
[5] ZHAO Z, WEI Q Y, HUA W W, et al.Hepatoprotective effects of berberine on acetaminophen-induced hepato-toxicity in mice[J]. Biomedicine and Pharmacotherapy, 2018, 103:1319-1326.
[6] AKAKPO J Y, RAMACHANDRAN A, ORHAN H, et al.4-methylpyrazole protects against acetaminophen-induced acute kidney injury[J]. Toxicology and Applied Pharmacology, 2020, 409:115317.
[7] NGUYEN N T, DU K, AKAKPO J Y, et al.Mitochondrial protein adduct and superoxide generation are prerequisites for early activation of c-Jun N-terminal kinase within the cytosol after an acetaminophen overdose in mice[J]. Toxicology Letters, 2020, 338:21-31.
[8] LUO G W, SHEN Y T, YANG L Z, et al.A review of drug-induced liver injury databases[J]. Archives of Toxicology, 2017, 91(3):3039-3049.
[9] KNOCKAERT L, DESCATOIRE V, VADROT N, et al.Mitochondrial CYP2E1 is sufficient to mediate oxidative stress and cytotoxicity induced by ethanol and acetaminophen[J]. Toxicology in vitro, 2011, 25:475-484.
[10] 陈克广.CYP2E1/microRNA-378a-3p途径在对乙酰氨基酚及雷公藤多苷诱导的肝损伤中的作用及机制研究[D].济南:山东大学, 2020. CHEN K G.Function and mechanism of CYP2E1/microRNA-378a-3p in acetaminophen and tripterygium glycosides-induced liver injury[D].Jinan:Shandong University, 2020.(in Chinese)
[11] 李会方.MBL在对乙酰氨基酚所致小鼠肝损伤中的保护作用及其机制研究[D].广州:南方医科大学, 2018. LI H F.Protective effects of MBL on APAP-induced liver injury and its potential mechanism[D].Guangzhou:Southern Medical University, 2018.(in Chinese)
[12] CHOWDHURY A, LU J H, ZHANG R M, et al.Mangiferin ameliorates acetaminophen-induced hepato-toxicity through APAP-Cys and JNK modulation[J]. Biomedicine & Pharmacotherapy, 2019, 117:109097.
[13] XIE W Y, JIANG Z H, WANG J, et al.Protective effect of hyperoside against acetaminophen (APAP) induced liver injury through enhancement of APAP clearance[J]. Chemico-Biological Interactions, 2016, 246:11-19.
[14] CAI Y, SUN W, ZHANG X X, et al.Osthole prevents acetaminophen-induced liver injury in mice[J]. Acta Pharmacologica Sinica, 2018, 39:74-84.
[15] MINSART C, RORIVE S, LEMMERS A, et al.N-acetylcysteine and glycyrrhizin combination:Benefit outcome in a murine model of acetaminophen-induced liver failure[J]. World Journal of Hepatology, 2020, 12:596-618.
[16] 国家药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社, 2015. NATIONAL PHARMACOPOEIA COMMITTE.Chinese Pharmacopoeia[M].Beijing:China Medical Science and Technology Press, 2015.(in Chinese)
[17] 郝敏.河南产连翘饮片的质量分析研究[D].郑州:河南中医药大学, 2016. HAO M.Study on the quality analysis of Forsythiae fructus from Henan province[D].Zhengzhou:Henan University of Chinese Medicine, 2016.(in Chinese)
[18] 王福男.中药连翘的化学成分研究[D].北京:北京协和医学院, 2009. WANG F N.Study on the chemical constituents of Forsythia suspense (Thunb.) vahl[D].Beijing:Peking Union Medical College, 2009.(in Chinese)
[19] 田燕泽.中药连翘化学成分分离及其抗氧化活性评价[D].北京:中央民族大学, 2011. TIAN Y Z.Isolation of chemical components of Chinese medicine Forsythia and evaluation of antioxidant activity[D].Beijing:Minzu University of China, 2011.(in Chinese)
[20] 曲欢欢.连翘化学成分和生物活性研究[D].西安:西北大学, 2008. QU H H.Chemical composition of Forsythia suspense and biological activity study[D].Xi'an:Northwest University, 2008.(in Chinese)
[21] 原江锋, 赵君峰, 孙军杰, 等.河南、山西连翘叶黄酮类和三萜酸类化合物含量比较[J]. 食品科学, 2015, 36(10):164-167. YUAN J F, ZHAO J F, SUN J J, et al.Determination of flavonoids and triterpene acids in Forsythia suspensa leaves from Henan and Shanxi provinces[J]. Food Science, 2015, 36(10):164-167.(in Chinese)
[22] YUAN A, GONG L H, LUO L, et al.Revealing anti-inflammation mechanism of water-extract and oil of Forsythiae fructus on carrageenan-induced Edema rats by serum metabolomics[J]. Biomedicine & Pharmacotherapy, 2017, 95:929-937.
[23] HU N H, GUO C C, DAI X Y, et al.Forsythiae fructuse water extract attenuates liver fibrosis via TLR4/MyD88/NF-κB and TGF-β/smads signaling pathways[J]. Journal of Ethnopharmacology, 2020, 262:113275.
[24] ZHAO P F, PIAO X S, PAN L, et al.Forsythia suspensa extract attenuates lipopolysaccharide-induced inflammatory liver injury in rats via promoting antioxidant defense mechanisms[J]. Animal Science Journal, 2017, 88:873-881.
[25] ZHANG Y, MIAO H, YAN H Y, et al.Hepatoprotective effect of Forsythiae Fructus water extract against carbon tetrachloride-induced liver fibrosis in mice[J]. Journal of Ethnopharmacology, 2018, 218:27-34.
[26] 薛璇玑, 罗俊, 张新新, 等.半仿生酶法提取柿叶中总黄酮的工艺筛选及优化[J]. 中国药房, 2017, 28(13):1813-1816. XUE X J, LUO J, ZHANG X X, et al.Screening and optimization of the extraction technology of total flavonoids in persimmon leaves by semi-bionic-enzyme method[J]. China Pharmacy, 2017, 28(13):1813-1816.(in Chinese)
[27] LAUSCHKE V M, Toxicogenomics of drug induced liver injury-from mechanistic understanding to early prediction[J]. Drug Metabolism Reviews, 2021, 53(2):245-252.
[28] TESCHKE R.Idiosyncratic DILI:Analysis of 46, 266 cases assessed for causality by RUCAM and published from 2014 to early 2019[J]. Frontiers in Pharmacology, 2019, 10:730.
[29] 刘文静, 高进贤, 钱倩, 等.白皮杉醇抑制内质网应激保护四氯化碳诱导的小鼠急性肝损伤[J]. 中成药, 2020, 42(12):3158-3165. LIU W J, GAO J X, QIAN Q, et al.Hepato protective effect of piceatannol against CCl4-induced acute liver injury in mice via ERS pathway inhibition[J]. Chinese Traditional Patent Medicine, 2020, 42(12):3158-3165.(in Chinese)
[30] 王瑞, 黄力勤, 张婧, 等.实施核酸检测后重新评估献血者ALT的检测功效[J]. 中国输血杂志, 2017, 30(9):1034-1038. WANG R, HUANG L Q, ZHANG J, et al.Reevaluation the effect of ALT on blood safety in nucleic acid era[J]. Chinese Journal of Blood Transfusion, 2017, 30(9):1034-1038.(in Chinese)
[31] 崔红权.血清ALT、AST、GGT检测在肝脏疾病诊断中应用研究[J]. 国际检验医学杂志, 2017, 38(16):2293-2295. CUI H Q.Study on the application of serum ALT, AST, GGT detection in the diagnosis of liver diseases[J]. International Journal of Laboratory Medicine, 2017, 38(16):2293-2295.(in Chinese)
[32] 孔文丽, 杨以良, 徐洪芹, 等.慢性丙型肝炎患者抗病毒治疗中肝功能与病毒学应答的关系[J]. 吉林大学学报(医学版), 2016, 42(01):99-103. KONG W L, YANG Y L, XU H Q, et al.Relationship between liver function and virological response in patients with chronic hepatitis C during antiviral treatment[J]. Journal of Jilin University(Medicine Edition), 2016, 42(01):99-103.(in Chinese)
[33] 张秀丽.ALT与AST比值预测慢性丙型肝炎患者肝脂肪变性的可行性分析[J]. 临床肝胆病杂志, 2017, 33(6):1096-1100. ZHANG X L.Feasibility of alanine aminotransferase/aspartate aminotransferase ratio in predicting hepatic steatosis in chronic hepatitis C patients[J]. Journal of Clinical Hepatology, 2017, 33(6):1096-1100.(in Chinese)
[34] MASSART J, BEGRICHE K, FROMENTY B.Cytochrome P4502E1 should not be neglected for acetaminophen-induced liver injury in metabolic diseases with altered insulin levels or glucose homeostasis[J]. Gastroentérologie Clinique et Biologique, 2021, 45:101470.
[35] 陈军宝, 徐智, 覃小艳, 等.扑热息痛肝损伤机制的研究进展[J]. 实用医学杂志, 2012, 28(20):3479-3480. CHEN J B, XU Z, QIN X Y, et al.Research progress on liver injury mechanism of paracetamol[J]. The Journal of Practical Medicine, 2012, 28(20):3479-3480.(in Chinese)
[36] MCGILL M R, JAESCHKE H.Metabolism and disposition of acetaminophen:Recent advances in relation to hepatotoxicity and diagnosis[J]. Pharmaceutical Research, 2013, 30(9):2174-2187.
[37] ANTOINE D J, WILLIAMS D P, PARK B K.Understanding the role of reactive metabolites in drug-induced hepatotoxicity:State of the science[J]. Expert Opinion on Drug Metabolism & Toxicology, 2008, 4(11):1415-1427.
[38] 任利君, 弥曼, 宁养红, 等.硝酸甘油减轻对乙酰氨基酚肝毒性的作用与机制[J]. 第四军医大学学报, 2008(17):1565-1568. REN L J, MI M, NING Y H, et al.Effects and mechanisms of glyceryl trinitrate on alleviation of acetaminophen hepatotoxicity[J]. Journal of the Fourth Military Medical University, 2008(17):1565-1568.(in Chinese)
[39] CAI C C, HUANG H, WHELAN S, et al.Benzyl alcohol attenuates acetaminophen-induced acute liver injury in a Toll-like receptor -4-dependent pattern in mice[J]. Hepatology, 2014, 60:990-1002.
[40] TORRES S, BAULIES A, INSAUSTI-URKIA N, et al.Endoplasmic reticulum stress-induced upregulation of STARD1 promotes acetaminophen-induced acute liver failure[J]. Gastroenterology, 2019, 157(2):552-568.
[41] 郭波, 唐秀玲, 石磊, 等.液相色谱-串联质谱法同时测定对乙酰氨基酚及其相关代谢产物[J]. 中国药师, 2017, 20(4):597-602. GUO B, TANG X L, SHI L, et al.Simultaneous determination of acetaminophen and its related metabolites by LC-MS/MS[J]. China Pharmacist, 2017, 20(4):597-602.(in Chinese)
[42] HOLT M P, JU C.Mechanisms of drug-induced liver injury[J]. AAPS Journal, 2006, 8(1):E48-54.
[43] CHENG D, GONG X Y, WU Q, et al.High-selectivity fluorescent reporter toward peroxynitrite in a coexisting nonalcoholic fatty liver and drug-induced liver diseases model[J]. Analytical Chemistry, 2020, 92(16):11396-11404.
[44] 黎明, 王妹芳, 吴宝珍, 等.白芷乙素通过抑制ERK1/2和NF-κB p65的活化缓解心肌缺血再灌注大鼠心肌损伤和氧化应激[J]. 安徽医科大学学报, 2021, 56(2):249-255. LI M, WANG M F, WU B Z, et al.Imperatorin alleviates myocardial injury and oxidative stress in myocardial ischemia-reperfusion rats via inhibiting the activation of ERK1/2 and NF-κB p65[J]. Acta Universitatis Medicinalis Anhui, 2021, 56(2):249-255.(in Chinese)
[45] 代国年, 王桂荣, 王萌, 等.车前子提取物抑制扑热息痛诱导小鼠肝损伤的作用研究[J]. 西北农林科技大学学报(自然科学版), 2020, 48(7):27-36. DAI G N, WANG G R, WANG M, et al.Inhibitory effect of active components of Plantago asiatica extraction on acetaminophen-induced liver injury in mice[J]. Journal of Northwest A&F University(Natural Science Edition), 2020, 48(7):27-36.(in Chinese)
[46] WU C T, DENG J S, HUANG W C, et al.Salvianolic acid C against acetaminophen-induced acute liver injury by attenuating inflammation, oxidative stress, and apoptosis through inhibition of the Keap1/Nrf2/HO-1 signaling[J]. Oxidative Medicine and Cellular Longevity, 2019, 2019:9056845.