目的：探究枳椇水提物对对乙酰氨基酚致小鼠急性肝损伤的相关机制。方法：采用对乙酰氨基酚建立小鼠急性肝损伤模型,将大鼠分为正常组、对乙酰氨基酚（APAP）组、N-乙酰半胱氨酸（NAC）组,枳椇 水提物低、中、高剂量组,计算各组小鼠的肝脏指数及肝质比指数;全自动生化仪检测血清 ALT、AST、ALP 水平变化;HE 染色法观察小鼠肝组织病理学变化的影响;TUNEL 法检测肝细胞凋亡情况;免疫组化法检 测Bax、Bcl-2 相关蛋白的表达;Westernblot 检测Csapase-3、Caspase-9 蛋白表达水平。结果：与正常组比较,APAP 组小鼠肝脏重量及肝指数显著升高（P<0.01）,ALT、AST、ALP 水平显著升高（P<0.05）,呈现明显肝组织病理损伤,TUNEL 阳性细胞数升高（P<0.05）,Bax 蛋白表达上调（P<0.05）,Bcl-2 蛋白表达下调（P<0.05）;与 APAP 组比较,枳椇水提物低、中、高剂量组小鼠肝脏重量及肝指数降低（P<0.05）,ALT、AST、 ALP 水平显著降低（P<0.05）,肝组织损伤、炎症细胞浸润明显减轻,肝组织坏死区域明显减少,TUNEL 阳性细胞数减少（P<0.05）,Bax 蛋白表达下调（P<0.05）,Bcl-2 蛋白表达上调（P<0.05）,Caspase-3、Caspase-9 蛋白表达水平降低（P<0.05）。结论：枳椇水提物对 APAP 致小鼠急性肝损伤有保护作用,其作用机制可能与抑制肝细胞凋亡,下调促凋亡蛋白 Bax 的表达,上调抑凋亡蛋白 Bcl-2 的表达有关。
Objective：To investigate the mechanism of water extract of Hovenia Dulcis Thunb on acetaminophen induced acute liver injury in mice. Methods：The acute liver injury model of mice was established by using paracetamol. The rats were divided into normal group,acetaminophen（APAP）group,N-acetylcysteine（NAC） group and water extract of Hovenia Dulcis Thunb low,medium and high dose groups,and the liver index and liver-to-mass ratio index of each group were calculated. The changes of serum ALT,AST and ALP were detected by automatic biochemical analyzer. HE staining was used to observe the effect of histopathological changes in the liver of mice. Apoptosis of hepatocytes was detected by TUNEL assay. Immunohistochemical method was used to detect the expression of Bax and Bcl-2 related proteins. Westernblot was used to detect the expression levels of Csapase-3 and Caspase-9. Results：Compared with the normal group,the liver weight and liver index of mice in the APAP group increased significantly（P<0.01）,the ALT,AST and ALP levels increased significantly（P<0.05）,and the pathological damage of liver tissues was obvious. The number of TUNEL positive cells increased（P<0.05）,the expression of Bax protein was up-regulated（P<0.05）,and the expression of Bcl-2 protein was down-regulated（P<0.05）. Compared with APAP group,water extract of Hovenia Dulcis Thunb low,medium and high dose group mice liver weight and liver index decreased（P<0.05）,ALT,AST,ALP level decreased significantly（P<0.05）, liver tissue injury,inflammatory cells infiltration,liver tissue necrosis area decreased significantly,and the number of TUNEL positive cells decreased（P<0.05）. The Bax protein expression（P<0.05）,the Bcl-2 protein expression（P<0.05）and the expression levels of Caspase-3 and Caspase-9 decreased（P<0.05）. Conclusion：Water extract of Hovenia Dulcis Thunb has a protective effect on acute liver injury induced by APAP in mice,and its mechanism may be related to inhibiting hepatocyte apoptosis,dow N-regulating the expression of pro-apoptotic protein Bax, and up-regulating the expression of anti-apoptotic protein Bcl-2.
[1] 于歆,李晓冰,何晓静,等.药物性肝损伤的机制[J].中国医院药学杂志,2017,37(10):895
YU X,LI XB,HE XJ,et al.The mechanisms of drug induced liver injury[J].Chin J Hosp Pharm,2017,37(10):895
[2] TEMPLE RJ,HIMMEL MH.Safety of newly approved drugs: implications for prescribing[J].JAMA,2002,287(17): 2273
[3] KAPLOWITZ N.Drug-induced liver disorders:implications for drug development and regulation[J].Drug Saf,2001,24(7): 483
[4] 苏毅. 急性肝衰竭的病因与治疗研究现状[J].中国实用医药,2019,14(19):194
SU Y.Current situation of etiology and treatment of acute liver failure[J].China Pract Med,2019,14(19):194
[5] 徐连民,梁侨丽.枳椇属植物化学成分与药理活性[J].国外医药(植物药分册),2001,16(5):190
XU LM,LIANG QL.Chemical components and pharmacological activity of the raisin tree[J].World Phytomed,2001,16(5): 190
[6] 王婷婷,李华,王华,等.拐枣的功效成分及加工利用[J].中国食物与营养,2018,24(10):20
WANG TT,LI H,WANG H,et al.Research progress on ingredients function of Hovenia dulcis and its processing and utilization[J]. Food Nutr China,2018,24(10):20
[7] FANG HL,LIN HY,CHAN MC,et al.Treatment of chronic liver injuries in mice by oral administration of ethanolic extract of the fruit of Hovenia dulcis[J].Am J Chin Med,2007,35(4):693
[8] JAESCHKE H.Acetaminophen:dose-dependent drug hepatotoxicity and acute liver failure in patients[J].Dig Dis,2015,33(4): 464
[9] CHEN SH,ZHAO XL,WAN J,et al.Dihydromyricetin improves glucose and lipid metabolism and exerts anti-inflammatory effects in nonalcoholic fatty liver disease:a randomized controlled trial[J]. Pharmacol Res,2015,99:74
[10] NI HM,MITCHELL RM,CHAO XJ,et al .Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice[J].J Hepatol,2016, 65(2):354
[11] KNOCKAERT L,D ESCATOIRE V ,V ADROT N ,e t al .Mitochondrial CYP2E1 is sufficient to mediate oxidative stress and cytotoxicity induced by ethanol and acetaminophen[J].Toxicol In Vitro,2011,25(2):475
[12] 屠梦珏,魏进歌,陈鑫,等.二氢杨梅素对 APAP 诱导小鼠急性肝损伤的保护作用[J].中国药理学通报,2018,34(12): 1707
TU MJ,WEI JG,CHEN X,et al.Effect of dihydromyricetin on acetaminophen induced liver injury in mice[J].Chin Pharmacol Bull,2018,34(12):1707
[13] 宛莹,吴艳玲,廉丽花,等.虎眼万年青通过抑制 CYP2E1 和 HIF-1α 保护对乙酰氨基酚诱导的急性肝损伤[J].中国天然药物,2012,10(3):177
WAN Y,WU YL,LIAN LH,et al.Protective effect of Ornithogalum saundersiae Ait (Liliaceae) against acetaminophen-induced acute liver injury via CYP2E1 and HIF-1α[J].Chin J Nat Med,2012, 10(3):177
[14] 楚胜,李传俊.枳椇水提液改善慢性酒精性肝损伤作用研究[J].智慧健康,2019,5(25):27
CHU S,LI CJ.Study on the effect of water extract on chronic alcoholic liver injury[J].Smart Healthcare,2019,5(25):27
[15] 刘立伟,赵新颜.药物性肝损伤临床与组织病理学特点[J].实用肝脏病杂志,2019,22(2):160
LIU LW,ZHAO XY.Clinicopathological features of drug-induced liver injury[J].J Pract Hepatol,2019,22(2):160
[16] 阮豪南,王露露,孙航,等.中药有效成分减轻对乙酰氨基酚肝损伤研究进展[J].中国药学杂志,2019,54(2):81
RUAN HN,WANG LL,SUN H,et al.Research advance of effective components of traditional chinese medicine on alleviating acetaminophen-induced liver injury[J].Chin Pharm J,2019,54(2):81
[17] BUDNITZ DS,LOVEGROVE MC,CROSBY AE.Emergency department visits for overdoses of acetaminophen-containing products[J].Am J Prev Med,2011,40(6):585
[18] JAESCHKE H,DUAN LQ,AKAKPO JY,et al.The role of apoptosis in acetaminophen hepatotoxicity[J].Food Chem Toxicol,2018,8(118):709
[19] YOULE RJ,STRASSER A.The BCL-2 protein family:opposing activities that mediate cell death[J].Nat Rev Mol Cell Biol,2008, 9(1):47
[20] 朱玉山,林家凌,郝小江,等.Bcl-2 家族蛋白调控线粒体膜通透性和细胞色素 C 释放的新机制[J].生命科学,2011,23(11): 1076
ZHU YS,LIN JL,HAO XJ,et al.Functional conversion of Bcl-2 into a pro-apoptotic molecule to regulate mitochondrial cytochrome C release[J].Chin Bull Life Sci,2011,23(11):1076
[21] SULEJCZAK D,CZARKOWSKA-BAUCH J,MACIAS M,et al.Bcl-2 and Bax proteins are increased in neocortical but not in thalamic apoptosis following devascularizing lesion of the cerebral cortex in the rat:an immunohistochemical study[J].Brain Res, 2004,1006(2):133
