乔松素对对乙酰氨基酚诱发的肝损伤小鼠模型的保护作用

目的探索乔松素(PIN)对对乙酰氨基酚(APAP)诱发肝损伤小鼠模型的保护作用。方法50只健康雄性C57BL/6J小鼠随机分成空白组、PIN(50 mg/kg)组、APAP(300 mg/kg)模型组、PIN(30 mg/kg)+APAP(300 mg/kg)组和PIN(50 mg/kg)+APAP(300 mg/kg)实验组,每组各10只。各组采取灌胃给药,空白对照组和模型组给予等量的生理盐水,PIN组及PIN+APAP组每日给药1次,连续给药7 d。末次给药2 h后,模型组和PIN+APAP组腹腔注射APAP 300 mg/kg 1次,空白组和PIN组腹腔注射等量生理盐水。收集血清,检测ALT、AST水平,肝组织匀浆检测丙二醛(MDA)、超氧化物歧化酶(SOD)及谷胱甘肽(GSH)生化指标,HE染色观察肝脏组织病理情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与空白组比校,APAP(300 mg/kg)模型组的ALT、AST水平显著升高(P<0.01),模型构建成功;PIN(30 mg/kg)+APAP(300 mg/kg)及PIN(50 mg/kg)+APAP(300 mg/kg)组的ALT、AST与APAP(300 mg/kg)模型组相比均显著降低(P值均<0.01)。与空白组比校,APAP(300 mg/kg)模型组小鼠肝脏中MDA的水平显著升高(P<0.01),且SOD活性及GSH水平显著降低(P<0.01);与APAP(300 mg/kg)模型组比较,PIN(30 mg/kg)+APAP(300 mg/kg)及PIN(50 mg/kg)+APAP(300 mg/kg)组的小鼠肝脏中MDA的水平显著降低(P值均<0.05),且SOD活性及GSH水平显著升高(P值均<0.05)。组织病理观察显示PIN可明显改善APAP对肝组织的损伤,使肝组织形态相对正常。结论PIN对APAP诱发的小鼠肝损伤具有明显的保护作用,其保护作用可能与抑制肝脏氧化应激相关。     

藏红花醛对脂多糖诱导的脓毒症相关肝损伤小鼠模型的作用及其机制

目的 探讨藏红花醛对脂多糖（LPS）诱导的脓毒症相关肝损伤（SRLI）小鼠模型的保护作用及其机制。方法 采用简单随机分组法将32只实验用C57BL/6雄性小鼠分为对照组、单药组、模型组和治疗组,每组各8只。单药组和治疗组腹腔注射藏红花醛（60 mg/kg）预处理7天,模型组和治疗组腹腔注射LPS(10 mg/kg)诱导急性肝损伤。检测各组小鼠血清ALT、AST活性,HE染色观察肝组织切片标本,免疫组化分析信号通路下游蛋白血红素加氧酶-1(HO-1)的表达差异,TUNEL法分析肝细胞凋亡情况,Western Blot法分析肝组织总蛋白[核因子NF-E2相关因子2(Nrf2)、HO-1]表达差异。采用藏红花醛（100μmol/L）预处理人类肝细胞系L02细胞,通过LPS 100 ng/mL诱导急性肝细胞损伤,DCFH-DA荧光标记氧自由基。结果 藏红花醛预处理后,治疗组小鼠ALT、AST明显低于模型组（P值均<0.01）,治疗组小鼠肝脏维持了较为完整的假小叶结构且坏死面积更小。治疗组小鼠经藏红花醛+LPS处理后肝组织Nrf2、HO-1表达水平相较于模型组明显升高（P值均<0.0...     

线粒体靶向抗氧化剂SS-31对脓毒症小鼠模型急性肝损伤的影响

目的 探究线粒体靶向抗氧化剂SS-31对脓毒症小鼠模型急性肝损伤的作用。方法 将24只C57BL/6J成年雄性小鼠随机分为control 组、control+SS-31组、脂多糖(LPS)组(脓毒症组)、LPS+SS-31组,每组6只。小鼠腹腔注射LPS(10 mg/kg)制备脓毒症急性肝损伤模型或对照组腹腔注射等体积PBS液,继之腹腔注射SS-31(10 mg/kg)进行治疗或对照组腹腔注射等体积生理盐水, 12 h后ELISA法检测ALT、AST、活性氧(ROS)、超氧化物歧化酶(SOD)、TNFα、IL-1β、IL-6水平,HE染色观察肝脏组织病理学。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 与LPS组相比,LPS+SS-31组小鼠血清中ALT[(140.05±12.22) U/L vs (102.64±8.75)U/L]、AST[(80.22±4.71)U/L vs (69.26±5.37)U/L],以及肝组织中ROS[(1 030.21±115.87) pg/mL vs (847.84±63.65) pg/mL]、TNFα[(767.18±...     

橙皮苷对扑热息痛和四氯化碳所致小鼠急性肝损伤的保护作用

目的 探讨橙皮苷（HDN）对扑热息痛（APAP）和四氯化碳（CCl4）所致小鼠急性肝损伤的保护作用.方法 将50只小鼠随机分为正常组、APAP组、CCl4组、HDN＋APAP组、HDN＋CCl4组,每组各10只.HDN＋APAP组和HDN＋ CCl4组首先均给予HDN悬浊液500 mg/kg灌胃,其他各组分别给予同体积的0.5％羧甲基纤维素钠（CMC-Na）溶液灌胃,均1次/d,连续10 d.末次给药2h后,APAP组和HDN＋ APAP组腹腔注射APAP 175mg/kg,CCI4组和HDN＋ CCl4组腹腔注射0.4％ CCl40.1 mL/10 g制备急性肝损伤模型;正常组腹腔注射同体积0.5％ CMC-Na溶液.制模16 h后处死各组大鼠,测定血清ALT、AST、LDH水平,光镜下观察肝组织病理组织学变化.结果 HDN＋APAP组、HDN＋CCl4组血清ALT、AST、LDH水平低于APAP组及CCl4组（P＜0.05或0.01）,肝组织病理损伤程度亦轻于APAP组、CCl4组.结论 HDN对APAP和CCl4导致的急性肝损伤均具有一定的保护作用.     

小鼠肝库普弗细胞铁蓄积对急性肝损伤的影响

目的探究肝库普弗细胞铁蓄积对CCl_4致小鼠急性肝损伤的影响。方法雄性FPN-/-cre小鼠(肝库普弗细胞铁蓄积小鼠)和FPN-/-小鼠(正常小鼠)各16只,分别随机分为两组,每组8只,两对照组腹腔注射橄榄油,两实验组腹腔注射CCl_4。计算小鼠肝脏指数;测定血清中ALT、AST水平和肝组织MDA、SOD、GSH含量;肝组织HE染色进行病理学观察。结果两对照组肝脏指数、ALT、AST、MDA、SOD、GSH差异无统计学意义(P0.05),FPN-/-cre实验组肝脏指数、ALT、AST、MDA高于FPN-/-实验组,差异有统计学意义(P0.05)。FPN-/-cre实验组SOD、GSH水平低于FPN-/-实验组,差异无统计学意义(P0.05)。两对照组肝细胞结构完整,排列有序;FPN-/-实验组肝细胞结构破裂、排列紊乱;FPN-/-cre实验组肝细胞结构破裂、溶解、排列杂乱、包浆松散。结论肝库普弗细胞铁蓄积会加重CCl_4所致的急性肝损伤,可能与铁的过氧化作用有关。     

LncRNA NEAT1缺乏增加对乙酰氨基酚诱导的急性肝损伤的机制研究

目的探讨长链非编码RNA(LncRNA)核富含丰富的转录本1(NEAT1)在对乙酰氨基酚(APAP)诱导小鼠急性肝损伤中的分子机制。方法 8只C57BL/6小鼠随机分为实验组和对照组,每组各四只。通过腹腔注射APAP诱导急性肝损伤,进行谷丙转氨酶(ALT)、谷草转氨酶(AST)检测以及组织学分析来评估APAP诱导的肝损伤的程度。提取小鼠肝组织RNA检测LncRNA NEAT1表达变化。提取小鼠原代肝细胞并培养,用小干扰RNA敲低LncRNA NEAT1,并用APAP刺激小鼠原代肝细胞,通过检测细胞培养上清ALT和AST的表达量来评估肝细胞损伤程度。结果在体实验显示,与对照组相比,APAP处理组小鼠血清中的ALT[(42.75±2.3)U/L比(7565±763.3)U/L,(t=9.854,P0.05)]和AST[(45.05±4.1)U/L比(7718±901.6)U/L,(t=8.510,P0.05)]显著增加;APAP处理组小鼠肝组织LncRNA NEAT1的表达量比对照组表达增高3倍(P0.05)。在体外实验中,原代肝细胞培养基加入APAP刺激24 h后提取RNA,LncRNA NEAT1的表达量显著增加;使用siRNA敲低肝细胞LncRNA NEAT1的表达并加入APAP刺激,与对照组相比,实验组肝细胞上清的ALT、AST显著增加。结论敲低LncRNA NEAT1的表达能够增加APAP诱导的急性肝损伤,提示LncRNA NEAT1可能具有防治APAP诱导的急性肝损伤的作用。     

血浆联合复方甘草酸苷注射液对LPS/D-GalN诱发的小鼠肝损伤的影响

目的:探讨血浆联合复方甘草酸苷注射液对肝损伤小鼠模型肝损伤的影响。方法:将40只清洁型ICR雄性小鼠随机分为4组(n=10):对照组:仅用生理盐水处理;LPS/D-galN组:仅用LPS/D-GalN处理(LPS50mg/ml,D-GalN 500mg/ml);LPS/DgalN+复方甘草酸苷(CG)组:在LPS/D-GalN诱导后2,4,8h腹腔注射5mg/ml的CG注射液3次;LPS/DgalN+复方苷草酸苷和血浆(CG和Plamsa)组:在LPS/D-GalN诱导后2,4,8h尾静脉注射150μl血浆3次,并腹腔注射CG注射液3次。12h后牺牲小鼠,收集血清和肝组织样本,利用AST和ALT检测试剂盒检测血清中AST和ALT的水平,ELISA法检测肝组织中IL-6、TNF-α和NF-κB的表达变化;肝组织进行HE染色,显微镜下观察肝组织病理学变化。结果:与LPS/D-GalN组对比,LPS/D-GalN+CG组和LPS/D-GalN+CG和Plasma组能够显著降低血清中AST和ALT水平(P0.05);炎症相关因子IL-6和TNF-α,转录因子NF-κB水平也明显降低(P0.05)。结论:血浆联合复方甘草酸苷注射液通过抑制炎症相关因子IL-6和TNF-α,并降低转录因子NF-κB的表达,改善LPS/D-GalN诱发的小鼠肝损伤。     

Toll样受体-4小干扰RNA预处理防治小鼠急性肝损伤

目的 观察Toll样受体(TLR)-4小干扰RNA(siRNA)对D-氨基半乳糖盐酸盐/月旨多糖(D-GalN/LPS)诱导的肝损伤小鼠的保护作用.方法 150只雄性C57BL/6小鼠均分为PBS预处理组、阴性对照质粒预处理组、TS4预处理组、TS6预处理组和TS7预处理组.腹腔内联合注射LPS(10 ng/g)及D-GalN(1 mg/g)诱导小鼠急性肝损伤.在D-GalN/LPS联合注射前24及48 h通过尾静脉高压水注射法导人siRNA质粒50 mg/L.末端脱氧核苷酸转移酶介导的dUTP缺口标记术(TUNEL)检测细胞凋亡水平,免疫组织化学法检测肝组织中TLR-4表达,RT-PCR检测TLR-4、TNF-α及巨噬细胞炎性蛋白(MIP)-2 mRNA水平,ELISA检测小鼠血清中MIP-2及TNF-α水平,标准自动分析仪检测血清中ALT及AST水平,苏木精-伊红染色观察肝脏组织学变化,Fisher确切概率法比较各组间小鼠存活率.结果 TLR-4 siRNA可减轻肝细胞坏死、减轻炎性反应,并可显著降低血清转氨酶水平.TS4预处理组(0.065±0.015)比PBS预处理组(0.346±0.062)的TUNEL标记指数(LI)明显降低(t=9.796,P<0.05).TLR-4 siRNA预处理下调TLR-4 mRNA及蛋白表达水平,显著降低TNF-α及MIP-2 mRNA表达及细胞因子水平,显著降低D-GalN/LPS联合诱导的急性肝损伤C57BL/6小鼠的死亡率和肝损伤.结论 TLR-4 siRNA抑制TLR-4表达在防治肝损伤方面可能具有潜在应用价值.     

氧化应激介导糖原合成酶激酶-3促进D-氨基半乳糖联合脂多糖诱导的急性肝衰竭肝损伤

目的探讨糖原合成酶激酶3(GSK3)在氧化应激促进D-氨基半乳糖联合脂多糖诱导小鼠急性肝衰竭肝损伤中的作用。方法以C57BL/6小鼠为研究对象,腹腔注射D-氨基半乳糖(D-Gal N)联合脂多糖(LPS)建立小鼠急性肝衰竭模型。氧化应激抑制剂N-乙酰半胱氨酸(NAC)或GSK3特异性抑制剂SB216763分别对急性肝衰竭小鼠模型进行干预。检测小鼠血清转氨酶ALT、AST评价肝脏功能,检测肝脏组织中谷胱甘肽(GSH)、超氧化物歧化酶活性(SOD)及丙二醛(MDA)水平评价氧化应激程度,免疫印迹方法检测肝脏组织p-GSK3、p-JNK蛋白表达。结果 D-Gal N/LPS诱导急性肝衰竭引起肝组织GSH和SOD水平降低,肝组织MDA水平上升。NAC干预改善急性肝衰竭损伤(血清ALT、AST水平明显下降),同时增加肝脏组织GSK3β的磷酸化水平(降低GSK3β的活性)。SB216763抑制GSK3β活性增加急性肝衰竭小鼠肝组织GSH和SOD含量,降低肝组织中MDA含量。GSK3β抑制下调肝脏中p-JNK的表达。结论 GSK3β是氧化应激促进急性肝衰竭损伤中的一种重要信号分子,抑制GSK3β活性,减轻氧化应激可以改善急性肝衰竭损伤。     

内毒素预适应减轻急性肝功能衰竭中热休克蛋白27的作用

目的研究热休克蛋白27（HSP27）介导内毒素（LPS）预适应减轻急性肝功能衰竭（ALF）的作用机制。方法实验动物为雄性C57BL/6小鼠。将小鼠分为5组,对照组为正常C57BL/6小鼠;肝功能衰竭组小鼠以LPS10μg/kg＋D-半乳糖胺700 mg/kg,用1 mL0.9%氯化钠溶液溶解后腹腔内注射,建立急性肝功能衰竭的模型;LPS预处理组小鼠经LPS预适应24 h后,建立急性肝功能衰竭的模型;HSP27干扰组小鼠和转染对照组小鼠分别以尾静脉注射包装有干扰HSP27表达的短发夹RNA的重组腺病毒以及对照空病毒,成功干扰HSP27表达后,经LPS预处理,建立急性肝功能衰竭模型。通过比较各组ALT和AST水平,评价肝组织病理损伤程度,以及检测肝组织内TNF-α、IL-6mRNA水平,观察HSP27对LPS预适应减轻急性肝功能衰竭的影响。结果 LPS预适应可明显减轻Galn/LPS所致的肝损伤,干扰HSP27的表达后,小鼠血清ALT和AST明显升高,肝组织病理损伤和炎性反应加重（P〈0.05）。下调HSP27的表达水平后,LPS预适应的保护作用几乎完全消失。结论 LPS预适应可以减轻小鼠的急性肝损伤,HSP27在这一效应中发挥重要作用。     

硫辛酰胺对db/db小鼠肝损伤的保护作用研究

目的观察硫辛酰胺(ALM)对db/db小鼠肝损伤的保护作用及可能机制。方法db/db小鼠随机分为糖尿病组(DM)和ALM组,C57BL/6J小鼠为正常对照组(NC),每组各6只。ALM组于第9周时予ALM[100 mg/(kg·d)]进行灌胃干预,干预8周后处死小鼠,检测生化指标及肝组织谷丙转氨酶(ALT)、谷草转氨酶(AST)、过氧化氢酶(CAT)活性及丙二醇(MDA)表达量,油红O、HE染色观察肝脏病理学改变,Western blot法检测肝组织中核因子E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)蛋白水平。结果与NC组比较,DM组体重、TG、TC、FBG升高,MDA含量升高[(0.73±0.04)vs(0.92±0.17)nmol/mg,P<0.05],CAT活性降低[(1.08±0.18)vs(0.52±0.14)U/mg,P<0.05],ALT、AST活性升高[(16.85±3.84)vs(22.42±4.56)U/g,(6.07±1.91)vs(8.19±1.51)U/g,P<0.05],Nrf2、HO-1的表达升高[(0.33±0.25)vs(1.81±0.34),(0.29±0.13)vs(1.25±0.19),P<0.05]。与DM组比较,ALM组体重、TG、TC降低,MDA降低[(0.92±0.17)vs(0.56±0.11)nmol/mg,P<0.05],CAT活性升高[(0.52±0.14)vs(0.91±0.20)U/mg,P<0.05],ALT、AST活性降低[(22.42±4.56)vs(17.08±5.08)U/g,(8.19±1.51)vs(5.10±0.46)U/g,P<0.05],Nrf2、HO-1表达降低[(1.81±0.34)vs(1.01±0.30),(1.25±0.19)vs(0.52±0.17),P<0.05]。结论ALM可抑制T2DM小鼠肝损伤的发生发展,其机制可能是通过改善肝组织脂质沉积、抑制氧化应激,调节Nrf2、HO-1蛋白表达以实现。     

Ethanol binging enhances hepatic microvascular responses to acetaminophen in mice

OBJECTIVE: Chronic alcoholism has been considered to be a risk for acetaminophen (APAP) hepatotoxicity, but little is known about the effect of binge alcohol drinking on APAP-induced liver injury. The present study was conducted to examine the effect of ethanol binging on APAP-induced hepatic microcirculatory dysfunction. METHODS: Male C57Bl/6 mice received 3 weekly ethanol binges (4 g/kg every 12 h x 5 doses/ week) or water binges. At 12 h after the last gavage, APAP (300 mg/kg) was given by oral gavage. In one group of mice, gadolinium chloride (GdCl3, 10 mg/kg) was intraperitoneally administered 2 and 1 days before the start of each weekly ethanol binge. RESULTS: Ethanol binging enhanced APAP-induced liver injury as indicated by ALT levels. Intravital microscopic study showed that APAP further increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space as well as Kupffer cell phagocytic activity in ethanol-binged mice when compared with water-binged mice, while no significant differences in sinusoidal perfusion and leukocyte adhesion were observed. ALT levels after APAP were exacerbated in ethanol-binged mice treated with GdCl3, but APAP-induced hepatic microcirculatory dysfunction was not changed significantly. CONCLUSIONS: These results suggest that ethanol binging increases APAP-induced liver injury by exacerbating infiltration of the Disse space with blood cells. Kupffer cells exert a protective role in the liver against APAP intoxication following ethanol binging.     

肝爽颗粒浸膏减轻对乙酰氨基酚所致肝细胞损伤的作用机制

目的研究肝爽颗粒浸膏减轻对乙酰氨基酚(N-乙酰基-对氨基苯酚,APAP)诱导的肝细胞毒性的能力,以及可能涉及的机制。方法设立5组细胞培养组,分别为正常对照组、APAP损伤组、3组不同肝爽颗粒浸膏浓度的损伤保护组。使用20 mmol/L APAP加入细胞培养液孵育24 h构建造体外药物肝损伤模型,损伤保护组提前加用不同浓度肝爽颗粒浸膏(0.2μg/ml、1μg/ml、5μg/ml)8 h孵育后加入APAP损伤24 h。检测不同组别的肝细胞损伤标志物(ALT、AST、LDH)、线粒体损伤标志物(线粒体膜电位、GDH)、抗氧化及氧化应激标志物(GSH、SOD、MDA、ROS)等。进一步针对实验结果进行机制探讨。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果肝爽颗粒浸膏可以减轻APAP引起的肝细胞毒性,肝爽颗粒浸膏可以提高细胞活力(P<0.001),降低上清中AST、ALT、LDH的含量(P值分别为<0.001、<0.001、<0.05);肝爽颗粒浸膏可以抑制APAP诱导的肝细胞氧化应激,肝爽颗粒浸膏组的氧化应激指标ROS、MDA较APAP组下降(P值均<0.01);肝爽颗粒浸膏可以剂量依赖性减轻APAP诱导的肝细胞线粒体膜电位丢失(P<0.05),降低了上清线粒体损伤标志物GDH的含量(P<0.001);肝爽颗粒浸膏可以抑制CYP2E1/1A2的表达(P值均<0.05);肝爽颗粒浸膏可以增加肝细胞Ⅱ相酶表达;肝爽颗粒浸膏可以诱导Nrf2及其下游基因NQO-1及GCLC的表达(P值均<0.05)。结论肝爽颗粒浸膏可通过两种途径预防APAP诱导肝脏损伤,第一种途径是肝爽颗粒浸膏下调CYP2E1/1A2的表达减少了APAP毒性产物NAPQI的生成;第二种途径是肝爽颗粒浸膏上调解毒通路的表达,激活Nrf2增加抗氧化酶(SOD、GSH)和Ⅱ相酶的表达,从而加速APAP的无害代谢。     

Stimulated hepatic tissue repair underlies heteroprotection by thioacetamide against acetaminophen-induced lethality

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that causes massive centrilobular hepatic necrosis at high doses, leading to death. The objectives of this study were to test our working hypothesis that preplaced cell division and hepatic tissue repair by prior thioacetamide (TA) administration provides protection against APAP-induced lethality and to investigate the underlying mechanism. Male Sprague-Dawley rats were treated with a low dose of TA (50 mg/kg, intraperitoneally [i.p.]) before challenge with a 90% lethal dose (1,800 mg/kg, i.p.) of APAP. This protocol resulted in a 100% protection against the lethal effect of APAP. Because TA caused a 23% decrease of hepatic microsomal cytochromes P-450, the possibility that TA protection may be caused by decreased bioactivation of APAP was examined. A 30% decrease in cytochromes P-450 induced by cobalt chloride failed to provide protection against APAP lethality. Time course of serum enzyme elevations (alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase) indicated that actual infliction of liver injury by APAP peaked between 12 to 24 hours after the administration of APAP, whereas the ultimate outcome of that injury depended on the biological events thereafter. Although liver injury progressed in rats receiving only APAP, it regressed in rats pretreated with TA. Acetaminophen t1/2 was not altered in TA-treated rats, indicating that significant changes in APAP disposition and bioactivation are unlikely. Moreover, hepatic glutathione was decreased to a similar extent regardless of TA pretreatment, suggesting that decreased bioactivation of APAP is unlikely to be the mechanism underlying TA protection. [³h]Thymidine incorporation studies confirmed the expected stimulation of S-phase synthesis, and proliferating cell nuclear antigen studies showed a corresponding stimulation of cell division through accelerated cell cycle progression. Intervention with TA-induced cell division by colchicine antimitosis ended the TA protection in the absence of significant changes in the time course of serum enzyme elevations during the inflictive phase of APAP hepatotoxicity. These studies suggest that hepatocyte division and tissue repair induced by TA facilitate sustained hepatic tissue repair after subsequent APAP-induced liver injury, producing recovery from liver injury and protection against APAP lethality.     

Mechanisms of protection by melatonin against acetaminophen-induced liver injury in mice

The present study was performed to determine whether melatonin protects mouse liver against severe damage induced by acetaminophen (APAP) administration and where melatonin primarily functions in the metabolic pathway of APAP to protect mouse liver against APAP-induced injury. Treatment of mice with melatonin (50 or 100 mg/kg, p.o.) 8 or 4 hr before APAP administration (750 mg/kg, p.o.) suppressed the increase in plasma alanine aminotransferase and aspartate aminotransferase activities in a dose- and a time-dependent manner. Melatonin treatment (100 mg/kg, p.o.) 4 hr before APAP administration remarkably inhibited centrilobular hepatic necrosis with inflammatory cell infiltration and increases in hepatic lipid peroxidation and myeloperoxidase activity, an index of tissue neutrophil infiltration, as well as release of nitric oxide and interleukin-6 into blood circulation at 9 hr after APAP administration. However, melatonin neither affected hepatic reduced glutathione (GSH) content nor spared hepatic GSH consumption by APAP treatment. Moreover, pretreatment with melatonin 4 hr before APAP administration did not influence the induction of hepatic heat shock protein 70 (HSP70) by APAP and melatonin alone did not induce HSP70 in mouse liver. These results indicate that exogenously administered melatonin exhibits a potent hepatoprotective effect against APAP-induced hepatic damage probably downstream of the activity of cytochrome P450 2E1, which works upstream of GSH conjugation in the pathway of APAP metabolism, via its anti-nitrosative and anti-inflammatory activities in addition to its antioxidant activity.     

臭氧化盐水对肝细胞Keap1-Nrf2-ARE通路的激活作用

目的 研究臭氧化盐水对肝组织细胞Keap1-核因子E2相关因子2(Nrf2)-抗氧化元件(ARE)通路中Nrf2的作用.方法 采用成年健康雄性Sprague-Dawley大鼠,随机分为正常对照组(NC组)、模型组、臭氧等渗盐水(OS)组,OS对照组(OSC组).OS组、OSC组分别予5 ml/kg OS,模型组予5 ml/kg氧气盐水每日尾静脉注射,连续15 d,第16天分别予OS组及模型组50%CCl4橄榄油溶液2ml/kg腹腔注射造肝损伤模型.NC组及OSC组予植物油2ml/kg腹腔注射,24 h后,检测大鼠血清ALT、AST、肝组织总抗氧化能力(TAOC)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GPx)、过氧化氢酶(CAT).再提取处死大鼠肝组织的核蛋白,应用Western blot测定其细胞核中Nrf2的含量,免疫荧光组织化学技术检测细胞内Nrf2的分布.结果 与模型组比较,OS组大鼠ALT、AST降低[(1240.4±188.2)U/L、(1245.4±176.9)U/L对比(539.8±175.3)U/L、(546.0±130.2)U/L)],差异有统计学意义(P<0.01),TAOC、GSH,GPx,CAT 活性升高,分别为(0.72±0.24)U/mg、(1.05±0.21)mg/g,(676.9±115.1)U/mg、(45.2±14.3)U/mg对比(1.37±0.19)U/mg、(2.23±0.55)mg/g、(1024.6±162.9)U/mg、(68.2±9.9)U/mg,差异有统计学意义(P<0.01).与NC组比较,OSC组大鼠肝组织TAOC、GSH、GPx,CAT活性升高,差异有统计学意义(P<0.01或P<0.05).Western blot及免疫荧光均显示O3能增强肝细胞核内Nrf2的表达,Keap1-Nrf2-ARE通路的激活在O3抗氧化过程中发挥了重要的作用.结论 臭氧化盐水静脉注射可减轻CCl4所致大鼠肝损伤.其机制可能通过激活Keap1-Nrf2-ARE通路及其下游基因,增强细胞抗氧化和抗自由基的能力.

Abstract：

Objective To study the effect of ozonized saline on the activation of the Keapl-Nrf2ARE signaling pathway in rat liver cells. Methods Twenty maleSprague-Dawley rats were randomly divided into ozonized saline(OS) group, model group, ozonized saline control (OSC) group and normal control (NC)group. The rats in OS group and model group were intravenously administered with OS or oxygen saline (5 ml/kg) respectively, once a day for 15 days, and then intraperitoneally injected with CCU dissolved in Oliver oil. The rats in OSC group were pretreated with OS for 15 days. The rats in NC group were fed normally for 15 days. On the 16th day, the rats in OSC group and NC group were intraperitoneally injected with Oliver oil (2 ml/kg) without CCU. After 24 hours of CCU or olive oil intraperitoneal injection, the serum levels of alanine transaminase (ALT) and aspertate aminotransferase (AST) were measured. The liver tissues were also collected for detection of total anti-oxygen capability (TAOC), glutathione (GSH), catalase (CAT), Glutathione peroxidase (GPx). Western Blot was used to detect Nrf2 and immunofluorescence staining assay to display intracelluar distribution of Nrf2. Results Compared with the rats in model group,the serum ALT and AST levels of rats in OS group were significantly lower (P < 0.01) ,which were (1240.4 ± 188.2) U/L and (1245.4 ± 176.9) U/L vs (539.8 ± 175.3) U/L and (546.0 ± 130.2) U/L, and the TAOC, CAT, GPx and GSH activity of rats in OS group were significantly higher, which were (0.72 ± 0.24) U/mg, (1.05 ±0.21) mg/g, (676.9 ± 115.1) U/mg and (45.2 ± 14.3) U/mg vs (1.37 ± 0.19) U/mg, (2.23 ± 0.55) mg/g,(1024.6 ± 162.9) U/mg and (68.2 ± 9.9) U/mg, respectively. In contrast with NC group, pretreatment of OS in OSC group elevated TAOC, CAT, GPx and GSH activity (P < 0.01 or P < 0.05). Ozonized saline can strengthen the Nrf2 expression in liver cells. Conclusions Preconditioning injection of ozonized saline can reduce rat's liver injury induced by CCl4- The ozonized saline, as a novel Nrf2 activator, can reduce the oxidative damage of radical oxygen species (ROS) and the deleterious substance by activating the KeaplNrf2-ARE signaling pathway and its downstream genes expression.     

Role of the coagulation system in acetaminophen-induced hepatotoxicity in mice

Acetaminophen (N-acetyl-p-aminophenol [APAP]) is one of the leading causes of acute liver failure, and APAP hepatotoxicity is associated with coagulopathy in humans. We tested the hypothesis that activation of the coagulation system and downstream protease-activated receptor (PAR)-1 signaling contribute to APAP-induced liver injury. Fasted C57BL/J6 mice were treated with either saline or APAP (400 mg/kg intraperitoneally) and were euthanized 0.5-24 hours later. Hepatotoxicity and coagulation system activation occurred by 2 hours after administration of APAP. Treatment with APAP also caused a rapid and transient increase in liver procoagulant activity. In addition, significant deposition of fibrin was observed in the liver by 2 hours, and the concentration of plasminogen activator inhibitor-1 in plasma increased between 2 and 6 hours. Pretreatment with heparin attenuated the APAP-induced activation of the coagulation system and hepatocellular injury and diminished hepatic fibrin deposition at 6 hours. Loss of hepatocellular glutathione was similar in APAP-treated mice pretreated with saline or heparin, suggesting that heparin did not diminish bioactivation of APAP. In mice deficient in tissue factor, the principal cellular activator of coagulation, APAP-induced liver injury, activation of coagulation, and hepatic fibrin deposition were reduced at 6 hours. Formation of the tissue factor-factor VIIa complex leads to the generation of thrombin that can activate cells through cleavage of PAR-1. Mice lacking PAR-1 developed less injury and hepatic fibrin deposits at 6 hours in response to APAP than control mice. CONCLUSION: Activation of the coagulation system and PAR-1 signaling contribute significantly to APAP-induced liver injury.     

Protective effect of chitosan treatment against acetaminophen-induced hepatotoxicity

Acetaminophen (APAP) is the most commonly reported toxic ingestion in the world. Severe liver injury resulting from overdose or chronic use of APAP remains a significant clinical problem. In recent years, the mechanisms underlying liver injury caused by APAP have become much better understood. We have studied the protective effect of chitosan supplementation against APAP-induced hepatotoxicity with respect to changes in the levels of total and lipid-bound sialic acid in the serum and in the liver tissue and changes in the activity of diagnostic marker enzymes, lipid peroxidation, and ceruloplasmin oxidase enzyme in normal and experimental groups of rats. During the experimental period, chitosan (200 mg/kg body weight per day) was administered to APAP + chitosan-treated rats by oral gavage. Results showed that treatment with APAP induced a significant increase in the serum alanine aminotransferase and alkaline phosphatase activities, in total and lipid-bound sialic acids levels, and in the liver lipid peroxide content. The administration of chitosan significantly prevented APAP-induced alterations in the levels of diagnostic marker enzymes, total sialic acid, lipid-bound sialic acid, and malondialdehyde in the experimental groups of rats. Furthermore, chitosan administration increased the activity of ceruloplasmin oxidase. In conclusion, our results suggest that chitosan has a protective effect on APAP-induced hepatic injury in rats. The study sheds light on the therapeutic potential of chitosan in an APAP-induced hepatotoxicity model.     

急性胰腺炎大鼠血和组织中降钙素原水平的变化

目的 观察急性胰腺炎(AP)大鼠血和组织中降钙素原(procalcitonin,PCT)水平的变化,并探讨其意义.方法 将102只雄性Wistar大鼠按数字表法随机分为正常对照组(6只)、脂多塘(LPS)组(24只)、急性水肿性胰腺炎(AEP)组(24只)、急性坏死性胰腺炎(ANP)组(24只)和ANP+LPS组(24只).皮下注射雨蛙素制备AEP模型,逆行胆胰管注射牛磺胆酸钠制备ANP模型.术后3、6、18、24 h分批处死动物,胰腺组织常规病理检查并评分;取血检测PCT水平;取肝、肺、脾、胰腺、小肠及大肠组织检测PCT含量.结果 AEP和ANP模型制备成功.术后6 h时,对照组、LPS组、AEP组、ANP组和ANP+LPS组血PCT水平分别为(0.0144±0.0082)ng/ml、(0.1722±0.0449)ng/ml、(0.4751±0.0572)ng/ml、(0.7070±0.1040)ng/ml和(1.1960±0.8644)ng/ml,各组间相差显著(P＜0.05).正常大鼠肝、肺、脾、胰腺、小肠及大肠组织均检测到PCT.与之相比,ANP组肝和胰腺PCT含量无明显变化;肺、脾及大肠PCT含量显著降低[分别为(5.63±0.62)ng/ml对(6.85±0.46)ng/ml,(4.73±1.27)ng/ml对(6.88±0.31)ng/ml,(1.08±0.52)ng/ml对(4.12±1.02)ng/ml,P值均＜0.01],而小肠组织明显升高[(2.51±0.90)ng/m1对(0.98±0.20)ng/ml,P＜0.01].结论 血清PCT水平与AP的严重程度及感染相关,AP时各组织PCT含量的不同变化可能与器官功能的改变有一定的关系.