凉血化瘀方对急性肝衰竭大鼠肝细胞凋亡的影响

目的：研究凉血化瘀方防治急性肝功能衰竭的作用机制。方法：将SD大鼠36只随机分为6组，除正常组外每组大鼠分别连续灌胃给药4天。末次给药后1小时，每组腹腔注射GaIN＋LPS，造成大鼠急性肝功能衰竭。6小时后采用流式细胞术检测大鼠肝细胞凋亡，同时采用原位末瑞标记（TUNEL）法半定量检测肝细胞凋亡情况。结果：流式细胞仪检测结果发现凉血化瘀方与阳性对照组细胞凋亡率比较模型组显著下降（P〈0．01，P〈0．05），并且模型组大量细胞阻滞在s期，G2期细胞减少，凉血化瘀方中剂量组与阳性药物对照组较模型组其细胞周期阻滞改善，凉血化瘀方组与模型组比较，差异有显著性意义（P〈0．01）。TUNEL半定量检测细胞凋亡与流式细胞仪结果基本一致，凉血化瘀方与模型组比较细胞凋亡率显著下降。结论：凉血化瘀方能够抑制急性肝损伤肝细胞凋亡，调节细胞周期阻滞，其机制可能为修复DNA复制损伤。     

解毒凉血方含药血清调节TGFβ1/Smad信号通路对急性肝衰竭肝细胞凋亡的抑制作用

目的:研究解毒凉血方含药血清对TNFα联合ActD诱导肝衰竭肝细胞凋亡的抑制作用及机制。方法:采用人正常肝细胞Chang liver及肿瘤细胞HepG2筛选解毒凉血方含药血清的有效比例。干预组采用有效比例的解毒凉血方含药血清预刺激24 h,然后采用TNFα联合ActD刺激Chang liver模拟急性肝衰竭肝细胞凋亡,流式细胞术检测各组细胞的凋亡率,免疫荧光实验检测凋亡肝细胞TBFβ1的表达及分布,免疫印迹法检测TGFβ1/Smad信号通路中TGFβ1、p-TβRⅡ、Smad7及p-Smad2/3的表达水平。结果:15%(体积比)解毒凉血方含药血清对TNFα联合ActD诱导的肝细胞凋亡具有抑制作用,15%解毒凉血方含药血清组细胞凋亡率显著低于模型组及空白血清组(P<0.01);与模型组及空白血清组比较,15%解毒凉血方含药血清预处理组胞质中TGFβ1、p-TβRⅡ以及Smad2/3磷酸化(p-Smad2/3)均低水平表达,而Smad7表达水平较高(均P<0.05)。结论:解毒凉血健脾方含药血清对TNFα联合ActD刺激诱导的急性肝衰竭肝细胞凋亡具有抑制作用,其机制可能与调节...     

痢疾杆菌及其L型内毒素与细胞凋亡

目的　探讨痢疾杆菌及其L型内毒素与细胞凋亡的关系。方法　将痢疾杆菌诱导为稳定L型 ,分别制备原菌内毒素和L型内毒素 ,用尾静脉注射法感染小鼠 ,观察小鼠的发病情况 ,并对死亡小鼠进行组织学及凋亡细胞检查。本实验共分三组 :A组原菌内毒素组 ,B组L型内毒素组 ,C组生理盐水组。结果　A组致小鼠细胞凋亡作用最强 ,以肝细胞凋亡最明显 ,A、B两组之间致凋亡的差异有显著性 (P <0 0 1)。结论　痢疾杆菌内毒素可致小鼠肝细胞凋亡 ,而其L型 ,由于失去细胞壁 ,内毒素丢失 ,致肝细胞凋亡能力大大降低     

正交设计优选凉血化瘀方及其防治肝衰竭的药效研究

目的:探讨凉血化瘀方防治急性肝衰竭的最佳配伍。方法:ICR小鼠85只被随机分成17组,包括正常组和16个用药组,根据L16(215)正交表将凉血化瘀方按四因素两水平安排成16种不同的搭配,制成煎剂分别灌胃给药,每组小鼠腹腔注射D-GaIN(500mg/kg) LPS(4.8μg/kg)造成急性肝功能衰竭,6小时后取血和肝组织,观察不同药物组小鼠的胆红素(TBil)、转氨酶(ALT、AST)水平和肝组织病理损伤程度。结果:赤芍、生地、大黄配伍组比模型组和其他用药组能够显著降低肝衰竭小鼠胆红素水平,减轻肝组织坏死程度。结论:凉血化瘀中药具有很好的防治肝衰竭,保护肝细胞的作用,且由赤芍、生地、大黄配伍组方疗效最佳。     

扑热息痛引起肝细胞损害的机制

目的:探讨肝细胞凋亡在扑热息痛(acetaminophen, AAP)肝损害中作用．方法:建立SD大鼠AAP肝损害模型;分别于给药后3,6,12,24 h处死大鼠,AAP组和对照组测定血清ALT水平,HE染色光镜下观察肝脏病理改变,原位末端标记(TUNEL)法和透射电镜检测肝细胞凋亡．结果:AAP组各时间点血清ALT(nkat／L)水平均显著高于对照组(3,6,12,24 h:1167±151 vs587±89,2154±255vs573±76,4221± 929vs751±82,13 203±1393 vs 780±161, P<0．01),随时间进行性升高,24 h达高峰; AAP组可见不同程度肝细胞坏死,随时间进行性加重,24 h达高峰,对照组未见肝细胞坏死; AAP组中央静脉周围可见大量凋亡细胞,12 h 达高峰,对照组罕见凋亡细胞,AAP组各时间点肝细胞凋亡指数(AI)均显著高于对照组 (3,6,12,24 h:13．1％±2．9％vs 1．8％±0．5％, 24．8％±5．3％vs 1．7％±0．5％,40．4％±3．7％vs 2．0％±0．4％,35．3％±3．5％vs1．92％±0．3％, P<0．01);透射电镜下观察,AAP组可见明显的肝细胞凋亡现象,12 h最易见到,对照组肝组织超微结构基本正常．结论:AAP肝损害过程中同时存在肝细胞坏死和凋亡,早期(给药后12 h内)以凋亡为主, 后期(给药后24 h)以坏死为主．     

内毒素诱导肝细胞凋亡及肝细胞生长因子的抑制作用观察

目的观察内毒素(LPS)诱导肝细胞凋亡的作用及肝细胞生长因子(HGF)对LPS的拮抗作用。方法HepG2细胞传代培养24 h,分三组。对照组加原培养液,LPS组加含LPS(20μg/ml)的培养液,干预组加含LPS(20μg/ml)、HGF(40 U/L)的培养液．继续培养16 h。Tunel标记法检测细胞凋亡。结果LPS组细胞凋亡率为98．65%±0．64%．干预组为35．97%±0．45%,两组相比,P＜0．01。结论LPS可诱导肝细胞凋亡,HGF可抑制LPS所诱导的肝细胞凋亡。     

解毒化瘀方对内毒素血症大鼠肝细胞线粒体氧化损伤的影响

目的:观察解毒化瘀方对内毒素血症( endotoxemia,ETM)大鼠肝细胞线粒体氧化损伤的影响.方法:将56只大鼠随机分为正常对照组及内毒素注射后6小时、12小时、24小时组和解毒化瘀方6小时、12小时、24小时组.眼眶取血,检测大鼠血清肿瘤坏死因子(TNF)-α水平,同时取肝组织分离线粒体,测定其丙二醛(MDA)的含量及超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性.结果:在注射内毒素6小时后,大鼠血清TNF-α水平及线粒体MDA的含量升高,线粒体SOD、GSH-Px活性下降,随着时间的延长,TNF-α水平及MDA的含量进一步升高,SOD、GSH-Px活性明显下降.与对应的内毒素组比较,经解毒化瘀方处理后,TNF-α水平及MDA的含量均降低,SOD、GSH-Px活性均升高.结论:解毒化瘀方对内毒素刺激Kupffer细胞释放TNF-α有抑制作用,同时减少内源性自由基的生成,提高机体的抗氧化损伤能力,具有保护肝细胞的作用.     

姜黄素对NASH肝细胞凋亡及JNK信号通路的影响

目的:探讨姜黄素对非酒精性脂肪性肝炎(NASH)的抗肝细胞凋亡作用及其对JNK信号通路的影响。方法:将HL-7702细胞分为对照组、模型组、低剂量治疗组及高剂量治疗组,对照组予普通培养液培养,模型组培养液中加入浓度为1.0 mmol/L的游离脂肪酸(FFA)诱导NASH模型,高、低剂量治疗组在模型组的基础上分别加入浓度为10μmol/L、20μmol/L的姜黄素干预,48 h后采用流式细胞术检测细胞凋亡情况,Western Blot法检测p-JNK蛋白的表达情况。结果:模型组肝细胞的早期及总凋亡率均明显高于对照组(均P<0.05),高、低剂量治疗组与模型组相比,肝细胞早期及总凋亡率均明显降低(均P<0.05)。模型组肝细胞的p-JNK蛋白表达明显高于对照组(P<0.05),治疗组的p-JNK表达较模型组显著下降(P<0.05),且呈剂量相关。结论:姜黄素可抑制JNK的活化,这可能是其抗细胞凋亡、延缓NASH进展的机制之一。     

小鼠暴发性肝衰竭肝细胞凋亡形态学及其调控机制

目的:研究在实验性暴发性肝衰竭(fulminant hepatic failure,FHF)中肝细胞凋亡的形态学变化以及一氧化氮(nitric oxide,NO)、Fas和Bcl-2对肝细胞凋亡的调控作用．方法:脂多糖(LPS)和D-氨基半乳糖(D-GalN)联合应用制备FHF小鼠模型;采用免疫组化方法检测肝组织Fas 及Bcl-2表达,分别采用硝酸还原酶法和RT-PCR法检测血清NO水平及肝组织iNOS mRNA表达;TUNEL法检测肝细胞凋亡;在用药后动态观察Fas及Bcl-2表达、血清NO水平及肝组织iNOS mRNA表达及肝细胞凋亡的变化,并对模型鼠给予iNOS的抑制剂L-NMMA,动态观察上述指标的变化．结果:在FHF模型小鼠中,用药后2 h开始血清NO水平及iNOs mRNA的表达即升高,于4 h达高峰;用药后2 h 开始Fas有少量表达,至8 h和12 h表达均明显增多,与 2 h组比较差异显著(100％ vs 20％,P<0．01),与4 h组比较差异也比较显著(100％ vs 40％,P<0．05)．模型组2 h Bcl-2有较多表达,4 h表达最多,4 h与2 h比较差异显著 (90％ vs 60％,P<0．05),与8,12h比较差异非常显著(90％ vs 20%,均P<0．01)．8 h可出现典型的肝细胞凋亡表现．与模型组各时间点比较,给予iNOS的抑制剂L-NMMA 后血清NO水平及iNOs mRNA的表达均为正常水平, Fas及Bcl-2表达均无显著差异(P>0．05),阻断后8 h亦可见典型的肝细胞凋亡表现,阻断NO可使病变较模型组更为严重．结论:在FHF中,Fas及Bcl-2的表达均增加,Fas的表达与肝细胞凋亡相一致,而Bcl-2的表达与肝细胞凋亡呈负相关．单纯应用NO拮抗剂对肝细胞凋亡及肝损伤无保护作用．     

PGE2在内毒素诱导的幼鼠急性胃黏膜损伤中的变化及PAF受体拮抗剂对其影响

目的:探讨前列腺素E2(PGE2)在内毒素(LPS) 腹腔注射(ip)诱导的幼鼠急性胃黏膜损伤中的保护作用．方法:18日龄Wistar大鼠,随机分为对照组、 LPS组、PAF受体拮抗剂预防组和治疗组四组.用内毒素(E coli O55:B5脂多糖)5 mg／kg ip 制备幼年大鼠内毒素血症模型,同等量生理盐水ip为对照组,于注射后1．5,3,6,24,48． 72 h处死动物,大体及光学显微镜下观察胃黏膜损伤情况,用放射免疫方法测定胃黏膜 PGE2浓度．观察分别于内毒素ip前和注射后 0．5h应用血小板活化因子(PAF)受体拮抗剂 BN52021(GinkgolideB)5 mg/kg ip对胃黏膜损伤和胃黏膜PGE2浓度影响．结果:内毒素组6h胃黏膜损伤最重,黏膜表面可见大片糜烂、出血、条索状坏死,光镜下上皮脱落,黏膜内有出血,核碎裂、固缩,凋亡小体出现;应用PAF受体拮抗剂后黏膜表面上皮仅见充血水肿,光镜下损伤仅限于黏膜上皮:内毒素组6h胃黏膜PGE2浓度最低,此时 PGE2浓度在LPS组(134．5±9.3μg／L)与对照组 (245．1±8．9 μg／L)间差异显著(P<0．01);在PAF 受体拮抗剂预防组(304．4±15．0μg／L)、PAF 受体拮抗剂治疗组(315．9±43．7 μg／L)与LPS 组(134．5±9．3 μg／L)间均差异显著(P<0．01); PAF受体拮抗剂预防组(304．4±15．0μg／L)和治疗组(315．9±43．7μg／L)与对照组(245．1± 8．9μg/L)间差异显著(P<0．05)．结论:内毒素血症时胃黏膜PGE2下降,PAF受体拮抗剂可以改善这种PGE2下降;PGE2对内毒素造成的幼鼠急性胃黏膜损伤有保护作用．     

肿瘤坏死因子α致暴发性肝功能衰竭小鼠肠上皮细胞凋亡的作用

目的研究肿瘤坏死因子α(TNFα)对暴发性肝功能衰竭(FHF)小鼠肠黏膜上皮细胞凋亡的作用。方法用D-氨基半乳糖(GalN) 脂多糖(LPS)或TNFα造FHF小鼠模型;酶联免疫吸附法测定血清TNFα含量;光学显微镜和电子显微镜观察肠组织;末端转移酶介导的dUTP缺口末端标记法检测肠组织细胞凋亡情况;免疫组织化学法检测肿瘤坏死因子受体I(TNFRⅠ)蛋白在肠组织的表达与分布。结果实验各组动物各时间点光学显微镜下肠黏膜上皮细胞结构均保持完整,电镜下可见肝功能衰竭组有典型的凋亡细胞;对照组、LPS组、GalN组血清TNFα水平几乎正常,凋亡率低且基本没有TNFRⅠ蛋白表达;GalN LPS组血清TNFα水平12h明显升高(P<0.01),且TNFRⅠ蛋白在6h(大肠:2.82e 4±4.60e 3;小肠:1.14e 4±2.13e 3)即有表达,此时肠上皮细胞凋亡不明显,9h和12hTNFRⅠ蛋白表达明显增加,同时肠上皮细胞凋亡也明显。用GalN TNFα造FHF模型,结果也与GalN LPS组类似。应用抗TNFα抗体,可降低TNFRⅠ蛋白表达和减少肠上皮细胞凋亡的发生。结论TNFα能诱导肝功能衰竭小鼠肠上皮细胞凋亡,抗TNFα抗体能阻断这一作用;在FHF的模型动物中,TNFRⅠ蛋白表达增多,TNFRⅠ蛋白表达与肠上皮细胞凋亡在一定程度上呈正相关。     

Tumor necrosis factor-alpha induces apoptosis of enterocytes in mice with fulminant hepatic failure

AIM: To explore the alterations of intestinal mucosa morphology, and the effects of tumor necrosis factor a (TNFα) on enterocyte apoptosis in mice with fulminant hepatic failure (FHF). METHODS: Liver damage was induced by lipopolysaccharide (LPS)/TNF-α in D-galactosamine (GaIN) sensitized BALB/c mice. There were 40 mice in normal saline (NS)-treated group, 40 mice in LPS-treated group, 40 mice in GaIN-treated group, 120 mice in GaIN/ LPS-treated group and 120 mice in GaIN/ TNFα-treated group. Each group was divided into five subgroups of eight mice each. Serum samples and liver, intestinal tissues were respectively obtained at 2, 6,9,12 and 24 h after administration. Anti-TNFa monoclonal antibody was injected intravenously into GaIN/LPS-treated mice. Serum TNFα levels were determined by enzyme linked immunosorbent assays (ELISA). Serum ALT levels were determined using an automatic analyzer. The intestinal tissues were studied under light microscope and electron microscope at 2, 6, 9,12 and 24 h in mice with fulminant hepatic failure, respectively. Enterocyte apoptosis was determined by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) method. The expression of tumor necrosis factor receptor 1 (TNFR1) in intestinal tissue was tested by immunohistochemistry Envision Two Steps. RESULTS: Gut mucosa was morphologically normal at all time points in all groups, but typical apoptotic cells could be seen in all experimental groups under electron microscope. Apoptosis rate of gut mucosal epithelial cells were significantly increased at 6, 9 and 12 h, peaked at 12 h in mice with fulminant hepatic failure. TNFa induced apoptosis of enterocytes in mice with FHF. The integrated OD (IOD) levels of TNFa receptor 1 protein expressed in the intestine of mice with GaIN/LPS and GaIN/ TNFα induced FHF at 2, 6, 9, 12 and 24 h after GaIN/LPS and GaIN/TNFα administration were 169.54±52.62/905.79±111.84,11 350.67±2 133.26/28 160.37±4 601.67, 25 781.00±2 277.75/122 352.30±49 412.40, 5 241.53±3 007.24/ 49 157.93±9 804.88, 7 086.13±1 031.15/3 283.45±127.67, respectively, compared with those in control groups (with NS, LPS and GaIN administration, respectively). IOD level of TNFR1 changed significantly at 6, 9 and 12 h after GaIN/LPS and GalN/TNFa administration. The expression of TNFR1 protein was significantly higher at 9 h after GaIN/LPS and GaIN/TNFα administration than that in control groups. Protein expression of TNFR1 was positively correlated with enterocyte apoptosis. CONCLUSION: TNFα can induce apoptosis of enterocytes in mice with FHF. Anti-TNFα IgG can inhibit this role.     

析因实验法建立规范化小鼠暴发性肝衰竭模型

目的:建立稳定有效的小鼠D-氨基半乳糖(D-GalN)和内毒素(LPS)致暴发性肝衰竭(FHF)模型.方法:采用析因实验法,选择两个影响模型成功率的因素:D-GalN和LPS的攻击剂量.两者各选取三个水平,以小鼠24 h病死率为评价指标,观察实验鼠的血清肝功能和肝组织病理学改变.结果:优选出两种较理想的给药方案:D-Ga1N600 mg/kg联合LPS 0.5 mg/kg和D-GalN 800mg/kg联合LPS 0.04 mg/kg腹腔注射.不同剂量的D-GalN和LPS对模型的病死率均有影响(F=36.878,P=0.000;F=32.386,P=0.000),均存在量效关系;而且D-Ga1N和LPS间存在交互作用(F=5.226,P=0.005),两种试剂合用可以明显提高模型的病死率.结论:建立了D-GalN和LPS致小鼠FHF的规范化模型.     

暴发性肝衰竭小鼠细菌侵袭肠黏膜方式的研究

目的 通过研究细菌侵袭肠黏膜屏障的方式,探讨暴发性肝衰竭(FHF)并发自发性腹膜炎(SBP)的机制.方法 取240只雄性BALB/c小鼠,分为等渗盐水(NS)组(40只)、脂多糖(LPS)组(40只)、氨基半乳糖(GalN)组(40只).FHF模型组(120只).分别腹腔注射相同体积的NS、LPS(10μg/kg)、GalN(800 mg/kg)、LPS(10μg/kg)/GalN(800 mg/kg).注射处理后,分别于2、6、9、12 h和24 h处死小鼠(每个时间点处死8只小鼠).实验小鼠均在相应时间点摘取眼球,留取血清,并断头处死动物,留肝脏及大肠组织标本.用全自动生物化学分析仪检测ALT;对肝组织和大肠组织进行HE染色检测;透射电镜观察大肠黏膜超微结构及细菌侵袭肠黏膜的方式.用SPSS13.0统计软件进行数据分析,两组间ALT水平的分析采用Mann-Whitney U检验.结果 FHF模型组ALT水平、肝组织病理学检测结果及病死率和临床表现均符合FHF的诊断标准.4组小鼠注射处理后9 h,HE染色发现大肠组织仅有轻微水肿及少量炎性细胞浸润,此时,透射电镜下观察发现FHF模型组肠上皮细胞微绒毛断裂、脱落、变短,紧密连接(TJs)不完整,细胞器变化明显,HE染色发现FHF模型组肝脏呈成片的出血性坏死,残存的肝细胞肿胀,出血坏死区见较多炎性细胞浸润,但NS组、LPS组、GalN组肝脏组织病理形态及大肠黏膜超微结构变化不明显.FHF模型组注射处理后6～9 h,细菌以胞饮的形式穿入肠壁,细菌穿入肠壁区域的肠道黏膜绒毛脱落,TJs出现断裂,注射处理后12 h发现穿入的细菌以囊胞的形式存在.结论 LPS(10μg/kg)/GalN(800 mg/kg)联合注射建立的FHF小鼠模型是成功的.FHF时,肠黏膜TJs的断裂可能为肠道内细菌进入肠黏膜提供了条件,TJs的断裂可能是FHF并发SBP的原因之一.

Abstract：

Objective To explore the mechanism of fulminate hepatic failure (FHF) complicated with spontaneous peritonitis (SBP) through the research of bacteria invading the intestinal mucosa barrier.Methods 240 BalB/c male mice were divided into four groups as isotonic NS group (n = 40), lipopolysaccharide (LPS) group (n = 40), galactosamine (GalN) group (n = 40) and FHF model group (n = 120). Each mouse received same volume of NS, LPS (10 μ g/kg), GalN (800 mg/kg) or LPS (10 μ g/kg)/GalN (800 mg/kg)intraperitoneal injection according to its group. 8 mice were executed at 2, 6, 9, 12 and 24 hours after injection, respectively, and the liver and intestinal tissue samples were taken at the same time. ALT was measured by automatic biochemical analyzer and was compared between groups using Mann-Whitney U test.Liver and intestinal tissue received HE staining. The ultrastructure of intestinal mucosa and the method by which bacteria invaded the intestinal mucosa were observed by transmission electron microscopy. All data were analyzed by SPSS13.0 statistic software. Results ALT level, results of hepatic pathology, mortality and clinical manifestations of mice in the FHF model group met the diagnostic criteria of FHF. Intestinal tissue was found with slight edema and little inflammatory cells infiltration through HE staining in all the 4 groups of mice 9 hours after injection. Microvilli were found broken, shed and shorten in the intestinal epithelial cells with incomplete tight junction (TJs) and obviously changed organelles in the FHF model group of mice observed by transmission electron microscope. Mass hemorrhagic necrosis of liver cells with remnant liver cells swelling and many inflammatory cells infiltration by HE staining in the FHF model group. But the changes in hepatic pathology and intestinal mucosa ultrastructure were not so obvious in the mice of NS, LPS and GalN groups. Bacteria penetrated the intestinal wall by pinocytosis 6-9 hours after injection in the FHF model group, the microvilli were broken off and TJs turned rupture in the areas that the bacteria penetrated.The bacteria were found in the form of cyst 12 hours after injection. Conclusions LPS (10 mg/kg)/GalN (800 mg/kg) combined injection was successful in establishing the FHF mice model. The rupture of TJs may provide conditions for intestinal bacteria to penetrate the intestinal mucosa in FHF. Rupture of TJs may be one of the reasons why FHF was complicated with SBP.     

Intraperitoneal Administration of Fetuin-A Attenuates d-Galactosamine/Lipopolysaccharide-Induced Liver Failure in Mouse

Background

Fulminant hepatic failure (FHF) is a devastating syndrome, which sometimes results in death or liver transplantation, in which inflammation would aggravate the development of fetuin-A which would act as an anti-inflammatory factor and may be an available approach to attenuate FHF.

Aims

The purpose of this study was to investigate the effects of fetuin-A on d-galactosamine/lipopolysaccharide (d-GalN/LPS)-induced liver failure in mice.

Methods

A mouse model of FHF induced by d-GalN/LPS was established and fetuin-A was injected intraperitoneally prior to d-GalN/LPS treatment. At different time points after d-GalN/LPS intervention, serum TNF-α and IL-6 levels were measured by ELISA. Fetuin-A mRNA and protein expression in liver tissues was assessed by RT-PCR, Western blotting and immunohistochemical staining. Besides, an observation of liver tissue injury, the apoptosis of hepatocytes, was analyzed by TUNEL assay.

Results

Expression of fetuin-A mRNA and protein in liver tissue were significantly and gradually decreased after d-GalN/LPS administration. A pre-intervention of exogenous fetuin-A significantly improved the liver function, decreased TNF-α and IL-6 expression in peripheral blood, and liver tissue inhibited hepatocyte apoptosis responded to d-GalN/LPS induction so as to decrease the mortality rates of FHF mouse. Meanwhile, fetuin-A was negatively correlated with the hepatic pathological score and TNF-α protein staining in FHF mouse.

Conclusions

An intraperitoneal injection of fetuin-A attenuates d-GalN/LPS-induced FHF in mice. Fetuin-A might be a protective agent of liver damage partly through inhibiting liver inflammatory response and hepatocyte apoptosis.     

Hepatocyte growth factor prevents endotoxin-induced lethal hepatic failure in mice.

Sepsis and endotoxemia are involved in the development of fulminant hepatic failure, the prognosis of which is extremely poor and the mortality is high, with no available effective therapy. Here, we report that hepatocyte growth factor (HGF) exerts potent antiapoptotic effects in vivo and effectively prevents endotoxin-induced fulminant hepatic failure in mice. The animals were intraperitoneally injected three times with 120 micrograms human recombinant HGF or saline 6 hours and 30 minutes before and 3 hours after an intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (GalN). Administration of LPS + GalN, without HGF, rapidly led to massive hepatocyte apoptosis and severe liver injury, and all mice died of hepatic failure within 8 hours. In contrast, administration of human recombinant HGF strongly suppressed extensive progress of hepatocyte apoptosis and the liver injury induced by LPS + GalN, and 75% of the HGF-treated mice survived. Moreover, HGF strongly induced Bcl-xL expression and blocked apoptotic signal transduction upstream of CPP32 (caspase-3) in the liver, thereby leading to inhibition of massive hepatocyte apoptosis. We suggest that HGF may well have the potential to prevent fulminant hepatic failure, at least through its potent antiapoptotic action.     

肿瘤坏死因子α对暴发性肝衰竭小鼠肠上皮细胞紧密连接蛋白表达的影响

目的 研究TNFα对暴发性肝衰竭（FHF）小鼠大肠上皮细胞紧密连接蛋白occludin表达的影响。方法 采用D-氨基半乳糖（GaIN）和内毒素（LPS）联合腹腔注射（ip）制备FHF小鼠动物模型,并设立TNFα组（TNFα10μs／kg,ip）,TNFα抗体组（注射LPS和GaIN前30min尾静脉注射TNFd抗体100μg/只）。在不同的时间点（6h,9h）处死动物,应用免疫组织化学技术、Westernblot及实时定量PCR检测各组小鼠大肠上皮细胞紧密连接蛋白occludin表达的变化。结果 在生理盐水（NS）对照组,几乎整张切片上均可见到沿大肠黏膜上皮细胞膜顶端呈线性分布的occludin阳性染色,但FHF组及TNFα组小鼠的阳性染色较之明显减弱,TNFα抗体组occludin的阳性反应与Ns对照组比较无明显减弱。Westernblot结果与免疫组织化学结果相一致,FHF组及TNFα组小鼠9h时occludin蛋白含量明显下降,与NS对照组相比差异有统计学意义（0．36±0．05,0．48±0．02比0．71±0．09,P〈0．05）,TNFα抗体组与NS对照组相比差异无统计学意义（0．74±0．03比0．71±0．09,P〉0．05）。实时定量PCR结果显示,FHF组与TNFα组小鼠6h时occludinmRNA水平最低,与NS对照组相比差异有统计学意义（0．72±0．04,0．81±0．03比1．00±0．05,P〈0．05）,TNFα抗体组与Ns对照组相比差异无统计学意义（1．01±0．10比1．00±0．05,P〉0．05）。结论 在小鼠暴发性肝衰竭过程中,TNFα介导大肠上皮细胞间紧密连接蛋白occludin表达的下降。     

Protective effect and mechanism of stronger neo-minophagen C against fulminant hepatic failure

AIM： To investigate the protective effect of stronger neo-minophafen C （SNMC） on fulminant hepatic failure （FHF） and its underlying mechanism.
METHODS： A mouse model of FHF was established by intraperitoneal injection of galactosamine （D-Gal N） and lipopolysaccharide （LPS）. The survival rate, liver function, inflammatory factor and liver pathological change were obtained with and without SNMC treatment. Hepatoo/te survival was estimated by observing the stained mitochondria structure with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate fluorescence nick end labeling （TUNEL） method and antibodies against cytochrome C （Cyt-C） and caspase-3.
RESULTS： The levels of plasma tumor necrosis factor alpha （TNF-α）, nitric oxide （NO）, ET-1, interleukin-6 （IL-6）, and the degree of hepatic tissue injury were decreased in the SNMC-treated groups compared with those in the model group （P 〈 0.01）. However, there were no differences after different dosages administered at different time points. There was a significant difference in survival rates between the SNMC-treated groups and the model group （P 〈 0.01）. The apoptosis index was 32.3% at 6 h after a low dose of SNMC, which was considerably decreased from 32.3% ± 4.7% vs 5% ± 2.83% （P 〈 0.05） to 5% on d 7. The expression of Cyt-C and caspase-3 decreased with the prolongation of therapeutic time. Typical hepatocyte apoptosis was obviously ameliorated under electron microscope with the prolongation of therapeutic time. CONCLUSION： SNMC can effectively protect liver against FHF induced by LPS/D-Gal N. SNMC can prevent hepatocyte apoptosis by inhibiting inflammatory reaction and stabilizing mitochondria membrane to suppress the release of Cyt-C and sequent activation of caspase-3.     

重组人生长激素对内毒素致肝细胞凋亡效应的抑制作用

目的观察内毒素（LPS）对肝细胞的直接作用及重组人生长激素（rhGH）的干预效果。方法内毒素诱导肝细胞损害,HepG2细胞传代培养24h后,换用含内毒素（20μg/ml）和/或rhGH、肝细胞生长因子（HGF）的新鲜培养液继续培养16h,观察药物对LPS作用的干预效果。结果HepG2细胞经LPS作用16h,透射电镜观察既有形态正常的细胞,同时也出现典型的凋亡细胞表现为胞浆浓缩核凝聚成块状,凋亡小体形成,细胞器（包括线粒体）结构基本正常;TUNEL标记后胞核呈棕褐色染色,而对照细胞无阳性染色信号,证实LPS作用16h后HepG2细胞已发生凋亡。将rhGH与LPS同时加入培养液中共同作用16h,rhGH可明显减少HepG2细胞经LPS诱导的细胞凋亡数量对照组为（99±0.8）%,rhHG处理组为（36±5.6）%,P＜0.001。结论重组人生长激素可以抑制LPS致肝细胞凋亡作用。