肿瘤坏死因子-α诱导肝细胞凋亡在暴发性肝衰竭中的作用

目的 研究肿瘤坏死因子－α（ｔｕｍｏｒ ｎｅｃｒｏｓｉｓ ｆａｃｔｏｒ－α,ＴＮＦ－α）诱导肝细胞凋亡细胞凋亡在暴发性肝衰竭中的作用机制。方法 分别注射脂多糖（ｌｉｐｏｐ ｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）和ＴＮＦ－α于Ｄ－氨基半乳糖（Ｄ－ｇａｌａｃｔｏｓａｍｉｎｅ,ＧａｌＮ）致敏的ＢＡＬＢ／ｃ小鼠造成暴发性肝衰竭模型,用脱氧核糖核酸转移酶介导的缺口原位末端标记（ｉｎ ｓｉｔｅ ｅｎｄ ｌａｂｅ     

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。     

肿瘤坏死因子受体相关因子2基因缺失小鼠引起暴发性肝功能衰竭及其机制

目的 探讨TNF受体相关因子基因缺失导致肝功能衰竭的可能性及其可能原因。方法 实验鼠分为TNF受体相关因子2（TRAF2）基因缺陷鼠TRAF2^lox／lox／Cre^＋组、对照鼠TRAF2^lox／lox／Cre^-组、敲除TNF基因的TRAF2基因缺陷鼠TRAF2^lox／lox／Cre^＋／TNF^-／-组和对照鼠TRAF2^lox／lox／Cre^-／TNF^-／-组，用PolyI：PolyC诱导敲除TRAF2基因，动态观察血清ALT的变化，观察肝组织病理学改变。胶原酶分离肝细胞，流式细胞术（FACS）检测Fas和CD40的表达，肝细胞加TNF培养后计算存活肝细胞的比率。结果 TRAF2^lox／lox／Cre^＋组鼠于注药后第2天ALT开始升高，第4天达高峰，为（234．80±12．34）U／L，TRAF2^lox／lox／Cre^＋／TNF^-／-组鼠ALT亦有升高，为（90．30±15．63）U／L，但病理组织学显示前者有明显的肝细胞坏死和炎性细胞浸润。培养的TRAF2^lox／lox／Cre^＋／TNF^-／-组鼠肝细胞在加入TNF后存活细胞明显低于对照鼠TRAF2^lox／lox／Cre^-／TNF^-／-组，且前者Fas表达高于后者，而CD40表达低于后者。结论 TRAF2基因的缺失可导致肝细胞坏死，从而引起暴发性肝功能衰竭。     

肿瘤坏死因子α及caspase-3表达与暴发性肝衰竭细胞凋亡

目的　研究肿瘤坏死因子α(TNFα)与caspase 3表达在实验性暴发性肝衰竭 (FHF)中对肝细胞凋亡的作用。方法　用脂多糖和D 氨基半乳糖制备FHF小鼠模型 ;ELISA和逆转录PCR法检测血清TNFα水平及肝组织TNFαmRNA表达 ;原位杂交法检测肝组织内caspase 3表达 ;DNA琼脂糖凝胶电泳和末端转移酶介导的dUTP缺口末端标记 (TUNEL)检测肝细胞凋亡。结果　用药后 2h开始 ,TNFαmRNA表达增加 ( 0 91± 0 75 ,正常值 0 32± 0 10 ) ,血清TNFα水平升高 [( 32 0 5 0± 86 5 7)ng/L ,正常值 ( 16 6 6± 7 0 1)ng/L],caspase 3少量表达 ;8h后 ,血清ALT和总胆红素 (TBil)水平显著增加 [分别为 ( 5 6 0 6 6± 6 0 2 0 )U/L和 ( 16 3 6 6± 34 5 1) μmol/L ,正常值为 ( 2 3 5 6± 8 0 3)U/L和 ( 14 90± 4 80 ) μmol/L],caspase 3表达至最高峰 ,并出现典型的肝细胞凋亡改变 ;12h后 ,血清ALT和TBil水平达最高峰 ,caspase 3表达较 8h减少 ,肝细胞坏死和凋亡同时存在。抗TNFα单抗阻断试验后 ,肝细胞凋亡和坏死明显减轻 ,TUNEL和caspase 3表达显著减少。 结论　TNFα有促进肝细胞凋亡与坏死作用 ,肝细胞凋亡与caspase 3的激活有关 ,在时间上肝细胞凋亡先于坏死。     

小鼠暴发性肝坏死模型建立及肿瘤坏死因子对其作用

小鼠暴发性肝坏死模型建立及肿瘤坏死因子对其作用朴文姬，俞红，周霞秋本文作者通过建立小鼠暴发性肝坏死模型，对其血清肿瘤坏死因子（ＴＮＦ）活性水平进行动态观察，进一步探讨ＴＮＦ在重型肝炎发病机制中的作用。材料和方法一、实验动物昆明鼠（上海中国科学院实验动...     

肿瘤坏死因子—α诱导体外培养大鼠肝细胞凋亡的作用

目的 了解肿瘤坏死因子－α（ＴＮＦ－α）对体外培养大鼠肝细胞凋亡的作用。方法 在半乳糖胺（ＧａＩＮ）或放线菌素Ｄ（ＡｃｔＤ）致敏情况下,加入ＴＮＦ－α,通过光镜、电镜及ＤＮＡ电泳等方法,观察ＴＮＦ－α对体外培养肝细胞凋亡的作用。结果 在大鼠注射ＧａｌＮ后分离肝细胞加入ＴＮＦ－α,ＡＬＴ上升,电镜下可见细胞坏死及凋亡;给正常鼠肝细胞ＡｃｔＤ加入ＴＮＦ－α,光镜下出现典型凋亡细胞,ＤＮＡ电泳出现凋亡的     

肿瘤坏死因子受体l在暴发性肝衰竭肠黏膜屏障功能损害中的作用

肠黏膜屏障对于抵抗肠道病原微生物的侵入具有重要作用。暴发性肝衰竭（FHF）时肠黏膜屏障的通透性增加^[1-4]，细菌及其毒性产物穿透肠壁进人体内，引起自发性腹膜炎甚至败血症。FHF时肠黏膜屏障通透性的增加与肠上皮细胞紧密连接的破坏有关。我们的前期研究发现，在FHF时，肠上皮细胞间紧密连接蛋白occludin表达下降，     

肿瘤坏死因子-α介导凋亡肝细胞内胱门蛋白酶-3的表达

目的 研究小鼠暴发性肝衰竭（FHF）动物模型凋亡肝细胞中凋亡基因胱门蛋白酶（cas-pase-3)mRNA及蛋白的表达及意义。方法 尾静脉注射肿瘤坏死因子-α（TNF-α）于D-氨基半乳糖（GalN)致敏的BALB/c小鼠,造成暴发性肝衰竭模型。用脱氧核糖核酸转移酶介导的缺口原位末端标记（ISEL）技术、电镜及琼脂糖凝胶电泳检测肝组织DNALadder观察肝细胞凋亡,分别用免疫组化和半定量逆转录-聚合酶链反应（RT-PCR）方法检测凋亡基因caspase-3蛋白及mRNA表达情况。结果 肝细胞凋亡率分别为0.0%(1.5h),0.3%(3.5h),3.15%(6h)和3.19%(9h)。肝组织中caspase-3mRNA和蛋白表达与肝细胞凋亡率成正相关趋势,结论 在TNF-α介导的小鼠暴发性肝衰竭模型中,caspase-3蛋白和mRNA表达上调并激活肝细胞凋亡的级联过程,最终导致肝细胞凋亡和坏死。     

微小RNA在肿瘤坏死因子α介导的急性肝功能衰竭小鼠体内的表达谱及其作用

目的 了解微小RNA(miRNA)在急性肝功能衰竭小鼠模型中的表达谱及对其发病机制的调控作用.方法 将BALB/c小鼠85只分为4组,模型组40只用D-氨基半乳糖(D-GalN)联合脂多糖(LPS)腹腔注射建立肝功能衰竭模型,D-GalN单药组20只、LPS单药组20只和对照组5只分别予D-GalN、LPS和0.9%NaCl溶液腹腔注射.在给药后0、5和7 h观察小鼠肝脏组织学变化,0、1、3、5、7和9 h留取小鼠血清和肝脏组织标本.采用实时定量PCR方法检测小鼠血清及肝组织中炎性细胞因子(TNF-α和IL-6)表达水平;采用miRNA微阵列(microarray)检测小鼠肝脏中miRNA的表达谱,实时定量PCR验证其表达.体外LPS诱导小鼠巨噬细胞Raw264.7活化,诱导不同时间点收集细胞检测细胞miRNA的表达情况.组间均数比较用单因素方差分析,相关性分析采用Pearson和Spearman相关分析.结果 miRNA微阵列发现小鼠急性肝功能衰竭发病过程中miRNA的表达谱发生了明显变化,与对照组相比,模型组有97个miRNA表达变化显著(P＜0.01),其中21个miRNA在给药后5 h和7 h均表达上调,27个miRNA均表达下调,进一步PCR验证得到miR-146a和miR-155随给药时间延长表现为持续性上调,且miR-155表达与TNF-α和IL-6表达均具有良好的相关性.体外实验进一步发现miR-146a和miR-155在活化的巨噬细胞中表达上调.结论 在小鼠急性肝功能衰竭发病过程中,伴随着miRNA表达谱的变化,炎性反应相关miR-146a和miR-155明显上调,可能在急性肝功能衰竭的免疫发病机制中发挥重要的调控作用.     

卡维地洛与阿替洛尔对肿瘤坏死因子α诱导内皮细胞凋亡的影响

本研究通过体外培养人肺静脉血管内皮细胞,观察卡维地洛与阿替洛尔对肿瘤坏死因子(TNF α)诱导内皮细胞凋亡的影响。一、资料与方法1.传4～6代生长良好的人肺静脉血管内皮细胞,消化,离心,用10 %的16 4 0培养液配成1×10 6/ml浓度的细胞悬液;6孔培养板每孔加入1×10 6/ml的细胞悬液1ml,再分别加入1mmol/L浓度卡维地洛(山东齐鲁制药厂) 0、2 5、5、10、15 μl(此时培养液中的卡维地洛浓度分别为0、2 5、5、10、15 μmol/L) ,然后加入10mg/LTNF α10 μl,每种处理5个复孔,置于37℃含5 %CO2 的饱和水汽培养箱中培养。阿替洛尔(Sigma公司)…     

Tumor necrosis factor alpha increases intestinal permeability in mice with fulminant hepatic failure

AIM:To determine the effect of tumor necrosis factor alpha(TNF-α) on intestinal permeability(IP) in mice with fulminant hepatic failure(FHF),and the expression of tight junction proteins.METHODS:We selected D-lactate as an index of IP,induced FHF using D-galactosamine/lipopolysaccharide and D-galactosamine/TNF-α,assessed the results using an enzymatic-spectrophotometric method,transmission electron microscopy,immunohistochemistry,Western blotting and real-time quantitative polymerase chain reaction.The effect of the administration of antiTNF-α immunoglobulin G(IgG) antibody,before the administration of D-galactosamine/lipopolysaccharide,on TNF-α was also assessed.RESULTS:IP was significantly increased in the mouse model of FHF 6 h after injection(13.57 ± 1.70 mg/L,13.02 ± 1.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.001).Electron microscopic analysis revealed tight junction(TJ) disruptions,epithelial cell swelling,and atrophy of intestinal villi.Expression of occludin and claudin-1 mRNA was significantly decreased in both FHF models(occludin:0.57 ± 0.159 fold vs baseline,P = 0.000;claudin-1:0.3067 ± 0.1291 fold vs baseline,P = 0.003),as were the distribution density of proteins in the intestinal mucosa and the levels of occludin and claudin-1 protein(occludin:0.61 ± 0.0473 fold vs baseline,P = 0.000;claudin-1:0.6633 ± 0.0328 fold vs baseline,P = 0.000).Prophylactic treatment with antiTNF-α IgG antibody prevented changes in IP(4.50 ± 0.97 mg/L vs 3.76 ± 0.67 mg/L,P = 0.791),intestinal tissue ultrastructure,and the mRNA levels of occludin and claudin-1 expression(occludin:0.8865 ± 0.0274 fold vs baseline,P = 0.505;claudin-1:0.85 ± 0.1437 fold vs baseline,P = 0.1),and in the protein levels(occludin:0.9467 ± 0.0285 fold vs baseline,P 0.05;claudin-1:0.9533 ± 0.0186 fold vs baseline,P = 0.148).CONCLUSION:Increased in IP stemmed from the downregulation of the TJ proteins occludin and claudin-1,and destruction of the TJ in the colon,which were induced by TNF-α in FHF mice.     

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.     

Tumor necrosis factor alpha in the pathogenesis of human and murine fulminant hepatic failure

BACKGROUND & AIMS: The tumor necrosis factor (TNF)-alpha/TNF receptor system is critical for liver development because hepatocytes undergo apoptosis if the antiapoptotic cascades resulting in RelA NF-kappaB activation are not effective. Therefore, we studied the role of TNF-alpha in fulminant hepatic failure (FHF) and developed a new therapeutic strategy. METHODS: Serum levels and hepatic expression of TNF-alpha and both TNF receptors were determined by enzyme-linked immunosorbent assay and immunohistochemistry. Adenoviral vectors were constructed expressing dominant-negative proteins interfering with intracellular TNF-alpha-dependent pathways. The relevance of these constructs was studied in primary mouse hepatocytes and in a murine model of FHF. RESULTS: Serum levels of TNF-alpha and TNF receptors are significantly increased in FHF; this increase correlates with patient prognosis. In livers of patients with FHF, infiltrating mononuclear cells express high amounts of TNF-alpha and hepatocytes overexpress TNF receptor 1 (TNF-R1). Apoptotic hepatocytes are significantly increased in FHF, and there is a strong correlation with TNF-alpha expression, which is even more pronounced in areas of mononuclear infiltrates. In an in vivo FHF model, the Fas-associated death domain (FADD), adenovirus selectively blocked the intracellular pathway, leading to mitochondrial cytochrome c release, caspase-3 activation, and, thus, apoptosis of hepatocytes. CONCLUSIONS: The results show that the TNF-alpha/TNF-R1 system is involved in the pathogenesis of FHF in humans. Studies in this animal model indicate that FADD may serve as a molecular target to prevent liver cell death in vivo.     

Role of cathepsin B-mediated apoptosis in fulminant hepatic failure in mice

AIM: To investigate the pathogenic role of cathepsin B and the protective effect of a cathepsin B inhibitor (CA-074Me) in fulminant hepatic failure in mice.METHODS: LPS/D-Gal N was injected into mice of the model group to induce fulminant hepatic failure;the protected group was administered CA-074me for 30 min before LPS/D-Gal N treatment; the normal group was given isochoric physiologic saline. Liver tissue histopathology was determined with HE at 2,4, 6 and 8 h after Lps/D-Gal injection. Hepatocyte apoptosis was examined by TUNEL method. The expression of cathepsin B in liver tissues was investigated by immunohistochemistry, Western blot and RT-PCR.RESULTS: Compared with the normal group, massive typical hepatocyte apoptosis occurred in the model group; the number of apoptotic cells reached a maximum 6 h after injection. The apoptosis index (AI) in the protected group was clearly reduced (30.4$$$$markedly increased in drug-treated mice compared with the normal group ( P < 0.01). Incubation with LPS/D-Gal N at selected time points resulted in a timedependent increase in cathepsin B activity, and reached a maximum by 8 h. The expression of cathepsin B was significantly decreased in the protected group ( P <0.01).CONCLUSION: Cathepsin B plays an essential role in the pathogenesis of fulminant hepatic failure, and the cathepsin B inhibitor CA-074me can attenuate apoptosis and liver injury.     

Tumor necrosis factor alpha and glutathione interplay in chronic heart failure

The pro-inflammatory cytokine, tumor necrosis factor alpha (TNF alpha), in concert with neurohormones, contributes to chronic heart failure (CHF) progression. This implies that TNF a antagonism may constitute an important target for CHF therapy. However, clinical trials in CHF patients using compounds that trap TNF alpha, comprising infliximab, an antibody directed to TNF alpha, and etanercept, a soluble recombinant receptor of TNF alpha, gave disappointing results bringing back to light the dual, short-term beneficial and long-term harmful effect of TNF alpha. This review focuses on the dual, concentration- and time-related effects of TNF alpha, the yin and yang action of TNF alpha in cardiac ischemia/reperfusion and contraction. Importantly, the harmful effects of TNF a are related to glutathione deficiency, a common hallmark to several other chronic inflammatory diseases. Recently, in rat models of CHF, oral administration of the glutathione precursor, N-acetylcysteine (NAC), was shown to hinder pathways of TNF alpha harmful signalling and to rescue cardiac structure and function. These results suggest that glutathione deficiency in association with TNF alpha activation may play a role in the pathophysiology of CHF and that NAC may represent a potential therapy in CHF.