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.     

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.     

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。     

Adiponectin deficiency exacerbates lipopolysaccharide/D-galactosamine-induced liver injury in mice

AIM: To examine the effects of adiponectin on the functions of Kupffer cells, key modulators of lipopolysaccha-ride (LPS) -induced liver injury. METHODS: D-galactosamine (GaIN) and LPS were injected intraperitoneally into adiponectin-/- mice and wild type mice. Kupffer cells, isolated from Sprague-Dawley rats, were preincubated with or without adiponectin, and then treated with LPS. RESULTS: In knockout mice, GalN/LPS injection significantly lowered the survival rate, significantly raised the plasma levels of alanine transaminase and tumor necrosis factor-α(TIMF-α) and significantly reduced IL-10 levels compared with wild type mice. TIMF-αgene expression in the liver was which higher and those of IL-10 were lower in knockout mice than in wild type mice. In cultured adiponectin-pre-treated Kupffer cells, LPS significantly lowered TNF-αlevels and raised IL-10 levels in the culture media and their respective gene expression levels, compared with Kupffer cells without adiponectin-pre-treatment. CONCLUSION: Adiponectin supresses TNF-αproduction and induces IL-10 production by Kupffer cells in response to LPS stimulation, and a lack of adiponectin enhances LPS-induced liver injury.     

Differential effects of pyrrolidine dithiocarbamate on TNF-alpha-mediated liver injury in two different models of fulminant hepatitis

BACKGROUND/AIMS: Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of nuclear factor kappa B (NF-kappaB) activation. The present study aimed to investigate the effects of PDTC on lipopolysaccharide (LPS)-induced liver injury in two different models of fulminant hepatitis. METHODS: Mice infected with Bacillus Calmette Guerin (BCG) were challenged with LPS (0.2 mg/kg) to induce the model of inflammatory liver injury. Mice were injected with D-galactosamine (GalN, 600 mg/kg) and LPS (20 microg/kg) to induce the model of apoptotic liver injury. In the treatment groups, mice were pre-treated with PDTC (100 mg/kg), initiated 24 h prior to LPS. RESULTS: PDTC pretreatment reduced the infiltration of inflammatory cells, inhibited NF-kappaB activation and the expression of tumor necrosis factor alpha (TNF-alpha), attenuated nitric oxide production, and alleviated hepatic glutathione depletion. Correspondingly, PDTC reduced serum alanine aminotransferase, improved hepatic necrosis, and prolonged the survival in the BCG/LPS model. Conversely, PDTC accelerated death and aggravated liver apoptosis in the GalN/LPS model, although it reduced nitric oxide production, attenuated glutathione depletion, and inhibited the expression of TNF-alpha in liver. CONCLUSIONS: PDTC protects mice against BCG/LPS-induced inflammatory liver injury through the repression of NF-kappaB-mediated TNF-alpha release, while it seems to be detrimental in GalN/LPS-induced apoptotic liver damage.     

Different approaches to therapy of esophageal granular cell tumor (Abrikossoff tumor)

Abstract

Objective. This study aimed to investigate the protective effects of echinacoside, one of the phenylethanoids isolated from the stems of Cistanche salsa, a Chinese herbal medicine, on D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced acute liver injury in mice. Methods. We administered GalN (650 mg/kg) together with LPS (30 μg/kg) to mice by intraperitoneal injection to induce acute liver damage. Echinacoside (60 mg/kg) was given intraperitoneally to mice at 1 h prior to GalN/LPS exposure. Mice were sacrificed at different time points following GalN/LPS treatment, and the liver and blood samples were collected for future analysis. Results. It showed that GalN/LPS treatment produced severe hepatic injury, evidenced by significantly elevated plasma alanine aminotransferase (ALT) levels and abnormal histological changes such as hepatocyte necrosis or apoptosis, hemorrhage, fatty degeneration, and neutrophil infiltration. Notably, pretreatment with echinacoside remarkably improved the survival rate of GalN/LPS-treated mice and attenuated acute hepatotoxicity, as demonstrated by decreased ALT levels and improved histological signs. Echinacoside shows both anti-apoptotic and anti-inflammatory properties, characterized by a substantial inhibition of hepatocyte apoptosis and a significant reduction in the inflammatory markers, including myeloperoxidase, extracellular nucleosomes, high-mobility group box 1, and inflammatory cytokines in the plasma of mice, which may be important mechanisms related to its protective effect. Conclusion. Our results suggest that echinacoside can provide a pronounced protection against GalN/LPS-induced acute liver injury in mice, which may complement the available strategies for management of acute liver damage in clinical settings.     

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。     

Urotensin Ⅱ在急性肝衰竭小鼠肝组织中的表达及损伤作用

目的:探讨Urotensin Ⅱ(UⅡ)在急性肝衰竭(acute liver failure,ALF)小鼠肝组织中的表达及损伤作用.方法:♂Balb/c小鼠随机分成4组(每组6只):正常对照组(A组)、预处理对照组(B组)、模型组(C组)和预处理模型组(D组).模型动物以脂多糖(lipopolysaccharide,LPS)/D-半乳糖胺(D-galactosamine,D-GalN)腹腔注射,预处理动物在造模前30min,用UⅡ受体拮抗剂Urantide0.6mg/kg尾静脉注射.LPS/D-GalN攻击12h后,采集血清和肝组织标本,并观察24h小鼠存活情况;采用Reitman-Frankel法检测血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天冬氨酸氨基转移酶(aspartate amino-transferase,AST)活性水平;采用HE染色显微镜观察肝组织损伤程度;RT-PCR法检测UⅡ及其受体UTmRNA的表达;ELISA法检测血清UⅡ多肽分泌水平;免疫组织化学方法检测肝组织UⅡ多肽及其UT受体蛋白质表达.结果:C组小鼠死亡率为66.7%,A、B和D组所有动物均存活;LPS/D-GalN攻击引起C和D组小鼠血清ALT和AST水平显著升高(P<0.01),而D组较C组显著降低(2271.09U/L±102.24U/Lvs1160.67U/L±258.32U/L,1569.42U/L±204.04U/Lvs1030.31U/L±108.09U/L,P<0.01);C组小鼠肝组织结构破坏明显,见大片出血性坏死及炎症表现,D组肝组织结构保持完整,仅有局灶性出血坏死,炎症明显减轻;C和D组小鼠血清UⅡ多肽水平较A和B组高(P<0.01),但D组较C组明显降低(3.73g/L±0.52g/Lvs1.90g/L±0.27g/L,P<0.01);LPS/D-GalN诱导了C和D组小鼠肝组织UⅡ和UT的mRNA及蛋白质高水平表达,而D组的表达水平较C组显著降低(P<0.01).结论:LPS/D-GalN可诱导ALF小鼠肝组织表达和分泌UⅡ,并促进肝组织UT受体的表达;UⅡ的表达与分泌可能存在正反馈调控机制;UⅡ/UT受体介导了LPS/D-GalN诱导的ALF的发生.     

Sialoadenectomy enhances hepatic injury induced by lipopolysaccharide/galactosamine in mice

Background: Submandibular salivary glands (SMGs) synthesize, accumulate and secrete a large amount of epidermal growth factor (EGF) in mice. It is known that surgical removal of SMG (sialoadenectomy) alters cell turnover in the liver and exacerbates liver injury induced by lipopolysaccharide/galactosamine (LPS/GalN). Results: Here we show that such increased hepatotoxicity is not the consequence of the lack of EGF production from SMG. On the contrary, it appears to be the consequence of an inadequate cytokine production by the liver of sialoadenectomized mice. Thus, we found that the increase of plasma tumour necrosis factor‐α and interleukin‐6 was slower in sialoadenectomized than in sham‐operated mice. This is because of a decreased rate of production of both cytokines by the liver. We found that the increase of plasma corticosterone (CS) concentration is lower in sialoadenectomized than that in sham‐operated mice. Adrenalectomy exacerbated liver injury induced by LPS/GalN. In these animals, sialoadenectomy did not further increase the effect of LPS/GalN. Conclusions: Our results suggest that the effect of sialoadenectomy on LPS/GalN‐induced liver toxicity may be the consequence of an altered cytokine production by the liver and a reduced CS release from adrenal glands.     

Deletion of the Ron receptor tyrosine kinase domain in mice provides protection from endotoxin-induced acute liver failure

The targeted deletion of the cytoplasmic domain of the Ron receptor tyrosine kinase (TK) in mice leads to exaggerated responses to injury in several murine models of inflammation as well as increased lethality in response to endotoxin (lipopolysaccharide [LPS]). Using a well-characterized model of LPS-induced acute liver failure (ALF) in galactosamine (GalN)-sensitized mice, we show that Ron TK(-/-) mice display marked protection compared with control Ron TK(+/+) mice. Whereas control mice have profound elevation of serum aminotransferase levels (a marker of hepatocyte injury) and hemorrhagic necrosis of the liver, in dramatic contrast, Ron TK(-/-) mice have mild elevation of aminotransferase levels and relatively normal liver histology. These findings are associated with a reduction in the number of liver cells undergoing apoptosis in Ron TK(-/-) mice. Paradoxically, treatment of Ron TK(-/-) mice with LPS/GalN leads to markedly elevated (3.5-fold) serum levels of tumor necrosis factor (TNF) alpha, a key inflammatory mediator in this liver injury model, as well as reduced amounts of interleukin (IL) 10 (a suppressor of TNF-alpha production) and interferon (IFN)-gamma (a TNF-alpha sensitizer). These results show that ablation of the TK activity of the Ron receptor leads to protection from the development of hepatocellular apoptosis in response to treatment with LPS/GalN, even in the presence of excessive levels of serum TNF-alpha. In conclusion, our studies show that the Ron receptor TK plays a critical role in modulating the response of the liver to endotoxin.     

雷帕霉素对急性肝功能衰竭大鼠Toll样受体-4表达的影响

目的 探讨雷帕霉素抑制Janus激酶/信号转导和转录激活子(JAK/STAT)通路对急性肝功能衰竭大鼠Toll样受体(TLR)-4基因表达的影响.方法采用腹腔注射D-氨基半乳糖(D-GalN)800 mg/kg和脂多糖(LPS)8 μg/只,建立急性肝功能衰竭大鼠模型,分别在注射D-GalN和LPS后2、6、12、24、48 h 5个时间点留取大鼠血及肝脏标本.SD大鼠分为对照组(n=6)、急性肝功能衰竭模型组(n=30)、STAT抑制剂雷帕霉素(RPM)干预组(n=30),在各不同时间点检测ALT、AST.ELISA法检测血清TNF-α、IL-6水平,RT-PCR法检测大鼠肝组织TLR-4 mRNA表达.数据行t检验.结果急性肝功能衰竭组大鼠在造模后2 h TNF-α、IL-6水平均显著升高,6 h达峰值,RPM可明显抑制TNF-α、IL-6水平.急性肝功能衰竭组大鼠6、12、24、48 h肝组织中TLR-4 mRNA分别为0.745±0.135、1.092±0.175、1.115±0.152和0.812±0.130,RPM干预后分别为0.545±0.118、0.798±0.124、0.857±0.109和0.595±0.152,各时间点两组比较,差异均有统计学意义(t值分别为2.726、3.349、3.382和2.567,均P＜0.05).TLR-4 mRNA表达与ALT、AST均呈正相关(r值分别为0.722、0.712,均P＜0.01).结论抑制JAK/STAT通路可明显下调急性肝功能衰竭大鼠肝组织TLR-4表达,JAK/STAT通路可能参与急性肝功能衰竭过程中TLR-4mRNA表达的调控.     

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     

Genipin prevents fulminant hepatic failure resulting in reduction of lethality through the suppression of TNF-alpha production.

Genipin is a metabolite derived from the herbal medicine Inchinko-to. Little is known about the mechanism of genipin action on acute liver injury through inflammatory cytokines. We examined the effects of genipin on production of TNF-alpha in vivo and in vitro. Mice were given GalN/LPS with or without genipin treatment. All mice not given genipin died within 12h. But in mice given genipin, 8 of 15 mice survived for 24h after GalN/LPS administration. Histologically, hepatic necrosis and inflammatory cells infiltration were significantly slight in mice given genipin. Serum AST and ALT activity were significantly lower in mice given genipin. Serum and liver homogenate TNF-alpha levels were significantly lower in mice given genipin. However, in IL-6 and IL-1beta, there were no significant differences in mice given and not given genipin. TNF-alpha, NF-kappaB activation and TNF-alpha mRNA expression in a cultured mouse macrophage-like cell line J774.1 were significantly suppressed by genipin administration. In conclusion, the present findings suggest that genipin, a metabolite derived form the herbal medicine Inchinko-to improved acute liver dysfunction by suppressive effect of TNF-alpha production.     

Treg及IL-4对重症急性胰腺炎时枯否细胞M2极化状态的调节作用

目的探讨雨蛙肽联合脂多糖诱导小鼠重症急性胰腺炎(severe acute pancreatitis,SAP)的方法和Treg(CD4+CD25+FoxP3+T调节淋巴细胞)及IL-4(白介素-4)对SAP时枯否细胞(Kupffer cells)M2极化状态的调节作用的比较。方法 (1)正常组予腹腔注射生理盐水(NS);SAP组予腹腔注射雨蛙肽联合脂多糖。SAP组细分为3组:造模后9 h、12 h和24 h组,比较胰腺病理情况。(2)比较正常组与模型组(SAP 8 h+NS组)肝脏炎症因子的基因表达水平。(3)模型组分3组:SAP生理盐水对照组(SAP16 h+NS组)、SAP IL-4治疗组(SAP 16 h+IL-4组)、SAP Treg治疗组(SAP 16 h+Treg组)。RT-PCR检测肝脏炎症因子IL-1β、TNF-α和IL-10 mRNA表达水平;免疫荧光技术检测肝脏CD163、CCR7的表达。结果 (1)HE病理结果造模后24 h可见胰腺片状坏死。(2)模型组IL-1βmRNA的表达显著高于正常组(P0.05)。(3)SAP Treg治疗组IL-1β、TNF-αmRNA的表达显著低于SAP生理盐水对照组(P0.05);SAP Treg治疗组IL-1βmRNA的表达显著低于SAP IL-4治疗组(P0.05)。结论雨蛙肽联合脂多糖成功诱导小鼠SAP。IL-4和Treg对SAP具有一定的治疗作用。     

Effect of hepatocyte apoptosis induced by TNF-alpha on acute severe hepatitis in mouse models

AIM:To study the effect of hepatocyte apoptosis and necrosis induced by TNF-alpha on the pathogenesis of acute severe hepatitis (ASH).METHODS:The model of ASH was prepared in D-galactosamine (GalN) sensitized BALB/c mice by injection of either endotoxin (ET) or tumor necrosis factor-alpha(TNF-alpha. Morphological changes of apoptotic hepatocytes were studied by both light and electron microscope and in site end labeling method (ISEL). Molecular biological changes of DNA ladder were observed by electrophoresis of extract from liver tissues. Biochemical changes were measured by alanine aminotransferase (ALT), aspartic aminotransferase (AST) and TNF-alpha. The relation between apoptosis and necrosis was evaluated simultaneously.RESULTS:The sequence of hepatocyte apoptosis, necrosis, and final death from ASH was observed both in GalN/ET and GalN/TNF-alpha group. Apoptosis was prominent at 3.5h and 6h after injection of inducer, while necrosis became dominant at 9h after challenge. The appearance of apoptosis was earlier in GalN/TNF-alpha group than that in GalN/ET group. Pretreatment of mice with antiTNF IgG1 may completely prevent the liver injury induced by GalN/ET.CONCLUSION:TNF-alpha can cause liver amage by inducing hepatic apoptosis and necrosis in mice with endotoxemia.     

Salvage effect of E5564, Toll‐like receptor 4 antagonist on d‐galactosamine and lipopolysaccharide‐induced acute liver failure in rats

Background and Aims: The transmembrane protein Toll‐like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro‐inflammatory cytokine release. Activation of the pro‐inflammatory cytokine cascade, including tumor necrosis factor‐alpha (TNF‐α), has a pivotal role in the progression of severe liver injury. D‐galactosamine (GalN) and lipopolysaccharide (LPS)‐induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF‐α plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin‐stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS‐induced acute liver failure (ALF) in rats. Methods: ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 µg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS‐induced ALF rats were compared between those with and without E5564 treatment. Results: The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF‐α levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS‐induced ALF rats at 24 h (8% vs 43%). Conclusions: TLR4 antagonist E5564 reduced GalN+LPS‐induced acute liver injury in rats and improved the overall survival rate of GalN+LPS‐induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin‐induced TNF‐α overproduction of macrophages.     

LPS对乙型肝炎肝功能衰竭患者PBMC分泌细胞因子功能的影响

目的 通过观察PBMC分泌细胞因子功能的变化,探索内毒素血症对乙型重型肝炎患者免疫状态的影响.方法 选择10例乙型肝炎肝功能衰竭、10例慢性乙型肝炎患者和10例健康人,分别抽取外周血10 mL,分离血浆和PBMC.采用鲎实验检测血浆中LPS的浓度.同时常规体外培养PBMC,分为LPS刺激组和LPS未刺激组两组,培养24 h后收集上清液,Luminex检测上清液中TNF-α、IFN-γ和IL-6的浓度.数据处理采用方差分析,t检验及LSD法.结果 1.乙型肝炎肝功能衰竭患者PBMC体外培养上清液中TNF-α为(2 729±778) pg/mL、IFN-γ为(33.97±14.33) pg/mL及IL-6为(30415±8 132) pg/mL,健康人组分别为(350±190)、(5.53±4.86)和(4532±538),慢性乙型肝炎组分别为(504±226)、(0.58±0.59)和(15 587±2 983),乙型肝炎肝功能衰竭组明显高于健康人及慢性乙型肝炎患者.2.乙型肝炎肝功能衰竭患者血浆LPS平均值为(31.16±20.15) pg/mL,明显高于健康人及慢性乙型肝炎患者.3.LPS刺激组PBMC培养24h后,在乙型肝炎肝功能衰竭患者组.PBMC分泌的TNF-α和IL-6刺激组与未刺激组相比有明显降低(P=0.015和P=0.004),差异有统计学意义.IFN-γ刺激组与未刺激组相比差异无统计学意义(P＞0.737).而健康对照组和慢性乙型肝炎患者无明显改变.结论 乙型肝炎肝功能衰竭患者存在明显的内毒血症,PBMC处于活化状态.长期内毒血症可能会引起患者出现内毒素耐受状态,加重患者免疫状态的紊乱,提示临床诊治过程中要积极关注并评估患者免疫状态.     

Antithrombin III injection via the portal vein suppresses liver damage

AIM: To investigate the effects of antithrombin III (AT III) injection via the portal vein in acute liver failure.METHODS: Thirty rats were intraperitoneally challenged with lipopolysaccharide (LPS) and D-galactosamine (GalN) and divided into three groups: a control group; a group injected with AT III via the tail vein; and a group injected with AT III via the portal vein. AT III (50 U/kg body weight) was administrated 1 h after challenge with LPS and GalN. Serum levels of inflammatory cytokines and fibrin degradation products, hepatic fibrin deposition, and hepatic mRNA expression of hypoxia-related genes were analyzed.RESULTS: Serum levels of alanine aminotransferase, tumor necrosis factor-α and interleukin-6 decreased significantly following portal vein AT III injection compared with tail vein injection, and control rats. Portal vein AT III injection reduced liver cell destruction and decreased hepatic fibrin deposition. This treatment also significantly reduced hepatic mRNA expression of lactate dehydrogenase and heme oxygenase-1.CONCLUSION: A clinically acceptable dose of AT III injection into the portal vein suppressed liver damage, probably through its enhanced anticoagulant and anti-inflammatory activities.