Apoptosis and Bcl-2 expression in the livers of patients with steatohepatitis

OBJECTIVES: Apoptosis may play a role in the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). In this study, we investigated the modulation of apoptosis-related liver proteins in steatohepatitis. METHODS: Hepatocyte apoptosis was evaluated by the TUNEL assay in liver tissue of 12 patients with NASH, 12 with ASH and in histologically normal controls. In addition, caspase-3 processing was evaluated by immunoblot analysis. Expression of death receptors, Bcl-2 family members, and NF-kappaB inhibitor (IkappaB) were determined by western blot. Liver biopsies were also graded for inflammation and fibrosis. RESULTS: Apoptotic hepatocytes were markedly increased in NASH (P<0.05) and ASH (P<0.001) as compared to controls. Active caspase-3 was also elevated in steatohepatitis (P<0.01), coinciding with upregulation of pro-apoptotic Bax (P<0.001). Further, production of tumour necrosis factor-receptor 1 was increased up to 4-fold (P<0.05). Degradation of IkappaB increased >70% in steatohepatitis (P<0.001). Notably, Bcl-2 was also strongly expressed (>100-fold; P<0.001). These data were significantly correlated with relative degrees of portal and lobular inflammation. CONCLUSION: The results show that liver injury in NASH and ASH is associated with apoptosis and NF-kappaB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.     

Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression

Background and Aims:  We examined extrinsic and intrinsic (endogenous) mitochondrial apoptosis pathways in experimental non-alcoholic steatohepatitis (NASH).
Methods:  To assess extrinsic pathways, we measured hepatic expression of death-inducing cytokine receptors (tumor necrosis factor-α-receptor (TNF-R)1, TNF-R2, Fas, and TNFα-related apoptosis-inducing ligand-receptor (TRAIL-R) mRNA, TUNEL, caspase 3 activation, liver injury and liver pathology in mice fed a methionine and choline deficient (MCD) diet. For endogenous stress pathways, we determined serum insulin-like growth factor-1 (IGF-1), hepatic p53, Bcl-XL, tBid and p21 expression.
Results:  Methionine and choline deficient feeding increased alanine aminotransferase (ALT) and apoptosis from day 10, without increases in TNF-R1, TNF-R2, and Fas. However, murine TRAIL receptors, particularly decoyTRAIL-R1/TNFRSFH23 and Killer/DR5 mRNA increased. MCD feeding enhanced hepatic p53 expression, corresponding to ∼50% fall in serum IGF-1, decreased Bcl-XL, enhanced Bid cleavage to tBid, and up-regulation of p21. Nutritional restitution experiments showed that correcting either methionine or choline deficiency suppressed liver inflammation (extrinsic pathway), but failed to correct apoptosis, IGF-1 or p53.
Conclusions:  Methionine and choline deficiency lower IGF-1 to de-repress p53 during induction of steatohepatitis. The p53 induced by nutritional stress is biologically active in mediating mitochondrial cell death pathways, but may also be responsible for TRAIL receptor expression, thereby linking intrinsic and exogenous apoptosis pathways in NASH.     

非酒精性脂肪性肝炎中细胞凋亡及相关基因表达的作用

目的探讨细胞凋亡在NASH发病中的作用,以及细胞凋亡相关基因Fas配体(FasL),Fas和半胱氨酸-天冬氨酸蛋白酶(caspase)-3,-8表达或激活在细胞凋亡及NASH病情进展中的作用。方法采用高脂、胆碱-蛋氨酸缺乏饮食(MCD-)建立小鼠进展性NASH模型(实验周期分别为2、5、10d、3周和8周),并以胆碱蛋氨酸充足饮食(MCD )设立对照组。HE染色观察肝脏脂肪变、炎症活动度及纤维化程度;TUNEL分析观察细胞凋亡情况;FasL、Fas及caspase-8 mRNA表达采用实时定量RT-PCR检测,蛋白质表达采用Western blot;caspase-3活性分析采用ApoAlert caspase-3分析试剂盒测定。结果实验组动物,实验5 d可见轻微肝细胞脂肪变性,10 d形成轻度肝脂肪变,可见炎性细胞浸润,3周形成中至重度肝脂肪变及明显的炎性细胞浸润,8周肝脂肪变、肝细胞坏死、炎性细胞浸润加重并可发生肝纤维化。TUNEL分析显示,实验3周、8周实验组细胞凋亡指数显著高于对照组(15.59%±4.87%对比5.17%±3.19%;11.29%±3.22%对比5.41%±1.54%,P<0.05)。肝组织FasL mRNA和蛋白质表达于实验10 d及3周实验组显著高于对照组(P<0.05和P<0.01);实验组Fas mRNA表达于实验3周及8周明显上调(P<0.01),而蛋白质表达以实验8周增强明显(P<0.01)。实验组caspase-8 mRNA表达于实验3周和8周显著增高(P<0.01和P<0.05),而caspase-8活化以8周为著(P<0.05)。除5 d组外,各实验组caspase-3活性均高于对照组(P<0.05)。结论肝细胞凋亡存在于NASH模型中,并与疾病的进展,如肝细胞炎症和肝纤维化有关,肝细胞凋亡与FasL/Fas及其下游的caspase信号转导通路激活有关,可能为NASH发病的重要机制之一。     

Apoptotic and anti-apoptotic seromarkers for assessment of disease severity of non-alcoholic steatohepatitis

Background and study aimsThis study aimed to find out non-invasive markers for the assessment of severity of non-alcoholic steatohepatitis (NASH) in an attempt to decrease the need for liver biopsy. It also aimed to evaluate the key role of apoptosis in the pathogenesis of the disease and the suggested role of anti-apoptotic factors in therapeutic modalities and disease prognosis.Patients and methodsThe serum levels of soluble Fas (s. Fas), s. Fas ligand, cytokeratin 18 (CK-18) fragment and Bcl-2 were measured in 80 patients and 15 non-hepatic subjects as control. The patients were divided based on histological examination of liver biopsy into three groups. Group I included 40 patients with NASH, group II had 40 patients with non-alcoholic fatty liver disease (NAFLD) non-NASH and group III had 15 non-hepatic subjects as control. Apoptosis of hepatocytes was assessed by morphological examination using a light microscope and expressed as number per square millimetre.ResultsThere was a significant increase in the serum levels of s. Fas, s. Fas ligand and CK-18 fragments in the NASH group. The anti-apoptotic protein Bcl-2 showed significantly low levels in NASH patients. Apoptosis of hepatocytes was significantly higher in the NASH group. The degree of apoptosis was inversely correlated with the level of Bcl-2. A significant correlation between both s. Fas and CK-18 fragment with liver histology with regard to lobular inflammation and ballooning was found.ConclusionsIncreased serum levels of s. Fas and CK-18 fragment in the NASH group and its correlation with the severity of disease suggested the key role of apoptosis in NASH pathogenesis which can be used for the assessment of the severity of NASH. A high level of anti-apoptotic Bcl-2 in NAFLD suggests its protective role in disease progress.     

Bile acid levels are increased in the liver of patients with steatohepatitis

BACKGROUND/AIMS: The pathogenesis of steatohepatitis remains largely unknown; however, bile acids may play a role as potential mediators of liver damage. The aim of this study was to characterize bile acid profiles in liver tissue of patients with steatohepatitis. METHODS: Bile acid composition was determined by gas-liquid chromatography in liver tissue from patients with nonalcoholic steatohepatitis (NASH; n=15), patients with alcoholic steatohepatitis (ASH; n=14), and controls (n=8). Liver biopsies were graded for steatosis, inflammation, and fibrosis. RESULTS: Bile acids were moderately increased in liver tissue of steatohepatitis patients compared with controls (P<0.05). Deoxycholic, chenodeoxycholic, and cholic acids were elevated by 92, 64, and 43%, respectively, in patients with steatohepatitis (P<0.05). Cholic acid was the prevailing bile acid in NASH patients and in controls. More hydrophobic bile acid species were elevated in ASH patients compared with controls (P<0.05). Significant correlations were found in NASH patients between hepatic chenodeoxycholic acid and fibrosis, and between cholic acid and trihydroxy/dihydroxy bile acids and inflammation (P<0.05). In patients with ASH, cholic acid and trihydroxy/dihydroxy bile acids were correlated with steatosis (P<0.01). CONCLUSION: This study shows a distinct pattern of bile acids in the liver of patients with steatohepatitis. Further, the association between bile acids and histological liver injury suggests an association of specific bile acids and disease progression, possibly through bile acid-induced liver injury.     

过氧化物酶体增殖物激活受体γ抗肝细胞凋亡的机制

目的 研究靶向调控过氧化物酶体增殖物激活受体γ(PPAR γ)的表达及活性在非酒精性脂肪性肝纤维化中抗肝细胞凋亡的作用及其机制.方法 用高脂、胆碱-蛋氨酸缺乏饮食(MCD)喂养小鼠8周,设立对照组:胆碱-蛋氨酸充足饮食;模型组:MCD喂养;PPAR γ激动剂组:用MCD加50mg·kg-1·d-1罗格列酮喂养; PPAR γ抑制剂组:用MCD喂养,腹腔注射PPAR γ抑制剂GW9662 1 mg/kg,每周3次; PPAR γ基因导入组:用MCD喂养,腹腔注射重组腺病毒质粒Ad-PPAR γ 1×1010 vp,每周3次;腺病毒空载体导入组:用MCD喂养,腹腔注射重组腺病毒质粒Ad-LacZ 1×1010 vp,每周3次; PPAR γ基因导入联合激动剂组;用MCD喂养,腹腔注射Ad-PPAR γ 1×1010 vp,每周3次,同时给予罗格列酮50 mg·kg-1·d-1.检测肝组织PPAR γ、Fas、Fas配体、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白及天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)mRNA及蛋白质表达.多组样本均数间差异比较用单因素方差分析,用LSD-t检验进行组间比较.结果 肝组织促凋亡基因Fas、Fas配体、Bcl-2相关X蛋白及caspase-3表达较对照组明显上调,mRNA相对表达量分别为3.59±0.35比1.11±0.37、4.37±1.03比1.09±0.33、4.27±0.48比1.03±0.10及4.93±0.67比1.12±0.24,两组比较,t值分别为2.49、3.28、3.25、3.80,P值均<0.01;蛋白质相对表达量分别为1.96±0.07比0.45±0.07、0.53±0.07比0.22±0.02、1.32±0.06比0.59±0.03及1.51±0.23比0.36±0.09,两组比较,t值分别为1.51、0.31、0.73、1.14,P值均<0.01.抑凋亡基因Bcl-2表达亦相应上调,mRNA相对表达量为3.95±0.34比1.20±0.19,两组比较,t=2.75,P<0.01,蛋白质相对表达量为0.57±0.01比0.29±0.01,两组比较,t=0.28,P<0.01.PPAR γ激动剂组Fas、Fas配体、Bcl-2相关X蛋白、caspase-3及Bcl-2 mRNA相对表达量分别为3.78±0.58、3.66±0.83、3.04±0.37、2.54±0.62、4.42±0.42,与模型组比较,t值分别为0.18、0.71、1.23、2.39、0.46,P值分别>0.05、>0.05、<0.01、<0.01、>0.05,蛋白质相对表达量分别为1.06±0.03、0.30±0.01、0.70±0.05、1.19±0.30、0.90±0.01,t值分别为0.90、0.23、0.62、0.31、0.34,P值分别<0.01、<0.01、<0.01、>0.05、<0.01;PPAR γ基因导入组mRNA相对表达量分别为2.31±0.16、2.71±0.23、2.52±0.27、1.79±0.32、5.97±0.72,与模型组比较,t值分别为1.28、1.66、1.75、3.13、2.02,P值均<0.05,蛋白质相对表达量分别为1.73±0.07、0.43±0.04、1.01±0.08、1.31±0.10、1.56±0.04,t值分别为0.23、0.10、0.30、0.20、0.99,P值分别<0.01、<0.01、<0.01、>0.05、<0.01.结论 PPAR γ靶向性激活和(或)PPAR γ基因导入可抑制促凋亡基因主导的信号转导通路激活,抑制肝细胞凋亡.

Abstract：

Objective To elucidate the effect of targeted gene modulation of peroxisome proliferator activated receptor gamma (PPAR γ) on hepatocellular apoptosis in nutritional fibrotic steatohepatitis in mice. Methods C57BL/6J mice were fed with high fat, methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. Mice fed the MCD diet were treated with adenovirus carrying PPAR γ (AdPPAR|(A)), adenovirus-beta-galactosidase (Ad-LacZ), Ad-PPAR γ plus PPAR γ agonist rosiglitazone, or PPAR γ antagonist 2-chloro-5-nitro- benzaniliden (GW9662), respectively. H&E stain was performed for observation of hepatocellular apoptosis, hepatic steatosis, inflammation and fibrosis in the liver sections. The expression levels of mRNA and protein of PPAR γ and apoptosis related genes, Fas, Fas Ligand (FasL), B celllymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine-containing aspartate-specific proteases-3 (caspase-3) were detected by real-time RT-PCR and Western blot assay, respectively. Results Mice fed with MCD diet for 8 weeks showed severe hepatic injury including steatosis, hepatocellular apoptosis, inflammatory infiltration and fibrosis, concomitancy with enhanced expression of pro-apoptosis genes, Fas, FasL, Bax and caspase-3 and increased expression of anti-apoptosis gene Bcl-2, by comparing with the control group. The mRNA expression levels of these genes were 3.59 ± 0.35 vs 1.11 ± 0.37, 4.37 ± 1.03 vs 1.09 ± 0.33,4.27 ± 0.48 vs 1.03 ± 0.10, 4.93 ± 0.67 vs 1.12 ± 0.24 and 3.95 ± 0.34 vs 1.20 ± 0.19,and LSD-t values were 2.49, 3.28, 3.25, 3.80 and 2.75, as compared with the control group, P < 0.01; the protein expression levels were 1.96 ± 0.07 vs 0.45 ± 0.07, 0.53 ± 0.07 vs 0.22 ± 0.02, 1.32 ± 0.06 vs 0.59 ± 0.03,1.51 ± 0.23 vs 0.36 ± 0.09 and 0.57 ± 0.01 vs 0.29 ± 0.01, and LSD-t values were 1.51, 0.31, 0.73, 1.14 and 0.28, P < 0.01. Administration of PPAR γ agonist rosiglitazone and/or Ad-PPAR γ significantly ameliorated hepatic steatosis, hepatocellular apoptosis, necroinflammation and fibrosis. These effects were associated with repressed expression of pro-apoptosis genes and up-regulated expression of anti-apoptosis gene. After rosiglitazone treatment, the mRNA expression levels were 3.78 ± 0.58, 3.66 ± 0.83, 3.04 ± 0.37, 2.54 ± 0.62 and 4.42 ± 0.42, and LSD-t values were 0.18, 0.71, 1.23,2.39 and 0.46, as compared with MCD group, the P values were 0.627,0.241, <0.01, <0.01 and 0.278; the protein expression levels were 1.06 ± 0.03, 0.30 ± 0.01, 0.70 ± 0.05, 1.19 ± 0.30 and 0.90 ± 0.01, and LSD-t values were 0.90,0.23,0.62,0.31 and 0.34, the P values were <0.01, <0.01, <0.01,0.122, <0.01. After Ad-PPAR γ treatment, the mRNA expression levels were 2.31 ± 0.16, 2.71 ± 0.23, 2.52 ± 0.27, 1.79 ± 0.32 and 5.97 ± 0.72, and LSD-t values were 1.28, 1.66,1.75, 3.13 and 2.02, as compared with MCD group, P < 0.05; the protein expression levels were 1.73 ± 0.07, 0.43 ± 0.04, 1.01 ± 0.08, 1.31 ± 0.10 and 1.56 ± 0.04, and LSD-t values were 0.23,0.10,0.30,0.20 and 0.99, with P values equal 0.009,0.01, <0.01,0.322 and <0.01. Conclusions This study provided evidences for the protective role of activation and overexpression of PPAR γ in ameliorating hepatocellular apoptosis in mice with hepatic fibrosing steatohepatitis.     

Predictors reflecting the pathological severity of non-alcoholic fatty liver disease: comprehensive study of clinical and immunohistochemical findings in younger Asian patients

BACKGROUND AND AIMS: The spectrum of non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to severe steatohepatitis (NASH). The aim of our study was to identify clinical predictors distinguishing NASH from steatosis and to study the pathogenesis of NASH in a young Korean population. METHODS: Clinical and biochemical variables from 39 biopsied NAFLD patients were retrospectively analyzed. All liver biopsy specimens were immunohistochemically examined for Kupffer cells (CD68, CD14), as well as expression of tumor necrosis factor-alpha (TNF-alpha) and mitochondrial uncoupling protein 2 (UCP-2). RESULTS: There were no significant differences in serum biochemistry between the two groups (15 steatosis vs 24 NASH). There was a significant difference between the body mass index (BMI) values (kg/m(2)) of the NASH (28.4 +/- 3.4 kg/m(2)) and steatosis (25.8 +/- 2.8 kg/m(2)) patients (P < 0.025), with a BMI of 28.9 kg/m(2) representing the best threshold for distinguishing NASH from steatosis patients. BMI was significantly related to the degree of fibrosis (P < 0.01). CD68+ Kupffer cells were more common in the livers of NASH patients (P < 0.05), and TNF-alpha and UCP-2 were expressed in all NASH specimens and were related with the severity of inflammation and fibrosis (P < 0.05). CONCLUSIONS: BMI could be used to distinguish NASH from steatosis in younger Korean patients. A high BMI with a low alanine aminotransferase (ALT) value tended to suggest the presence of severe fibrosis in NASH, while the number of CD68+ Kupffer cells and the staining intensity of TNF-alpha and UCP-2 were correlated with general pathologic severity in patients with NAFLD.     

非酒精性脂肪性肝炎大鼠肝组织Bcl-2和Bax的表达及其意义

目的探讨凋亡调控蛋白Bel-2和Bax表达在大鼠非酒精性脂肪性肝炎发病中的作用。方法将20只Wistar大鼠随机分为普通饲料喂养组（正常组）和高脂饲料喂养组（模型组）,连续喂养12周后,测定血清甘油三酯（TG）、总胆固醇（TC）、丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）,观察肝组织病理学变化和炎症活动度计分,采用westem blot法检测肝组织Bcl-2和Bax的表达。结果模型组大鼠肝小叶内肝细胞呈弥漫性脂肪变,伴大量炎性细胞浸润及散在性点状坏死;模型组动物肝组织Bax蛋白表达较正常组显著性升高（1．02±0．33对0．51±0．03,P〈0．01）,而Bel-2蛋白较正常组则明显下降（0．15±0．31对0．52±0．01,P〈0．01）,Bel-2／Bax比值降低（0．14±0．01对1．03±0．06,P〈0．01）。结论凋亡调控蛋白Bel-2和Bax的表达变化可能参与了NASH的发生和发展过程。     

The role of immune mechanisms in alcoholic and nonalcoholic steatohepatitis: a 2015 update

So far, innate immune mechanisms have been recognized as the main responsible for the evolution of both alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH). However, increasing evidence points toward the possible role of adaptive immune responses, as an additional factor in promoting hepatic inflammation in steatohepatitis. In this article, we discuss recent data involving circulating antibodies and lymphocyte-mediated responses in sustaining the progression of ASH and NASH to fibrosis, as well as the possible mechanisms implicated in favoring the onset of adaptive immunity in the setting of steatohepatitis.     

Molecular mechanisms of apoptosis in hepatocellular carcinoma cells induced by ethanol extracts of Solanum lyratum Thumb through the mitochondrial pathway

AIM To explore the induction effects and mechanism of Solanum lyratum Thumb(ST) on human hepatocellularcarcinoma SMMC-7721 cells through the mitochondrial pathway.METHODS The experiments were conducted on three groups: an experimental group (with ST ethanol extracts' concentration being 2.5, 5 and 10 mg/L), a negative control group (with only nutrient solution, 0 mg/L ST ethanol extracts), and a positive control group (2.5 mg/L DDP). The inhibition rate of cell proliferation was checked by using the methyl thiazolyl tetrazolium method, and cell apoptosis was tested by TUNEL method. Furthermore, RT-PCR was used to examine m RNA expression of Fas, Fas L, caspase-8, caspase-3, p53 and Bcl-2 genes.RESULTS Compared with the negative control group, the inhibition and apoptosis rates of the experimental group with different concentrations of ST extracts on human hepatocellular carcinoma SMMC-7721 cells significantly increased(P0.05). Besides, the m RNA expression of Fas L and Bcl-2 significantly decreased(P0.05) while the m RNA expression of Fas, caspase-8, caspase-3 and p53 increased significantly. When compared with the positive control group, the experimental groups with 5 mg/L ST ethanol extracts showed effects similar to the positive control group.CONCLUSION ST ethanol extracts induced the apoptosis of hepatocellular carcinoma SMMC-7721 cells through up-regulated Fas, caspase-8, caspse-3 and p53, and down-regulated Fas L and Bcl-2 in the mitochondrial pathway.     

Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis

BACKGROUND & AIMS: The pathogenesis of nonalcoholic steatohepatitis (NASH) remains poorly understood. Although apoptosis is a common mechanism of liver injury, the extent and clinical significance of apoptosis in NASH has not been examined. Thus, the aims of this study were to quantify hepatocyte apoptosis in NASH, correlate it with disease severity, and identify possible mechanisms of apoptosis induction. METHODS: Hepatocyte apoptosis was assessed in NASH, simple steatosis, alcoholic hepatitis, and controls without liver disease using the TUNEL assay and immunohistochemistry for activated caspases 3 and 7. Liver specimens were also graded according to the magnitude of inflammation and fibrosis. RESULTS: TUNEL-positive cells were significantly increased in liver biopsy specimens from patients with NASH compared with simple steatosis and controls. Unexpectedly, TUNEL-positive cells were also greater in NASH vs. alcoholic hepatitis. Immunohistochemistry demonstrated active caspases 3 and 7 in NASH specimens, confirming the occurrence of apoptosis in this disease. A positive correlation was observed between hepatocyte apoptosis and hepatic fibrosis and inflammatory activity, respectively. The Fas receptor was strongly expressed in hepatocytes in liver specimens from NASH patients as compared with controls. CONCLUSIONS: Hepatocyte apoptosis is significantly increased in patients with NASH and correlates with disease severity, suggesting that antiapoptotic therapy may be useful in this syndrome.     

The pathway of estradiol-induced apoptosis in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a disease with unknown etiology. The pathologic role of sex hormones and apoptosis in SLE has often been discussed. We studied the effects of estradiol in the pathway of induced apoptosis in Iranian SLE patients. T lymphocytes from 35 SLE patients and 20 age-matched controls were isolated and cultured in the presence of 10⁻⁸ M 17-β estradiol. The expression levels of Fas, Fas ligand (FasL), Bcl-2, caspase-8, and caspase-9 mRNAs were determined semiquantitatively in comparison to the expression level of beta actin RNA. Estradiol exposure did not have any significant effects on the expression levels of Fas, Bcl-2, and caspase-9 in SLE patients and controls. However, the expression levels of FasL and caspase-8 were significantly increased in SLE patients, but not in controls. This suggests the probable involvement of extrinsic apoptosis pathway in estradiol-induced apoptosis in SLE.     

Hepatocyte apoptosis is a pathologic feature of human alcoholic hepatitis

BACKGROUND/AIMS: The pathogenesis of alcoholic hepatitis (AH) remains poorly understood. Although apoptosis is now recognized as a mechanism of liver injury, the extent and mechanisms of apoptosis in human AH remain unknown. Thus, our aims were to quantify hepatocyte apoptosis in patients with AH, correlate it with disease severity, and identify the mechanisms of apoptosis induction. METHODS: Hepatocyte apoptosis was assessed in 26 patients with AH and 27 controls without liver disease using the TUNEL assay and immunohistochemistry for activated caspase 3. Liver specimens were also graded for disease severity. The expression of the death receptors, Fas and tumor necrosis factor-alpha receptor 1 (TNF-R1), was assessed by immunohistochemistry. RESULTS: In contrast to normal livers, TUNEL- and caspase 3-positive hepatocytes were readily observed in the livers of patients with AH. In the AH group, hepatocyte apoptosis was significantly higher in patients with a serum bilirubin of > 3 mg/dl. Apoptosis was also greater in grade 4 steatohepatitis. The Fas receptor was strongly expressed in hepatocytes in AH, but not in normal livers; the TNF-R1 expression was comparable in both groups. CONCLUSIONS: The present results demonstrate that hepatocyte apoptosis is significantly increased in human AH and justify therapeutic strategies aimed at inhibiting apoptosis in this disease.     

NALP3炎性体在小鼠非酒精性脂肪性肝炎发病中的作用

目的 探讨NALP3炎性体在非酒精性脂肪性肝炎(NASH)小鼠发病中的作用.方法 予C57BL小鼠高脂饮食(高脂组)及脂多糖建立非酒精性脂肪肝模型(NASH组),同时设立正常饮食组作为对照.观察各组小鼠血清ALT、AST值与计算肝指数,评定脂肪性肝炎程度,ELISA法测定肝脏TNF-α水平,蛋白质印迹法测定NF-κB、NALP3、caspase-1、ASC的表达;实时荧光定量反转录-PCR检测NALP3、caspase-1、ASC mRNA的表达水平.数据处理采用单因素方差分析及LSD法.结果 NASH组小鼠ALT、AST及肝指数明显高于高脂组,差异有统计学意义(P＜0.05),高脂组和NASH组的NALP3 mRNA表达高于正常对照组(P＜0.05).NASH组caspase-1和ASC mRNA表达水平明显高于正常对照组及高脂组(P＜0.01),高脂组caspase-1和ASC mRNA表达高于正常对照组(P＜0.05).NASH组NALP3、caspase-1和ASC表达水平明显高于正常对照组(P＜0.01),NASH组NALP3、caspase-1和ASC表达水平明显高于高脂组(P＜0.05),高脂组NALP3、caspase-1和A SC表达水平与正常对照组无明显差异(P＞0.05).结论 NALP3炎性体参与胰岛素抵抗的形成,可能是影响NASH发生和发展的重要因素.     

Nuclear factor-kappaB translocation mediates double-stranded ribonucleic acid-induced NIT-1 beta-cell apoptosis and up-regulates caspase-12 and tumor necrosis factor receptor-associated ligand (TRAIL)

The mechanism of induction of apoptosis by double-stranded RNA (dsRNA) is not fully characterized. The dsRNA is normally present in extremely low quantities in cells, but following infection with RNA viruses, large quantities of the dsRNA viral replicative intermediate may be produced triggering the antiviral response as well as cell death. In this report, transfection of polyinosinic-polycytidylic acid [poly(I:C)] into NIT 1 cells has been used as a model of intracellular dsRNA-induced beta-cell apoptosis. At 18 h post transfection, 45% of the cells were apoptotic as indicated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining, and this was accompanied by an increase in nuclear factor kappaB (NF-kappaB) p50/p65 nuclear translocation and cleavage of caspases 3 and 8. The NF-kappaB inhibitor peptide, SN50, significantly reduced caspase-3 activity and the percentage of TUNEL-positive cells, substantiating a role for NF-kappaB in inducing intracellular dsRNA-mediated apoptosis. Concomitantly, RNA-dependent protein kinase activity was observed at 3 h post transfection along with phosphorylation and degradation of inhibitory kappaB-alpha. Expression of TRAIL (TNF-related apoptosis-inducing ligand), Fas, IL-15, and caspase-12 mRNAs was up-regulated in the presence of poly(I:C) but not when SN50 was also added. In contrast, there was no change detected in Fas, Fas-associated death domain, Bcl-2, Bcl-xl, Bax, p53, or XIAP(X-linked inhibitor of apoptosis protein) expression up to 12 h after poly(I:C) transfection. In addition, caspase-12 was cleaved, and phosphorylation of eukaryotic initiation factor 2alpha occurred, suggesting that an endoplasmic reticulum stress pathway was involved in addition to NF-kappaB induction of an extrinsic pathway, possibly mediated by TNF-related apoptosis-inducing ligand.     

在Fas诱导Bel-7402细胞凋亡中p38MAPK调节Bcl-2的表达

目的:探讨p38MPAK是否参与Fas和AD诱导Bel-7402细胞的凋亡过程,以及p38MPAK和bcl-2的关系,进一步揭示p38MAPK的凋亡途径.方法:在Fas和AD作用24h后,用MTT法检测Bel-7402细胞的活力,用Western-blot和RT- PCR法检测p38MAPK,p-p38MAPK和Bcl-2 expression,用免疫荧光法对p-p38MAPK进行细胞定位.结果:随着Fas浓度的增加,Bel-7402细胞的活力明显抑制,p38MAPK和p-p38MAPK表达明显增高(P<0.01),且p-p38MAPK由胞质易位到胞核.Bcl-2的表达明显降低(P<0.01),并且这种降低趋势被p38MAPK抑制剂SB203580所阻止.结论:p38MAPK参与Fas诱导的凋亡途径,以磷酸化形式激活后抑制Bcl一2的表达,进而促进细胞凋亡.     

ASH and NASH

Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) have a similar pathogenesis and histopathology but a different etiology and epidemiology. NASH and ASH are advanced stages of non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD). NAFLD is characterized by excessive fat accumulation in the liver (steatosis), without any other evident causes of chronic liver diseases (viral, autoimmune, genetic, etc.), and with an alcohol consumption ≤20-30 g/day. On the contrary, AFLD is defined as the presence of steatosis and alcohol consumption >20-30 g/day. The most common phenotypic manifestations of primary NAFLD/NASH are overweight/obesity, visceral adiposity, type 2 diabetes, hypertriglyceridemia and hypertension. The prevalence of NAFLD in the general population in Western countries is estimated to be 25-30%. The prevalence and incidence of NASH and ASH are not known because of the impossibility of performing liver biopsy in the general population. Up to 90% of alcoholics have fatty liver, and 5-15% of these subjects will develop cirrhosis over 20 years. The risk of cirrhosis increases to 30-40% in those who continue to drink alcohol. About 10-35% of alcoholics exhibit changes on liver biopsy consistent with alcoholic hepatitis. Natural histories of NASH and ASH are not completely defined, even if patients with NASH have a reduced life expectancy due to liver-related death and cardiovascular diseases. The best treatment of AFLD/ASH is to stop drinking, and the most effective first-line therapeutic option for NAFLD/NASH is non-pharmacologic lifestyle interventions through a multidisciplinary approach including weight loss, dietary changes, physical exercise, and cognitive-behavior therapy.     

Abnormal electron microscopic findings of nonalcoholic steatohepatitis and related factors]

BACKGROUND/AIMS: In spite of increasing interests about nonalcoholic steatohepatitis (NASH), there are few reports about the ultrastructure of hepatocyte in this disease. The aim of this study was to clarify abnormal electron microscopic (EM) findings and related factors in NASH. METHODS: Total of fourteen patients who underwent liver biopsy due to steatohepatitis were included. Precise personal history was taken and variable blood tests such as liver function test, lipid profile, and serum iron study were done. Pathologic examination with light and electron microscopy was done by single pathologist. RESULTS: Eleven men and three women were included and mean age was 33.7+/-12.8 years. Nine patients drinking less than 40 g/week was grouped as "NASH group" and other 5 patients drinking more than 40 g/week and body mass index less than 25 was grouped as "ASH (Alcoholic Steatohepatitis) group". Polymorphism of mitochondria such as megamitochondria or loss of cristae was major abnormal EM findings and was more common in "NASH group" than "ASH group" (p=0.027). There was no significant clinical or pathological factors related with the presence of these abnormal EM findings. CONCLUSIONS: Polymorphism of mitochondria is major abnormal EM finding of steatohepatitis and is more common in NASH than ASH. And there is no significant clinical or pathological factors which could predict the presence of these abnormal EM findings.     

Caspase-8在慢性肝炎患者肝组织中的表达及意义

目的探讨Caspase-8在慢性乙型肝炎、慢性丙型肝炎和非酒精性脂肪性肝炎中的作用及机制。方法应用免疫组化方法对70例慢性乙型肝炎、50例慢性丙型肝炎、50例非酒精性脂肪性肝炎和15例正常肝组织进行Caspase-8检测。结果Caspase-8在慢性乙型肝炎、慢性丙型肝炎、非酒精性脂肪性肝炎中的表达（58.6%、64.0%、82.0%）高于其在正常肝组织中表达（13.3%）,差异有统计学意义（P〈0.05）;并与肝细胞凋亡密切相关（r=0.409,P〈0.001）。Caspase-8在重型肝炎中的表达阳性率高,轻型肝炎表达阳性率最低,差异有统计学意义（P〈0.05）。Caspase-8在不同ALT水平肝炎患者肝组织中的表达差异无统计学意义（P〉0.05）;结论 Caspase-8活性增强导致细胞凋亡增多,在肝病理生理过程中起着重要作用。