miR-200s在中药丹芍化纤干预大鼠肝纤维化过程中的表达变化

 目的：观察肝组织中微小RNA-200家族成员（miR-200s）在肝纤维化形成及中药丹芍化纤胶囊干预过程中的表达变化并探讨其机制。方法：雄性Wistar大鼠皮下注射四氯化碳（CCl4）制备肝纤维化模型,干预组在给予CCl₄造模同时给予丹芍化纤胶囊（0.5 g/kg）灌胃,分别在4周和8周处死大鼠,测定肝脏指数和血清谷丙/谷草转氨酶(ALT和AST)活性,观察肝组织病理改变,real-time PCR方法分别检测肝组织miR-200a、-200b、-200c、-141和-429表达变化。结果：模型组及干预4周组大鼠肝脏指数、血清ALT和AST活性显著高于正常对照组（P<0.01）,8周模型组肝纤维化明显,并且肝组织中miR-200a、-200b、-200c、-141和-429表达较正常组显著增加（P<0.05）。丹芍化纤胶囊干预8周组肝功能生化指标及病理学改变与对照组无明显差异,且肝组织中miR-200a、-200b、-200c、-141和-429表达均低于8周模型组。结论：miR-200s在肝纤维化形成及中药丹芍化纤胶囊干预过程中的表达变化,提示其参与肝纤维化的发生发展并可能是潜在的中药作用靶点。     

肝炎相关性标志物检测在婴儿肝炎综合征的变化及临床意义

目的检测肝炎血清相关标志物水平，探讨该项指标在婴幼儿肝炎综合征中的变化及诊疗价值。方法临床确诊的婴儿肝炎综合征53例，采用全自动化学法分析技术，用杜邦全自动生化分析仪对53例肝炎综合征患儿及38例正常对照组婴幼儿进行血清TBA、ALP、ALT、AST、TBIL、DBIL肝功能指标测定。结果ALT、AST、ALP在该综合征时均值比正常上限升高约2—4倍；DBIL、TBIL升高3—6倍；而TBA则平均升高约8—10倍，患儿组血清TBA（80．84±48．06）μmol／L，异常率达94，3％，与正常对照组有显著性差异；TBA与垂清碱性磷酸酶（ALP）、直接胆红素（DBIL）检测水平有良好的相关性。结论TBA的灵敏度和特异性均优于常规指标，血清TBA测定在婴幼儿肝炎综合征的诊断、疗效观察及预后判断中均有重要临床意义，与ALP、DBIL联合检测，曼有利于婴儿肝炎综合征的诊疗。     

Lipid and carbohydrate parameters in children with chronic hepatitis C

PurposeHCV chronic infection still presents a very serious epidemiological and clinical problem. Apart from its cytopathic effect on liver parenchyma, its detrimental effect on lipid and carbohydrate metabolism has recently been emphasized. The aim of the study was to assess lipid and carbohydrate parameters in children with chronic HCV-related hepatitis.Material/MethodsThe study comprised 41 children with chronic hepatitis C (CHC) aged between 7 and 18 years, and 30 healthy controls. The anthropometric measurements of the subjects were taken, and, after overnight fasting, serum glucose, insulin, total bilirubin, AST, ALT, total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride levels were investigated. The HOMA IR insulin resistance index was also calculated.ResultsThe values for mean body mass index (BMI), glucose, insulin, bilirubin, LDL-cholesterol, HDL-cholesterol, triglycerides, HDL/C index and HOMA IR levels did not differ significantly between the two groups. AST and ALT were significantly higher in the control group. The serum levels of cholesterol and LDL-cholesterol showed a tendency toward lower values in the control group. We found positive correlation between serum levels of insulin and HOMA IR with staging (respectively r=0.336, P < 0.04 and r=0.386, P < 0.02).ConclusionsIn children with CHC and a relatively short duration of the disease, lipid and glucose disorders are not observed. Correlations between insulin and HOMA IR with staging suggest the ability of HCV to contribute to fibrosis through interference with glucose metabolism.     

miR-30a-5p启动子区DNA甲基化在同型半胱氨酸致肝损伤中的作用

目的:探讨微小RNA-30a-5p(miR-30a-5p)启动子区DNA甲基化在肝损伤中的作用。方法:随机选取4周龄胱硫醚β-合成酶(CBS)基因正常(CBS+/+)小鼠(n=12)及单基因敲除(CBS+/-)小鼠(n=12),均给予高蛋氨酸饮食8周。HL-7702细胞体外常规培养,分为对照(control)组、同型半胱氨酸(Hcy)组和Hcy+5-氮杂胞苷(AZC)组。全自动生化分析仪检测小鼠血清Hcy、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平;微板法测定肝细胞ALT和AST水平;小鼠肝脏石蜡切片行HE染色观察肝脏损伤情况;细胞活力染色检测肝细胞活力;RT-qPCR法检测小鼠肝脏组织和肝细胞中miR-30a-5p的表达;Pearson相关性分析肝脏miR-30a-5p表达与血清ALT和AST水平的相关性;巢式降落式甲基化特异性PCR(nMS-PCR)检测小鼠肝脏组织以及肝细胞中miR-30a-5p启动子区DNA甲基化水平的变化。结果:与CBS+/+对照组相比,CBS+/-组小鼠血清的Hcy、ALT和AST水平显著升高(P<0.05);HE染色显示肝细胞肿胀,可见核碎裂、溶解;肝脏miR-30a-5p表达明显降低(P<0.01);小鼠肝脏组织中miR-30a-5p的表达与血清ALT和AST的水平呈负相关(r2=0.4557,P=0.0003;r2=0.4626,P=0.0003);miR-30a-5p启动子区DNA的甲基化水平升高(P<0.01)。在细胞水平,与control组相比,Hcy组的ALT和AST水平升高(P<0.05,P<0.01),细胞活力显著降低,肝细胞miR-30a-5p启动子区DNA的甲基化水平升高(P<0.01),而使用AZC后miR-30a-5p启动子区DNA的甲基化水平降低(P<0.05),miR-30a-5p的表达上调(P<0.05)。结论:miR-30a-5p启动子区DNA高甲基化可能在肝损伤中发挥重要作用。     

Lack of association of hepatic estrogen receptor-alpha expression with histopathological and biochemical findings in chronic hepatitis C

Estrogens exert a protective effect against hepatic steatosis and fibrosis. Loss of estrogen receptor-alpha (ER-α) in the liver is associated with hepatic steatosis and inflammation in animal models. We conducted a study in order to investigate the presence and extent of ER-α expression in HCV infection, and its relationship with histological and biochemical findings. Ninety biopsy-proven chronic hepatitis C (CHC) patients were enrolled in the study. Liver biopsy specimens were immunohistochemically stained for ER-α expression. Nuclear ER-α expression percentage was calculated. ER-α was positive in 69 of the patients (76%). ER-α positive and negative groups were not significantly different in terms of age, gender, necroinflammatory activity, fibrosis, steatosis, serum levels of AST, ALT, ALP, GGT, and bilirubin. ER-α expression percentage was not correlated with fibrosis, steatosis, necroinflammatory activity and biochemical findings. Although estrogens are known to be protective against fibrosis and steatosis in animal models, we did not find any significant correlation between ER-α expression and histopathological and biochemical findings in CHC patients. These findings should be verified in further large scale studies.     

血清PAB、CHE、TBA和ALT的含量变化与肝炎病理分级的关系探讨

目的:探讨慢性乙型病毒性肝炎(乙肝)患者血清前白蛋白(PAB)、胆碱酯酶(CHE)、总胆汁酸(TBA)及丙氨酸氨基转移酶(ALT)水平的变化及其与肝脏炎症分级及纤维化分期之间的相关性。方法:观察93例经肝活检证实的慢性乙肝患者血清PAB、CHE、TBA及ALT水平的变化,并将其与肝活检组织的炎症分级和纤维化分期进行相关性分析。结果:肝脏病理组织炎症分为G1～G4级,纤维化分为S0～S4期。炎症轻重两组间PAB、CHE、TBA及ALT均有显著性差异(P<0.01)。随着纤维化程度的增加,ALT变化无规律,PAB、CHE逐渐下降,而TBA逐渐升高,且S4及S0、S1、S2和S3比较有显著性差异(P<0.01)。PAB、CHE、TBA及ALT与炎症分级之间,CHE、TBA和PAB与纤维化分期之间,相关性均非常显著(P均<0.01)。结论:PAB、CHE、TBA及ALT能较好地反映慢性乙肝肝脏的炎症活动水平,CHE、TBA和PAB能较敏感地反映慢性乙肝肝脏的纤维化程度;四个指标在一定程度上可以提示早期肝硬化。     

血清谷氨酰转肽酶含量测定在慢性乙型肝炎中的临床意义

目的探讨血清谷氨酰转肽酶（GGT）含量变化在慢性乙型肝炎（CHB）不同程度肝脏病理损害中的变化规律及临床意义。方法测定70例CHB患者血清ALT、AST、GGT水平，同时行肝活体组织检查，对肝脏进行炎症分级和纤维分期。分析ALT、AST、GGT与CHB之间的关系。结果（1）ALT、AST、GGT随炎症程度和纤维化程度的上升而上升，但到G4和s4后则下降。GGT随ALT、AST的升高而升高，ALT、AST和GGT的相关系数分别为：0．322、0．328（P〈0．05）。在保肝治疗后，ALT较快降至正常且GGT保持在一个较低水平的为轻度CHB，而随着ALT下降，GGT仍持续在一个较高水平的为中度及重度CHB，其中重度CHB的GGT水平有所波动。结论血清GGT比ALT、AST更准确的反映肝脏的炎症程度，GGT的活动度给临床判断慢乙肝的炎症提供了重要的判断依据。     

Evaluation of serum procollagen aminoterminal propeptide III, laminin, and hydroxyproline as predictors of severe fibrosis in patients with chronic hepatitis C

In an attempt to identify biochemical analytes that could enhance the discrimination between the patients with severe liver fibrosis (F3-F4) and mild fibrosis (F1-F2) based on absolute values of biochemical markers, we measured 12 analytes, including procollagen III aminoterminal propeptide (PIIINP), laminin, proline, hydroxylproline, glycine, AST, ALT, alkaline phosphatase, albumin, total bilirubin, total protein, and prothrombin time in 252 individuals with chronic hepatitis C infection (CHC). PIIINP and laminin were determined by radio-immunoassay; the degraded amino acids were determined using high performance liquid chromatography. Statistical analyses were performed by logistic regression, and receiver operating characteristic (ROC) curves. The best linear combination of blood markers was selected by multivariate discriminant analysis (MDA) for construction of the fibrosis discriminant score (FDS). FDS, an index of five markers (PIIINP, laminin, hydroxyproline, prothrombin activity, and AST/ALT) correctly classified 82% of the patients with severe liver fibrosis at a discriminant cut-off score=-0.5 (i.e., less than -0.5 indicated severe liver fibrosis and greater than -0.5 indicated mild liver fibrosis with sensitivity (76%) and specificity (89%). This result was reproduced in a validation study with no significant difference. In conclusion, FDS is useful for identifying severe liver fibrosis in patients with CHC.     

Low Rate of Adverse Hepatic Events Associated with Fosamprenavir/Ritonavir-Based Antiretroviral Regimens

Abstract

Purpose: To appraise the incidence of liver toxicity in a population of patients receiving fosamprenavir/ritonavir (FPV/r) with a high frequency of viral hepatitis co-infection. Method: 636 patients, 341 (54%) with HCV antibodies and 38 (5.6%) bearing serum HBsAg, were recruited. All of them received FPV/r 700/100 twice every day. 93 (27%) patients who tested positive for HCV antibodies showed an AST to platelet ratio index (APRI) higher than 1.5, consistent with significant liver fibrosis. Results: After a median (range) follow-up time of 6.91 (0.46–20.66) months, 3 (0.47%) patients developed grade ?3 ALT elevation. All the former patients were hepatitis virus co-infected, 2 with hepatitis C virus and 1 with hepatitis B virus. The frequency of grade ?3 ALT elevation in patients with HCV antibodies was 0.58% and in those harbouring HBsAg it was 2.63%. 4 (0.62%) patients suffered from a liver decompensation and 1 died due to a hepatic cause while on follow-up. No patients with APRI equal to or higher than 1.5 showed grade ?3 ALT elevation. Conclusion: The incidence of adverse hepatic events in patients receiving FPV/r including combinations seems to be low, even in subjects co-infected with hepatitis virus and in those with significant liver fibrosis.     

Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection

ABSTRACT: BACKGROUND: Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122). METHODS: Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection. RESULTS: We found that monocytes from chronic HCV infected treatment-naive (cHCV) but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFalpha, and had increased TNFalpha production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFalpha production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels. CONCLUSION: In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate increased miR-155 and TNFalpha production in chronic HCV infection. The positive correlation between serum miR-155 and miR-122 increase in cHCV may be an indicator of inflammation-induced hepatocyte damage.     

护肝合剂对肝星形细胞的活化及胶原表达的作用和机制

目的观察护肝合剂对肝损害模型大鼠血清TGF-β1的影响及护肝合剂含药血清对HSC—T6细胞的增殖、Ⅰ型胶原和Ⅲ型胶原的表达的影响,探讨该复方对肝纤维化的可能作用及其机制。方法设立正常组、护肝合剂组、模型组,采用卡介苗和脂多糖进行肝损害造模,然后观察3组ALT、AST和血清TGF-β1的变化。制备各组大鼠的血清,进行HSC-T6细胞的培养,采用MTT方法观察该细胞的增殖情况．并用RT—PCR的方法观察Ⅰ型胶原和Ⅲ型胶原的表达。结果模型组造模前后的ALT、AST及TGF-β1都显著升高（P〈0．01）,护肝合剂组上述指标较模型组显著下降（P〈0．01）,护肝合剂组的HSC-T6细胞增殖较模型组显著下降（P〈0．01）,且没有Ⅰ型胶原和Ⅲ型胶原的表达。结论护肝合剂能在一定程度上通过改善肝功能、降低TGF-β1水平抑制肝星形细胞的增殖,改善肝纤维化程度。     

慢乙肝患者HBV-DNA和Pre-S1Ag及HBVM与肝脏功能的检测意义

目的:了解乙型肝炎病毒HBV-DNA、Pre-S1Ag、乙肝标志物(HBVM)和肝脏功能之间的关系及临床意义.方法:采用荧光定量聚合酶链式反应(FQ-PCR)和ELISA分别检测169例乙肝病人血清HBV-DNA含量和乙肝标志物及Pre-S1Ag与肝功能,并对结果进行对比分析.结果:各种不同类型乙肝HBsAg的阳性率均高于91.1%,HBeAg、Pre-S1Ag的阳性率随HBV-DNA拷贝数的升高而升高,但肝功能和HBV-DNA拷贝数之间不存在相关关系.结论:同时检测血清乙肝标志物、Pre-S1Ag、HBV-DNA和肝脏功能对临床HBV感染、复制及传染性的判断以及肝功能损伤程度均有重要意义.     

Frequency and distribution of DNA fragmentation as a marker of cell death in chronic liver diseases

 To study the early stages of cell death in various types of chronic liver injury, liver biopsies from a total of 26 patients, including 7 with chronic hepatitis C(CHC), 4 with chronic hepatitis B(CHB), 7 with alcoholic liver disease (ALD), 4 with autoimmune or drug hepatitis(AI/DH), and 4 with primary biliary cirrhosis(PBC), were examined by an in situ nucleotidyl transferase assay (ISNTA), which detects DNA fragmentation. Positive nuclei in hepatocytes and sinusoidal lining cells were counted in all parenchymal areas, excluding triads and areas of fibrosis, using a computer with Sigmascan software. The number of positive hepatocytes/mm² was similar in the biopsies of patients with CHC, CHB, ALD and AI/DH, but significantly lower in PBC. The number of positive sinusoidal lining cells/mm² was significantly greater in biopsies with CHC compared to CHB, ALD, AI/DH and PBC. Double staining revealed that the ISNTA-positive sinusoidal lining cells were also CD68 positive, indicating that they were Kupffer cells. The frequency of ISNTA positivity did not correlate with serum AST or ALT levels, steatosis, cell swelling or cirrhosis. ISNTA-positive hepatocytes were more frequent than acidophilic bodies in every disease category. We conclude that apoptosis may be a common pathway of cell death in different liver diseases, that the high frequency of DNA fragmentation in Kupffer cells in CHC suggests that during chronic hepatitis C infection activated Kupffer cells may be subject to regulatory control by apoptosis and that ISNTA is more sensitive than acidophilic bodies in assessing the degree of cell injury in the liver. Received: 17 February 1997 / Accepted: 18 March 1997     

慢性重型乙型肝炎患者血清IL-23水平检测及其意义

目的 检测慢性重型乙型肝炎患者血清中IL-23表达,探讨IL-23水平与丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)及HBV DNA载量的相关性.方法 用酶联免疫吸附法(ELISA)检测50例慢性重型乙型肝炎患者(重肝组)及18名健康人(对照组)血清中IL-23表达,并与患者ALT、AST、TBil、HBV DNA载量进行相关性分析.结果 重肝组患者血清中IL-23表达高于对照组,两组之间的差异有统计学意义(P＜0.05);IL-23水平与ALT、AST呈正相关(P＜0.05),与TBil、HBV DNA载量无相关性(P＞0.05).结论 慢性重型乙型肝炎患者血清中IL-23表达增高,与炎症程度相关,可能参与慢性重型乙型肝炎的发病.     

Description of liver disease in a cohort of HIV/HBV coinfected patients

BackgroundFactors associated with advanced liver disease have been incompletely explored in HIV/HBV coinfected patients.ObjectivesTo describe liver-related morbidity, mortality, and related risk factors, in HIV/HBV coinfected patients.Study designWe followed-up 107 consecutive HIV/HBV coinfected patients. Clinical, biological and virological data were collected every 3 months. Liver-related mortality and a composite score were used to define advanced liver disease.ResultsThe patients were mainly sub-Saharan Africans (61%) or Europeans (33%). Forty-four percent of patients had liver biopsy, 78% of patients received lamivudine. Advanced liver disease (ALD) was diagnosed in 19/107 patients during follow-up (mean 4.8 years): 10 extensive fibrosis, 5 cirrhosis, 3 hepatocellular carcinoma resulting from cirrhosis, and 1 fulminant hepatitis following lamivudine withdrawal. Eleven patients died, 4 from HBV-related liver disease. In univariate analysis, male gender, mean HIV and HBV viral loads, and raised AST/ALT transaminases were associated with increased risk of ALD. The strongest associations, in a multivariate model, were mean AST transaminase and cumulated time receiving lamivudine, with a favourable effect. 39% of patients with increased mean AST presented with ALD, versus 7% when normal mean AST (Relative Risk 5.5).ConclusionsDuring HIV/HBV coinfection, transaminase levels are strongly associated with ALD. Normal mean AST has a high negative predictive value, contrary to previously reported data in HIV/HCV patients.     

Role of serum adiponectin level in the development of liver cirrhosis in patients with hepatitis C virus

Adiponectin possesses anti-inflammatory and insulin-sensitizing properties. Little is known about the role of adiponectin in hepatitis-C-related liver disease. The aim of our study was to find a relationship between serum adiponectin levels and different grades of steatohepatitis in HCV-infected patients and to correlate it with the severity of liver disease. Sixty HCV-infected patients were divided into two groups according to the presence/absence of steatosis proofed by abdominal ultrasonography and liver biopsy was selected. We evaluated the biochemical parameters for all patients including: ALT, AST, total bilirubin, direct bilirubin, alkaline phosphatase, serum albumin, prothrombin time, CBC, lipid profile, fasting blood glucose, fasting insulin, and serum adiponectin; HOMA-IR was calculated as [fasting insulin (mIU/l) × fasting glucose (mmol/l)]/22.5. We found that adiponectin was significantly lower in HCV-infected patients with steatosis than in those without steatosis. BMI, fasting blood glucose and HOMA-IR were significantly higher in HCV-infected patients with steatosis than in those without steatosis. Furthermore, it was found that steatosis correlates directly with fibrosis index, BMI, HOMA-IR, fasting blood glucose and ALT. Serum adiponectin levels inversely correlates with the grade of steatosis, histological activity index and the stage of fibrosis.     

MiR-34c promotes hepatic stellate cell activation and Liver Fibrogenesis by suppressing ACSL1 expression

Normally, there are multiple microRNAs involved in the pathogenesis of liver fibrosis. In our work, we aimed at identifying the role of miR-34c in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. Our results have shown that during natural activation of HSC, the level of miR-34c was increased significantly whereas acyl-CoA synthetase long-chain family member-1(ACSL1), which is a key enzyme can affect fatty acid(FA) synthesis, was decreased. A double fluorescence reporter assay further confirmed that ACSL1 is a direct target gene of miR-34c. Moreover, the inhibition of miR-34C can attenuate the synthesis of collagen in HSC-T6. In our rescue assay, ACSL1 expression was 1.49-fold higher compared to normal control cells which were transfected with the miR-34c inhibitor in a stable low expression ACSL1 cell line. While at the same time, α-SMA and Col1α expression decreased by 18.22% and 2.58%, respectively. Moreover, we performed an in vivo model using dimethylnitrosamine (DMN) in conjunction with the miR-34c agomir, combined with the treatment of DMN and the miR-34c agomir can increase liver fibrosis. Meanwhile, the degree of hepatic fibrosis was increased and lipid droplets reduced dramatically in rats and HSC-T6 cell treated with miR-34c mimics alone compared to untreated groups. Our results indicate that miR-34c plays an essential role in liver fibrosis by targeting ACSL1 closely associated with lipid droplets, and it might be used as a potential therapeutic target.