丙氨酸-乙醛酸转氨酶2类似物1对人肝癌97H和HCCLM3细胞增殖、凋亡和周期的影响

目的 观察丙氨酸-乙醛酸转氨酶2类似物1(AGXT2L1)对肝癌细胞增殖、凋亡和周期的调控作用。方法 实时定量反转录聚合酶链反应(RT-qPCR)检测肝癌细胞株97H、HCCLM3和正常肝细胞株LO2中AGXT2L1的表达水平;设计并构建AGXT2L1的靶向siRNA,将AGXT2L1-siRNA转染97H和HCCLM3细胞株构建AGXT2L1靶向敲低组和阴性对照组,通过RT-qPCR验证敲低效果;采用细胞计数试剂盒(CCK-8)实验检测各组细胞在0、12、24、48、72、96 h的增殖能力;采用流式细胞周期实验检测各组细胞在G1、S、G2期的分布情况;采用原位缺口末端标记法(TUNEL)实验检测各组细胞的凋亡率。结果 AGXT2L1在肝癌97H和HCCLM3细胞中相对表达高于正常肝细胞LO2(1 350.04±91.36、1 710.64±92.34,t97H=25.574,P<0.01;tHCCLM3=32.068,P<0.01)。转染AGXT2L1-siRNA后,对比对照组及空白组,肝癌97H和HCCLM3细胞内AGXT2L1相对表达量明显降低(0.19±0.04比1.10±0.04,t97H=-28.722,P<0.01;0.20±0.01比0.93±0.09,tHCCLM3=-13.887,P<0.01)。CCK-8实验结果显示,敲低AGXT2L1后,97H和HCCLM3细胞的增殖率明显下降(0.71±0.01比0.95±0.12,t97H=-31.243,P<0.01;0.67±0.13比0.87±0.17,tHCCLM3=-17.083,P<0.01),差异有统计学意义。细胞周期实验结果显示,干扰AGXT2L1表达后,S期细胞比例低于对照组[(13.45±1.20)%比(18.73±0.28)%,t97H=-7.451,P<0.01;(15.26±0.52)%比(20.45±1.08)%,tHCCLM3=-7.483,P<0.01];G2期细胞比例低于对照组[(16.90±0.54)%比(19.99±0.54)%,t97H=-6.992,P<0.01;(16.23±0.28)%比(23.36±0.39)%,tHCCLM3=-25.720,P<0.01],抑制细胞从G1期向S期转变,抑制肝癌细胞增殖。细胞凋亡实验显示:干扰AGXT2L1表达后,97H和HCCLM3细胞凋亡率明显升高[(13.06±1.86)%比(1.06±0.75)%,t97H=14.673,P<0.01;(14.41±2.21)%比(0.80±0.32)%,tHCCLM3=14.947,P<0.01],差异有统计学意义。结论 敲低AGXT2L1的表达抑制肝癌97H和HCCLM3细胞株增殖,促进细胞凋亡。

Serum γ-glutamyltransferase,alanine aminotransferase,and aspartate aminotransferase activity in Iranian healthy blood donor men

AIM: To determine serum γ-glutamyltransferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activity, and to assess their correlation with demographic and clinical findings in healthy blood donors. METHODS: This cross-sectional study was performed in 934 male blood donors, aged 18 to 68 years, who consecutively attended Tehran blood transfusion service in 2006. All participants were seronegative for HBV or HCV infections, non alcohol users, and all underwent a standard interview and anthropometric tests. Clinical and biochemical parameters including AST, ALT, and GGT activities were determined. Patients taking drugs known to cause hepatic fat deposition were excluded. For AST, ALT, and GGT variables, we used 33.33 and 66.66 percentiles, so that each of them was divided into three tertiles. RESULTS: Mean AST, ALT, and GGT activities were 25.26 ± 12.58 U/L (normal range 5-35 U/L), 33.13 ± 22.98 (normal range 5-35 U/L), and 25.11 ± 18.32 (normal range 6-37 U/L), respectively. By univariate analyses, there were significant associations between increasing AST, ALT, or GGT tertiles and age, body weight, body mass index, and waist and hip circumferences (P < 0.05). By multiple linear regression analyses, ALT was found to be positively correlated with dyslipidemia (B = 6.988, P = 0.038), whereas ALT and AST were negatively correlated with age. AST, ALT, and GGT levels had positive correlation with family history of liver disease (B = 15.763, P < 0.001), (B = 32.345, P < 0.001), (B =24.415, P < 0.001), respectively.CONCLUSION: Although we did not determine the cutoffs of the upper normal limits for AST, ALT, and GGT levels, we would suggest screening asymptomatic patients with dyslipidemia and also subjects with a family history of liver disease.

A dose-up of ursodeoxycholic acid decreases transaminases in hepatitis C patients

AIM:To examine whether a dose-up to 900 mg of ursodeoxycholic acid(UDCA) decreases transaminases in hepatitis C patients.METHODS:From January to December 2007,patients with chronic hepatitis C or compensated liver cirrhosis with hepatitis C virus(HCV)(43-80 years old) showing positive serum HCV-RNA who had already taken 600 mg/d of UDCA were recruited into this study.Blood parameters were examined at 4,8 and 24 wk after increasing the dose of oral UDCA from 600 to 900 mg/d.RESULTS:Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),and gamma-glutamyl transpeptidase(GGT) levels were signifi cantly decreased following the administration of 900 mg/d as compared to 600 mg/d.The decrease in ALT from immediately before the dose-up of UDCA to 8 wk after the dose-up was 14.3 IU/L,while that for AST was 10.5 IU/L and for GGT was 9.8 IU/L.Platelet count tended to increase after the dose-up of UDCA,although it did not show a statistically signifi cant level(P=0.05).Minor adverse events were observed in 3 cases,although no drop-outs from the study occurred.CONCLUSION:Oral administration of 900 mg/d of UDCA was more effective than 600 mg/d for reducing ALT,AST,and GGT levels in patients with HCV-related chronic liver disease.

Metformin prevents hormonal and metabolic disturbances and 1,2-dimethylhydrazine-induced colon carcinogenesis in non-diabetic rats

Effects of two doses of the anti-diabetic drug, metformin （MF）, on hormonal and metabolic levels of serum of non-diabetic male Wistar rats with 1,2-dimethylhydrazine （DMH）-induced colon tumor adenocarcinomas were studied. Carcinogenesis in the animals was also observed. Rats with DMH-induced colon adenocarcinomas had elevated levels of serum glucose, insulin, insulin- like growth factor-l, total cholesterol, triglycerides, catalase, malonic dialdehyde, glycated hemoglobin, aspartate aminotransferase, and alanine aminotransferase and decreased hemoglobin. Treatment with two doses of MF normalized maiority of these changes in DMH-treated rats, whereas the drug was ineffective in rats without DMH treatment. The only exception was the decreased triglyceride levels in MF-treated rats. A 100 mg/kg dose of MF increased DMH-induced exophytic colon carcinomas and decreased endophytic tumors compared with untreated rats. Moreover, both MF doses increased DMH-induced and highly differentiated tumors and decreased the invasiveness of colon carcinomas compared with rats provided with DMH and water. Therefore, effects of MF on metabolic homeostasis are critical for preventing colon cancer.

Pathological evolution of hepatitis C virus-“Healthy carriers”

AIM： To determine factors associated with fibrosis progression in hepatitis C virus （HCV）-infected patients without significant initial pathological lesions. METHODS： Seventy six untreated HCV-infected patients with initially normal liver as defined by a Knodell score ≤ 3, with 2 liver biopsies and detectable HCVRNA were included. Markers of fibrosis progression were assessed. RESULTS： Median duration of infection and time between paired biopsies was 13 （95% CI： 1-28） and 4 （95% CI： 2-16） years respectively. Alaninetransaminase （ALT） activity was normal in 43.4% of cases. 50% demonstrated progression of the necroinflammation and 34% of fibrosis after a median time evolution of 4 years （95% CI： 2-16）. The median difference in the necro-inflammation and fibrosis score between biopsies was low, 1.5 and 0.0 respectively. Univariate analysis showed there was no difference between fibrosis activity or evolution according to genotype or viral load. A higher fibrosis progression （P = 0.03） was observed in patients with body mass index （BMI） 〉 25. Fibrosis progression correlated with the time interval between biopsies （P = 0.01）. A significant progression of activity （1.7 vs 0.4, P 〈 0.05） or fibrosis （0.9 vs 0.0, P 〈 0.01） was observed in patients with elevated ALT. There was a significant correlation between activity progression and fibrosis progression（P = 0.003）. Multivariate analysis demonstrated that fibrosis progression was associated with elevated ALT, BMI 〉 25 and the time interval between 2 biopsies. CONCLUSION： There is no fibrosis progression in 66% of patients without significant initial histopathological lesion. Fibrosis progression is associated with elevated ALT and BMI 〉 25.

Protective effects of C-phycocyanin on alcohol-induced acute liver injury in mice

Excessive alcohol consumption leads to liver disease. Extensive evidence suggests that C-phycocyanin(C-PC), a chromophore phycocyanobilin derived from Spirulina platensis, exerts protective eff ects against chemical-induced organ damage. In this study, we investigated whether C-PC could protect against ethanol-induced acute liver injury. Serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG), total cholesterol(CHOL), low-density lipoprotein(LDL), liver homogenate malondialdehyde(MDA), superoxide dismutase(SOD) content were measured, and pathological examination of liver sections were examined. C-PC showed obvious inhibitory eff ects on serum ALT, AST, TG, CHOL, LDL and MDA, and SOD content significantly increased in the liver. The structure of hepatic lobules was clear, liver sinus returned to normal, and liver cell cords were arranged in neat rows. Cloudiness, swelling, inflammatory cell infiltration and spotty necrosis of liver cells were significantly reduced. Therefore, C-PC can significantly protect against ethanol-induced acute liver injury.

Prevention of Carbon Tetrachloride （CCl4）-Induced Liver Damage in Guinea Pigs by Cyphostemma digitatum

Cyphostemma digitatum （Vitaceae） is a perennial, climbing, succulent undershrub with compound fleshy leaves and tendrils. The plant is used mainly as a food flavoring, but it is also a main constituent of traditional Yemeni soup （Marak）. Besides that, it has been described to be used as a medicinal plant. The aim of this work was to study the hepatoprotective effect of the aqueous extract of C. digitatum against CCl4-induced liver injury in Guinea pigs. Animals were divided into four groups. Group I, served as normal control. Group II received 2 mL CCl4/kg b.w. diluted with olive oil, at 1：1 ratio on day 11. Group III （test group） was pre-treated orally with 100 mg/kg b.w. aqueous leaves extract of C. digitatum for 10 days followed by subcutaneous injection of CC14 （2 mL/kg b.w.）, once on day 11. Group IV were orally given Liv-52 （100 mg/kg b.w.） once daily for 10 days followed by subcutaneous injection of CC14. Our results show that, the activity of serum hepatic enzymes （alanine aminotranferase （ALT）, aspartate aminotranferase （AST）, and alkaline phosphatase （ALP）） were significantly elevated in Guinea pigs treated with CCl4, while both the C. digitatum extract and Liv-52 reduced significantly these enzymes activity. Also, the levels of glucose, urea, cholesterol and triglycerides were decreased when compared with intoxicated control. Histopathological examination of intoxicated animals showed fatty changes, inflammation and necrosis indicating liver damage, while the animals received C. digitatum or Liv-52 showed less pathological effects or normal liver when compared to animals treated with CC14 alone. Biochemical and histological results confirm the hepatoprotective effect of aqueous extract of C. digitatum.

EFFICACY OF URSODEOXYCHOLIC ACID IN TREATMENT OF ISCHEMIA-REPERFUSION INJURY IN LIVER TRANSPLANTATION

Objective To investigate the efficacy of ursodeoxycholic acid in treatment of ischemia-reperfusion injury （IRI） in liver transplantation. Methods Eighty liver transplantation adult recipients were preoperatively enrolled and randomized into the ursodeoxycholic acid （ UDCA ） （42 cases） and control （ 38 cases ） groups between May 2005 and June 2006. The two groups were statistically compared in liver biochemical parameters on post- transplant d 1, 7, 14, and 21. Rates of severe IRI-induced liver graft dysfunction, acute cellular rejection （ ACR ） episode, drug-induced hepatotoxicity, viral hepatitis, and recurrence of primary liver disease were measured within 3 weeks post-transplantation; and rates of vascular, biliary complications, and death were also measured within 3 months post-transplantation. Results In the UDCA group, serum levels of alanine aminotransferase （ ALT） on post-transplant d 7, 14, and 21 were significantly lower than those in the control group （ P = 0. 002,0. 030, 0. 049, respectively）. Compared with the control group, serum levels of aspartate aminotransferase （ AST） and y-Glutamyltranspeptidase （ GGT） on d 7 were also lower in the UDCA group （ P =0. 012 and 0. 025）. The cases of severe IRI- induced liver graft dysfunction in the UDCA group were significantly fewer than those in the control group （ 17. 5% vs. 26.3%, P =0. 048）. There were no significant differences in rates of ACR episode, histological Banff grading, or drug-induced hepatotoxicity within 3 weeks post-transplantation as well as rates of vascular, biliary complications, and death within 3 months post-transplantation between the two groups. We did not find any case of viral hepatitis or recurrence of primary liver disease in the study. Conclusion UDCA treatment can improve graft IRI early after liver transplantation. It significantly decreased serum ALT level and incidence of severe IRl-induced liver dysfunction within post-transplant 3 weeks. Cytoprection of hepatocytes by UDCA was more outstanding than that of bile duct when cold ischemia time was beneath 12 h. Vascular and biliary complications within 3 months post-transplantation can not be affected by UDCA administration in the study.

Relationship between alanine aminotransferase levels and metabolic syndrome in nonalcoholic fatty liver disease

Objective： To investigate the relationship between alanine aminotransferase （ALT） levels and metabolic syndrome （MS） in nonalcoholic fatty liver disease （NAFLD）. Methods： A total of 26527 subjects who received medical health checkup in our hospital from January 2005 to July 2007 were enrolled in the study. The diagnosis of fatty liver was based on ultrasound imaging. MS was defined according to the criteria of the Adult Treatment Panel III. ALT, triglyceride （TG）, high density lipoprotein cholesterol （HDL-c）, fasting plasma glucose （FPG）, height, weight, waist circumference （WC）, systolic blood pressure （SBP） and diastolic blood pressure （DBP） were measured in each subject to analyze the relationship between MS and ALT activity Results： （1） The prevalence of NAFLD in men （30.94%） was significantly higher than that in women （15.65%）; （2） The incidence of MS in NAFLD （33.83%） was significantly greater than that in non-NAFLD （10.62%）; （3） Of the 6470 subjects with NAFLD, in the age-adjusted partial correlation analysis, there were statistically significant correlations between the ALT levels and most metabolic risk factors in each sex （P〈0.01）, except that ALT levels multiple stepwise regression analysis, SBP lost its significance, and had no correlation with HDL-c in women. Moreover, in the WC, body mass index （BMI）, age, DBP, TG and FPG were independently associated with ALT levels in both sexes （P〈0.05）. HDL-c remained significant and was independently related to ALT levels in men; （4） ALT levels were significantly higher in subjects with MS compared to those without MS （P〈0.001）. Mean ALT levels increased with the number of MS components in each sex （P〈0.05 for trend）. Conclusion： We found a strong relationship between ALT levels and MS in NAFLD and revealed that the cluster of MS components might be the predictor for ALT elevations.

Noninvasive Parameters and hepatic fibrosis scores in children with nonalcoholic fatty liver disease

AIM:To evaluate the noninvasive parameters and hepatic fibrosis scores in obese children with nonalcoholic fatty liver disease(NAFLD).METHODS:A total of 77 children diagnosed with NAFLD via liver biopsy were included and divided into 2 subgroups according to the histopathologic staging of hepatic fibrosis:mild(stage 0-1)vs significant fibrosis(stage 2-4).Clinical and laboratory parameters were evaluated in each patient.The aspartate aminotransferase(AST)/alanine aminotransferase(ALT)ratio,AST/platelet ratio index(APRI),PGA index,Forns index,FIB-4,NAFLD fibrosis score,and pediatric NAFLD fibrosis index(PNFI)were calculated.RESULTS:No clinical or biochemical parameter exhibited a significant difference between patients with mild and significant fibrosis.Among noninvasive hepatic fibrosis scores,only APRI and FIB4 revealed a significant difference between patients with mild and significant fibrosis(APRI:0.67±0.54 vs 0.78±0.38,P=0.032 and FIB4:0.24±0.12 vs 0.31±0.21,P=0.010).The area under the receiving operating characteristic curve of FIB4 was 0.81,followed by Forns index(0.73),APRI(0.70),NAFLD fibrosis score(0.58),AST/ALT ratio(0.53),PGA score(0.45),and PNFI(0.41).CONCLUSION:APRI and FIB4 might be useful noninvasive hepatic fibrosis scores for predicting hepatic fibrosis in children with NAFLD.

Healthy ranges of serum alanine aminotransferase levels in Iranian blood donors

AIM: The healthy ranges for serum alanine aminotransferase(ALT) levels are less well studied. The aim of this study was to define the upper limit of normal (ULN) for serum ALT levels, and to assess factors associated with serum ALT activity in apparently healthy blood donors. METHODS: A total of 1 939 blood donors were included.ALT measurements were performed for all cases using the same laboratory method. Healthy ranges for ALT levels werecomputed from the population at the lowest risk for liver disease. Univariate and multivariate analyses were performed to evaluate associations between clinical factors and ALT levels.RESULTS: Serum ALT activity was independently associated with body mass index (BMI) and male gender, but not associated with age. Association of ALT with BMI was more prominent in males than in females. Upper limit of normal for non-overweight women (BMI of less than 25) was 34 U/L,and for non-overweight men was 40 U/L.CONCLUSION: Serum ALT is strongly associated with sex and BMI. The normal range of ALT should be defined for male and female separately.

Melatonin ameliorates experimental hepatic fibrosis induced by carbon tetrachloride in rats

AIM:To investigate the protective effects of melatonin on carbon tetrachloride(CCl4)-induced hepatic fibrosis in experimental rats.METHODS:All rats were randomly divided into normal control group,model control group treated with CCl4 for 12 wk,CCl4+NAC group treated with CCl4+NAC(100 mg/kg,i.p.)for 12 wk,CCl4+MEL-1 group treated with CCl4+melatonin(2.5 mg/kg)for 12 wk,CCl4+MEL-2 group treated with CCl4+ melatonin(5.0 mg/kg)for 12 wk,and CCl4+MEL-3 group treated with CCl4+melatonin(10 mg/kg).Rats in the treatment groups were injected subcutaneously with sterile CCl4(3 mL/kg,body weight)in a ratio of 2:3 with olive oil twice a week.Rats in normal control group received hypodermic injection of olive oil at the same dose and frequency as those in treatment groups.At the end of experiment,rats in each group were anesthetized and sacrificed.Hematoxylin and eosin(HE)staining and Van Gieson staining were used to examine changes in liver pathology.Serum activities of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and protein concentration weremeasured with routine laboratory methods using an autoanalyzer.Hydroxyproline(HYP)content in liver and malondialdehyde(MDA)and glutathione peroxidase(GPx)levels in liver homogenates were assayed by spectrophotometry.Serum hyaluronic acid(HA),laminin(LN),and procollagenⅢN-terminal peptide(PⅢNP)were determined by radioimmunoassay.RESULTS:Pathologic grading showed that the fibrogenesis was much less severe in CCl4+MEL3 group than in model control group(u=2.172,P<0.05),indicating that melatonin(10 mg/kg)can significantly ameliorate CCl4-induced hepatic fibrotic changes.The serum levels of ALT and AST were markedly lower in CCl4+MEL treatment groups(5,10 mg/kg)than in model control group(ALT:286.23 ±121.91 U/L vs 201.15±101.16 U/L and 178.67 ±103.14 U/L,P=0.028,P=0.007;AST:431.00 ±166.35 U/L vs 321.23±162.48 U/L and 292.42 ±126.23 U/L,P=0.043,P=0.013).Similarly,the serum laminin(LN)and hyaluronic acid(HA)levels and hydroxyproline(HYP)contents in liver were significantly lower in CCl4+MEL-3 group(10 mg/kg)than in model control group(LN:45.89±11.71μg/L vs 55.26± 12.30μg/L,P=0.012;HA:135.71±76.03μg/L vs 201.10±68.46μg/L,P=0.020;HYP:0.42±0.08 mg/g tissue vs 0.51±0.07 mg/g tissue,P=0.012).Moreover,treatment with melatonin(5,10 mg/kg)significantly reduced the MDA content and increased the GPx activity in liver homogenates compared with model control group(MDA:7.89±1.49 noml/mg prot vs 6.29±1.42 noml/mg prot and 6.25±2.27 noml/mg prot,respectively,P=0.015,P=0.015;GPx:49.13± 8.72 U/mg prot vs 57.38±7.65 U/mg prot and 61.39± 13.15 U/mg prot,respectively,P=0.035,P=0.003).CONCLUSION:Melatonin can ameliorate CCl4-induced hepatic fibrosis in rats.The protective effect of melatonin on hepatic fibrosis may be related to its antioxidant activities.

Clinical impact of selective transarterial chemoembolization on hepatocellular carcinoma:A cohort study

AIM： To prospectively long term clinical impact evaluate the short and of selective transarterial chemoembolization （TACE） on fiver function in patients with hepatocellular carcinoma （HCC）. To assess side effects in relation to treatments. To analyze the overall survival and HCC progression free survival probability. METHODS： One hundred and seventeen cirrhotic patients with HCC were enrolled. Baseline liver function included Child-Pugh score and serum levels of alanine- aminotransferase （ALT）, prothrombin time （PT） and bilirubin. According to Cancer Liver Italian Program （CLIP） and Barcelona Clinic Liver Cancer （BCLC） staging systems, 71 patients were eligible for TACE; 32 had previously received treatment for HCC. No significant differences in liver function were observed between previously treated and not treated patients. TACE was performed by selective catheterization of the arteries nourishing the lesions. While hospitalized, patients underwent clinical, hematologic and ultrasonographic assessments. One month after TACE a CT scan was performed to assess tumor response. A second TACE was performed ＂on demand＂ Liver function tests were checked in all patients every four months. RESULTS： After first TACE, the mean Child-Pugh score increased from a mean baseline 5.62 ±1.12 to 6.11 ±1.57 at discharge time （P 〈 0.0001）, decreasing after four months to 5.81 ± 0.73 （not significant）. ALT, PT and bilirubin significantly （P 〈 0.0001） increased 24 h after TACE and progressively decreased until discharge. After the second TACE, variations in Child-Pugh score, ALT, PT and bilirubin were comparable to that described after the first TACE. No major complications were observed. The mean follow-up was 14.7 ＋ 6.3 mo （median： 16 mo）. Only one patient died. No other patient experienced important long term worsening of clinical status. The overall survival probability at twenty-four months was 98.18% with a correspondent HCC progression free survival probability of 69%. CONCLUSION： Selective TACE may produce significant, but transitory increases in ALT values, with no major impact on liver function and Child-Pugh score. Preservation of liver function is achievable also in patients previously treated with other therapeutic modalities and in patients undergoing multiple TACE cycles. Liver function can remain stable in the long-term, with optimal medium term survival. This result can be achieved through rigorous patient selection on the basis of tumour characteristics and clinical conditions.

An Experimental Study on Drugs for Improving Blood Circulation and Removing Blood Stasis in Treating Mild Chronic Hepatic Damage

Large and small doses of drugs for improving blood circulation and removing blood stasis were used in model rats to treat mild chronic hepatic damage induced by carbon tetrachloride (CCl4). The results show that large dose of Dang Gui ([symbol: see text] Radix Angelicae Sinensis) and Dan Shen ([symbol: see text] Radix Salviae Miltiorrhizae) (drugs for regulating blood flow) and small dose of Yu Jin ([symbol: see text] Radix Curcumae) and Niu Xi ([symbol: see text] Radix Achyranthis Bidentatae) (drugs for activating blood flow) can significantly elevate the activity of SOD (P

Dynamic tracking of stem cells in an acute liver failure model

AIM： To investigate a dual labeling technique, which would enable real-time monitoring of transplanted em- bryonic stem cell （ESC） kinetics, as well as long-term tracking. METHODS： Liver damage was induced in C57/BL6 male mice （n = 40） by acetaminophen （APAP） 300 mg/kg administered intraperitoneally. Green fluores- cence protein （GFP） positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbo- cyanine iodide （DiR） immediately before transplantationinto the spleen. Each of the animals in the cell therapy group （n = 20） received 5 x 106 ESCs 4 h following treatment with APAP. The control group （n = 20） re- ceived the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS lumina-2 at 30 rain post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohisto- chemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine amino- transferase （ALT） was measured as an indication of liver damage.RESULTS： DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradu- ally moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imag- ing, and confirmed that the highest photon emission was in the liver （P 〈 0.0001）. At 2 wk, the DiRsignal was no longer detectable in vivo; however, immuno- histochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP ＋ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP ＋ve cells incorporated within the sinu- soidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP ＋re cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly （P = 0.016） lower and al- bumin （P = 0.009） was significantly higher when com- pared with the control group at the 2 wk time-point;however there was no difference in mortality between the two groups. CONCLUSION： Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribu- tion, survival and engraftment of transplanted cells, and could be used for cell therapy models.

A better parameter in predicting insulin resistance:Obesity plus elevated alanine aminotransferase

AIM:To investigate the association of obesity and elevated alanine aminotransferase with insulin resistance and compare these factors with metabolic syndrome. METHODS:We enrolled a total of 1308 male workers aged from 22 to 63 years.Data was extracted from the workers'periodic health check-ups in hospitals.All cases were from the community of northern Taiwan. This was a cross-sectional observational study from July to September in 2004.We grouped all cases into four groups,based on the quartile of homeostasis model assessment.The top fourth quartile group was defined as the group with insulin resistance.We performed multivariate logistic regression analysis for the odds ratio of the risk factors for insulin resistance. RESULTS:Compared with metabolic syndrome,the coexistence of both factors had a 4.3-fold(95%CI: 2.7-6.8)increased risk,which was more than metabolic syndrome with a 3.6-fold(95%CI:2.6-5.0)increased risk.The two factors had a synergistic effect.The synergistic index of obesity and elevated alanine aminotransferase(ALT)was 2.1(95%CI:1.01-4.3).CONCLUSION:Obesity and elevated ALT are associatedwith insulin resistance.The effects are synergistic. Coexistence of them is better than metabolic syndrome in predicting insulin resistance.

Decreased mitochondrial deoxyribonucleic acid and increased oxidative damage in chronic hepatitis C

AIM： To determine whether alteration of the mito- chondria DNA （mtDNA） copy number and its oxidative damage index （mtDNA△CT） can be detected by analysis of peripheral blood cells in hepatitis C virus （HCV）- infected patients. METHODS： This study enrolled two groups of pa- tients aged 40-60 years： a control group and an HCV- infected group in Department of Gastroenterology and Hepatology in Changhua Christian Hospital. Patients with co-infection with hepatitis B virus or human im- munodeficiency virus, autoimmune disease, malignant neoplasia, pregnancy, thyroid disease, or alcohol con- sumption 〉 40 g/d were excluded. HCV-infected pa- tients who met the following criteria were included： （1） positive HCV antibodies for 〉 6 mo; （2） alanine aminotransferase （ALT） levels more than twice the upper lim- it of normal on at least two occasions during the past 6 mo; and （3） histological fibrosis stage higher than F1. The mtDNA copy number and oxidative damage index of HCV mtDNA （mtDNA△CT） were measured in periph- eral blood leukocytes. The association between mtDNA copy number and mtDNA△CT was further analyzed using clinical data. RESULTS： Forty-seven normal controls （male/female： 26/21, mean age 50.51 ± 6.15 years） and 132 HCV- infected patients （male/female： 76/61, mean age 51.65 ± 5.50 years） were included in the study. The geno- types of HCV-infected patients include type 1a （n = 3）, type 1b （n = 83）, type 2a （n = 32）, and type 2b （n = 14）. Liver fibrosis stages were distributed as follows： F1/F2/F3/F4 = 1/61/45/25 and activity scores were A0/ A1/A2/A3 = 7/45/55/25. There were no age or gender differences between the two groups. HCV-infected pa- tients had higher hepatitis activity （aspartate transami- nase levels 108.77 ± 60.73 vs 23.19 ± 5.47, P 〈 0.01; ALT levels 168.69 ± 93.12 vs 23.15 ± 9.45, P 〈 0.01） and lower platelet count （170.40±58.00 vs 251.24 ± 63.42, P 〈 0.01） than controls. The mtDNA copy num- ber was lower in HCV-infected patients than in controls （173.49 vs 247.93, P 〈 0.05）. The mtDNA△CT was higher in HCV-infected patients than in controls （2.92 vs 0.64, P 〈 0.05）. To clarify the clinical significance of these results in HCV-infected patients, their association with different clinical parameters among HCV-infected pa- tients was analyzed. A negative association was found between mtDNA copy number and elevated aspartate transaminase levels （r = -0.17, P 〈 0.05）. Changes in mtDNA copy number were not associated with HCV RNA levels, HCV genotypes, liver fibrosis severity, or inflammatory activity in the liver biopsy specimen. How- ever, a correlation was observed between mtDNA△CT and platelet count （r = -0.22, P 〈 0.01）, HCV RNA level （r = 0.36, P 〈 0.01）, and hepatitis activity （r = 0.20, P = 0.02）. However, no difference in the change in mtDNA△CT was observed between different fibrosis stages or HCV CONCLUSION： Oxidative stress and mtDNA dam- age are detectable in patient＇s peripheral leukocytes. Increased leukocyte mtDNA△CT correlates with higher HCV viremia, increased hepatitis activity, and lower platelet count.

Hepatoprotective evaluation of Anogeissus latifolia:In vitro and in vivo studies

AIM： To evaluate the hepatoprotective activity of a hydroalcoholic extract of the bark of Anogeissus latifolia; in vitro in primary rat hepatocyte monolayer culture and in vivo in the liver of Wistar rats intoxicated by carbon tetrachloride （CCl4）. METHODS： In the in vitro study, a primary hepatocyte monolayer culture was treated with CCh and extract of Anogeissus latifolia. Hepatoprotective activity was demonstrated in the CCh damaged primary monolayer culture. In the in vivo study, the hepatoprotective activity of a hydroalcoholic extract ofAnogeissus latifolia was analyzed in liver injured CCh-treated rats. Biochemical parameters including serum transaminases [aspartate aminotransferase （AST） and alanine aminotransferase （ALT）] and alkaline phosphatase （ALP） in serum wereanalyzed. The biochemical findings were supplemented with histopathological examination of rat liver sections. RESULTS： In vitro： primary hepatocyte monolayer cultures were treated with CCh and extract of Anogeissus latifolia. A protective activity could be demonstrated in the CCh damaged primary monolayer cultUre. In vivo： Hydroalcoholic extract of Anogeissus latifolia （300 mg/kg） was found to have protective activity in rats with CCh-induced liver damage as judged from serum marker enzyme activity. CONCLUSION： The above findings lead to the conclusion that the hydroalcoholic extract of Anogeissus latifol/a is hepatoprotective. Hence, we suggest that the inclusion of this plant in the management of liver disorders is justified.

Intrahepatic expression of genes related to metabotropic receptors in chronic hepatitis

AIM： To screen for genes related to metabotropic re- ceptors that might be involved in the development of chronic hepatitis. METHODS： Assessment of 20 genes associated with metabotropic receptors was performed in liver speci- mens obtained by punch biopsy from 12 patients with autoimmune and chronic hepatitis type B and C. For this purpose, a microarray with low integrity grade and with oligonucleotide DNA probes complementary to target transcripts was used. Evaluation of gene expression was performed in relation to transcript level, correlation between samples and grouping of clinical parameters used in chronic hepatitis assessment. Clini- cal markers of chronic hepatitis included alanine and aspartate aminotransferase, ~,-glutamyltranspeptidase, alkaline phosphatase and cholinesterase activity, levels of iron ions, total cholesterol, triglycerides, albumin, glucose, hemoglobin, platelets, histological analysis of inflammatory and necrotic status, fibrosis according to METAVIR score, steatosis, as well as anthropometric body mass index, waist/hip index, percentage of adi- pose tissue and liver size in ultrasound examination. Gender, age, concomitant diseases and drugs were also taken into account. Validation of oligonucleotide microarray gene expression results was done with the use of quantitative real-time polymerase chain reaction （qRT-PCR）. RESULTS： The highest （0.002 〈 P 〈 0.046） expres- sion among genes encoding main components of metabotropic receptor pathways, such as the a subunit of G-coupled protein, phosphoinositol-dependent pro- tein kinase or arrestin was comparable to that of an- giotensinogen synthesized in the liver. Carcinogenesis suppressor genes, such as chemokine ligand 4, tran- scription factor early growth response protein 1 and lysophosphatidic acid receptor, were characterized by the lowest expression （0.002 〈 P 〈 0.046）, while the factor potentially triggering hepatic cancer, transcrip- tion factor JUN-B, had a 20-fold higher expression. The correlation between expression of genes of protein kinases PDPK1, phosphoinositide 3-kinase and protein kinase A （Spearman＇s coefficient range： 0.762-0.769） confirmed a functional link between these enzymes. Gender （P = 0.0046） and inflammation severity, mea- sured by alanine aminotransferase activity （P = 0.035）, were characterized by diverse metabotropic receptor gene expression patterns. The Pearson＇s coefficient ranging from -0.35 to 0.99 from the results of qRT-PCR and microarray indicated that qRT-PCR had certainlimitations as a validation tool for oligonucleotide mi- croarray studies. CONCLUSION： A microarray-based analysis of hepa- tocyte metabotropic G-protein-related gene expression can reveal the molecular basis of chronic hepatitis.

Routine blood tests to predict liver fibrosis in chronic hepatitis C

AIM: To verify the usefulness of FibroQ for predicting fi brosis in patients with chronic hepatitis C, compared with other noninvasive tests. METHODS: This retrospective cohort study included 237 consecutive patients with chronic hepatitis C who had undergone percutaneous liver biopsy before treatment. FibroQ, aspartate aminotransferase (AST)/alanine aminotransferase ratio (AAR), AST to platelet ratioindex, cirrhosis discriminant score, age-platelet index (API), Pohl score, FIB-4 index, and Lok's model were calculated and compared. RESULTS: FibroQ, FIB-4, AAR, API and Lok's model results increased significantly as fibrosis advanced (analysis of variance test: P < 0.001). FibroQ trended to be superior in predicting signifi cant fi brosis score in chronic hepatitis C compared with other noninvasive tests. CONCLUSION: FibroQ is a simple and useful test for predicting signifi cant fi brosis in patients with chronic hepatitis C.