Objective To invastigate the anti-proliferating and apoptotic effect of LY294002 and 1,25-dihydroxyvitamin D3（1,25-（OH）2D3） on hepatic stellate （HSC-T6） cells in vitro. MethodsThe HSC-T6 cells were divided into five groups, e.g. control, DMSO, 1,25-（OH）2D3,LY294002 and 1,25-（OH）2D3 plus LY294002. The inhibitory effects were detected by MTT assay; The cell morphology was observed under microscopy; The apoptosis was detected by FCM, and the expression of Ser473,AKT and caspase-3 protein were assayed by Western blotting.Results The inhibitory rate in 1,25-（OH）2D3 plus LY294002 group was 87.5%,much higher than 50.3% in 1,25-（OH）2D3 or 40.3% in LY294002 group （P<0.05） after 48 h intervention;the apoptotic rate in 1,25-（OH）2D3 plus LY294002 group was 92.12%,much higher than 71.0% in 1,25-(OH）2D3 or 34.0% in LY294002-intervented group（P<0.05）;the ratio of Bax/Bcl-2 increased (P<0.05）,and the expression of caspase-3 also increased obviously in 1,25-（OH）2D3 plus LY294002 group(P<0.05）.ConclusionLY294002 has an anti-fibrotic effect with the assistance of 1,25-（OH）2D3 on T6 cells in vitro.

Objective To investigate the expression of multifunctional protein apurinic/ apyrimidinic endonuclease 1（APE1/Ref1） in different parts of liver cells in rats with acute-on-chronic liver failure （ACLF）. Methods 30 SD rats were random1y divided into normal control （n=15）,and ACLF model group （n=15）. ACLF models were established by intraperitoneal injections of CCL₄ combined with D-GalN and LPS and the establishment of liver failure model was confirmed by ultrasonography. Serum ALT,AST,and TBIL were detected by automatic biochemistry analyzer,pathologic changes in liver were revealed by HE staining,and APE1/Ref1 expression in cytoplasmprotein,nucleoprotein and total protein were detected by immunohistochemistry(IHC） and western blot. Results Success rate of model establishment screened by B ultrasound in this series was as high as 80%;serum ALT, AST and TBIL in control group were significantly lower than those in the model group [（56.5±16.0） U/L vs. （620.4±347.5） U/L,（178.7±36.5） vs. （1077.7±666.1） U/L,（3.2±0.6） μmol/L vs. （21.2±16.9） μmol /L,respectively,P<0.05 for all];the expression of APE1/Ref1 in model group was significantly lower than in the control[（6.8±1.3） IOD vs. （8.1±1.2） IOD,P<0.05];APE1/Ref1 in cytoplasmprotein in model group was significantly higher than that in control group[（0.91±0.08） IOD vs.（0.18±0.01） IOD,P<0.01],while APE1/Ref1 in nucleoprotein and total protein in model group were lower than those in control group[（0.71±0.01） IOD vs. （1.41±0.04） IOD,and （0.92±0.03） vs. （1.15±0.01） IOD,respectively,P<0.05 for both]. Conclusion APE1/Ref1 are co-expressed in hepatocellular cytoplasm and nucleus,presenting transferred expression from intranucleus to intracytoplasm with a declined expression manner.

Objective To construct eukaryotic expression vector of human hepatocyte nuclear factor (HNF）-4α gene and identify its expression in vitro. MethodsThe total RNA was isolated from the liver tissues of two patients with hepatocellular carcinoma (HCC） who had underwent surgical operation, and HNF4α cDNA was synthesized by reverse transcription and amplified with the existence of specific primers. Then, the HNF4α fragment was directionally linked to pcDNA3.1 positive-eukaryotic expression vector. After antibiotic screening, the sequence analysis was conducted. The vector was transfected into HepG2 cells, and the expression of target protein after 48-hour incubation was detected by Western blot. Results Enzyme digestion and sequencing illustrated that pcDNA3.1-4α positive-eukaryotic expression vector was successfully constructed, and the results of Western blot revealed that obvious band of fusion protein was present at 53 kDa position. Conclusion The eukaryotic expression vector of HNF4α gene is successfully constructed, and it works well in HepG2 cells in vitro, which is good for further study of HNF4α protein.

Objective To explore the effect of Shuganjianpi decoction in the prevention of alcohol-induced steatosis in HepG2 cells in vitro. Methods Cellular injury of HepG2 cells were induced by alcohol treatment, and HepG2 cells were divided into four groups, e.g. control group (group A）, alcohol-treated group (group B）, serum containing Shuganjianpi decoction treated group (group C） and normal serum treated group (group D）. Cellular morphology and growth were observed. Cell viability was tested by MTT assay. The degree of steatosis in viable cells was evaluated by Oil red O staining, and the expression of peroxisome proliferator-activated receptor-γ(PPAR-γ） was detected by Western blotting assay. ResultsThe cells in group A and group C had similar morphological manifestations and good performance of adherence, and similar mild extent of steatosis; Oil red O staining revealed that after the decolorization by isopropyl alcohol solution, absorbance value in group B (0.942 ± 0.051） and group D (0.928±0.049） were significantly higher than those in group C (0.619±0.043） or in group A [(0.621±0.050）, P<0.05]; MTT assay showed that the absorbance value in group A(1.021±0.071） and group C (0.962±0.066） were significantly higher than in group B (0.561 ± 0.035） and group D [(0.572±0.041）, P<0.05]; The expression of PPARγ protein decreased in group A and group C than in group B and group D did (P<0.05）. Conclusions The effect of Shuganjianpi decoction is effective in the prevention of alcohol-induced steatosis in HepG2 cells.

Objective To construct the eukaryotic expression vector,to identify and to investigate its expression of HEV ORF3 fusion protein His label. Methods HEV RNA was isolated from the serum of a male patient with hepatitis E, and the HEV ORF3 cDNA was synthesized by reverse transcription and specific primers,then directionally ligated into pcDNA3.1（-） his in the eukaryotic expression vector,and the sequence of the enzyme digestion and sequencing was detected. The vector plasmid was transfected into HepG2 cells,which was used to detect the target protein 4 days later,and the target protein was detected by Western blot with anti-His. Results The HEV RNA type I were successfully isolated;after antibiotic screening and enzyme digestion,the pcDNA3.1（-）-HEF3-His eukaryotic expression vector was successfully constructed. The vector was transfected into HepG2 cells,and after 96 h incubation, an 15 kDa protein was demonstrated suggesting the fusion protein existed. Conclusion The eukaryotic expression vector of HEV ORF3 and His tag fusion are successfully constructed,and transfected into HepG2 cells,which might lay a foundation for further study of HEV ORF3 protein.

Objective To investigate the hypophosphatemia in patients with chronic hepatitis B (CHB) and hepatitis B-induced liver cirrhosis （LC） during nucleos（t）ide analogs （NUCs） treatment. Methods The patients with CHB and LC in our hospital from March 2012 to March 2015 was retrospectively analyzed. The serum phosphorus levels was routinely detected and hypophosphatemia was defined as<0.82 mmol/L for at least two times at the interval of 2 months. The Logistic analysis was estimated by SPSS21.0 installation packages for the factors of hypophosphatemia. Results 318 patients （197 CHB and 121 LC） were recruited in this study, and 217 of them took adefovir dipivoxil（ADV） alone or in combination with other NUCs and 101 took other NUCs without ADV. The occurrence of hypophosphatemia in patients with CHB was 11.7%,no significant difference to 10.7%（P>0.05） in patients with LC;Twenty （15.9%） out of one hundred and twenty-six CHB patients with ADV treatment had,while 3（4.2%） out of 71 without ADV had hypophosphatemia（x²=5.83,P<0.05）;11（12.1%）had hypophosphatemia in 91 patients with LC taking ADV,while 2 out of 30 without ADV had hypophosphatemia（x²=0.29,P>0.05）;8 （24.2%）out of 33 patients younger than 35 yr-old had hypophosphatemia, while 3 （9.1%）out of 33 older than 55 yr old had hypophosphatemia;Logistic analysis showed that gender （OR=7.36,P=0.007）and taking ADV（OR=3.51,P=0.015）were the risk factors of hypophosphatemia. Conclusion The clinician should take consideration that young adult patients taking ADV might have hypophosphatemia.

Objective To explore the effect of polyene phosphatidylcholine combined with vitamin E in treatment of patients with nonalcoholic fatty liver disease and diabetes type 2. Methods Eighty three patients with nonalcoholic fatty liver disease and diabetes type 2 were randomly divided into two groups. Patients in the experimental group were given polyene phosphatidylcholine and vitamin E regularly for eight weeks,and patients in the control group received polyene phosphatidylcholine alone for eitht weeks. Then,the efficacy in the two groups as respect to normalization of blood biochemistry index and serum lipids were compared. ResultsAt the end of treatment,the blood glucose levels in experimental group and in the control group decreased,with a average of 7.6 mmol/L in patients of experimental group and 7.8 mmol/L in patients of the control group;Serum ALT and AST levels in experimental group were （43.8±18.2） IU/L and（42.3±9.6） IU/L,respectively,which were significantly lower than those in the controls[（59.0±10.8） IU/L and （51.5±12.2） IU/L,P<0.05];Blood TG and TC levels in experimental group were（1.50±0.57） mmol/L and （5.09±1.18） mmol/L,respectively,which were significantly lower than those in the controls[（3.00±0.29） mmol/L and（5.78±1.23） mmol/L,P<0.05];The B-mode ultrasound examination index of liver in the experimental group was（1.2±0.3）,significantly lower than that in the controls（1.6±0.3）;The effective rate in the experimental group was 79.5%,significantly higher than that in the controls（54.5%,P<0.05）. Conclusion Polyene phosphatidylcholine combined with vitamin E is more effective in the treatment of patients with nonalcoholic fatty liver disease and diabetes type 2 than polyene phosphatidylcholine alone.

Objective To study the correlation between serum glycosylated hemoglobin（HbA1c） and non-alcoholic fatty liver diseases（NAFLD）. Methods A total of 12381 adult residents aged above 18 years in Kunming were enrolled in this study. The data including the waistline, body mass index（BMI）,blood pressure, fasting blood-glucose,blood lipids,glutamyltranspeptidase,HbA1c and the results of epigastrial B ultrasonography were recorded. According to the quartile of HbA1c levels,the participants were divided into four groups,e.g.,the HbA1c≤5.2% in Q1 group,5.2%<Q2 group≤5.4%,5.4%<Q3 group≤5.6%,and>5.6% in Q4 group. Logistic regression was conducted to predict independent risk factors for NAFLD. ResultsThe overall incidence of NAFLD in our series was 27.2% with 31.9% in male and 21.0% in female,which was significantly different （P<0.001）;The incidences of NAFLD in Q1,Q2,Q3 and Q4 group were 18.5% （534/2883）,22.8% （555/2436）,25.6%（840/3285） and 38.1%（1440/3777）,respectively,suggesting that the incidence of NAFLD increased with the rising HbA1c level;The systolic pressure,total cholesterol, low density lipoprotein cholesterol and fasting blood-glucose in 3369 NAFLD patients also elevated as blood HbA1c levels increased;Multiple Logistic regression analysis indicated that high HbA1c level was the risk factor for the incidence of NAFLD（OR=1.7,95%CI 1.2~2.4,P=0.007）. ConclusionBlood HbA1c levels is an independent risk factor for NAFLD and both of them are closely related to disorders of blood lipid metabolism.

Objective To investigate the changes and risk factors of arterial stiffness in patients with nonalcoholic fatty liver diseases（NAFLD）. Methods A total of 2,382 healthy persons were enrolled for physical examination,and the NAFLD was diagnosed by ultrasound monitoring. Brachial ankle pulse wave velocity （baPWV）was determined by a domestic BP-203RPE III arterial stiffness detector,and the baPWV level≥1400 cm/s was arbitrarily believed as the increase of arterial stiffness. Risk factors of baPWV elevation were assessed by using logistic regression analysis. Results Overall,The prevalence of NAFLD was 39.3% among 2382 subjects,45.5% in 1595 men and 26.6% in 787 women,respectively;In individuals between 20 to 39 years old,the baPWV level was significantly higher in NAFLD group than in those without NAFLD[（1340.0±180.7） cm/s vs （1203.9±155.2） cm/s,P<0.001],and the increases baPWV rate was 31.4% vs. 10.5%（P<0.001）;In individuals between 40 to 59 years old,the baPWV was also significantly different between NAFLD patients and non-NAFLD subjects [（1437.1±232.6） cm/s vs.（1355.8±217.9） cm/s,P<0.001],and the increased baPWV rate was 50.1% vs. 33.1%（P<0.001）; However,such difference was not significant among 330 cases over 60 years old;In addition,the effect of NAFLD on baPWV increase was higher in female patients than in males;Logistic analysis showed that NAFLD,age,gender and serum total cholesterol levels were independent risk factors for baPWV increase in individuals with normal blood pressure. Conclusion The baPWV level is higher in individuals with NAFLD,especially in middle-aged ones. NAFLD is an independent risk factor for the baPWV elevation.

Objective To study the effects of plasma exchange （PE） combined with plasma perfusion （PP） to treat patients with liver failure. Methods 46 patients with liver failure from June 2012 to July 2015 were selected as observation group,and the patients were treated by using PE combined with PP,and another 46 patients with liver failure from January 2007 to May 2008 were selected as control group,and they were treated by using PE alone. Total serum bilirubin（TBIL）,albumin（ALB）,thrombin original time international normalized ratio （INR）,ammonia （NH3） were detected routinely,and serum C-reactive protein （CRP）,tumor necrosis factor -α（TNF-α）,interleukin-6（IL-6） were detected by ELISA. Results The effective rates and total effective rates （41.3%,93.47%） in the observation group were significantly higher than in control group（21.74%,78.26%,P<0.05）;Serum TBIL,INR,NH₃,CRP,TNF-α and IL-6 levels in the observation group was（308.3±35.3） μmol/L, （1.6±0.2）,（214.3±22.7） μmol/L,（7.4±1.1） mg/L,（1128.3±345.3） ng/L and（115.5±12.0） ng/L,significantly lower than in the control group [（326.1±38.4） μmol/L,（1.9±0.8）,（267.5±26.1） μmol/L,（10.3±1.3） mg/L,（2012.3±318.4） ng/L,（184.3±20.1） ng/L,P<0.05];Serum ALB level in the observation group was （34.3±4.9）g/L,significantly higher than in the control group[（31.4±3.9）g/L,P<0.05];The incidence of complications in the observation group was 19.6%, significantly lower than in the control group（36.1%,P<0.05）. Conclusions Plasma exchange and plasma perfusion are helpful to clear inflammatory cytokines,and improve liver function in patients with liver failure.

Objective To investigate the clinical characteristics of patients with hepatitis B-induced acute-on-chronic liver failure（ACLF） and the effects of plasma exchanges（PE） on their prognosis. MethodsAccording to the guideline for diagnosis and treatment of liver failure,the clinical data of 52 patients with hepatitis B related ACLF were collected. The relationship between the laboratory indexes at admission and the prognosis of patients, and the effects of complications on the prognosis were analyzed. In addition,the laboratory indexes before and after PE were also explored to evaluate its efficacy. ResultsSerum levels of PT,APTT,INR and blood ammonia at admission in dead patients were significantly higher than those in survived cases[（48.8±11.7） s vs.（42.7±14.0） s,（65.8±19.0） s vs.（48.0±11.4） s,（2.4±1.0） vs.（1.7±0.4）,（100.1±74.7） μmol/L vs. （47.9±21.5） μmol/L,respectively,P<0.05 for all],while serum levels of PTA,PLT,ALB and potassium at admission in dead cases were significantly lower than those in survived cases [（31.8±12.9） % vs. （47.9±21.2）%, （85.6±61.3）×10⁹/L vs.（133.4±50.7）×10⁹/L,（29.2±4.1） g/L vs.（32.8±4.7） g/L,（3.8±0.5） μmol/L vs.（4.1±0.6） μmol/L,respectively,P<0.05 for all];the hepatic encephalopathy,ascites,spontaneous peritonitis,electrolyte disorder and more than one of them in dead patients were higher than in survived cases（37.5% vs. 2.8%, 68.7% vs. 30.3%,25.0% vs. 2.8%, 62.5% vs. 11.1%,62.5% vs. 11.1%, respectively,P<0.05 for all）;Lab indexes like PTA,WBC,ALB and Urea after the PE were significantly higher than those before treatment [（44.8±23.5） % vs. （36.6±14.6）%,（8.0±3.6）×10⁹/L vs. （5.9±2.8）×10⁹/L,（36.4±3.6） g/L vs. （33.7±4.1）g/L,（7.1±4.6） mmol/L vs. （5.4±3.8） mmol/L, respectively,P<0.05 for all];RBC,Hb,ALT,AST,TBIL and DBIL after the PE were remarkably lower than those before treatment [（3.9±0.7） ×10⁹/L vs. （4.2±0.8）×10⁹/L,（119.5±18.2） g/L vs. （130.6±23.8） g/L,（100.6±67.9） U/L vs. （300.0±302.3） U/L,（120.0±62.8） U/L vs. （227.2±174.6） U/L,（335.7±121.3） μmol/L vs. （410.8±129.8） μmol/L,（226.3±77.9） μmol/L vs. （290.4±100.5） μmol/L,respectively,P<0.05 for all）. Conclusion PT,PLT and ALB are sensitive laboratory indexes for the prediction of the prognosis in patients with hepatitis B related ACLF,and patients with complications have poor prognosis. PE could temporarily improve the blood coagulation function and the liver function,but has no influence on the survival in the patients with hepatitis B related ACLF.

Objective To evaluate the effects of the small diameter splenocaval shunt plus devascularization procedure on changes to hepatic hemodynamics and functional reserve in patients with portal hypertension. Methods Clinical data of 86 patients （58 underwent shunt plus devascularization procedure and 28 received devascularization procedure only） with portal hypertension of cirrhosis from Aug 2010 to Jul 2013 were analyzed retrospectively. Portal vein hemodynamics was studied by monitoring the free portal pressure （FPP） intra-operatively and color Doppler flow imaging. Hepatic functional reserve was estimated by the indo-cyanine green retention ratio at fifteen minutes（ICGR15） and the functional hepatic flow（FHF）. Clinical effects were followed-up and compared between two groups. Results In combinational therapy group,the postoperative FPP, portal venous flow（PVF）,FHF and ICGR15 were（31.4±2.4）cmH2O,（900±350） ml/min,（551±246） ml/min and （31.2±13.8）%,respectively,which were significantly different from those before operation [（38.2±3.6） cmH2O, （1250±360） ml/min,（696±300） ml/min and （23.6±11.9）%,P<0.05]. In devascularization group, the postoperative FPP,portal venous flow（PVF）,FHF and ICGR15 were （32.8±3.2） cmH2O,（980±250） ml/min,（507±140） ml/min and （27.4±13.0）%,respectively,which also different from those before operation [（36.9±3.9） cmH2O,（1320±320） ml/min,（625±158） ml/min and（22.2±13.4）%,（P<0.05）]. Compared to the devascularization group, the decrease of FPP in combinational therapy group was greater（P<0.05）,however,there were no differences in PVF,FHF and ICGR15 between the two groups. There were four （1 in combinational therapy and 3 in devascularization group） and 3 patients（2 in combinational therapy and 1 in devascularization group） experienced re-hemorrhage and encephalopathy,respectively,during postoperative follow-up. Conclusion Changes in the hemodynamics are sensible because the combined procedure would not only decrease the FPP but also maintain the portal flow to the liver,sustain the hepatic reserve and protect the liver function from the risk of liver failure. This indicates that the small diameter splenocaval shunt plus devascularization procedure is safe and effective for the treatment of portal hypertension.

Objective To investigate the clinical characteristics and disposal experiences of entecavir-induced peripheral neuropathy in three patients with HBV related liver cirrhosis. Methods We had a retrospective analysis about peripheral neuropathy caused by using entecavir in three patients with HBV related liver cirrhosis in our hospital from July 2011 to July 2014. The clinical characteristics of peripheral neuropathy were summarized. The clinical index,serum HBV DNA and neuro-electromyography were routinely carried out. Results The three patients were middle-aged men,the symptoms of peripheral neuropathy appeared from 3 days to 52 weeks after entecavir administration. Numbness and pain in distal limbs were main clinical manifestations. Neuro-electromyography showed peripheral nerves were injured. Electromyogram were normal in the three patients. Entecavir were discontinued and adefovir were given in the three patients. Vitamin B,combined coenzyme, alprostadil,salvia miltiorrhiza and physical exercise were also carried out in this three patients. The symptoms of peripheral neuropathy disappeared 1 to 5 weeks after entecavir discontinuation and the function of peripheral nerves recovered 16 to 61 weeks later. During follow-up,the three patients were not showed recurrence. Conclusion Peripheral neuropathy can be caused by using entecavir in patients with HBV related liver cirrhosis. The clinical manifestation in each patient differs a little. When a patient like this is met,the discontinuation of suspected medicine is recommended and an appropriate treatment should be given. Then the patient would have a good prognosis.

Objective To evaluate the therapeutic efficacy of transcatheter arterial chemoembolization （TACE） in patients with small hepatocellular carcinoma and underlying liver cirrhosis of Child-Pugh class C. Methods Retrospective analysis of 53 patients with cirrhosis of Child-Pugh class C complicated by small hepatocellular carcinoma who had underwent TACE in our Department from December 2002 to December 2012 was conducted. Each patient received TACE for at least two times with an interval of 30 to 40 days,and was followed after the second TACE for 2 years. Liver function,serum AFP,coagulation function and abdominal CT scan or MRI scan were monitored at 1,3,6,12 and 24 months after the treatment. Results Effective rates of TACE in this series at 1,3,6,12 and 24 months after TACE were 100.0%,100.0%,83.0%,54.7%,41.5%, respectively;There was no significant differences in liver function and coagulation function between baseline and those at 1 month after TACE; However, serum ALT levels significantly declined at 24 months after the treatment [（67.9±20.4） U/L vs.（89.4±21.2） U/L,P<0.05];And the major cause of death was fundamental liver disease. Conclusion The clinical efficacy TACE in patients with liver cirrhosis of Child-Pugh class C complicated by small hepatocellular carcinoma is reliable,and TACE markedly reduces death due to exacerbation of hepatocellular carcinoma.

Systematic and local hypoxia in liver is common in patients with nonalcoholic fatty liver diseases （NAFLD）. Studies on the relationship between obstructive sleep apnea and NAFLD show that the extent of hypoxia is positively correlated with insulin resistance,liver steatosis,and hepatic inflammation and fibrosis. Experiments in animals and molecular researches demonstrate that hypoxia and the hypoxia-inducible factors could promote the occurrence and progression of hepatic steatosis,inflammation,fibrosis,and even induce carcinoma in liver.

Acute-on-chronic liver failure （ACLF） is common type of liver failure,with poor prognosis and high mortality. Hepatitis B virus（HBV）infection is the major cause of ACLF in China. HBV precore/core promoter（PC/CP） region gene mutation is closely related to the occurrence of ACLF. In this article,we reviewed the structure and functions, biological change by mutations of HBV precore/core promoter, and immunopathogenetic mechanisms of ACLF.

A tons of studies have shown that the incidence of intestinal disorder or excessive growth of flora is very high in patients with liver diseases,especially with severe liver diseases,such as liver cirrhosis,liver cancer,and there is a close relationship between the intestinal dysbacteriosis and the occurrence of hapatocellular carcinoma. Lipopolysaccharide （LPS） produced by the intestinal flora can bond its recognition receptor-Toll-like receptor 4（TLR4）,through cell internal signaling pathways,mediating a series of immune inflammatory response,leading to furtherliver damages,and promoting inflammation related to the occurrence of hepatocellular carcinoma（HCC）. Antibotics maybe retard the occurrence and development of HCC by regulating intestinal flora or reducing the endotoxin production.

Hepatocellular carcinoma （HCC） is the fifth most common cancer and the third most common cause of the tumor-related death worldwide. Because of the absence of clinical symptoms at the early stage,the majority of patients are diagnosed at the intermediate/advanced stage when the options of curative treatments are limited.The approaches for the treatment of primary liver cancer are transcatheter arterial chemoembolization, ablation radiotherapy, chemotherapy,and targeted molecular therapy. These therapeutic methods, either alone or in combination, have been proven to control the tumor growth, prolong survival, and improve quality of life to some extent.

Hepatitis C virus （HCV） infection is causally associated with diabetes mellitus type Ⅱ（T2DM）. Various mechanisms have been proposed explaining involvement of HCV infection in the process of T2DM occurrence,including the interference of insulin signaling pathway,overexpression of tumor necrosis factor-α,lipid and iron metabolism inbalance,resulting in pancreatic cell damage and so on. T2DM is one of various complications associated with HCV infection. T2DM may result in hepatic steatosis,fibrosis,failure to anti-viral treatment and hepatocellular carcinoma. In this review,we summarize some available information related to HCV infection and T2DM.