Staging of liver fibrosis in chronic hepatitis B patients with a composite predictive model:A comparative study

AIM:To evaluate the efficacy of 6 noninvasive liver fibrosis models and to identify the most valuable model for the prediction of liver fibrosis stage in chronic hepatitis B(CHB) patients.METHODS:Seventy-eight CHB patients were consecutively enrolled in this study.Liver biopsy was performed and blood serum was obtained at admission.Histological diagnosis was made according to the METAVIR system.Significant fibrosis was defined as stage score ≥ 2,severe fibrosis as stage score ≥ 3.The diagnostic accuracy of 6 noninvasive liver fibrosis models,including serum aspartate aminotransferase(AST) to platelet ratio index(APRI),FIB-4,Forn's index,Fibrometer,Hepascore,and Shanghai Liver Fibrosis Group's index(SLFG),was investigated.RESULTS:The APRI,FIB-4 and Forn's index under receiver operating characteristic curve(AUROC) for sig-nificant fibrosis were 0.71,0.75 and 0.79,respectively,with a diagnosis accuracy of 67%,77% and 80%,respectively,and 0.80,0.87 and 0.86,respectively,under the AUROC for severe fibrosis.The Hepascore,SLFG,and Fibrometer were 0.80,0.83 and 0.85,respectively under the AUROC for significant fibrosis(P < 0.01).The diagnosis accuracy of Hepascore and SLFG was 86% and 88%,respectively.The Hepascore,SLFG,and Fibrometer were 0.95,0.93,and 0.94,respectively,under the AUROC for severe fibrosis(P < 0.01).CONCLUSION:The models containing direct serum markers have a better diagnostic value than those not containing direct serum markers.

Correlation between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models

AIM： To evaluate serum TIMP-1 level and the correlation between TIMP-1 expression and liver fibrosis in immuneinduced and CCL4-induced liver fibrosis models in rats. METHODS： Immune-induced and CCL4-induced liver fibrosis models were established by dexamethasone （0.01 mg） and CCL4 respectively. Serum TIMP-1 level was detected with ELISA, while histopathological grade of liver biopsy was evaluated. Spearman rankcorrelation test was used to analyse the difference of the correlation between the TIMP-1 expression and hepatic fibrosis in the two fibrosis models. Furthermore,in situ hybridization was used to determine the expression difference of TIMP-1 mRNA in the two models. RESULTS： Positive correlation existed between serum TIMP-1 level of immune induced group and the histopathological stages of fibrosis liver of corresponding rats （Spearman rank-correlation test, rs = 0.812, P 0.05）, and the positive in situ hybridization signal of TIMP-1 mRNA was strong. In CCL4-induced liver fibrosis model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant（Spearman rank-correlation test, rs = 0.229, P 〉 0.05）. And compared with immune-induced model, the positivein situ hybridization signal of TIMP-1 mRNA was weaker, while the expression variation was higher in hepatic fibrosis of the same severity. CONCLUSION： The correlations between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models are different. In immune-induced model, serum TIMP-1 level could reflect the severity of liver fibrosis, while in CCL4-induced model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant.

Antifi brotic effects of green tea on in vitro and in vivo models of liver fibrosis

AIM: To examine the protective effect of green tea extract (GT) on hepatic fi brosis in vitro and in vivo in dimethylnitrosamine (DMN)-induced rats.METHODS: HSC-T6, a rat hepatic stellate cell line, was used as an in vitro assay system. Cell proliferation, collagen content, and type 1 collagen expression were examined in activated HSC-T6 cells. Collagen was determined by estimating the hydroxyproline content. In rats with DMN-induced hepatic fi brosis, serum aspartate aminotransferase and alanine aminotransferase concentrations, liver hydroxyproline and lipid peroxides were determined. Pathologic changes were examined by hematoxylin & eosin staining.RESULTS: GT administration prevented the development of hepatic fibrosis in the rat model of DMN-induced liver fi brosis. These results were confi rmed both by liver histology and by quantitative measurement of hepatic hydroxyproline content, a marker of liver collagen deposition. Accordingly, inhibition of proliferation, reduced collagen deposition, and type 1 collagen expression were observed in activated HSC-T6 cells following GT treatment. These results imply that GT reduced the proliferation of activated HSC and down regulated the collagen content and expression of collagen type 1, thereby ameliorating hepatic fibrosis.CONCLUSION: This study demonstrates that greentea administration can effectively improve liver fibrosis caused by DMN, and may be used as a therapeutic option and preventive measure against hepatic f ibrosis.

A novel pulmonary fibrosis murine model with immune-related liver injury

Idiopathic pulmonary fibrosis(IPF),characterized by aggravated alveolar destruc-tion and fibrotic matrix deposition,tendentiously experiences the stage called acute exacerbation IPF(AE-IPF)and progresses to multiple organ damage,especially liver injury.Recent studies have found a variety of immune microenvironment disorders associated with elevated IPF risk and secondary organ injury,whereas current animal models induced with bleomycin(BLM)could not completely reflect the pathologi-cal manifestations of AE-IPF patients in clinic,and the exact underlying mechanisms are not yet fully explored.In the current study,we established an AE-IPF model by tracheal administration of a single dose of BLM and then repeated administrations of lipopolysaccharide in mice.This mouse model successfully recapitulated the clinical features of AE-IPF,including excessive intrapulmonary inflammation and fibrosis and extrapulmonary manifestations,as indicated by significant upregulation of Il6,Tnfa,Il1b,Tgfb,fibronectin,and Col1a1 in both lungs and liver and elevated serum aspartate transaminase and alanine transaminase levels.These effects might be attributed to the regulation of Th17 cells.By sharing this novel murine model,we expect to pro-vide an appropriate experimental platform to investigate the pathogenesis of AE-IPF coupled with liver injury and contribute to the discovery and development of targeted interventions.

Multiparameter magnetic resonance imaging of liver fibrosis in a bile duct ligation mouse model

BACKGROUND Bile duct ligation(BDL)in animals is a classical method for mimicking cholestatic fibrosis.Although different surgical techniques have been described in rats and rabbits,mouse models can be more cost-effective and reproducible for investigating cholestatic fibrosis.Magnetic resonance imaging(MRI)has made great advances for noninvasive assessment of liver fibrosis.More comprehensive liver fibrotic features of BDL on MRI are important.However,the utility of multiparameter MRI to detect liver fibrosis in a BDL mouse model has not been assessed.AIM To evaluate the correlation between the pathological changes and multiparameter MRI characteristics of liver fibrosis in a BDL mouse model.METHODS Twenty-eight healthy adult male balb/c mice were randomly divided into four groups:sham,week 2 BDL,week 4 BDL,and week 6 BDL.Multiparameter MRI sequences,included magnetic resonance cholangiopancreatography,T1-weighted,T2-weighted,T2 mapping,and pre-and post-enhanced T1 mapping,were performed after sham and BDL surgery.Peripheral blood and liver tissue were collected after MRI.For statistical analysis,Student’s t-test and Pearson’s correlation coefficient were used.RESULTS Four mice died after BDL surgery;seven,six,five and six mice were included separately from the four groups.Signal intensities of liver parenchyma showed no difference on TI-and T2-weighted images.Bile duct volume,ΔT1 value,T2 value,and the rate of liver fibrosis increased steadily in week 2 BDL,week 4 BDL and week 6 BDL groups compared with those in the sham group(P<0.01).Alanine aminotransferase and aspartate transaminase levels initially surged after surgery,followed by a gradual decline over time.Strong correlations were found between bile duct volume(r=0.84),T2 value(r=0.78),ΔT1 value(r=0.62),and hepatic fibrosis rate(all P<0.01)in the BDL groups.CONCLUSION The BDL mouse model induces changes that can be observed on MRI.The MRI parameters correlate with the hepatic fibrosis rate and allow for detection of cholestatic fibrosis.

Evaluation of hepatic fibrosis parameter model and elastic modulus of liver and spleen for the diagnosis of hepatic fibrosis in chronic hepatitis b

Objective Toinvestigate the diagnostic value of hepatic fibrosis parameter model and elastic modulus of liver and spleen in hepatic fibrosis of chronic hepatitis b.Methods 77 patients with hepatic fibrosis of chronic hepatitis in the infection clinic were recruited from July 2016 to December 2018.According to the classification of hepatic fibrosis,23 patients were classified as S1,20 as S2,18 as S3 and 16 as S4.The serum indexes of liver function in all patients were tested,FIB-4,APRI and GPR model indexes were calculated.SWE values of liver and spleen were evaluated,and the correlation between FIB-4,APRI,GPR and SWE was analyzed.Results The SWE values of liver and spleen in the study group were significantly higher than those in the normal group(P<0.01),and the differences in serum GGT,PLT,AST and portal vein velocity between the two groups were statistically significant(P<0.01).GGT and PLT were correlated with SWE values of liver and spleen,which were statistically significant(P<0.01).The model indexes of fib-4,APRI and GPR in the study group were all higher than those in the normal group,with statistically significant differences(P<0.01).Pearson correlation analysis showed that liver SWE value and spleen SWE value were positively correlated with fib-4,APRI and GPR,and the differences were significant(P<0.01),with a higher correlation with GPR.Conclusion GGT,PLT and GPR are positively correlated with SWE of liver and spleen,and combined detection can improve the early diagnosis accuracy of liver fibrosis.

Adult human liver slice cultures:Modelling of liver fibrosis and evaluation of new anti-fibrotic drugs

BACKGROUND Liver fibrosis can result in end-stage liver failure and death.AIM To examine human liver fibrogenesis and anti-fibrotic therapies,we evaluated the three dimensional ex vivo liver slice(LS)model.METHODS Fibrotic liver samples(F0 to F4 fibrosis stage according to the METAVIR score)were collected from patients after liver resection.Human liver slices(HLS)were cultivated for up to 21 days.Hepatitis C virus(HCV)infection,alcohol(ethanol stimulation)and steatosis(palmitate stimulation)were examined in fibrotic(F2 to F4)liver slices infected(or not)with HCV.F0-F1 HLS were used as controls.At day 0,either ursodeoxycholic acid(choleretic and hepatoprotective properties)and/or α-tocopherol(antioxidant properties)were added to standard of care on HLS and fibrotic liver slices,infected(or not)with HCV.Expression of the biomarkers of fibrosis and the triglyceride production were checked by quantitative reverse transcription polymerase chain reaction and/or enzymelinked immunosorbent assay.RESULTS The cultures were viable in vitro for 21 days allowing to study fibrosis inducers and to estimate the effect of anti-fibrotic drugs.Expression of the biomarkers of fibrosis and the progression to steatosis(estimated by triglycerides production)was increased with the addition of HCV and/or ethanol or palmitate.From day 15 of the follow-up studies,a significant decrease of both transforming growth factorβ-1 and Procol1A1 expression and triglycerides production was observed when a combined anti-fibrotic treatment was applied on HCV infected F2-F4 LS cultures.CONCLUSION These results show that the human three dimensional ex vivo model effectively reflects the in vivo processes in damaged human liver(viral,alcoholic,nonalcoholic steatohepatitis liver diseases)and provides the proof of concept that the LS examined model permits a rapid evaluation of new anti-fibrotic therapies when used alone or in combination.

Globulin-platelet model predicts minimal fibrosis and cirrhosis in chronic hepatitis B virus infected patients

AIM： To establish a simple model consisting of the rou- tine laboratory variables to predict both minimal fibrosis and cirrhosis in chronic hepatitis B virus （HBV）-infected patients. METHODS： We retrospectively investigated 114 chron- ic HBV-infected patients who underwent liver biopsy in two different hospitals. Thirteen parameters were analyzed by step-wise regression analysis and correla- tion analysis. A new fibrosis index [globulin/platelet （GP） model] was developed, including globulin （GLOB） and platelet count （PLT）. GP model = GLOB （g/mL） x 100/PLT （x 109/L）. We evaluated the receiver operating characteristics analysis used to predict minimal fibrosis and compared six other available models. RESULTS： Thirteen clinical biochemical and hemato- logical variables [sex, age, PLT, alanine aminotransfer- ase, aspartate aminotransferase （AST）, albumin, GLOB, total bilirubin （T.bil）, direct bilirubin （D.bil）, glutamyl-transferase, alkaline phosphatase, HBV DNA and pro- thrombin time （PT）] were analyzed according to three stages of liver fibrosis （F0-F1, F2-F3 and F4）. Bivariate Spearman＇s rank correlation analysis showed that six variables, including age, PLT, T.bil, D.bil, GLOB and PT, were correlated with the three fibrosis stages （FS）. Cor- relation coefficients were 0.23, -0.412, 0.208, 0.220, 0.314 and 0.212; and P value was 0.014, 〈 0.001, 0.026, 0.018, 0.001 and 0.024, respectively. Univariate analysis revealed that only PLT and GLOB were signifi- cantly different in the three FS （PLT： F = 11.772, P 〈 0.001; GLOB： F = 6.612, P = 0.002）. Step-wise multiple regression analysis showed that PLT and GLOB were also independently correlated with FS （R2 = 0.237）. By Spearman＇s rank correlation analysis, GP model was significantly correlated with the three FS （r = 0.466, P 〈 0.001）. The median values in F0-F1, F2-F3 and F4 were 1.461, 1.720 and 2.634. Compared with the six available models （fibrosis index, AST-platelet ratio, FIB-4, fibrosis-cirrhosis index and age-AST model and age-PLT ratio）, GP model showed a highest correlation coefficient. The sensitivity and positive predictive value at a cutoff value 〈 1.68 for predicting minimal fibrosis F0-F1 were 72.4% and 71.2%, respectively. The speci- ficity and negative predictive value at a cutoff value 〈 2.53 for the prediction of cirrhosis were 84.5% and 96.7%. The area under the curve （AUC） of GP model for predicting minimal fibrosis and cirrhosis was 0.762 [95% confidence interval （CI）： 0.676-0.848] and 0.781 （95% CI： 0.638-0.924）. Although the differences were not statistically significant between GP model and the other models （P all 〉 0.05）, the AUC of GP model was the largest among the seven models. CONCLUSION： By establishing a simple model using available laboratory variables, chronic HBV-infected patients with minimal fibrosis and cirrhosis can be di- agnosed accurately, and the clinical application of this model may reduce the need for liver biopsy in HBV- infected patients.

Serum N-glycan markers for diagnosing liver fibrosis induced by hepatitis B virus

BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion.Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis;however,this method is invasive and prone to clinical sampling error.In order to address these issues,we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis.AIM To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes.METHODS N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed.Significant changed N-glycan levels (peaks)(P <0.05) in differentfibrosis stages were selected in the modeling group,and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis.The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models.These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI),fibrosis index based on the four factors (FIB-4),glutamyltranspeptidase platelet albumin index (S index),GlycoCirrho-test,and GlycoFibro-test.Furthermore,we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power.In addition,the diagnostic accuracy of N-glycan models was also verified in the validation group of patients.RESULTS Multiparameter diagnostic models constructed based on N-glycan peak 1,3,4and 8 could distinguish between different stages of liver fibrosis.The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752,respectively differentiating fibrosis F0-F1 from F2-F4,and F0-F2 from F3-F4,and surpassing other serum panels.However,AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4.In combination with ALT and PLT,the multiparameter models showed better diagnostic power (AUROC=0.912,0.829,0.885,respectively) when compared with other models.In the validation group,the AUROCs of the three combined models (0.929,0.858,and 0.867,respectively) were still satisfactory.We also applied the combined models to distinguish adjacent fibrosis stages of 432patients (F0-F1/F2/F3/F4),and the AUROCs were 0.917,0.720 and 0.785.CONCLUSION Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV,especially in combination with ALT and PLT.

Efficacy of Chinese medicine Yi-gan-kang granule in prophylaxis and treatment of liver fibrosis in rats

AIM:To investigate the efficacy of a Chinese medicine, Yi-gan-kang granule (granules for benefiting the liver), in prophylaxis and treatment of liver fibrosis in rats and its possible mechanism. METHODS: One hundred and forty Sprague-Dawley rats were randomly divided into seven groups (20 each): group 1, blank control group without any interference during the study; group 2, CCI4-induced liver fibrosis group; group 3, pig serum-induced liver fibrosis group; group 4, prophylaxis group of CCl4-induced liver fibrosis by Yi-gan-kang; group 5, prophylaxis group of pig serum-induced liver fibrosis by Yi-gan-kang; group 6, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang; group 7, treatment group of CCI4-induced liver fibrosis by Yi-gan-kang. At wk 6,10,14 and 20 (baseline for CCl4., or big serum induction), five rats in each group were anesthetized and their livers were removed for pathological studies including immunohistochemical studies for α-SMA, type I collagen and In situ hybridization of tissue inhibitor of metalloproteinase-1 (TTMP-1) mRNA of hepatic stellate cells (HSCs). Anti-lipid peroxidation in isolated mitochondria and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay for proliferation and terminal deoxynucleotidyl transferase-medicated dUTP-biotin nick end-labeling (TUNEL), flow cytometry and electron microscopy for apoptosis in isolated HSCs were also studied. RESULTS: The mean number of pseudolobuli at wk 10, 14 and 20 in the prophylaxis group was significantly less than that in the control group (P<0.05 or 0.01). The effect of prophylaxis at wk 14 in CCI4 rats and at wk 10 in pig serum-induced rats was much better than that of treatment group (P<0.01). The thickness (in μm) of fibers both in pig serum-induced prophylaxis and in treatment groups at wk 14 and. 20 was significantly less than that in control group (P<0.05). The number of fibers both in prophylaxis and in treatment groups from wk 10 or 14 to 20 was significantly less than that in control group (P<0.05 or P<0.01). The tissue HSC positive rates of type I collagen, α-SMA and TIMP-1 mRNA, which represented the active phenotype of HSCs in tissues, remained very high from wk 6 to the end of model making in control group. While in prophylaxis group, they were at a relatively low level. In treatment group, there was a gradual decreasing trend. Time- and dose-dependent effects of anti-lipid peroxidation on isolated mitochondria, cell proliferation and apoptosis in cultured HSCs were also observed during the study. CONCLUSION: Yi-gan-kang can effectively inhibit or inverse the course of liver fibrogenesis in CCI4- and pig serum-induced rat models.

Effect of liver inflammation on accuracy of FibroScan device in assessing liver fibrosis stage in patients with chronic hepatitis B virus infection

BACKGROUND Transient elastography(FibroScan)is a new and non-invasive test,which has been widely recommended by the guidelines of chronic hepatitis B virus(HBV)management for assessing hepatic fibrosis staging.However,some confounders may affect the diagnostic accuracy of the FibroScan device in fibrosis staging.AIM To evaluate the diagnostic value of the FibroScan device and the effect of hepatic inflammation on the accuracy of FibroScan in assessing the stage of liver fibrosis in patients with HBV infection.METHODS The data of 416 patients with chronic HBV infection who accepted FibroScan,liver biopsy,clinical,and biological examination were collected from two hospitals retrospectively.Receiver operating characteristic(ROC)curves were used to analyze the diagnostic performance of FibroScan for assessing the stage of liver fibrosis.Any discordance in fibrosis staging by FibroScan and pathological scores was statistically analyzed.Logistic regression and ROC analyses were used to analyze the accuracy of FibroScan in assessing the stage of fibrosis in patients with different degrees of liver inflammation.A non-invasive model was constructed to predict the risk of misdiagnosis of fibrosis stage using FibroScan.RESULTS In the overall cohort,the optimal diagnostic values of liver stiffness measurement(LSM)using FibroScan for significant fibrosis(≥F2),severe fibrosis(≥F3),and cirrhosis(F4)were 7.3 kPa[area under the curve(AUC)=0.863],9.7 kPa(AUC=0.911),and 11.3 kPa(AUC=0.918),respectively.The rate of misdiagnosis of fibrosis stage using FibroScan was 34.1%(142/416 patients).The group of patients who showed discordance between fibrosis staging using FibroScan and pathological scores had significantly higher alanine aminotransferase and aspartate aminotransferase levels,and a higher proportion of moderate to severe hepatic inflammation,compared with the group of patients who showed concordance in fibrosis staging between the two methods.Liver inflammation activity over 2(OR=3.53)was an independent risk factor for misdiagnosis of fibrosis stage using FibroScan.Patients with liver inflammation activity≥2 showed higher LSM values using FibroScan and higher rates of misdiagnosis of fibrosis stage,whereas the diagnostic performance of FibroScan for different fibrosis stages was significantly lower than that in patients with inflammation activity<2(all P<0.05).A non-invasive prediction model was established to assess the risk of misdiagnosis of fibrosis stage using FibroScan,and the AUC was 0.701.CONCLUSION Liver inflammation was an independent risk factor affecting the diagnostic accuracy of FibroScan for fibrosis stage.A combination of other related noninvasive factors can predict the risk of misdiagnosis of fibrosis staging using FibroScan.

Contrast-enhanced ultrasound for quantitative assessment of portal pressure in canine liver fibrosis

AIM: To explore the feasibility of non-invasive quantitative estimation of portal venous pressure by contrast-enhanced ultrasound(CEUS) in a canine model.METHODS: Liver fibrosis was established in adult canines(Beagles; n = 14) by subcutaneous injection of carbon tetrachloride(CCl4). CEUS parameters, including the area under the time-intensity curve and intensity at portal/arterial phases(Qp/Qa and Ip/Ia, respectively), were used to quantitatively assess the blood flow ratio of the portal vein/hepatic artery at multiple time points. The free portal venous pressures(FPP) were measured by a multi-channel baroreceptor using a percutaneous approach at baseline and 8, 16, and 24 wk after CCl4 injections in each canine. Liver biopsies were obtained at the end of 8, 16, and 24 wk from each animal, and the stage of the fibrosis was assessed according to the Metavir scoring system. A Pearson correlation test was performed to compare the FPP with Q p/Q a and I p/I a.RESULTS: Pathologic examination of 42 biopsies from the 14 canines at weeks 8, 16, and 24 revealed that liver fibrosis was induced by CCl4 and represented various stages of liver fibrosis, including F0(n = 3), F1(n = 12), F2(n = 14), F3(n = 11), and F4(n = 2). There were significant differences in the measurements of Qp/Qa(19.85 ± 3.30 vs 10.43 ± 1.21, 9.63 ± 1.03, and 8.77 ± 0.96) and Ip/Ia(1.77 ± 0.37 vs 1.03 ± 0.12, 0.83 ± 0.10, and 0.69 ± 0.13) between control and canine fibrosis at 8, 16, and 24 wk, respectively(all P < 0.001). There were statistically significant negative correlations between FPP and Q p/Q a(r =-0.707, P < 0.001), and between FPP and Ip/Ia(r =-0.759, P < 0.001) in the canine fibrosis model. Prediction of elevated FPP based on Q p/Q a and I p/I a was highly sensitive, as assessed by the area under the receiveroperating curve(0.866 and 0.895, respectively).CONCLUSION: C E U S i s a p o t e n t i a l m e t h o d t o accurately, but non-invasively, estimate portal venous pressure through measurement of Qp/Qa and Ip/Ia parameters.

Preventive effect of Qianggan-Rongxian Decoction on rat liver fibrosis

AIM: To study the preventive effects of Qianggan-Rongxian Decoction on liver fibrosis induced by dimethylnitrosamine (DMN) in rats. METHODS: Male Wistar rats were randomly divided into hepatic fibrosis model group, control group and 3 treatment groups (12 rats in each group). Except for the normal control group, all the rats received 1% DMN (10 μL/kg body weight, i.p), 3 times a week for 4 wk. The rats in the 3 treatment groups including a high-dose DMN group (10 mL/kg), a medium-dose DMN group (7 mL/kg), and a low-dose DMN group (4 mL/kg) were daily gavaged with Qianggan-Rongxian Decoction, and the rats in the model and normal control groups were given saline vehicle. Enzyme-linked immunosorbent assay (ELISA) was used to determine the changes in serum hyaluronic acid (HA), laminin (LN), and type IV collagen levels. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using routine laboratory methods. Pathologic changes, particularly fibrosis, were examined by hematoxylin and eosin (HE) and Sirius red staining. Hepatic stellate cells (HSC) were examined by transmission electron microscopy. RESULTS: Compared with the model control group, the serum levels of HA, LN, type Ⅳ collagen, ALT and AST were decreased markedly in the other groups after treatment with Qianggan-Rongxian Decoction, especially in the medium-dose DMN group (P < 0.05).Moreover, the area-density percentage of collagen fibrosis was lower in the Qianggan-Rongxian Decoction treatment groups than in the model group, and a more significant drop was observed in the medium-dose DMN group (P < 0.05). CONCLUSION: Qianggan-Rongxian Decoction can inhibit hepatic fibrosis due to chronic liver injury, delay the development of cirrhosis, and notably ameliorate liver function. It may be used as a safe and effective thera-peutic drug for patients with fibrosis.

Serum ceruloplasmin can predict liver fibrosis in hepatitis B virusinfected patients

BACKGROUND The presence of significant liver fibrosis in hepatitis B virus(HBV)-infected individuals with persistently normal serum alanine aminotransferase(PNALT)levels is a strong indicator for initiating antiviral therapy.Serum ceruloplasmin(CP)is negatively correlated with liver fibrosis in HBV-infected individuals.AIM To examine the potential value of serum CP and develop a noninvasive index including CP to assess significant fibrosis among HBV-infected individuals with PNALT.METHODS Two hundred and seventy-five HBV-infected individuals with PNALT were retrospectively evaluated.The association between CP and fibrotic stages was statistically analyzed.A predictive index including CP[Ceruloplasmin hepatitis B virus(CPHBV)]was constructed to predict significant fibrosis and compared to previously reported models.RESULTS Serum CP had an inverse correlation with liver fibrosis(r=-0.600).Using CP,the areas under the curves(AUCs)to predict significant fibrosis,advanced fibrosis,and cirrhosis were 0.774,0.812,and 0.853,respectively.The CPHBV model was developed using CP,platelets(PLT),and HBsAg levels to predict significant fibrosis.The AUCs of this model to predict significant fibrosis,advanced fibrosis,and cirrhosis were 0.842,0.920,and 0.904,respectively.CPHBV was superior to previous models like the aspartate aminotransferase(AST)-to-PLT ratio index,Fibrosis-4 score,gamma-glutamyl transpeptidase-to-PLT ratio,Forn’s score,and S-index in predicting significant fibrosis in HBV-infected individuals with PNALT.CONCLUSION CPHBV could accurately predict liver fibrosis in HBV-infected individuals with PNALT.Therefore,CPHBV can be a valuable tool for antiviral treatment decisions.

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.

Calcitriol attenuates liver fibrosis through hepatitis C virus nonstructural protein 3-transactivated protein 1-mediated TGF β1/Smad3 and NF-κB signaling pathways

BACKGROUND Hepatic fibrosis is a serious condition,and the development of hepatic fibrosis can lead to a series of complications.However,the pathogenesis of hepatic fibrosis remains unclear,and effective therapy options are still lacking.Our group identified hepatitis C virus nonstructural protein 3-transactivated protein 1(NS3TP1) by suppressive subtractive hybridization and bioinformatics analysis,but its role in diseases including hepatic fibrosis remains undefined.Therefore,additional studies on the function of NS3TP1 in hepatic fibrosis are urgently needed to provide new targets for treatment.AIM To elucidate the mechanism of NS3TP1 in hepatic fibrosis and the regulatory effects of calcitriol on NS3TP1.METHODS Twenty-four male C57BL/6 mice were randomized and separated into three groups,comprising the normal,fibrosis,and calcitriol treatment groups,and liver fibrosis was modeled by carbon tetrachloride(CCl4).To evaluate the level of hepatic fibrosis in every group,serological and pathological examinations of the liver were conducted.TGF-β1 was administered to boost the in vitro cultivation of LX-2 cells.NS3TP1,α-smooth muscle actin(α-SMA),collagen I,and collagen Ⅲ in every group were examined using a Western blot and real-time quantitative polymerase chain reaction.The activity of the transforming growth factor beta 1(TGFβ1)/Smad3 and NF-κB signaling pathways in each group of cells transfected with pcDNA-NS3TP1 or siRNA-NS3TP1 was detected.The statistical analysis of the data was performed using the Student’s t test.RESULTS NS3TP1 promoted the activation,proliferation,and differentiation of hepatic stellate cells(HSCs)and enhanced hepatic fibrosis via the TGFβ1/Smad3 and NF-κB signaling pathways,as evidenced by the presence of α-SMA,collagen I,collagen Ⅲ,p-smad3,and p-p65 in LX-2 cells,which were upregulated after NS3TP1 overexpression and downregulated after NS3TP1 interference.The proliferation of HSCs was lowered after NS3TP1 interference and elevated after NS3TP1 overexpression,as shown by the luciferase assay.NS3TP1 inhibited the apoptosis of HSCs.Moreover,both Smad3 and p65 could bind to NS3TP1,and p65 increased the promoter activity of NS3TP1,while NS3TP1 increased the promoter activity of TGFβ1 receptor I,as indicated by coimmunoprecipitation and luciferase assay results.Both in vivo and in vitro,treatment with calcitriol dramatically reduced the expression of NS3TP1.Calcitriol therapy-controlled HSCs activation,proliferation,and differentiation and substantially suppressed CCl4-induced hepatic fibrosis in mice.Furthermore,calcitriol modulated the activities of the above signaling pathways via downregulation of NS3TP1.CONCLUSION Our results suggest that calcitriol may be employed as an adjuvant therapy for hepatic fibrosis and that NS3TP1 is a unique,prospective therapeutic target in hepatic fibrosis.

Growth and Reproduction Characteristics of TLR4 Knockout Mice Used for Liver Fibrosis Experiments

[Objectives]This study was conducted to investigate the similarity and differences between TLR4 knockout mice and C57 BL/6 mice used in liver fibrosis research in terms of growth rate and reproduction ability.[Methods]Twenty TLR4 knockout mice and C57 BL/6 mice,half male and half female,were selected to compare the growth rates of body weight and body length of mice from the 4th to 12th weeks;and 20 pairs of male and female mice of the same strain were compared for the number of baby mice of the second litter.[Results]The growth rates of body weight and body length of the TLR4 knockout mice were significantly lower than those of C57 BL/6 mice(P<0.05)(except for the 4th and 5th weeks when there was no significant difference in body length);and in terms of reproductive ability,the TLR4 knockout mice were significantly lower than the C57 BL/6 mice(the ratio of the total number of baby mice in the second litter of the two strains,72∶147).[Conclusions]Knockout of the TLR4 gene has a significant impact on the growth and reproduction of mice.

Effects of interleukin-18 and Anti-interleukin-18-mAb on Experimental immunological Liver Fibrosis induced by Repeatedly Administered Concanavalin A and its Mechanism

Objective To explore the prevention of IL-18 or anti-IL-18-m Ab to the immune liver fibrosis model induced by repeated injection of concanavalin A in BALB/c mice and its mechanism.Methods Total of 120 BALB/c mice were divided into four groups, control group mice(Ga) were injected weekly with normal saline, concanavalin A group was divided into Gb, Gc, Gd. All mice were injected with concanavalin A(15 mg/kg) once a week. Moreover, Gc, Gd mice were injected weekly with IL-18(7.5 mg/kg) and anti-IL-18-m Ab(10 mg/kg) 2 hours before treatment with concanavalin A, respectively. Twenty-four hours after concanavalin A challenge at 1, 5, 12 and 20 weeks, 3 mice were killed by vena orbitalis, repectively. The sera were storaged at 4℃ for detecting of up TNF-α and IFN-γ by ELISA. The liver of mice in different groups were excised and fixed in 10% formalin for HE staining and Masson staining or frozen in liquid nitrogen for immunohistochemical staining for α-SMA. After extracting of total RNA from liver tissue, MMP-2 and TIMP-1A messenger RNA were amplified by reverse transcription polymerase chain reaction(PCR). Products were electrophoresed on agrose gel containing ethidium bromide and visualized under ultraviolet light. Densitometric RT-PCR data were standardized with β-actin signals. Results After experiment, the number of dead mice of Ga, Gb, Gc and Gd were 0, 6, 15 and 3, respectively. There were significant difference on each group(P < 0.05). At the fifth week of experiment, hepatocellular necrosis in IL-18 administered group mice had become widespread throughout the lobule. Evidence of liver fibrosis was observed during this period. However, at the twelfth week of experimemt, bridging fibrosis and large fibrosis strip in the parenchyma with hepatocellular necrosis was detectable in Gb, but at twentieth week, only the small fibrosis strip had been found in anti-IL-18-mA b administered group mice by HE staining and Masson staining. The serum levels of TNF-α and IFN-γ in IL-18 administered group were higher than that in concanavalin A group and anti-IL-18 administered groups(P < 0.05). Moreover, immunohistochemical staining for α-SMA indicated that the semi-quantu scores in IL-18 administered group were more than concanavalin A group and anti-IL-18-mA b administered groups(P < 0.05). MMP-2-mR NA, TIMP-1- mR NA expression levels increased signifigantly compared with concanavalin A group and anti-IL-18-mA b administered group(P < 0.05).Conclusions The immune liver fibrosis model induced by repeated injection of concanavalin A in BALB/c mice could be worsened by IL-18 administration and block by anti-IL-18 mA b administraion.

Hepatocellular carcinoma risk after viral response in hepatitis C virus-advanced fibrosis: Who to screen and for how long?

Hepatitis C virus(HCV)chronic infection is associated with fibrosis progression,end-stage liver complications and HCC.Not surprisingly,HCV infection is a leading cause of liver-related morbidity and mortality worldwide.After sustained virological response(SVR),the risk of developing hepatocellular carcinoma is not completely eliminated in patients with established cirrhosis or with advanced fibrosis.Therefore,lifelong surveillance is currently recommended.This strategy is likely not universally cost-effective and harmless,considering that not all patients with advanced fibrosis have the same risk of developing HCC.Factors related to the severity of liver disease and its potential to improve after SVR,the molecular and epigenetic changes that occur during infection and other associated comorbidities might account for different risk levels and are likely essential for identifying patients who would benefit from screening programs after SVR.Efforts to develop predictive models and risk calculators,biomarkers and genetic panels and even deep learning models to estimate the individual risk of HCC have been made in the direct-acting antiviral agents era,when thousands of patients with advanced fibrosis and cirrhosis have reached SVR.These tools could help to identify patients with very low HCC risk in whom surveillance might not be justified.In this review,factors affecting the probability of HCC development after SVR,the benefits and risks of surveillance,suggested strategies to estimate individualized HCC risk and the current evidence to recommend lifelong surveillance are discussed.

BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of β-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis

BACKGROUND BIR repeat-containing ubiquitin conjugating enzyme(BRUCE)is a liver tumor suppressor,which is downregulated in a large number of patients with liver diseases.BRUCE facilitates DNA damage repair to protect the mouse liver against the hepatocarcinogen diethylnitrosamine(DEN)-dependent acute liver injury and carcinogenesis.While there exists an established pathologic connection between fibrosis and hepatocellular carcinoma(HCC),DEN exposure alone does not induce robust hepatic fibrosis.Further studies are warranted to identify new suppressive mechanisms contributing to DEN-induced fibrosis and HCC.AIM To investigate the suppressive mechanisms of BRUCE in hepatic fibrosis and HCC development.METHODS Male C57/BL6/J control mice[loxp/Loxp;albumin-cre(Alb-cre)-]and BRUCE Alb-Cre KO mice(loxp/Loxp;Alb-Cre+)were injected with a single dose of DEN at postnatal day 15 and sacrificed at different time points to examine liver disease progression.RESULTS By using a liver-specific BRUCE knockout(LKO)mouse model,we found that BRUCE deficiency,in conjunction with DEN exposure,induced hepatic fibrosis in both premalignant as well as malignant stages,thus recapitulating the chronic fibrosis background often observed in HCC patients.Activated in fibrosis and HCC,β-catenin activity depends on its stabilization and subsequent translocation to the nucleus.Interestingly,we observed that livers from BRUCE KO mice demonstrated an increased nuclear accumulation and elevated activity ofβ-catenin in the three stages of carcinogenesis:Pre-malignancy,tumor initiation,and HCC.This suggests that BRUCE negatively regulatesβ-catenin activity during liver disease progression.β-catenin can be activated by phosphorylation by protein kinases,such as protein kinase A(PKA),which phosphorylates it at Ser-675(pSer-675-β-catenin).Mechanistically,BRUCE and PKA were colocalized in the cytoplasm of hepatocytes where PKA activity is maintained at the basal level.However,in BRUCE deficient mouse livers or a human liver cancer cell line,both PKA activity and pSer-675-β-catenin levels were observed to be elevated.CONCLUSION Our data support a“BRUCE-PKA-β-catenin”signaling axis in the mouse liver.The BRUCE interaction with PKA in hepatocytes suppresses PKA-dependent phosphorylation and activation ofβ-catenin.This study implicates BRUCE as a novel negative regulator of both PKA andβ-catenin in chronic liver disease progression.Furthermore,BRUCE-liver specific KO mice serve as a promising model for understanding hepatic fibrosis and HCC in patients with aberrant activation of PKA andβ-catenin.