Naringin from Ganshuang granule inhibits inflammatory to relieve liver fibrosis through TGF-ß-Smad signaling pathway.

OBJECTIVE: The present study aims to investigate the specific protective effects and underlying mechanisms of Ganshuang granule (GSG) on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rat models. METHODS: Hepatic fibrosis was experimentally evoked in rats by DMN administration, and varying dosages of GSG were employed as an intervention. Hepatocellular damage was assessed by measuring serum levels of aminotransferase and bilirubin, accompanied by histopathological examinations of hepatic tissue. The hepatic concentrations of platelet-derived growth factor (PDGF) and transforming growth factor-ß1 (TGF-ß1) were quantitated via enzyme-linked immunosorbent assay (ELISA). The expression of α-smooth muscle actin (α-SMA) within hepatic tissue was evaluated using immunohistochemical techniques. The levels of hepatic interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and a spectrum of interleukins (IL-2, IL-4, IL-6, IL-10) were quantified by quantitative real-time PCR (qRT-PCR). Additionally, hepatic stellate cells (HSCs) were cultured in vitro and exposed to TNF-α in the presence of naringin, a principal component of GSG. The gene expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase-1 (MMP-1) in these cells were also quantified by qRT-PCR. Proliferative activity of HSCs was evaluated by the Cell Counting Kit-8 assay. Finally, alterations in Smad protein expression were analyzed through Western blotting. RESULTS: Administration of GSG in rats with fibrosis resulted in reduced levels of serum aminotransferases and bilirubin, along with alleviation of histopathological liver injury. Furthermore, the fibrosis rats treated with GSG exhibited significant downregulation of hepatic TGF-ß1, PDGF, and TNF-α levels. Additionally, GSG treatment led to increased mRNA levels of IFN-γ, IL-2, and IL-4, as well as decreased expression of α-SMA in the liver. Furthermore, treatment with naringin, a pivotal extract of GSG, resulted in elevated expression of MMP-1 and decreased levels of TIMP-1 in TNF-α-stimulated HSCs when compared to the control group. Additionally, naringin administration led to a reduction in Smad expression within the HSCs. CONCLUSION: GSG has the potential to mitigate fibrosis induced by DMN in rat models through the regulation of inflammatory and fibrosis factors. Notably, naringin, the primary extract of GSG, may exert a pivotal role in modulating the TGF-ß-Smad signaling pathway.

ATP7B R778L mutant hepatocytes resist copper toxicity by activating autophagy and inhibiting necroptosis.

Wilson's disease (WD) is an inherited disease characterized by copper metabolism disorder caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B). Currently, WD cell and animal model targeting the most common R778L mutation in Asia is lacking. In addition, the mechanisms by which hepatocytes resist copper toxicity remain to be further elucidated. In this study, we aimed to construct a novel WD cell model with R778L mutation and dissected the molecular basics of copper resistance. A novel HepG2 cell line stably expressing the ATP7B R778L gene (R778L cell) was constructed. The expression of necroptosis- and autophagy-related molecules was detected by PCR and Western blot (WB) in wild-type (WT) HepG2 and R778L cells with or without CuSO4 treatment. In addition, we detected and compared the levels of autophagy and necroptosis in CuSO4-treated R778L cells with the activation and inhibition of autophagy. Moreover, the mRNA and protein levels of autophagy and necroptosis signaling molecules were compared in R778L cells with the overexpression and knockdown of Unc-51 Like Autophagy Activating Kinase 1 (ULK1) and Autophagy Related 16 Like 1 (ATG16L1). We successfully constructed an R778L mutation HepG2 cell line. CuSO4 triggered the enhanced expression of autophagy and necroptosis signaling molecules in WT HepG2 cells and R778L cells. Remarkably, higher levels of autophagy and necroptosis were observed in R778L cells compared with those in WT cells. Autophagy activation led to weakened necroptosis mediated by RIPK3 and MLKL, conversely, autophagy inhibition brought about enhanced necroptosis. At the molecular level, ULK1- and ATG16L1 overexpression resulted in reduced necroptosis levels and vice versa. ULK1- and ATG16L1-mediated autophagy activation protects hepatocytes against RIPK3- and MLKL-mediated necroptosis in our new WD cell model treated with CuSO4. Targeted therapy by autophagy activation or necroptosis inhibition may be a novel and effective strategy to treat WD.

Consensus on the tertiary prevention of primary liver cancer.

To effectively prevent recurrence, improve the prognosis and increase the survival rate of primary liver cancer (PLC) patients with radical cure, the Chinese Society of Hepatology, Chinese Medical Association, invited clinical experts and methodologists to develop the Consensus on the Tertiary Prevention of Primary Liver Cancer, which was based on the clinical and scientific advances on the risk factors, histopathology, imaging finding, clinical manifestation, and prevention of recurrence of PLC. The purpose is to provide a current basis for the prevention, surveillance, early detection and diagnosis, and the effective measures of PLC recurrence.

Comparison of NK cell subsets, receptors and functions induced by radiofrequency ablation and microwave ablation in HBV-associated primary hepatocellular carcinoma.

Background: Topical therapy has been shown to induce an immune response in patients with hepatocellular carcinoma (HCC). In this study, a prospective parallel group control experiment was conducted to compare the differences between radiofrequency ablation and microwave ablation in inducing the immune regulation of NK cells. Methods: Sixty patients with clinically and pathologically confirmed hepatitis B-associated hepatocellular carcinoma (HCC) were selected for thermal ablation. Patients were randomly assigned into the MWA group (n = 30) and the RFA group (n = 30). Patient's peripheral blood was isolated on days D0, D7, and month M1. NK cell subsets, receptors, and killing function were detected by flow cytometry and LDH. Student t test and rank sum test were used to compare the statistical differences between the RFA (radio frequency) and MWA (microwave) groups. The Kaplan-Meier curve and log-rank test were used to calculate the difference between the two survival curves. Results: Comparison of the frequency of CD3-CD56+ and CD3-CD56+CD16+ in NK cells between the RFA and WMA groups showed that there was no difference in the D0, D7, M1, D7-D0, M1-D0, and M1-D7 groups. The changes of the inhibitory NK cell receptor CD159A were significantly different at D7 (P<0.05). CD107a were compared between the RFA and WMA groups, indicating that CD107a changes induced by NK cells were significantly different at D7-D0 (P<0.05). Comparison of NK cell lysis activity of target K562 cells between the RFA and WMA groups showed that there was no difference at D0, D7, D7-D0. There was no difference in recurrence-free survival (RFS) between the RFA and WMA groups (P=0.11). Conclusions: The difference between MWA and RFA-induced NK cell changes was mainly manifested in the inhibitory receptors CD159a and CD107a 1 week after surgery, with microwave-induced changes being more severe. Comparison of the NK cell lysis activity of the target K562 cells between the RFA and WMA groups showed that there was no difference in D0, D7, D7- D0. Survival analysis showed that these differences did not affect the recurrence-free survival (RFS) in the two groups.

Dynamic changes of phenotype and function of natural killer cells in peripheral blood before and after thermal ablation of hepatitis B associated hepatocellular carcinoma and their correlation with tumor recurrence.

BACKGROUND: Thermal therapy induces an immune response in patients with hepatocellular carcinoma (HCC), but the dynamic characteristics of the natural killer (NK) cell immune response post-thermal ablation remain unclear. We conducted a prospective longitudinal cohort study to observe the dynamic changes of phenotype and function of NK cells in peripheral blood before and after thermal ablation of hepatitis B-associated HCC and their correlation with tumor recurrence. METHODS: Fifty-six patients clinically and pathologically confirmed with hepatitis B-associated HCC were selected for thermal ablation. Peripheral blood was collected on day 0, day 7, and month 1. NK cell subsets, receptors, and killing function were detected by flow cytometry, and the LDH levels were examined. Overall recurrence and associated variables were estimated using Kaplan-Meier, log-rank, and Cox proportional-hazards analyses. RESULTS: The frequency of CD3-CD56+ cells was increased on day 7 (P < 0.01) without significant differences between D0 and M1. NKG2D, NKp44, NKp30, CD159a, and CD158a expression was increased on M1 (all P < 0.05). The granzyme B and IFN-γ expression in NK cells were higher on M1 vs. D0 (P < 0.05). On day 7, the NK cell lysis activity of the target K562 cells was increased (P < 0.01) but decreased on M1 (P < 0.05). Survival analysis showed that CD158a expression and IFN-γ and perforin release on day 0 were associated with the risk of HCC recurrence. Cox regression analysis showed that the expression changes in CD56, NKp46, granzyme B, and perforin (D7-D0) induced by thermal ablation were associated with recurrence-free survival (RFS) of patients with HCC. CONCLUSION: Thermal ablation increased the frequency and function of CD3-CD56+ NK cells in the peripheral blood of patients with HCC. These cells tended to be more differentiated and activated. Notably, expression levels of NK cell receptors NKp46, perforin, and granzyme B were associated with RFS.

Serum HBsAg and HBcrAg is associated with inflammation in HBeAg-positive chronic hepatitis B patients.

Backgrounds & aims: Liver inflammation is the main risk factor for developing liver fibrosis, cirrhosis, and even hepatocellular carcinoma in chronic hepatitis B (CHB) patients. To replace biopsy, additional non-invasive biomarkers to diagnose and grade liver necroinflammation are urgently required in clinical practice. Method: Ninety-four CHB patients, including 74 HBeAg-positive and 20 HBeAg-negative patients, were enrolled and started entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), ALT and AST levels, as well as intrahepatic HBV DNA and cccDNA were measured at baseline and during treatment. Liver inflammation was assessed at baseline and month 60 by liver biopsy. Inflammation regression was defined as a ≥1-grade decrease according to the Scheuer scoring system. Results: In HBeAg-positive CHB patients, at baseline, serum HBsAg and HBcrAg levels negatively correlated with inflammation grade, while ALT and AST levels positively correlated with inflammation grade. AST plus HBsAg exhibited excellent diagnostic ability for significant inflammation with an AUROC of 0.896. After 60 months of antiviral treatment, almost all the patients' liver inflammation ameliorated to G1, and no patients had inflammation progression. Conclusion: Besides ALT and AST, serum HBsAg and HBcrAg correlated with inflammation grade in HBeAg-positive CHB patients before NAs treatment. Moreover, the combination of HBsAg and AST exhibited excellent diagnostic ability for significant inflammation.

Programmed cell death and lipid metabolism of macrophages in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) has now become the leading chronic liver disease worldwide with lifestyle changes. This may lead to NAFLD becoming the leading cause of end-stage liver disease in the future. To date, there are still no effective therapeutic drugs for NAFLD. An in-depth exploration of the pathogenesis of NAFLD can help to provide a basis for new therapeutic agents or strategies. As the most important immune cells of the liver, macrophages play an important role in the occurrence and development of liver inflammation and are expected to become effective targets for NAFLD treatment. Programmed cell death (PCD) of macrophages plays a regulatory role in phenotypic transformation, and there is also a certain connection between different types of PCD. However, how PCD regulates macrophage polarization has still not been systematically elucidated. Based on the role of lipid metabolic reprogramming in macrophage polarization, PCD may alter the phenotype by regulating lipid metabolism. We reviewed the effects of macrophages on inflammation in NAFLD and changes in their lipid metabolism, as well as the relationship between different types of PCD and lipid metabolism in macrophages. Furthermore, interactions between different types of PCD and potential therapeutic agents targeting of macrophages PCD are also explored.

Serum HBV RNA is associated with liver fibrosis regression in HBeAg-positive chronic hepatitis B patients treated with nucleos(t)ide analogues.

Noninvasive methods for assessing hepatic fibrosis are clinically necessary. This study aims to explore HBV markers correlated with liver fibrosis and capable of diagnosing significant fibrosis and predicting fibrosis regression. Seventy-four HBeAg-positive chronic hepatitis B (CHB) patients were enrolled and started on entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg and hepatitis B core-related antigen (HBcrAg) levels were measured at baseline and during treatment. Liver fibrosis was assessed at baseline and month 60 by liver biopsy. Fibrosis regression was defined as Ishak fibrosis score decreased ≥1-point. At baseline, HBsAg, HBcrAg and HBV RNA levels had a stronger correlation with Ishak fibrosis score (r = -.441, p = .002; r = -.469, p = .001; r = -.398, p = .001) than APRI and FIB-4 (r = .321 p = .006; r = .371, p = .001). HBsAg >4 log10 IU/ml plus HBcrAg >7 log10 IU/ml or HBsAg >4 log10 IU/ml plus HBV RNA >5 log10 copies/ml exhibited the same excellent diagnostic ability for significant fibrosis with the AUROC of 0.857. After 60 months of antiviral treatment, 66.7% of patients who suffered significant fibrosis at baseline achieved fibrosis regression, and an HBV RNA decline from baseline to month 6 greater than 0.63 log10 copies/ml could predict the fibrosis regression at month 60. In conclusion, serum HBsAg, HBcrAg and HBV RNA are potential markers for predicting significant liver fibrosis. HBV RNA measurement would be particularly useful for monitoring hepatic fibrosis changes in HBeAg-positive CHB patients.

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance. METHODS: Taking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. RESULTS: The success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90-100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. CONCLUSIONS: Through the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.

Association between serum sphingolipids and necroinflammation of liver tissue pathology in chronic hepatitis B.

Background & Aims: Accurately identifying liver necroinflammation was essential for the timely implementation of antiviral therapy in chronic hepatitis B(CHB) patients. The sphingolipids were involved in various chronic inflammatory processes. This study aimed to evaluate the association between serum sphingolipids and liver necroinflammation in CHB patients. Methods: The study prospectively enrolled patients with a diagnosis of chronic hepatitis B who were subsequently treated with nucleos(t)ide analogs (NAs). Liver biopsy was performed at baseline and 5-year follow-up, and serum sphingolipid levels were measured by ultra-high-performance liquid chromatography tandem mass spectrometry. Results: A total of 70 CHB patients were enrolled with baseline liver necroinflammation of 27(38.6%) G1, 23(32.9%) G2, and 20(28.6%) G ≥ 3, respectively. A total of 126 liver biopsies were performed on the study population over a 5-year period, of which 80 (63.5%) G<2 and 46 (36.5%) G≥2. Serum ALT, ALP, SM d16:0/16:1, SM d16:0/17:1, SM d18:0/17:0 and Cer d18:2/22:0 showed significant differences between two groups (P<0.01). Multivariate analysis showed that serum ALT (OR 1.006, 95% CI: 1.000-1.011), SM d16:0/16:1 (OR 1.552, 95% CI: 1.150-2.093), Cer d18:2/22:0 (OR 0.003, 95% CI: 0.000-0.173) were associated with G ≥ 2. In the subgroup of patients with normal serum ALT, serum Cer d18:2/22:0 was lower in patients with G ≥ 2 than that with G < 2. After 5 years, alleviated inflammation was accompanied by decreased serum SM d16:0/16:1 and increased serum Cer d18:2/22:0 in patients with baseline G ≥ 2. Conclusions: Lower serum Cer d18:2/22:0 could reflect hepatic necroinflammation (G ≥ 2) in CHB patients including those with normal serum ALT, and its elevation predicts the inflammation improvement after NAs treatment.

Direct-acting Antiviral-induced Transient Recovery of NK Cells in Early-stage Treatment of Chronic Hepatitis C Patients.

Background and Aims: The rapid clearance of hepatitis C virus induced by direct-acting antivirals (DAAs) affects natural killer (NK) cells, but the reported results are not consistent, and the relative mechanism was unclear. This study focused on the dynamic changes of NK cells during and after DAA treatment and analyzed the reasons. Methods: Peripheral blood from 35 chronic hepatitis C patients who were treated with DAAs were collected at baseline and weeks 1, 2, 4, 12, and post-treatment week-12. The frequency, subset, and phenotype of NK cells were assayed by flow cytometry. Lactate dehydrogenase assays were used to evaluate the cytotoxicity of NK cells. Cytokine concentrations were measured with Luminex kits. Results: All patients achieved a sustained viral response (SVR), and the NK cell frequencies were not changed significantly during DAA therapy. However, the cytotoxicity of NK cells recovered significantly early in week 1, and then continuously decreased below normal levels. The changes of genotypes including NKp30+, NKp46+, and NKG2A+ NK cells were parallel to NK function. The subset of CD56dim NK cells continuously increased and did not return to normal even at 12 weeks after treatment. Interleukin (IL)-2, IL10, IL15, interferon-gamma, and tumor necrosis factor-alpha all increased after week 4, peaked at the end of therapy, and then exhibited varying degrees of reduction with time. Conclusions: DAA treatment led to transient functional recovery of NK cells in the early stage of treatment, and then continuously decreased to below normal levels. Alterations of NK subsets, phenotypes, and the microenvironment may be involved in the changes.

Galectin-3 critically mediates the hepatoprotection conferred by M2-like macrophages in ACLF by inhibiting pyroptosis but not necroptosis signalling.

We previously documented that M2-like macrophages exert a hepatoprotective effect in acute-on-chronic liver failure (ACLF) by inhibiting necroptosis signalling. Nevertheless, the molecular mechanism behind this hepatoprotection still needs to be further dissected. Galectin-3 (GAL3) has been reported to be critically involved in the pathogenesis of multiple liver diseases, whereas the potential role of GAL3 in ACLF remains to be explored. Herein, we hypothesised that GAL3 plays a pivotal role in the hepatoprotection conferred by M2-like macrophages in ACLF by inhibiting necroptosis. To test this hypothesis, we first assessed the expression of GAL3 in control and fibrotic mice with or without acute insult. Second, loss- and gain-of-function experiments of GAL3 were performed. Third, the correlation between GAL3 and M2-like macrophage activation was analysed, and the potential role of GAL3 in M2-like macrophage-conferred hepatoprotection was confirmed. Finally, the molecular mechanism underlying GAL3-mediated hepatoprotection was dissected. GAL3 was found to be obviously upregulated in fibrotic mice with or without acute insult but not in acutely injured mice. Depletion of GAL3 aggravated hepatic damage in fibrotic mice upon insult. Conversely, adoptive transfer of GAL3 provided normal mice enhanced resistance against acute insult. The expression of GAL3 is closely correlated with M2-like macrophage activation. Through adoptive transfer and depletion experiments, M2-like macrophages were verified to act as a major source of GAL3. Importantly, GAL3 was confirmed to hold a pivotal place in the hepatoprotection conferred by M2-like macrophages through loss- and gain-of-function experiments. Unexpectedly, the depletion and adoptive transfer of GAL3 resulted in significant differences in the expression levels of pyroptosis but not necroptosis signalling molecules. Taken together, GAL3 plays a pivotal role in the hepatoprotection conferred by M2-like macrophages in ACLF by inhibiting pyroptosis but not necroptosis signalling. Our findings provide novel insights into the pathogenesis and therapy of ACLF.

Gender differences in demographic and clinical characteristics in patients with HBV-related liver diseases in China.

Background: The gender differences in demographic and clinical characteristics were examined in patients with hepatitis B virus (HBV)-related liver diseases. Methods: Overall, 634 patients (44.7 ± 13.8 years) were consecutively included. Data of demographic and clinical characteristics were collected during an assessment interview. Comparisons between male and female patients in terms of demographic and clinical data were carried out using univariate analyses. The independent associations between the demographic and clinical variables and gender were examined with either logistic regression or analysis of covariance as appropriate. Results: The study sample consisted of 452 male and 182 female patients. Multiple logistic regression analyses revealed that being employed (OR = 3.4), personal monthly income <3,000 yuan (OR = 0.3), being current alcohol users (OR = 6.4), Cirrhosis (OR = 5.9), Hepatocellular Carcinoma (HCC) (OR = 8.5) and having less severe insomnia (OR = 0.6) were independently associated with male gender. The analysis of covariance revealed that after controlling for other potential confounding variables, later onset of HBV-related diseases (F = 4.5, p = 0.03) and older age (F = 6.7, p = 0.009) were independently associated with male gender. Conclusions: Given the significant clinical differences in male and female patients with HBV-related liver diseases, more attention should be given to gender-specific treatment and prevention for this population.

The Measurement of 25-Hydroxyvitamin-D in Chronic HBV Patients Using LC-MS/MS.

BACKGROUND: Vitamin D deficiency is universal among patients with chronic liver disease. Vitamin D may be involved in the regulation of immune function of chronic hepatitis B and related to disease progression. METHODS: The study was a cross-sectional study. The level of vitamin 25(OH)D was detected in patients with chronic hepatitis B, hepatitis B cirrhosis, hepatitis B cancer, and healthy groups by isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). At the same time, the clinical data, biochemical indexes, and T lymphocyte subsets were collected to study the relationship between vitamin 25(OH)D deficiency and clinical indexes of hepatitis B patients. RESULTS: The prevalence of vitamin D deficiency (< 20 ng/mL) was higher in patients with liver cancer group (96.97%, 10.59 ± 3.06 ng/mL) and cirrhosis group (93.18%, 11.85 ± 2.66 ng/mL) than in the healthy group (76.92%, 16.38 ± 5.53 ng/mL) and chronic hepatitis B group (77.83%, 15.06 ± 4.91 ng/mL). There were significant differences in vitamin 25(OH)D levels between the cirrhosis groups and the healthy groups, the liver cancer groups and the healthy groups, the hepatitis B cirrhosis groups and the chronic hepatitis B groups, the liver cancer groups and the chronic hepatitis B groups (p < 0.05). There was no significant difference in vitamin 25 (OH)D level between liver cancer group and hepatitis B cirrhosis group, healthy group and chronic hepatitis B group (p > 0.05). Vitamin 25(OH)D level was correlated with age (r = -0.24, p = 0.015), lymphocyte (r = 0.24, p = 0.015), hemoglobin (r = 0.28, p = 0.005), platelet (r = 0.27, p = 0.006), PTA (r = 0.33, p = 0.001), albumin (r = 0.30, p = 0.002), prealbumin (r = 0.39, p = 0.001), cholinesterase (r = 0.29, p = 0.003), CD3+ (r = 0.20, p = 0.04), CD3+ CD8+ (r = 0.20, p = 0.04), CD45+ (r = 0.24, p = 0.017), but none correlated with liver function and HBV-DNA. CONCLUSIONS: Vitamin D deficiency existed in patients with hepatitis B, which was related to the clinical progress of hepatitis B and may be involved in the regulation of immune function in patients with chronic HBV infection.

Current status of and barriers to the treatment of advanced-stage liver cancer in China: a questionnaire-based study from the perspective of doctors.

BACKGROUND: Liver cancer is a severe public health problem worldwide, and it creates a relatively higher disease burden in China than in the Western world. Despite achieving notable progress in China, potential differences in some aspects of medical services for liver cancer may persist across different regions and hospitals. This warrants serious consideration of the actual status of and barriers to liver cancer treatment. We intended to explore the present status of and obstacles in liver cancer treatment especially for advanced-stage liver cancer. METHODS: In February 2021, a national multicenter cross-sectional study was conducted among 1500 doctors from 31 provinces of mainland China using a self-administered online questionnaire. Participants completed the questionnaire about their general information, perspectives on the current status of liver cancer treatment, and expectations for future treatment. Chi-square and logistic regression analyses were performed to explore the differences associated with the regions, doctors' professional ranks, and hospital levels. RESULTS: Treatment conditions, medications, and treatment strategies were inconsistent across different economic regions and hospital of different levels. With respect to obstacles in treatment, 76.6% of the doctors were unsatisfied with the current treatment for liver cancer. Important factors that influenced their satisfaction with the treatment for liver cancer included early diagnosis and the disclosure of true conditions to patients. CONCLUSIONS: There persists differences in the treatment of liver cancer in China, besides barriers to treatment. More attention should be paid to the detection and treatment of liver cancer and the propagation of novel progress among doctors in underdeveloped areas.

Digital Droplet PCR for Detection and Quantitation of Hepatitis Delta Virus.

INTRODUCTION: Hepatitis delta virus (HDV) far exceeds our expected level. There remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet polymerase chain reaction (ddPCR) for HDV quantitative detection. METHODS: With plasmid (pMD19T) containing HDV full genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed with hepatitis D and 14 controls were examined using enzyme-linked immunosorbent assay, reverse-transcriptase PCR (RT-PCR), and ddPCR. A total of 728 hepatitis B virus-related patients, including 182 patients with chronic hepatitis B, 182 with liver cirrhosis, 182 with hepatocellular carcinoma, and 182 with liver failure, were screened for HDV infection. RESULTS: The detection limit of ddPCR for HDV is significantly low, with lower limit of detection and lower limit of quantitation of 0.29 IU/mL (95% confidence interval: 1.93 × 10-3-1.22 IU/mL) and 8.76 IU/mL (95% confidence interval: 1.83-1.03 × 106 IU/mL), respectively. Among the 44 samples, the enzyme-linked immunosorbent assay detected 30 cases positive, ddPCR reported 24 samples, and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV were 1.1%, 3.3%, 2.7%, and 7.1% in patients with chronic hepatitis B, liver cirrhosis, hepatocellular carcinoma, and liver failure, respectively; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4%, and 20%, respectively. However, the detection rates of ddPCR were 0%, 33.33%, 30.77%, and 60%, respectively. DISCUSSION: We establish a high sensitivity and specificity quantitative HDV RNA detection method based on ddPCR. Hepatitis B virus-related end-stage liver diseases, especially liver failure, are associated with a remarkably high rate of HDV infection.

Serum HBV DNA plus RNA reflecting cccDNA level before and during NAs treatment in HBeAg positive CHB patients.

Background & Aims: Correlations between serum viral markers and intrahepatic cccDNA in patients undergoing long-term nucleos(t)ide analogues (NAs) treatment haven't been fully explored. In this study, we evaluate the correlation between intrahepatic cccDNA and other serum viral markers and intrahepatic HBV DNA in HBeAg positive chronic hepatitis B (CHB) patients during 60-month treatment with NAs. Methods: Fifty-four HBeAg positive CHB patients received long-term NAs treatment were included in this study. Serial serum samples were regularly collected and quantitatively analyzed for HBsAg, HBV DNA, HBV RNA and HBcrAg. Histological samples from liver biopsy at baseline and month 60 were analyzed for intrahepatic HBV DNA and cccDNA. Results: At baseline, serum HBV DNA plus RNA was positively associated with intrahepatic cccDNA in multivariate regression analysis (ß=0.205, P<0.001). In the correlation analysis between cccDNA and serum viral markers, HBV DNA plus RNA had the highest correlation coefficient (r=0.698, P<0.001), followed by serum HBV DNA (r=0.641, P<0.001), HBV RNA (r=0.590, P<0.001), and HBcrAg (r=0.564, P<0.001). At month 60, correlations between these serum viral markers and cccDNA were not observed (P>0.05). Multivariate regression analysis showed that only the decreased HBV DNA plus RNA was positively associated with cccDNA decline (ß=0.172, P =0.006). Changes of HBV DNA plus RNA (r=0.525, P=0.001) was better correlated with cccDNA decline as compared to HBV RNA (r=0.384, P=0.008), HBV DNA (r=0.431, P=0.003), and HBsAg (r=0.342, P=0.029). Conclusions: Serum HBV DNA plus RNA better correlated with intrahepatic cccDNA than other viral makers before and during NAs treatment in HBeAg positive CHB patients.

Inducible nitric oxide synthase regulates macrophage polarization via the MAPK signals in concanavalin A-induced hepatitis.

INTRODUCTION: Acute liver inflammatory reactions contribute to many health problems; thus, it is critical to understand the underlying pathogenic mechanisms of acute hepatitis. In this study, an experimental in vivo model of concanavalin A (ConA)-induced hepatitis was used. MATERIALS AND METHODS: C57BL/6 (wild-type, WT) or inducible nitric oxide synthase-deficient (iNOS-/- ) mice were injected with PBS or 15 mg/kg ConA via tail vein. Detection of liver injury by histological examination and apoptosis, and flow cytometry to detect the effect of immune cells on liver injury. RESULTS: iNOS-/-  mice had lower levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase, suggesting that they were protected against ConA-induced pathological liver injury and that iNOS participated in the regulation of hepatitis. Furthermore, iNOS deficiency was found to lower CD86 expression and suppressed the messenger RNA levels of inflammatory factors in the liver. In vitro experiments also demonstrated that iNOS deficiency suppressed the sequential phosphorylation of the mitogen-activated protein kinase pathway cascade, thereby inhibiting the M1 polarization of macrophages and consequently suppressing the transcription of inflammation factors. CONCLUSION: iNOS may contribute to ConA-induced inflammation by promoting the activation of proinflammatory macrophages.

Phase IIa, randomised, double-blind study of GSK3389404 in patients with chronic hepatitis B on stable nucleos(t)ide therapy.

BACKGROUND & AIMS: Bepirovirsen, an antisense oligonucleotide targeting pregenomic and mRNA transcripts of HBV, has been conjugated to N-acetyl galactosamine (GSK3389404) to enhance hepatocyte delivery. This dose-finding study was the first to assess GSK3389404 for chronic HBV infection. METHODS: This phase IIa, randomised, double-blind, placebo-controlled, 2-part study was conducted in 22 centres in Asia (NCT03020745). Pharmacokinetic findings from Part 1 informed Part 2 dosing. In Part 2, patients with chronic hepatitis B on nucleos(t)ide analogue therapy were randomised 11:2 to GSK3389404 (30, 60, 120 mg weekly or 120 mg bi-weekly) or placebo until Day 85. Coprimary endpoints included HBsAg response (≥1.5 log10 IU/ml reduction from baseline) rate, safety and pharmacokinetics. RESULTS: Parts 1 and 2 included 12 (9 GSK3389404, 3 placebo) and 66 patients (56 GSK3389404, 10 placebo), respectively. In Part 2, one patient each in the 60 mg weekly, 120 mg weekly and 120 mg bi-weekly arms achieved a HBsAg response. HBsAg reductions were dose-dependent (Day 85: mean 0.34 [60 mg weekly] to 0.75 log10 IU/ml [120 mg weekly]) and occurred in hepatitis B e antigen-positive and -negative patients. No patient achieved HBsAg seroclearance. 43/56 (77%) GSK3389404- and 9/10 (90%) placebo-treated patients reported adverse events. No deaths were reported. Alanine aminotransferase flares (>2x upper limit of normal) occurred in 2 GSK3389404-treated patients (120 mg weekly, 120 mg bi-weekly); both were associated with decreased HBsAg, but neither was considered a responder. GSK3389404 plasma concentrations peaked 2-4 hours post dose; mean plasma half-life was 3-5 hours. CONCLUSIONS: GSK3389404 showed an acceptable safety profile and target engagement, with dose-dependent reductions in HBsAg. However, no efficacious dosing regimen was identified. CLINICAL TRIAL NUMBER: NCT03020745. LAY SUMMARY: Hepatitis B virus (HBV) can result in chronic HBV infection, which may ultimately lead to chronic liver disease, primary liver cancer and death; HBV proteins may prevent the immune system from successfully controlling the virus. GSK3389404 is an investigational agent that targets HBV RNA, resulting in reduced viral protein production. This study assessed the safety of GSK3389404 and its ability to reduce the viral proteins in patients with chronic HBV infection. GSK3389404 showed dose-dependent reduction in hepatitis B surface antigen, with an acceptable safety profile. While no clear optimal dose was identified, the findings from this study may help in the development of improved treatment options for patients with chronic HBV infections.

The Role of Autophagy in the Mother-to-Child Transmission of Pregnant Women With a High Level of HBV DNA.

Background: Mother-to-child transmission (MTCT) is the most common propagation mode of hepatitis B virus (HBV) transmission. Exploring the mechanisms of HBV MTCT is the key to protect infant from infection. In this study, we aim to clarify the important role of autophagy complicated in HBV MTCT. Methods: A total of 169 placental samples were collected in this study, includes 144 HBV positive pregnant women and 25 normal pregnant women. In vitro, JEG-3 cells were treated with serum contained different HBV viral loads. Electron microscope was used to observed the number of autophagosome. RT-qPCR and western blotting were used to measure the expression level of autophagy relative genes and proteins respectively. Immunofluorescence was used to analyzed the expression of LC-3 of the frozen section of placental tissue. Results: According to the number of autophagosomes and the expression level of autophagic genes mRNA and protein, autophagy was increased in HBV maternal placenta. Among the control, low viral load, medium viral load and high viral load groups, autophagy was significantly up-regulated with the increase of HBV viral loads. Also, autophagy was increased in the HBeAg positive pregnant women compared with their HBeAg negative counterparts. Also, autophagy in infant-infected group was up-regulated compared with infant-uninfected group. In vitro, choriocarcinoma JEG-3 cells were treated with the different HBV viral loads or different time incubation, the mRNA and protein of autophagy related genes was maximum expression in the medium viral load or treatment in a short period, but decreased in the high viral load treatment or with long-term HBV exposure. Conclusion: Our study determines the high levels of viremia could be the cause of both increase autophagy activities and MTCT. Autophagy was significantly up-regulated in pregnant women with high viral load or HBeAg positive, which plays an important part in the HBV MTCT.