Identification of PRMT5 as a therapeutic target in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a very difficult-to-treat cancer. Chemotherapies are little effective and response to immune checkpoint inhibitors is limited. Therefore, new therapeutic strategies need to be identified. OBJECTIVE: We characterised the enzyme protein arginine-methyltransferase 5 (PRMT5) as a novel therapeutic target in CCA. DESIGN: We evaluated the expression of PRMT5, its functional partner MEP50 and methylthioadenosine phosphorylase (MTAP)-an enzyme that modulates the sensitivity of PRMT5 to pharmacological inhibitors-in human CCA tissues. PRMT5-targeting drugs, currently tested in clinical trials for other malignancies, were assessed in human CCA cell lines and organoids, as well as in two immunocompetent CCA mouse models. Transcriptomic, proteomic and functional analyses were performed to explore the underlying antitumoural mechanisms. RESULTS: PRMT5 and MEP50 proteins were correlatively overexpressed in most CCA tissues. MTAP was absent in 25% of intrahepatic CCA. PRMT5-targeting drugs markedly inhibited CCA cell proliferation, synergising with cisplatin and gemcitabine and hindered the growth of cholangiocarcinoma organoids. PRMT5 inhibition blunted the expression of oncogenic genes involved in chromatin remodelling and DNA repair, consistently inducing the formation of RNA loops and promoting DNA damage. Treatment with PRMT5-targeting drugs significantly restrained the growth of experimental CCA without adverse effects and concomitantly induced the recruitment of CD4 and CD8 T cells to shrinking tumourous lesions. CONCLUSION: PRMT5 and MEP50 are frequently upregulated in human CCA, and PRMT5-targeting drugs have significant antitumoural efficacy in clinically relevant CCA models. Our findings support the evaluation of PRMT5 inhibitors in clinical trials, including their combination with cytotoxic and immune therapies.

Application of cardiovascular interventions to decrease blood loss during hepatectomy: a systematic review and meta-analysis.

BACKGROUND: Perioperative bleeding and allogeneic blood transfusion are generally thought to affect the outcomes of patients. This meta-analysis aimed to determine the benefits and risks of several cardiovascular interventions in patients undergoing hepatectomy. METHODS: In this systematic review and meta-analysis, randomised controlled trials (RCTs) were searched in the Cochrane Library, Medline, Embase, and Web of Science to February 02, 2023. RCTs focused on cardiovascular interventions aimed at reducing blood loss or blood transfusion requirements during hepatectomy were included. The primary outcomes were perioperative blood loss amount, number of patients requiring allogeneic blood transfusion and overall occurrence of postoperative complications. The secondary outcomes were operating time, perioperative mortality rate, postoperative liver and kidney function and length of hospital stay. RESULTS: Seventeen RCTs were included in the analysis. A total of 841 patients who underwent hepatectomy in 10 trials were included in the comparative analysis between low central venous pressure (CVP) and control groups. The forest plots showed a low operative bleeding volume [(mean difference (MD): -409.75 mL, 95% confidence intervals (CI) -616.56 to -202.94, P < 0.001], reduced blood transfusion rate [risk ratio (RR): 0.47, 95% CI 0.34 to 0.65, P < 0.001], shortened operating time (MD: -13.42 min, 95% CI -22.59 to -4.26, P = 0.004), and fewer postoperative complications (RR: 0.76, 95% CI 0.58 to 0.99, P = 0.04) in the low CVP group than in the control group. Five and two trials compared the following interventions, respectively: 'acute normovolaemic haemodilution (ANH) vs control' and 'autologous blood donation vs control'. ANH and autologous blood donation could not reduce the blood loss amount but greatly decreased the number of patients requiring allogeneic blood transfusion. No benefits were found in the rate of mortality and length of postoperative hospital stay in any of the comparisons. CONCLUSION: Lowering the CVP seems to be effective and safe in adult patients undergoing hepatectomy. ANH and autologous blood donation should be used as a part of blood management for suitable patients in certain circumstances. TRIAL REGISTRATION: PROSPERO, CRD42022314061.

Analysis of HBV X gene quasispecies characteristics by next-generation sequencing and cloning-based sequencing and its association with hepatocellular carcinoma progression.

OBJECTIVES: This study aimed to describe the differences between next-generation sequencing (NGS) and cloning-based sequencing (CBS) in HBX quasispecies research and primitively investigate the relationship between the dominant HBX quasispecies and hepatocellular carcinoma (HCC). METHODS: A total of 12 serum samples were collected. Serum hepatitis B virus (HBV) DNA was extracted, and the HBV X-region (HBX) was amplified by nested polymerase chain reaction (PCR). The PCR products were simultaneously tested with NGS and CBS to detect quasispecies of the HBX. RESULTS: A total of 9348 eligible quasispecies sequences were obtained by NGS, which were much larger than the 98 of that by CBS. By the phylogenetic tree, the dominant quasispecies sequence of each sample could be found, although they had several nucleotides differences between the dominant quasispecies sequences found by CBS and NGS. By comparing the quasispecies heterogeneity, it was found that the quasispecies complexity value of HBV X-region obtained by NGS was higher than CBS (P < 0.05). The diversity values, including d, dS, dN, an d d N/ dS obtained by NGS were lower than by CBS (all of P < 0.01). The relativity of Spearman(rs) in d, dS, and dN were statistically significant (rs_ d = 0.865, P = 0.001; rs_ dS = 0.722, P = 0.014; and rs_ dN = 0.738, P = 0.011, respectively). There were 21 different bases between the HBX quasispecies of case A and control B. CONCLUSION: The results of this can be used as guidance when researchers plan to choose a suitable method to study quasispecies, especially the HBV X gene quasispecies. Some high-risk mutations of HBX quasispecies were also found in this study and their relationship with HCC need deeper exploration.

Inhibition of ALK3-mediated signalling pathway protects against acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is one of the most prevalent causes of acute liver failure (ALF). We assessed the role of the bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 in APAP-induced hepatotoxicity. The molecular mechanisms that regulate the balance between cell death and survival and the response to oxidative stress induced by APAP was assessed in cultured human hepatocyte-derived (Huh7) cells treated with pharmacological inhibitors of ALK receptors and with modulated expression of ALK2 or ALK3 by lentiviral infection, and in a mouse model of APAP-induced hepatotoxicity. Inhibition of ALK3 signalling with the pharmacological inhibitor DMH2, or by silencing of ALK3, showed a decreased cell death both by necrosis and apoptosis after APAP treatment. Also, upon APAP challenge, ROS generation was ameliorated and, thus, ROS-mediated JNK and P38 MAPK phosphorylation was reduced in ALK3-inhibited cells compared to control cells. These results were also observed in an experimental model of APAP-induced ALF in which post-treatment with DMH2 after APAP administration significantly reduced liver tissue damage, apoptosis and oxidative stress. This study shows the protective effect of ALK3 receptor inhibition against APAP-induced hepatotoxicity. Furthermore, findings obtained from the animal model suggest that BMP signalling might be a new pharmacological target for the treatment of ALF.

Fecal microbiota transplantation from female donors restores gut permeability and reduces liver injury and inflammation in middle-aged male mice exposed to alcohol.

Background: Alcohol misuse, binge drinking pattern, and gender-specific effects in the middle-aged population has been clearly underestimated. In the present study, we focused on understanding gender-specific effects of alcohol exposure on the gut-liver axis and the role of gut microbiota in modulating gender-specific responses to alcohol consumption. Methods: Fifty-two-week-old female and male C57BL/6 mice were fasted for 12 h, and then administered a single oral dose of ethanol (EtOH) (6 g/kg). Controls were given a single dose of PBS. Animals were sacrificed 8 h later. Alternatively, fecal microbiota transplantation (FMT) was performed in 52-week-old male mice from female donors of the same age. Permeability of the large intestine (colon), gut microbiota, liver injury, and inflammation was thoroughly evaluated in all groups. Results: Middle-aged male mice exposed to EtOH showed a significant increase in gut permeability in the large intestine, evaluated by FITC-dextran assay and ZO-1, OCCLUDIN and MUCIN-2 immuno-staining, compared to PBS-treated animals, whilst female mice of the same age also increased their gut permeability, but displayed a partially maintained intestinal barrier integrity. Moreover, there was a significant up-regulation of TLRs and markers of hepatocellular injury, cell death (AST, TUNEL-positive cells) and lipid accumulation (ORO) in male mice after EtOH exposure. Interestingly, FMT from female donors to male mice reduced gut leakiness, modified gut microbiota composition, ameliorated liver injury and inflammation, TLR activation and the senescence phenotype of middle-aged mice. Conclusion: Our findings highlighted the relevance of gender in middle-aged individuals who are exposed to alcohol in the gut-liver axis. Moreover, our study revealed that gender-specific microbiota transplantation might be a plausible therapy in the management of alcohol-related disorders during aging.

Pilot-scale study of step-feed anaerobic coupled four-stage micro-oxygen gradient aeration process for treating digested swine wastewater with low carbon/nitrogen ratios.

This study developed an innovative step-feed anaerobic coupled four-stage micro-oxygen gradient aeration process to treat digested swine wastewater. An anaerobic zone was used for prepositive denitrification; four micro-oxygen reactors (zones O1-O4) were used for simultaneous partial nitrification and denitrification through low-dissolved oxygen gradient control, step-feed, and swine wastewater-digested swine wastewater distribution. The nitrogen-removal efficiency was satisfactory (93 ± 3 %; effluent total nitrogen, 53 ± 19 mg/L). Mass balance coupled with quantitative polymerase chain reaction analysis revealed that simultaneous partial nitrification and denitrification was achieved in four micro-oxygen zones. Zones O1 were the major denitrification zones for nitrogen removal; nitrification was primary happened in zones O2 and O3. Correlation analysis confirmed that low-dissolved oxygen gradient control was the key to achieving efficient nitrogen removal. This study provides a low oxygen energy consumption method to treat digested swine wastewater with a low carbon/nitrogen ratio (<3).

A Novel Mouse Model of Combined Hepatocellular-Cholangiocarcinoma Induced by Diethylnitrosamine and Loss of Ppp2r5d.

Primary liver cancer (PLC) can be classified in hepatocellular (HCC), cholangiocarcinoma (CCA), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The molecular mechanisms involved in PLC development and phenotype decision are still not well understood. Complete deletion of Ppp2r5d, encoding the B56δ subunit of Protein Phosphatase 2A (PP2A), results in spontaneous HCC development in mice via a c-MYC-dependent mechanism. In the present study, we aimed to examine the role of Ppp2r5d in an independent mouse model of diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Ppp2r5d deletion (heterozygous and homozygous) accelerated HCC development, corroborating its tumor-suppressive function in liver and suggesting Ppp2r5d may be haploinsufficient. Ppp2r5d-deficient HCCs stained positively for c-MYC, consistent with increased AKT activation in pre-malignant and tumor tissues of Ppp2r5d-deficient mice. We also found increased YAP activation in Ppp2r5d-deficient tumors. Remarkably, in older mice, Ppp2r5d deletion resulted in cHCC-CCA development in this model, with the CCA component showing increased expression of progenitor markers (SOX9 and EpCAM). Finally, we observed an upregulation of Ppp2r5d in tumors from wildtype and heterozygous mice, revealing a tumor-specific control mechanism of Ppp2r5d expression, and suggestive of the involvement of Ppp2r5d in a negative feedback regulation restricting tumor growth. Our study highlights the tumor-suppressive role of mouse PP2A-B56δ in both HCC and cHCC-CCA, which may have important implications for human PLC development and targeted treatment.

JNKs protect from cholestatic liver disease progression by modulating Apelin signalling.

Background & Aims: Cholestatic liver injury is associated with c-Jun N-terminal kinases (JNK) activation in distinct cell types. Its hepatocyte-specific function during cholestasis, however, has not yet been established. Therefore, in our present study, we investigated the role of JNK1/2 during cholestasis and dissected its hepatocyte-specific function. Methods: A cohort of patients with primary biliary cholangitis (n = 29) and primary sclerosing cholangitis (n = 37) was examined. Wild-type, hepatocyte-specific knockout mice for Jnk2 (Jnk2Δhepa) or Jnk1 and Jnk2 (Jnk1Δhepa/2Δhepa) were generated. Mice were subjected to bile duct ligation (BDL) or carbon tetrachloride (CCl4) treatment. Finally, Apelin signalling was blocked using a specific inhibitor. As an interventional approach, Jnk1/2 were silenced in wild-type mice using lipid nanoparticles for small interfering RNA delivery. Results: JNK activation was increased in liver specimens from patients with chronic cholestasis (primary biliary cholangitis and primary sclerosing cholangitis) and in livers of Mdr2-/- and BDL-treated animals. In Jnk1Δhepa/2Δhepa animals, serum transaminases increased after BDL, and liver histology demonstrated enhanced cell death, compensatory proliferation, hepatic fibrogenesis, and inflammation. Furthermore, microarray analysis revealed that hepatocytic Jnk1/2 ablation induces JNK-target genes involved in oxidative stress and Apelin signalling after BDL. Consequently, blocking Apelin signalling attenuated BDL-induced liver injury and fibrosis in Jnk1Δhepa/2Δhepa mice. Finally, we established an interventional small interfering RNA approach of selective Jnk1/2 targeting in hepatocytes in vivo, further demonstrating the essential protective role of Jnk1/2 during cholestasis. Conclusions: Jnk1 and Jnk2 work together to protect hepatocytes from cholestatic liver disease by controlling Apelin signalling. Dual modification of JNK signalling in hepatocytes is feasible, and enhancing its expression might be an attractive therapeutic approach for cholestatic liver disease. Impact and Implications: The cell-specific function of Jnk genes during cholestasis has not been explicitly explored. In this study, we showed that combined Jnk1/2, but not Jnk2 deficiency, in hepatocytes exacerbates liver damage and fibrosis by enhancing Apelin signalling, which contributes to cholestasis progression. Combined cell-specific Jnk targeting may be a new molecular strategy for treating cholestatic liver disease.

Genetic and pharmacological inhibition of XBP1 protects against APAP hepatotoxicity through the activation of autophagy.

Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1f/f) and hepatocyte-specific knockout Xbp1 mice (Xbp1∆hepa) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1-2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.

Fibrotic Events in the Progression of Cholestatic Liver Disease.

Cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are associated with active hepatic fibrogenesis, which can ultimately lead to the development of cirrhosis. However, the exact relationship between the development of liver fibrosis and the progression of cholestatic liver disease remains elusive. Periductular fibroblasts located around the bile ducts seem biologically different from hepatic stellate cells (HSCs). The fibrotic events in these clinical conditions appear to be related to complex crosstalk between immune/inflammatory mechanisms, cytokine signalling, and perturbed homeostasis between cholangiocytes and mesenchymal cells. Several animal models including bile duct ligation (BDL) and the Mdr2-knockout mice have improved our understanding of mechanisms underlying chronic cholestasis. In the present review, we aim to elucidate the mechanisms of fibrosis in order to help to identify potential diagnostic and therapeutic targets.

Activation of the Unfolded Protein Response (UPR) Is Associated with Cholangiocellular Injury, Fibrosis and Carcinogenesis in an Experimental Model of Fibropolycystic Liver Disease.

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 (Jnk1/2) knockout mice. Floxed JNK1/2 (Jnkf/f) and Jnk∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines (Tnf, Tgfß1) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk∆hepa compared with Jnkf/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk∆hepa animals compared with Jnkf/f littermates. Finally, thioacetamide (TAA) administration to Jnk∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl4-treated Jnk∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA.

Optimum O2:CH4 Ratio Promotes the Synergy between Aerobic Methanotrophs and Denitrifiers to Enhance Nitrogen Removal.

The O2:CH4 ratio significantly effects nitrogen removal in mixed cultures where aerobic methane oxidation is coupled with denitrification (AME-D). The goal of this study was to investigate nitrogen removal of the AME-D process at four different O2:CH4 ratios [0, 0.05, 0.25, and 1 (v/v)]. In batch tests, the highest denitrifying activity was observed when the O2:CH4 ratio was 0.25. At this ratio, the methanotrophs produced sufficient carbon sources for denitrifiers and the oxygen level did not inhibit nitrite removal. The results indicated that the synergy between methanotrophs and denitrifiers was significantly improved, thereby achieving a greater capacity of nitrogen removal. Based on thermodynamic and chemical analyses, methanol, butyrate, and formaldehyde could be the main trophic links of AME-D process in our study. Our research provides valuable information for improving the practical application of the AME-D systems.

Associations between serum HBX quasispecies and their integration in hepatocellular carcinoma.

HBV quasispecies are closely related to the course and outcome of liver disease. However, whether the complexity and diversity of HBX quasispecies affects its integration in the liver cell and thereby enhances the resultant carcinogenesis is still not clear. 15 HCC patients were recruited; genomic DNA and HBV DNA were extracted from liver cancer tissue and serum respectively. The integrated HBX fragment in liver cancer tissue was amplified by Alu repeat sequence-polymerase chain reaction (Alu-PCR) and sequenced. The serum HBX gene was amplified by nested PCR and sequenced. Quasispecies complexity and diversity, phylogenetic characteristics, lymphocyte count and survival time between HBX-integrated and HBX-unintegrated patients were evaluated. Results showed that the integrated HBX fragment was detected in the tumor tissue of nine patients, and the integration rate was 60.00% (9/15). Compared with the HBX-unintegrated patients, the HBX-integrated patients had a higher quasispecies complexity (P=0.028 and 0.004, at the nucleotide and amino acid levels, respectively). The HBX-integrated patients had a tendency of higher quasispecies diversity, lower lymphocyte count and the survival time. A total of 12 mutation sites were revealed in the HBX-integrated fragment after alignment with the reference sequence. In these, the HBX-integrated groups had significantly higher mutation frequencies at C1497T, A1630G, G1721A, A1762T/G1764A and A1774G. This study revealed influence factors of HBX integration both in virus and the host. The increased complexity and diversity of HBX quasispecies might destroy the host immune balance, and lead to HBX integration ultimately.