MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells.

Autoimmune hepatitis (AIH) is an inflammatory liver disease driven by the hyperactivation of various intrahepatic antigen-specific T cells due to a breach of immune tolerance. Studies in immunometabolism demonstrate that activated T cells harbor increased levels of reactive oxygen species that cause oxidative DNA damage. In this study, we assessed the potential of DNA damage repair enzyme MutT homolog 1 (MTH1) as a therapeutic target in AIH and karonudib as a novel drug for patients with AIH. We report herein that MTH1 expression was significantly increased in liver samples from patients with AIH compared to patients with chronic hepatitis B and nonalcoholic fatty liver disease and from healthy controls. In addition, the expression of MTH1 was positively correlated with AIH disease severity. We further found abundant T cells that expressed MTH1 in AIH. Next, we found that karonudib significantly altered T-cell receptor signaling in human T cells and robustly inhibited proliferation of human T cells in vitro. Interestingly, our data reflected a preferential inhibition of DNA damage repair in activated T cells by karonudib. Moreover, MTH1 was required to develop liver inflammation and damage because specific deletion of MTH1 in T cells ameliorated liver injury in the concanavalin A (Con A)-induced hepatitis model by inhibiting T-cell activation and proliferation. Lastly, we validated the protective effect of karonudib on the Con A-induced hepatitis model. Conclusion: MTH1 functions as a critical regulator in the development of AIH, and its inhibition in activated T cells reduces liver inflammation and damage.

Case Report: Response With Immunotherapy in a Patient With Mixed Neuroendocrine Non-Neuroendocrine Neoplasms of the Gallbladder.

BACKGROUND: Primary neuroendocrine tumors of the gallbladder (GB-NETs) are rare, accounting for 2% of all gallbladder cancers. Among GB-NETs, mixed neuroendocrine non-neuroendocrine neoplasms of the gallbladder (GB-MiNENs) are sporadic. CASE PRESENTATION: A 56-year-old woman admitted to our hospital due to right upper abdominal pain of 3 days duration. She underwent positron emission tomography/computed tomography, which showed multiple metastatic tumors and was unsuitable for operation. Initially, the patient was diagnosed with gallbladder adenocarcinoma. She underwent PD-1 inhibitor or combined with chemotherapy considering the PD-L1 high positive score. In the latter, the patient has the opportunity of surgery, and the new diagnosis was MiNENs. She achieved long-term disease control and has been alive from the first diagnosis. CONCLUSION: This case might support the strategy that PD-1 inhibitor could provide a feasible treatment option for MiNENs of gallbladder patients with the positive expression of PD-L1 in the future.

XRCC2 repairs mitochondrial DNA damage and fuels malignant behavior in hepatocellular carcinoma.

The effects of DNA damage repair (DDR) and mitochondrial dysfunction associated with HCC have been investigated, but the functional role of mitochondrial DDR in HCC remains elusive. We studied the DDR genes and identified XRCC2 as a potential prognostic marker for HCC. XRCC2 overexpression was detected in HCC cells and shown to promote the malignant behavior of cancer cells. XRCC2 depletion in HCC cells led to DNA damage accumulation at the replication site in the nucleus. Additionally, XRCC2-depleted HCC cells exhibited impaired mitochondrial respiration and reduced complex I (CI) activity as XRCC2 was responsible for elimination of mitochondrial DNA (mtDNA) damage and maintenance of mtDNA-encoded CI-related genes' transcription in a RAD51-dependent manner. We showed that tunicamycin (Tm)-activated sXBP1 bound to the TGTCAT domain and suppressed XRCC2 expression. In HCC patients, we observed a negative correlation between XBP1 and XRCC2 expression. Moreover, XRCC2 inhibition by Tm led to genomic and mtDNA damage, which impaired the transcription of mtDNA-encoded CI-related genes and prevented tumor proliferation in vivo. We described the role of XRCC2 in mtDNA damage repair and HCC progression while unveiling the potential anti-tumor effect of Tm.

Karonudib is a promising anticancer therapy in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is generally caused by viral infections or consumption of mutagens, such as alcohol. While liver transplantation and hepatectomy is curative for some patients, many relapse into disease with few treatment options such as tyrosine kinase inhibitors, for example, sorafenib or lenvatinib. The need for novel systemic treatment approaches is urgent. METHODS: MTH1 expression profile was first analyzed in a HCC database and MTH1 mRNA/protein level was determined in resected HCC and paired paracancerous tissues with polymerase chain reaction (PCR) and immunohistochemistry. HCC cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA. 8-oxoG was measured by the modified comet assay. The effect of MTH1 inhibition on tumor growth was explored in HCC xenograft in vivo models. RESULTS: MTH1 protein level is elevated in HCC tissue compared with paracancerous liver tissue and indicates poor prognosis. The MTH1 inhibitor Karonudib (TH1579) and siRNA effectively introduce toxic oxidized nucleotides into DNA, 8-oxoG, and kill HCC cell lines in vitro. Furthermore, we demonstrate that HCC growth in a xenograft mouse model in vivo is efficiently suppressed by Karonudib. CONCLUSION: Altogether, these data suggest HCC relies on MTH1 for survival, which can be targeted and may open up a novel treatment option for HCC in the future.

Inhibition of glycogen synthase kinase 3ß protects liver against ischemia/reperfusion injury by activating 5' adenosine monophosphate-activated protein kinase-mediated autophagy.

AIM: Autophagy has been found to play an important role in hepatic ischemia/reperfusion (I/R) injury. Our previous study has also clarified that rictor deficiency aggravated hepatic I/R injury by suppressing autophagy. Here, we explore whether autophagy participates in glycogen synthase kinase 3ß (GSK3ß)-mediated cytoprotection in liver I/R. METHODS: Mice were treated with SB216763 to inhibit GSK3ß before being subjected to hepatic I/R. Liver injury was evaluated by liver and blood samples. Autophagy was measured by detecting expression of microtubule-associated protein 1 light chain-3B (LC3B) II and autophagy protein 5 (ATG-5), as well as the number of autophagosomes by transmission electron microscope. Primary hepatocytes pretreated with SB216763 for 2 h were subjected to hypoxia/reoxygenation to induce autophagy. The lactate dehydrogenase level was used to evaluate cell death and survival. Autophagy inhibitors and 5' adenosine monophosphate-activated protein kinase (AMPK) inhibitor were given in vivo or in vitro. RESULTS: SB216763 significantly increased the number of autophagosomes and the protein levels of LC3B II and ATG-5 in liver I/R models, which was accompanied by a decline of hepatic necrosis and apoptosis. Consistent with the in vivo study, autophagy and cytoprotection were induced by the inhibition of GSK3ß in the in vitro study. Moreover, pretreatment with autophagy inhibitors attenuated the cytoprotective role of autophagy in the GSK3ß-treated liver I/R models. Further analysis showed that pretreatment with an AMPK inhibitor increased mammalian target of rapamycin (mTOR) activity, decreased autophagy, and abrogated GSK3ß- mediated liver protection. CONCLUSION: Autophagy was induced by GSK3ß inhibition through the AMPK/mTOR pathway and could substantially ameliorate liver I/R injury. Therefore, our findings strongly renew the therapeutic value of the GSK3ß/autophagy axis in hepatic I/R injury.

Rictor Deficiency Aggravates Hepatic Ischemia/Reperfusion Injury in Mice by Suppressing Autophagy and Regulating MAPK Signaling.

BACKGROUND/AIMS: The role of Rictor in hepatic ischemia/reperfusion (I/R) injury remains unknown. Here, we comprehensively examined the role of Rictor in hepatic I/R injury. METHODS: We studied the expression of Rictor during hepatic I/R injury. The regulatory effects of Rictor on inflammatory responses, cytokine and chemokine release, apoptotic and anti-apoptotic responses, and autophagy induction during hepatic I/R injury were identified via the shRNA-mediated knockdown of Rictor. Subsequently, we collected the liver and blood samples of these mice to evaluate liver injury, mRNA and protein levels. Additionally, the signaling pathways induced by Rictor were investigated. Furthermore, the extent of activation of MAPKs in response to Rictor deficiency was investigated in lipopolysaccharide (LPS)-treated RAW264.7 cells. The mRNA expression levels were analyzed by real-time PCR, and the protein expression levels were analyzed using Western blot, immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of Rictor was increased during hepatic I/R injury in vivo and hypoxia/reoxygenation (H/R) injury in vitro. Rictor deficiency enhanced the extent of liver injury by increasing macrophage and neutrophil infiltration, promoting cytokine and chemokine release, aggravating hepatocyte apoptosis, suppressing anti-apoptotic responses, and inhibiting autophagy induction during both hepatic I/R and H/R injury. Rictor was associated with the activation of hepatic I/R injury-induced MAPK signaling. In addition, Rictor deficiency affected MAPK activation in LPS-treated RAW264.7 cells. CONCLUSION: Rictor can substantially ameliorate I/R-induced liver injury. Therefore, our findings strongly suggest a therapeutic value of the Rictor/mTORC2 axis in hepatic I/R injury.

Inhibition of GSK-3ß induces AP-1-mediated osteopontin expression to promote cholestatic liver fibrosis.

The contribution of glycogen synthase kinase-3ß (GSK-3ß) to cholestatic liver disease (CLD) remains unknown. We investigated the role and mechanism of GSK-3ß in vivo in liver tissues of patients with CLD and the bile duct ligation (BDL) mouse model and in vitro using a hepatic progenitor cell (HPC) and hepatic stellate cell (HSC) coculture system. In liver tissues of patients with CLD, expression of the inactive form of GSK-3ß, phospho-GSK-3ß(Ser9), was increased in HPCs. GSK-3ß inhibition by SB216763 treatment aggravated liver fibrosis and elevated the expression of osteopontin (OPN) in the BDL mouse model. OPN was significantly overexpressed in liver tissues and serum from patients with CLD. In an HPC and HSC coculture system, inhibition of GSK-3ß induced OPN production, which activated HSCs in a cholestatic environment. The expression of activator protein 1 (AP-1), an important downstream transcription factor of GSK-3ß, was significantly increased in liver tissues of patients with CLD and SB216763-treated BDL mice. Finally, OPN expression was directly modulated by AP-1. These observations indicate that GSK-3ß inhibition up-regulates OPN expression via AP-1 activation, which accelerates the progression of cholestatic liver fibrosis in patients with CLD and in BDL mice.-Zhuang, S., Hua, X., He, K., Zhou, T., Zhang, J., Wu, H., Ma, X., Xia, Q., Zhang, J. Inhibition of GSK-3ß induces AP-1-mediated osteopontin expression to promote cholestatic liver fibrosis.

Hypoxia-inducible factor-2α promotes hepatocyte apoptosis during cholestasis.

AIM: Hypoxia-inducible factor-2α (HIF-2α) has been reported to play an important role in a host of pathophysiological processes, including cellular survival. This study explores the role of HIF-2α in cholestasis-mediated hepatocyte apoptosis. METHODS: Hypoxia-inducible factor-2α expression was measured by immunohistochemistry and confocal microscopy. Hepatic apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling. The cholestatic mouse model was treated with bile duct ligation. The c-myc, p53, and Bax protein levels were measured with Western blot analysis. RESULTS: In pediatric and murine cholestatic liver tissues, HIF-2α protein was widely expressed in the nucleus of parenchymal cells as well as in stromal cells. Hepatocyte HIF-2α expression was significantly elevated at the early stage of pediatric cholestasis and decreased at the late stage. In both in vivo and in vitro murine studies, HIF-2α deletion could alleviate cholestasis-mediated hepatocyte apoptosis and regulate the expression of c-myc, p53, and Bax proteins. CONCLUSION: These findings implied the contribution of HIF-2α to cholestasis-mediated hepatocyte apoptosis.

Contrast-enhanced micro-computed tomography using ExiTron nano6000 for assessment of liver injury.

AIM: To explore the potential of contrast-enhanced computed tomography (CECT) using ExiTron nano6000 for assessment of liver lesions in mouse models. METHODS: Three mouse models of liver lesions were used: bile duct ligation (BDL), lipopolysaccharide (LPS)/D-galactosamine (D-GalN), and alcohol. After injection with the contrast agent ExiTron nano6000, the mice were scanned with micro-CT. Liver lesions were evaluated using CECT images, hematoxylin and eosin staining, and serum aminotransferase levels. Macrophage distribution in the injury models was shown by immunohistochemical staining of CD68. The in vitro studies measured the densities of RAW264.7 under different conditions by CECT. RESULTS: In the in vitro studies, CECT provided specific and strong contrast enhancement of liver in mice. CECT could present heterogeneous images and densities of injured livers induced by BDL, LPS/D-GalN, and alcohol. The liver histology and immunochemistry of CD68 demonstrated that both dilated biliary tracts and necrosis in the injured livers could lead to the heterogeneous distribution of macrophages. The in vitro study showed that the RAW264.7 cell masses had higher densities after LPS activation. CONCLUSION: Micro-CT with the contrast agent ExiTron nano6000 is feasible for detecting various liver lesions by emphasizing the heterogeneous textures and densities of CECT images.

Risk factors for new onset diabetes mellitus after liver transplantation: A meta-analysis.

AIM: To determine the risk factors for new-onset diabetes mellitus (NODM) after liver transplantation by conducting a systematic review and meta-analysis. METHODS: We electronically searched the databases of MEDLINE, EMBASE and the Cochrane Library from January 1980 to December 2013 to identify relevant studies reporting risk factors for NODM after liver transplantation. Two authors independently assessed the trials for inclusion and extracted the data. Discrepancies were resolved in consultation with a third reviewer. All statistical analyses were performed with the RevMan5.0 software (The Cochrane Collaboration, Oxford, United Kingdom). Pooled odds ratios (OR) or weighted mean differences (WMD) with 95% confidence intervals (CIs) were calculated using either a fixed effects or a random effects model, based on the presence (I (2) < 50%) or absence (I (2) > 50%) of significant heterogeneity. RESULTS: Twenty studies with 4580 patients were included in the meta-analysis, all of which were retrospective. The meta-analysis identified the following significant risk factors: hepatitis C virus (HCV) infection (OR = 2.68; 95%CI: 1.92-3.72); a family history of diabetes (OR = 1.69, 95%CI: 1.09-2.63, P < 0.00001); male gender (OR = 1.53; 95%CI: 1.24-1.90; P < 0.0001); impaired fasting glucose (IFG; OR = 3.27; 95%CI: 1.84-5.81; P < 0.0001); a family history of diabetes (OR = 1.69; 95%CI: 1.09-2.63; P = 0.02); use of tacrolimus (OR = 1.34; 95%CI: 1.03-1.76; P = 0.03) and body mass index (BMI)(WMD = 1.19, 95%CI: 0.69-1.68, P < 0.00001). Other factors, such as hepatitis B virus infection and alcoholism, were not found to be associated with the incidence of NODM. CONCLUSION: The study showed that HCV infection, IFG, a family history of diabetes, male gender, tacrolimus and BMI are risk factors for NODM after liver transplantation.

A Potential Profibrogenic Role of Biliary Epithelium-Derived Cardiotrophin-1 in Pediatric Cholestatic Liver Disease.

As a cytokine of the interleukin-6 family, cardiotrophin-1 (CT-1) has been shown to be an important endogenous protector in liver injury. Our study aimed to investigate the role of CT-1 in liver fibrosis in pediatric cholestatic liver disease (PCLD). CT-1 mRNA and protein expression levels were upregulated in PCLD liver biopsy tissues compared with controls. Immunohistochemistry and confocal microscopy of liver sections showed that CT-1 was predominantly expressed by biliary epithelium cells. Serum CT-1 was elevated significantly in the children with PCLD compared with controls. Serum CT-1 levels exhibited a moderate positive correlation with the Scheuer stage of hepatic fibrosis and serum TB levels and a weak correlation with serum ALP levels. In vitro analysis indicated that LX-2 cells preconditioned with CT-1 exhibited significant increments in proliferation and accumulation of extracellular matrix components, while also positively regulating the STAT3 and p38MAPK pathways. In conclusion, biliary epithelium-derived CT-1 may exert a profibrogenic potential in PCLD.