A Case of Wilson's Disease Suspected to Involve a Novel Genetic Mutation of ATP7B Gene, Requiring a Differential Diagnosis with Non-alcoholic Steatohepatitis.

A 19-year-old Japanese man was referred for a further evaluation of liver dysfunction. Despite the absence of symptoms or obesity, the liver biopsy results were consistent with non-alcoholic steatohepatitis. Subsequent investigations revealed low serum ceruloplasmin, increased urinary copper excretion, and a known mutation c.3809A>G (p.Asn1270Ser) in the copper-transporting enzyme P-type ATPase (ATP7B) gene, leading to a diagnosis of Wilson's disease. A previously unreported variant, i.e., c.3866A>T (p.Asp1289Val) was detected on the patient's other allele and was considered a novel mutation, classified as 'likely pathogenic' according to the American College of Medical Genetics guidelines.

Hepatocellular carcinoma in a transplanted donor liver and colon cancer developing in a patient with a complex background: A case report.

The development of tumors in livers transplanted from hepatitis B virus (HBV)-negative donors to patients with hepatitis B and cirrhosis is rare. The present study describes the case of a woman in her 60s who developed hepatocellular carcinoma (HCC) in her grafted liver, 19 years after transplantation, as well as a metachronous colorectal tumor. The pathological findings, including clinical, immunohistochemical and molecular results, are described in the present case report. The liver tumor was a conventional HCC and the colorectal tumor comprised a tubular adenocarcinoma. Immunohistochemistry of both tumors showed a loss of expression of mutL homolog 1 and postmeiotic segregation increased 2 in the tumor cells, confirming microsatellite instability-high (MSI-H) status. Furthermore, a molecular study detected the presence of genes located on the Y chromosome in the normal and tumor tissues of the liver, proving that the HCC occurred in the grafted liver. The present report also discusses that prolonged use of immunosuppressive drugs to prevent post-transplant rejection, poorly controlled diabetes mellitus and MSI-H may have contributed to the risk of tumor development.

Impact of Post-progression Survival on Outcomes of Lenvatinib Treatment for Unresectable Hepatocellular Carcinoma: A Systematic Review and Retrospective Cohort Study.

BACKGROUND/AIM: Lenvatinib is a tyrosine kinase inhibitor (TKI) more effective against hepatocellular carcinoma (HCC) than sorafenib, making lenvatinib a first-line treatment option for patients with unresectable HCC. In patients treated with sorafenib, post-progression survival (PPS) rather than progression-free survival (PFS) is essential for overall survival (OS). However, the importance of PPS for OS in patients treated with lenvatinib is uncertain, and optimal treatment after lenvatinib failure has not yet been established. PATIENTS AND METHODS: The present study investigated the correlations of PFS and PPS with OS in studies of HCC patients treated with lenvatinib by weighted linear regression analysis. Furthermore, the contribution of treatment regimens after lenvatinib failure to OS were evaluated in daily clinical practice. RESULTS: An analysis of 20 studies with 4,054 patients found that PPS had a stronger correlation with OS (r=0.869, p<0.001) than did PFS (r=0.505, p=0.007). Analysis of 79 patients with unresectable HCC treated with first-line lenvatinib showed that subsequent treatment was the most significant contributor to OS. Second-line sorafenib was administered to 25 patients, with late transition to third-line treatment being highest among patients who received second-line treatment. CONCLUSION: PPS contributes significantly to OS in HCC treatment with TKIs, with multi-sequential treatment being a key determinant of longer OS.

Direct-acting antivirals for hepatitis C virus-infected patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) in hepatitis C virus (HCV)-infected patients has a high risk of recurrence. Although eradication of HCV is expected to reduce this risk, the risk in patients with a history of HCC may be high after treatment with direct-acting antivirals (DAAs). AIM: To determine the risk factors for HCC recurrence in patients with HCV and a history of HCC. METHODS: The risk of HCC recurrence in patients with a history of HCC and/or of HCC occurrence in patients without a history of HCC after DAA therapy was retrospectively analyzed in 311 HCV patients treated at our institution and several neighboring hospitals. The frequency and predictors of HCC recurrence/ occurrence after DAA treatment were included in these analyses. The clinical course of HCC before and after DAA treatment was also evaluated. RESULTS: HCV patients with a history of HCC were older and had greater progression of liver fibrosis and diabetes than patients without a history of HCC. Median recurrence-free survival (RFS) was 1092 d in patients with a history of HCC, and post-DAA HCC recurrence/occurrence was observed in 29 patients (53.7%) with and 5 (1.9%) without a history of HCC over 6 years (P < 0.001). RFS in patients with a history of HCC did not differ significantly before and after DAA treatment. The frequency of HCC recurrence/occurrence in patients with a history of HCC was lower after than before DAA treatment. Multivariate analysis showed that the incidence rate of HCC recurrence/occurrence before DAA treatment was the only independent predictor of HCC recurrence/occurrence after DAA treatment. Liver function was well preserved and clinical course was good in patients with HCC recurrence/occurrence after DAA therapy. CONCLUSION: DAA therapy in patients infected with HCV is also effective in patients with a history of HCC. Curative treatment for HCC is desirable before DAA therapy. The frequency of HCC recurrence/occurrence before DAA therapy was associated with a significantly increased risk of HCC recurrence after DAA therapy. Careful observation after DAA therapy is required in patients with a history of HCC.

Chronic liver disease questionnaire to manage patients with chronic liver diseases.

BACKGROUND AND AIMS: Although patients with chronic liver disease (CLD) usually show few symptoms, they exhibit decreased health-related QOL (HRQOL) with occurrence complications including hepatocellular carcinoma (HCC). Health-related QOL is an important indicator in the management of CLD. The Chronic Liver Disease Questionnaire (CLDQ) was established as a tool for assessment of HRQOL. In this study, we evaluate its usefulness for the management of daily clinical practice. METHODS: Patients (N = 190, median age 70 years old) treated between 2016 and 2019 were registered and prospectively followed-up with annual CLDQ. Associations of liver function and development of factors for admission or death were evaluated. RESULTS: Of the 190 patients registered, median age 70 years old, 140 were Child-A, 121 were Fib-4 index >2.67 and showed 80 HCC. All 6 domains including Systemic Symptoms (SS) were negatively correlated with Child-Pugh score more than with albumin-bilirubin score and Fib-4 index. A hundred four admission events and 49 deaths were found during observation period, and median event-free survival was 34.3 months. Treatment for HCC was the most frequent cause of admission, and 37 liver-related deaths were found. Systemic Symptoms score 2 years after registration was decreased in both HCC- and non-HCC cohort. Systemic Symptoms decreased and SS < 4 might be predictive for event occurrence. CONCLUSIONS: CLDQ is useful to assess HRQOL in patients with CLD and is well correlated with liver function especially Child-Pugh. Chronic Liver Disease Questionnaire might be useful to predict the prognosis of CLD and can be a tool of management in clinical practice.

Endoscopic ultrasound-guided fine-needle biopsy for the diagnosis of gastric linitis plastica.

We report two cases of patients with gastric linitis plastica (GLP), in which the histopathological diagnosis was made by endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) using a Franseen-tip needle. Esophagogastroduodenoscopy findings showed mucosal swelling and poor distensibility of the gastric antrum. Abdominal computed tomography findings showed significant thickening of the gastric wall at the antrum. Conventional endoscopic and bite-on-bite biopsy were attempted but resulted in failure to diagnose the lesions. We performed EUS-FNB to obtain histopathological samples from a deeper site, which confirmed the diagnosis. We considered this method safe and effective for the diagnosis of GLP.

A Mesenchymal-Epithelial Transition Factor-Agonistic Antibody Accelerates Cirrhotic Liver Regeneration and Improves Mouse Survival Following Partial Hepatectomy.

Small-for-size syndrome (SFSS) is a common complication following partial liver transplantation and extended hepatectomy. SFSS is characterized by postoperative liver dysfunction caused by insufficient regenerative capacity and portal hyperperfusion and is more frequent in patients with preexisting liver disease. We explored the effect of the Mesenchymal-epithelial transition factor (MET)-agonistic antibody 71D6 on liver regeneration and functional recovery in a mouse model of SFSS. Male C57/BL6 mice were exposed to repeated carbon tetrachloride injections for 10 weeks and then randomized into 2 arms receiving 3 mg/kg 71D6 or a control immunoglobulin G (IgG). At 2 days after the randomization, the mice were subjected to 70% hepatectomy. Mouse survival was recorded up to 28 days after hepatectomy. Satellite animals were euthanized at different time points to analyze liver regeneration, fibrosis, and inflammation. Serum 71D6 administration significantly decreased mouse mortality consequent to insufficient regeneration of the cirrhotic liver. Analysis of liver specimens in satellite animals revealed that 71D6 promoted powerful activation of the extracellular signal-regulated kinase pathway and accelerated liver regeneration, characterized by increased liver-to-body weight, augmented mitotic index, and higher serum albumin levels. Moreover, 71D6 accelerated the resolution of hepatic fibrosis as measured by picrosirius red, desmin, and α-smooth muscle actin staining, and suppressed liver infiltration by macrophages as measured by CD68 and F4/80 staining. Analysis of gene expression by reverse-transcription polymerase chain reaction confirmed that 71D6 administration suppressed the expression of key profibrotic genes, including platelet-derived growth factor, tissue inhibitor of metalloproteinase 3, and transforming growth factor-ß1, and of key proinflammatory genes, including tumor necrosis factor-α, interleukin-1ß, chemokine (C-C motif) ligand 3, and chemokine (C-C motif) ligand 5. These results suggest that activating the MET pathway via an hepatocyte growth factor-mimetic antibody may be beneficial in patients with SFSS and possibly other types of acute and chronic liver disorders.

Discrepant Diagnostic Results of Nested Polymerase Chain Reaction-based Genotyping in a Patient with Hepatitis C Virus and Human Immunodeficiency Virus Coinfection.

Accurate genotyping is important to improve the treatment of hepatitis C virus (HCV) infection. We herein report a 44-year-old Japanese man with hemophilia A and coinfection of HCV and human immunodeficiency virus (HIV) who was diagnosed with HCV genotype 4 by direct sequencing. Two genotyping tests based on the nested polymerase chain reaction method that we used misdiagnosed his genotype as 2b and 1b. Although several HCV genotyping tests are available in Japan, it is important to recognize that some cannot detect genotype 4. Care should be taken when genotyping HCV patients who have received non-heated coagulation factor preparations or were infected abroad.

Hydrogen-rich water protects against liver injury in nonalcoholic steatohepatitis through HO-1 enhancement via IL-10 and Sirt 1 signaling.

Nonalcoholic steatohepatitis (NASH) could progress to hepatic fibrosis in the absence of effective control. The purpose of our experiment was to investigate the protective effect of drinking water with a high concentration of hydrogen, namely, hydrogen-rich water (HRW), on mice with nonalcoholic fatty liver disease to elucidate the mechanism underlying the therapeutic action of molecular hydrogen. The choline-supplemented, l-amino acid-defined (CSAA) or the choline-deficient, l-amino acid-defined (CDAA) diet for 20 wk was used to induce NASH and fibrosis in the mice model and simultaneously treated with the high-concentration 7-ppm HRW for different periods (4 wk, 8 wk, and 20 wk). Primary hepatocytes were stimulated by palmitate to mimic liver lipid metabolism during fatty liver formation. Primary hepatocytes were cultured in a closed vessel filled with 21% O2 + 5% CO2 + 3.8% H2 and N2 as the base gas to verify the response of primary hepatocytes in a high concentration of hydrogen gas in vitro. Mice in the CSAA + HRW group had lower serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and milder histological damage. The inflammatory cytokines were expressed at lower levels in the HRW group than in the CSAA group. Importantly, HRW reversed hepatocyte fatty acid oxidation and lipogenesis as well as hepatic inflammation and fibrosis in preexisting hepatic fibrosis specimens. Molecular hydrogen inhibits the lipopolysaccharide-induced production of inflammation cytokines through increasing heme oxygenase-1 (HO-1) expression. Furthermore, HRW improved hepatic steatosis in the CSAA + HRW group. Sirtuin 1 (Sirt1) induction by molecular hydrogen via the HO-1/adenosine monophosphate activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα)/peroxisome proliferator-activated receptor γ (PPAR-γ) pathway suppresses palmitate-mediated abnormal fat metabolism. Orally administered HRW suppressed steatosis induced by CSAA and attenuated fibrosis induced by CDAA, possibly by reducing oxidative stress and the inflammation response.NEW & NOTEWORTHY The mRNA expression of inflammatory cytokines in the HRW group was lower than in the CSAA group. HRW reversed hepatocyte apoptosis as well as hepatic inflammation and fibrosis in NASH specimens. Molecular hydrogen inhibits LPS-induced inflammation via an HO-1/interleukin 10 (IL-10)-independent pathway. HRW improved hepatic steatosis in the CSAA + HRW group. Sirt1 induction by molecular hydrogen via the HO-1/AMPK/PPARα/PPARγ pathway suppresses palmitate-mediated abnormal fat metabolism.

Diagnostic Fine-Needle Biopsy of Small Solid Pancreatic Lesions Using a Franseen Needle during Endoscopic Ultrasound Examination.

BACKGROUND AND AIM: During endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNB), Franseen needles can help collect sufficient tissue to permit histopathological assessment. However, its efficacy might be limited by the size of the targeted lesion. This study aimed to evaluate the feasibility of histopathological assessment of small solid pancreatic lesions using a 22-gauge Franseen needle during EUS-FNB. METHODS: This retrospective study evaluated data from all patients who underwent EUS-FNB using a Franseen needle for solid pancreatic lesions at the University of Toyama Hospital between June 2018 and April 2020. RESULTS: The study included 159 patients who had 152 malignant lesions and 7 benign lesions. The malignant lesions included pancreatic cancers (n = 134), neuroendocrine neoplasms (n = 15), metastatic tumors (n = 2), and a solid pseudopapillary neoplasm (n = 1). The diagnostic accuracy of EUS-FNB (combining histology and cytology) was 98.7%. However, the histopathological diagnosis was only confirmed for 64.3% of small lesions (<10 mm), relative to 97.2% for larger lesions. Multivariate analysis also revealed that lesion size of <10 mm predicted a less accurate histopathological diagnosis (odds ratio: 6.97, 95% confidence interval: 1.02-47.67; p = 0.041). Further analyses revealed a failed histological diagnosis in 4 patients with lesions of <5 mm in size and accurate diagnoses in 9 out of 10 patients with lesions of 5-10 mm in size. CONCLUSIONS: The diagnostic accuracy for small lesions (<10 mm), especially for lesions of <5 mm, based on histological examination alone, was significantly lower than that for others (>10 mm). Furthermore, multivariate analysis revealed that only lesion size was an independent predictor of histopathological diagnosis accuracy.

Efficacy of on-demand intrahepatic arterial therapy in combination with sorafenib for advanced hepatocellular carcinoma.

PURPOSE: The purpose of this study was to evaluate the effectiveness and tolerability of "on-demand" combination therapy with sorafenib and hepatic arterial treatments, such as transarterial chemoembolization and hepatic arterial infusion chemotherapy, in patients with hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Eighty consecutive patients with advanced HCC, 58 administered sorafenib monotherapy and 22 administered on-demand combination therapy, were retrospectively evaluated. RESULTS: The disease control rate was significantly higher in the combination group than in the monotherapy group (86.3% vs 51.7%, p=0.01). Elevated alanine aminotransferase levels were significantly more frequent in the combination group (40.9% vs 12.1%, p=0.01), but it was tolerable. Progression-free survival (180 vs 45 days, p=0.045) and overall survival (983 vs 452 days, p=0.004) were significantly longer in the combination group, as was the duration of sorafenib treatment (367 vs 66 days, p<0.001). Multivariate analysis showed that hepatitis C virus infection, disease control, and combination therapy were positive independent prognostic factors for survival, whereas alpha-fetoprotein >400 ng/mL was negatively prognostic. In patients receiving combination therapy, male sex, hepatitis B virus infection, performance status deterioration, Barcelona clinic liver cancer-B, and major vascular invasion were prognostic of survival. CONCLUSION: On-demand combination therapy was tolerated and may be a therapeutic option for patients with advanced HCC.

Combined hepatocellular-cholangiocarcinoma successfully treated with sorafenib: case report and review of the literature.

Sorafenib, a multiple kinase inhibitor, has been established as first-line standard systemic chemotherapy for patients with advanced hepatocellular carcinoma (HCC). We encountered a patient with combined hepatocellular and cholangiocarcinoma (CHC) who achieved complete remission in response to sorafenib treatment. A 58-year old man with hepatitis C virus (HCV)-induced liver cirrhosis was diagnosed with CHC in segments 6th and 7th of the liver and underwent partial surgical resection. Three months later, CHC recurred as metastases at multiple intrahepatic sites, lymph nodes, and bones, making surgery impossible. Treatment with sorafenib was initiated at 400 mg b.i.d., later reduced to 400 mg/day. After 6 months of sorafenib administration, he no longer showed abnormal uptake on fluorodeoxyglucose positron emission tomography. He was continued on sorafenib for 2.5 years, but later discontinued due to adverse events. He has shown no evidence of tumor recurrence more than 1 year after sorafenib discontinuation. His HCV was eradicated by direct-acting antivirals, and he remains in good health.

Survival Benefit of Tolvaptan for Refractory Ascites in Patients with Advanced Cirrhosis.

AIMS: The study aimed to evaluate the effects of tolvaptan treatment on survival of patients with decompensated liver cirrhosis with refractory ascites. METHODS: This multicenter, retrospective, observational study included patients with cirrhosis who were treated with tolvaptan for hepatic ascites refractory to conventional diuretics. Patients who could and could not decrease accompanying diuretics within 1 month after tolvaptan administration were defined as the "Decreased" and "Not-decreased" groups, respectively. RESULTS: Median body weight change 1 week after tolvaptan treatment was -1.95 kg, with the 50% of patients experiencing a 2 kg/week reduction. Spot urinary sodium was found to be a better predictor of tolvaptan response than liver function and liver fibrosis markers. Median survival was significantly longer (not reached versus 116 days, p = 0.005) and serum creatinine concentrations 12 weeks after tolvaptan administration significantly lower (0.99 vs. 1.55 mg/dL, p < 0.05) in the Decreased than in the Not-decreased group. Multivariate analysis showed that the presence of viable hepatocellular carcinoma (hazards ratio [HR] 2.14, p = 0.02) and a decrease in diuretics were independently prognostic of survival (HR 0.36, p < 0.01). CONCLUSIONS: The maintenance of renal function is essential in enhancing survival of patients with cirrhosis. Doses of diuretics should be adjusted appropriately during tolvaptan treatment.

A Patient with Non-alcoholic Steatohepatitis Complicated by Multiple Myeloma.

A 68-year-old woman with liver dysfunction was diagnosed with nonalcoholic steatohepatitis (NASH) stage 1. Three years later, she showed massive ascites and jaundice. A trans-jugular liver biopsy confirmed advanced cirrhosis, suggesting that her liver fibrosis had progressed rapidly. At the same time, she was diagnosed with multiple myeloma (MM). In this case, the plasma levels of osteopontin (OPN), a proinflammatory cytokine that promotes liver fibrosis progression through the hedgehog pathway and is increased in patients with MM, were increased. This increased OPN expression was accompanied by the upregulation of the hedgehog pathway in this patient, suggesting that the MM-associated increase in OPN had promoted the progression of liver fibrosis through the hedgehog pathway. The progression of liver fibrosis should be monitored in patients with NASH if other diseases, such as MM, are present.

Astaxanthin prevents ischemia-reperfusion injury of the steatotic liver in mice.

Steatosis has a low tolerance against ischemia-reperfusion injury (IRI). To prevent IRI in the steatotic liver, we attempted to elucidate the protective effect of astaxanthin (ASTX) in the steatotic liver model by giving mice a methionine and choline-deficient high fat (MCDHF) diet. Levels of lipid peroxidation and apoptosis, the expression of inflammatory cytokines and heme oxygenase (HO)-1, in the liver were assessed. Reactive oxygen species (ROS), inflammatory cytokines, apoptosis-related proteins and members of the signaling pathway were also examined in isolated Kupffer cells and/or hepatocytes from the steatotic liver. ASTX decreased serum ALT and AST levels, the amount of TUNEL, F4/80, or 4HNE-positive cells and the mRNA levels of inflammatory cytokines in MCDHF mice by IRI. Moreover, HO-1 and HIF-1α, phosphorylation of Akt and mTOR expressions were increased by ASTX. The inflammatory cytokines produced by Kupffer, which were subjected to hypoxia and reoxygenation (HR), were inhibited by ASTX. Expressions of Bcl-2, HO-1 and Nrf2 in hepatocytes by HR were increased, whereas Caspases activation, Bax and phosphorylation of ERK, MAPK, and JNK were suppressed by ASTX. Pretreatment with ASTX has a protective effect and is a safe therapeutic treatment for IRI, including for liver transplantation of the steatotic liver.

Neutrophil-to-lymphocyte ratio predicts recurrence after radiofrequency ablation in hepatitis B virus infection.

BACKGROUND AND AIM: Radiofrequency ablation (RFA) is an established treatment for small hepatocellular carcinoma (HCC) wherein non-recurrence is essential for long-term survival. Recently, neutrophil-to-lymphocyte ratio (NLR), a marker of systemic inflammation that is associated with tumor-associated macrophages (TAMs), was suggested to be a prognostic marker of HCC treated with RFA. Therefore, we evaluated predictive factors, including NLR, associated with recurrence after curative RFA. METHODS: A total of 163 patients initially diagnosed with HCC and treated with RFA were enrolled. We retrospectively analyzed factors associated with recurrence and survival after RFA. Furthermore, TAMs were evaluated using surgically resected specimens. RESULTS: Hepatitis C virus (HCV) infection was the most frequent cause of HCC in this population (111 cases, 68.1%), whereas hepatitis B virus (HBV) infection accounted for 26 cases (16.0%). Recurrence, mostly intrahepatic distant recurrence, was found in 101 cases (61.9%). Recurrence and posttreatment NLR were independent prognostic factors related to survival, and male sex, HCV infection, serum des-γ-carboxy prothrombin >  40 AU/L, and posttreatment NLR were associated with recurrence. Pretreatment NLR showed no association with recurrence, whereas posttreatment NLR showed prognostic value. Interestingly, pretreatment NLR >  2.5 was significantly associated with recurrence in HBV-HCC patients (odds ratio 3.439, P = 0.037) not but HCV-HCC (odds ratio 1.430, P = 0.17). Furthermore, TAMs were increased in the peripheral area of HCCs with HBV infection compared with those with HCV. CONCLUSIONS: Recurrence of HCC after RFA was strongly associated with survival. NLR is useful as a predictive marker of recurrence, especially in HBV-HCC patients.

5-Aminolevulinic acid combined with ferrous iron ameliorate ischemia-reperfusion injury in the mouse fatty liver model.

BACKGROUND: The fatty liver could increase the risk of serious acute ischemia reperfusion (I/R) injury, and hepatic steatosis is indeed a major risk factor for hepatic failure after grafting a fatty liver. MATERIALS & METHODS: Fatty liver models of methionine- and choline-deficient high-fat mice were subjected to I/R injury with or without 5-aminolevulinic acid (5-ALA)/sodium ferrous citrate (SFC) treatment. Levels of hepatic enzymes, lipid peroxidation and apoptosis, inflammatory cytokines and heme oxygenase (HO)-1, and the carbon monoxide (CO) in the liver, and reactive oxygen species (ROS), inflammatory cytokines and members of the signaling pathway in isolated Kupffer were assessed. RESULTS: Alanine aminotransferase and aspartate aminotransferase levels, the number of necrotic areas, thiobarbituric acid reactive substance content, TUNEL-positive cells, infiltrated macrophages, and the inflammatory cytokine expression after I/R injury were dramatically decreased, whereas the endogenous CO concentrations and the HO-1 expression were significantly increased by 5-ALA/SFC treatment. The expression of toll-like receptors 2 and 4, NF-κB and inflammatory cytokines and ROS production in Kupffer cells were significantly decreased with 5-ALA/SFC treatment. CONCLUSION: 5-ALA/SFC significantly attenuates the injury level in the fatty liver after I/R injury.

Dihydroquercetin (DHQ) ameliorated concanavalin A-induced mouse experimental fulminant hepatitis and enhanced HO-1 expression through MAPK/Nrf2 antioxidant pathway in RAW cells.

Autoimmune hepatitis represents a ubiquitous human health problem and has a poor prognosis. Dihydroquercetin (DHQ), a well-known antioxidant, significantly inhibits fulminant hepatitis through anti-oxidant and anti-inflammation mechanisms. In this study, we show that administration of DHQ ameliorated concanavalin A (ConA)-induced mouse liver injury by increasing the survival rate, reducing the serum ALT and AST level, preventing histopathological injuries and decreasing pro-inflammatory cytokine mRNA expression in hepatic tissue. As macrophages/Kupffer cells in oxidative stress and pro-inflammatory mediators play an important role in the pathogenesis of immune-mediated hepatitis, we further exposed mouse RAW264 macrophage cell lines to ConA in vitro and found that DHQ significantly inhibited mRNA expression and secretion of IFN-γ and TNF-α in cell culture supernatant. In addition, DHQ significantly enhanced heme oxygenase-1 (HO-1) expression in a dose- and time-dependent manner via increased Nrf2 expression in cytoplasm and nuclear translocation. Furthermore, DHQ enhanced phosphorylation of three members of the mitogen-activated protein kinase (MAPK) family, and cell treatment with MEK/ERK (PD98059), p38 (SB203580) and JNK (SP600125) inhibitors reduced DHQ-induced HO-1 expression. These results indicate that DHQ possesses hepatoprotective properties against ConA-induced liver injury, which are attributed to its ability to scavenge oxidative stress and to inhibit the release of inflammatory mediators via upregulation of HO-1 activity through the MAPK/Nrf2 signaling pathway in macrophages/Kupffer cells.

IFN-γ deficiency attenuates hepatic inflammation and fibrosis in a steatohepatitis model induced by a methionine- and choline-deficient high-fat diet.

Cytokines play important roles in all stages of steatohepatitis, including hepatocyte injury, the inflammatory response, and the altered function of sinusoidal cells. This study examined the involvement of a major inflammatory cytokine, interferon-γ (IFN-γ), in the progression of steatohepatitis. In a steatohepatitis model by feeding a methionine- and choline-deficient high-fat (MCDHF) diet to both wild-type and IFN-γ-deficient mice, the liver histology, expression of genes encoding inflammatory cytokines, and fibrosis-related markers were examined. To analyze the effects of IFN-γ on Kupffer cells in vitro, we examined the tumor necrosis factor-α (TNF-α) production by a mouse macrophage cell line. Forty two days of MCDHF diet resulted in weight loss, elevated aminotransferases, liver steatosis, and inflammation in wild-type mice. However, the IFN-γ-deficient mice exhibited less extensive changes. RT-PCR revealed that the expression of tumor necrosis factor-α (TNF-α), transforming growth factor-ß, inducible nitric oxide synthase, interleukin-4 and osteopontin were increased in wild-type mice, although they were suppressed in IFN-γ-deficient mice. Seventy days of MCDHF diet induced much more liver fibrosis in wild-type mice than in IFN-γ-deficient mice. The expression levels of fibrosis-related genes, α-smooth muscle actin, type I collagen, tissue inhibitor of matrix metalloproteinase-1, and matrix metalloproteinase-2, were dramatically increased in wild-type mice, whereas they were significantly suppressed in IFN-γ-deficient mice. Moreover, in vitro experiments showed that, when RAW 264.7 macrophages were treated with IFN-γ, they produced TNF-α in a dose-dependent manner. The present study showed that IFN-γ deficiency might inhibit the inflammatory response of macrophages cells and subsequently suppress stellate cell activation and liver fibrosis. These findings highlight the critical role of IFN-γ in the progression of steatohepatitis.

Matrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in the injured liver.

Effective mobilization of hematopoietic stem cells (HSCs) in injured organs has not been established. Matrix metalloproteinase-9 (MMP-9) is known to release HSCs from bone marrow (BM) into the peripheral blood, but its role in the recruitment of HSCs to injured organs is unclear. In this study we tried to clarify the role of the host MMP-9 in trafficking of HSCs toward the injured liver, especially the relation of MMP-9 with the chemokine receptor 4 (CXCR4)-chemokine ligand 12 (CXCL12) axis, and to examine whether MMP-9 deficiency affects BM cell trafficking to the injured liver in mice. In vitro, we investigated the effect of MMP-9 on migration activity and CXCR4 expression on lineage-negative (Lin(-)) BM cells. In vivo, we induced acute and chronic liver injury in MMP-9 knockout (KO) and control mice by inoculation of carbon tetrachloride, followed by transplantation of Lin(-) BM cells obtained from enhanced green fluorescent protein (EGFP)-transgenic mice, and counted the BM cells mobilized in the injured liver. In a migration assay, active MMP-9, but not proMMP-9, increased the number of migrated Lin(-) BM cells, which was inhibited by tissue inhibitor of metalloproteinase-1 or a MMP inhibitor. This chemoattractant function by MMP-9 was synergistic when cotreated with CXCL12. CXCR4 expression on Lin(-) BM cells was dose- and time-dependently increased by active MMP-9. At the same time, treatment with MMP-9 enhanced CXCL12 expression, and CXCL12 reciprocally increased MMP-9 expression in BM cells. In in vivo studies, many EGFP(+) cells were seen in control recipient mice. In contrast, few EGFP(+) cells were observed in MMP-9 KO mice. BM cells tended to differentiate into desmin(+) cells. In conclusion, MMP-9 contributes to the mobilization of BM cells in the injured liver by upregulating the expression of CXCR4 on Lin(-) BM cells and attracting BM cells along its gradient of CXCL12. Therefore, host MMP-9 plays an important role in BM cell migration in the injured liver.