3,3′-Diindolylmethane rapidly and selectively inhibits hepatocyte growth factor/c-Met signaling in breast cancer cells

3,3′-Diindolylmethane (DIM), an indole derivative from vegetables of the Brassica genus, has antiproliferative activity in breast cancer cells. Part of this activity is thought to be due to DIM inhibition of Akt signaling, but an upstream mechanism of DIM-induced Akt inhibition has not been described. The goals of this study were to investigate the kinetics of inhibition of Akt by physiologically relevant concentrations of DIM and to identify an upstream factor that mediates this effect. Here we report that DIM (5–25 μM) inhibited Akt activation from 30 min to 24 h in tumorigenic MDA-MB-231 cells but did not inhibit Akt activation in non-tumorigenic preneoplastic MCF10AT cells. DIM inhibited hepatocyte growth factor (HGF)-induced Akt activation by up to 46%, cell migration by 66% and cell proliferation by up to 54%, but did not inhibit induction of Akt by epidermal growth factor or insulin-like growth factor-1. DIM decreased phosphorylation of the HGF receptor, c-Met, at tyrosines 1234 and 1235, indicating decreased activation of the receptor. This decrease was reversed by pretreatment with inhibitors of p38 or calcineurin. Our results demonstrate the important role of HGF and c-Met in DIM's anti-proliferative effect on breast cancer cells and suggest that DIM could have preventive or clinical value as an inhibitor of c-Met signaling.     

18F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model

Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-¹⁸F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide (¹⁸F-FBHGal), a new ¹⁸F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P <0.0001) was observed using confocal microscopy when co-incubated with 0.5 μM of asialofetuin, a well known ASGPR blocking agent. Animal studies showed the accumulation of ¹⁸F-FBHGal in fibrosis liver (14.84 ± 1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50 ± 1.51 %ID/g, P <0.01) at 30 min post-injection. The receptor indexes (liver/liver-plus-heart ratio at 30 min post-injection) of hepatic fibrosis mice derived from both microPET imaging and biodistribution study were significantly lower (P <0.01) than those of normal mice. The pharmacokinetic parameters (T_1/2α, T_1/2β, AUC and Cl) derived from microPET images revealed prolonged systemic circulation of ¹⁸F-FBHGal in hepatic fibrosis mice compared to that in normal mice. The findings in biological characterizations suggest that ¹⁸F-FBHGal is a feasible agent for PET imaging of hepatic fibrosis in mice and may provide new insights into ASGPR-related liver dysfunction.     

The significance of CD14+ monocytes in peripheral blood stem cells for the treatment of rat liver cirrhosis

Background aimsCirculating monocytes have been exploited as an important progenitor cell resource for hepatocytes in vitro and are instrumental in the removal of fibrosis. We investigated the significance of monocytes in peripheral blood stem cells (PBSC) for the treatment of liver cirrhosis.MethodsRat CD14⁺ monocytes in PBSC were mobilized with granulocyte-colony-stimulating factor (G-CSF) and harvested by magnetic cell sorting (MACS). Female rats with carbon tetrachloride (CCl₄)-induced liver cirrhosis were injected CM-DiI-labeled monocytes, CD14^? cells (1 × 10⁷ cells/rat) or saline via the portal vein.ResultsRat CD14⁺ and CD11b⁺ monocytes in PBSC were partly positive for CD34, CD45, CD44, Oct3/4 and Sox2, suggesting monocytes with progenitor capacity. Compared with CD14^? cell-infused and saline-injected rats, rats undergoing monocyte transplantation showed a gradually increased serum albumin level and decreased portal vein pressure, resulting in a significantly improved survival rate. Meanwhile, monocyte transplantation apparently attenuated liver fibrosis by analysis for fibronectin, α2-(1)-procollagen, α-smooth muscle aorta (SMA) and transforming growth factor (TGF)-β. Transplanted monocytes mainly clustered in periportal areas of liver, in which 1.8% cells expressed hepatocyte marker albumin and CK18. The expression level of hepatocyte growth factor (HGF), TGF-α, extracellular matrix (EGF) and vascular endothelial growth factor (VEGF) increased, while monocyte transplantation enhanced hepatocyte proliferation. On the other hand, the activities and expression of matrix metalloproteinases (MMP) increased while tissue inhibitor of metalloproteinase (TIMP)-1 expression significantly reduced in monocyte-transplanted livers. Some transplanted monocytes expressed MMP-9 and -13.ConclusionsThe data suggest that CD14⁺ monocytes in PBSC contribute to hepatocyte regeneration and extracellular matrix (ECM) remodeling in rat liver cirrhosis much more than CD14^? cells, and might offer a therapeutic alternative for patients with liver cirrhosis.     

Design,synthesis and biological evaluation of 4-aminopyrimidine-5-cabaldehyde oximes as dual inhibitors of c-Met and VEGFR-2

The synergistically collaboration of c-Met/HGF and VEGFR-2/VEGF leads to development of tumor angiogenesis and progression of various human cancers. Therefore, inhibiting both HGF/c-Met and VEGF/VEGFR signaling may provide a novel and effective therapeutic approach for treating patients with abroad spectrum of tumors. Toward this goal, we designed and synthesized a series of derivatives bearing 4-aminopyrimidine-5-cabaldehyde oxime scaffold as potent dual inhibitors of c-Met and VEGFR-2. The cell proliferation assay in vitro demonstrated most target compounds have inhibition potency both on c-Met and VEGFR-2 with IC₅₀ values in nanomolar range, especially compound 14i, 18a and 18b. Based on the further enzyme assay in vitro, compound 18a was considered as the most potent one, the IC₅₀s of which were 210 nM and 170 nM for c-Met and VEGFR-2, respectively. Following that, we docked the compound 10 and 18a with the proteins c-Met and VEGFR-2, and interpreted the SAR of these analogs. All the results indicate that 18a is a dual inhibitors of c-Met and VEGFR-2 that holds promising potential.     

Quercetin in vesicular delivery systems: Evaluation in combating arsenic-induced acute liver toxicity associated gene expression in rat model

Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. The theme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbal polyphenolic antioxidant Quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosis associated upregulation of its gene expression and plasma TGF ß (transforming growth factor ß) in rat model.A single dose of Arsenic (sodium arsenite-NaAsO₂, 13 mg/kg b.wt) in oral route causes the generation of reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepatic plasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis associated elevation of its gene expression in liver, plasma TGF ß (from normal value 75.2 ± 8.67 ng/ml to 196.2 ± 12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery systems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 ml of nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1 h after arsenic administration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidative stress associated gene expression of liver. Histopathological examination also confirmed the pathological improvement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation in combating arsenic induced upregulation of gene expression of liver fibrosis through a complete protection against oxidative attack in hepatic cells of rat liver.     

PKC mediates fluctuant ERK-paxillin signaling for hepatocyte growth factor-induced migration of hepatoma cell HepG2

Hepatocyte growth factor (HGF) is critical for triggering metastasis of hepatocellular carcinoma cell (HCC). Extracellular signal-regulated kinase (ERK) mediates HGF-induced cell migration via focal adhesion signaling. Protein kinase C (PKC) is a negative regulator of ERK activation, however, both PKC and ERK were required for HGF-induced cell migration. To address this intriguing issue, the signal mechanisms for HGF-induced HepG2 cell migration were investigated in a long-term fashion. HGF-induced phosphorylations of ERK, Src (at Tyr 416) and paxillin (at Ser178 and Tyr31) were up and down for 3 times within 24 h. HGF also induced fluctuant PKC activation and Rac degradation. Consistently, HGF induced intermittent actin polarization within 24 h, which can be blocked by the inhibitors of PKC (Bisindolymaleimide) and ERK. Inhibitor studies revealed that ERK was required for HGF-induced paxillin phosphorylation at Ser178, whereas PKC and Rac-1 may suppress HGF-induced phosphorylation of ERK and paxillin (at Ser178) and upregulate phosphorylation of paxillin at Tyr31. Based on shRNA technique, PKCα and δ were responsible for suppressing HGF-induced phosphorylation of ERK and paxillin (at Ser178), whereas PKC ε and ζ were required for phosphorylation of paxillin at Tyr31. The HGF-induced fluctuant signaling is reminiscent of c-Met endocytosis. Using Concanavalin A, an inhibitor of endocytosis, we found that c-Met endocytosis was required for PKC to suppress ERK phosphorylation. Moreover, HGF-induced c-Met degradation was also fluctuant, which can be prevented by Bisindolymaleimide. In conclusion, PKC is critical for mediating HGF-induced fluctuant ERK-paxillin signaling during cell migration, probably via triggering endosomal degradation of c-Met.     

Met signaling in cardiomyocytes is required for normal cardiac function in adult mice

Hepatocyte growth factor (HGF) and its receptor, Met, are key determinants of distinct developmental processes. Although HGF exerts cardio-protective effects in a number of cardiac pathologies, it remains unknown whether HGF/Met signaling is essential for myocardial development and/or physiological function in adulthood. We therefore investigated the requirement of HGF/Met signaling in cardiomyocyte for embryonic and postnatal heart development and function by conditional inactivation of the Met receptor in cardiomyocytes using the Cre-α-MHC mouse line (referred to as α-MHCMet-KO). Although α-MHCMet-KO mice showed normal heart development and were viable and fertile, by 6 months of age, males developed cardiomyocyte hypertrophy, associated with interstitial fibrosis. A significant upregulation in markers of myocardial damage, such as β-MHC and ANF, was also observed. By the age of 9 months, α-MHCMet-KO males displayed systolic cardiac dysfunction. Mechanistically, we provide evidence of a severe imbalance in the antioxidant defenses in α-MHCMet-KO hearts involving a reduced expression and activity of catalase and superoxide dismutase, with consequent reactive oxygen species accumulation. Similar anomalies were observed in females, although with a slower kinetics. We also found that Met signaling down-regulation leads to an increase in TGF-β production and a decrease in p38MAPK activation, which may contribute to phenotypic alterations displayed in α-MHCMet-KO mice. Consistently, we show that HGF acts through p38α to upregulate antioxidant enzymes in cardiomyocytes. Our results highlight that HGF/Met signaling in cardiomyocytes plays a physiological cardio-protective role in adult mice by acting as an endogenous regulator of heart function through oxidative stress control.     

Necrotic cell death and suppression of T-cell immunity characterized acute liver failure due to drug-induced liver injury

Background & aims: The aim of this study was to investigate the clinical characteristics and pathophysiology of drug-induced liver injury (DILI) – acute liver failure (ALF). Methods: The patients with acute liver injury (ALI) including ALF from 2009 to 2014 were analyzed. The hepatic encephalopathy (HE) development rate was compared with the findings from a national survey in Japan. The serum cytokines levels and the findings of a liver function test were evaluated in the DILI patients. Results: The HE development rate substantially decreased for autoimmune hepatitis (AIH) – and undetermined cause-induced ALI owing to the early prediction system, but not in DILI-ALI. Among the DILI-ALF and AIH-ALF cases, the CK-18 fragment (1480.1 U/L, 3945.4 U/L), IL-8 (82.9 pg/mL, 207.5 pg/mL), IP-10 (1379.6 pg/mL, 3731.2 pg/mL) and MIP-1β (1017.7 pg/mL, 2273.3 pg/mL) levels were lower in the DILI-ALF cases. Among the DILI-ALI and DILI-ALF cases, IL-4 (19.8 pg/mL, 25.4 pg/mL) and RANTES (14028.0 pg/mL, 17804.7 pg/mL) were higher in DILI-ALI, and HMGB-1 (397.1 pg/μL, 326.2 pg/μL) and HGF (2.41 ng/mL, 0.55 ng/mL) were higher in DILI-ALF. We observed that HGF independently associated with DLI-ALF development. Conclusions: Despite the low grade apoptosis and inflammation, DILI patients progressed to ALF comparable with that of the AIH patients.     

Bombesin and neurotensin exert antiproliferative effects on oval cells and augment the regenerative response of the cholestatic rat liver

The regenerative capacity of the cholestatic liver is significantly attenuated. Oval cells are hepatic stem cells involved in liver's regeneration following diverse types of injury. The present study investigated the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation as well as on hepatocyte and cholangiocyte proliferation and apoptosis in the cholestatic rat liver. Seventy male Wistar rats were randomly divided into five groups: controls, sham operated, bile duct ligated (BDL), BDL + BBS (30 μg/kg/d), BDL + NT (300 μg/kg/d). Ten days later, alpha-fetoprotein (AFP) mRNA (in situ hybridization), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphologic features of oval cells that were cytokeratin-19(+) and AFP mRNA(+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes and cholangiocytes were determined. Alanine aminotransferase (ALT) levels and hepatic oxidative stress (lipid peroxidation and glutathione redox state) were also estimated. The neuropeptides BBS and NT significantly reduced ALT levels and hepatic oxidative stress. Both agents exerted similar and cell type-specific effects on oval cells, hepatocytes and cholangiocytes: (a) oval cell proliferation and accumulation in the cholestatic liver was attenuated, (b) hepatocyte proliferation was increased along with a decreased rate of their apoptosis and (c) cholangiocyte proliferation was attenuated and their apoptosis was increased. These observations might be of potential value in patients with extrahepatic cholestasis.     

Polaprezinc prevents ongoing thioacetamide-induced liver fibrosis in rats

AimsCirrhotic patients commonly have a liver zinc deficiency, which may aggravate liver fibrosis due to the lack of antioxidative effects of zinc. This study examined the ability of polaprezinc, N-(3-aminopropionyl)-l-histidinato zinc, to prevent fibrosis in a rat model of thioacetamide (TAA)-induced hepatic fibrosis.Main methodsLiver cirrhosis was induced by orally administering TAA for 20 weeks. The rats were cotreated with one of the following for the last 10 weeks of TAA treatment: (1) polaprezinc (50 or 200 mg/kg/day); (2) l-carnosine (155 mg/kg/day), which contained equal amounts of l-carnosine as 200 mg/kg/day polaprezinc; (3) zinc sulfate (112 mg/kg/day) or (4) zinc-l-aspartic complex (317.8 mg/kg/day). Both zinc supplementations contained equal amounts of zinc as high-dose polaprezinc.Key findingsHepatic zinc levels fell significantly in rats treated with TAA for 20 weeks. Cotreating with high-dose polaprezinc and zinc-l-aspartic complex for 10 weeks prevented hepatic zinc loss. Hepatic hydroxyproline and tissue inhibitor of metalloproteinases-1 (TIMP-1) were significantly higher in rats treated with TAA for 20 weeks than 10 weeks, whereas polaprezinc prevented changes in these fibrosis markers and reduced hepatic transforming growth factor-β1 protein concentration, macroscopic and histologic changes. TAA caused oxidative stress-related changes in the liver that were prevented by high-dose polaprezinc and partially by zinc-l-aspartic complex. Treatment with l-carnosine, low-dose polaprezinc or zinc sulfate for 10 weeks did not affect liver fibrosis progression or oxidative stress-related changes.SignificancePolaprezinc may prevent ongoing fibrosis by preventing zinc depletion, oxidative stress and fibrosis markers in cirrhotic livers.     

Fasting reduces liver fibrosis in a mouse model for chronic cholangiopathies

Chronic cholangiopathies often lead to fibrosis, as a result of a perpetuated wound healing response, characterized by increased inflammation and excessive deposition of proteins of the extracellular matrix. Our previous studies have shown that food deprivation suppresses the immune response, which led us to postulate its beneficial effects on pathology in liver fibrosis driven by portal inflammation. We investigated the consequences of fasting on liver fibrosis in Abcb4^?/? mice that spontaneously develop it due to a lack of phospholipids in bile. The effect of up to 48 h of food deprivation was studied by gene expression profiling, (immuno)histochemistry, and biochemical assessments of biliary output, and hepatic and plasma lipid composition. In contrast to increased biliary output in the wild type counterparts, bile composition in Abcb4^?/? mice remained unchanged with fasting and did not influence the attenuation of fibrosis. Markers of inflammation, however, dramatically decreased in livers of Abcb4^?/? mice already after 12 h of fasting. Reduced presence of activated hepatic stellate cells and actively increased tissue remodeling further propelled a decrease in parenchymal fibrosis in fasting. This study is the first to show that food deprivation positively influences liver pathology in a fibrotic mouse model for chronic cholangiopathies, opening a door for new strategies to improve liver regeneration in chronic disease.     

Effect of Corilagin on the miR-21/smad7/ERK signaling pathway in a schistosomiasis-induced hepatic fibrosis mouse model

This study sought to investigate the anti-fibrotic effect of Corilagin via interference with the miR-21/smad7/ERK signaling pathway in a schistosomiasis-induced hepatic fibrosis mouse model. Mice were infected with Schistosoma japonicum cercaria to establish the mouse model of schistosomiasis-induced hepatic fibrosis. At four weeks after infection, the groups were given different medications. The living conditions were observed. Real-time PCR was employed to detect the mRNA levels of miR-21, smad7 and connective tissue growth factor (CTGF), and western blotting was used to examine the protein levels of smad7, CTGF, smad1, p-smad1, smad2, p-smad2, ERK1/2, p-ERK1/2 and TGF-β receptor I. Immunohistochemistry was used to examine the expression of CTGF. Compared with the model group, increasing concentrations of Corilagin improved the quality of life, inhibited the mRNA expression of miR-21, promoted smad7 protein expression, and inhibited CTGF protein expression (p < 0.05 or 0.01). Moreover, Corilagin significantly reduced the protein levels of p-smad1, p-smad2, p-ERK1/2, and TGF-β receptor I (p < 0.05 or 0.01). CTGF staining in the cytoplasm was markedly decreased by Corilagin (p < 0.05 or 0.01). In conclusion, Corilagin inhibited schistosomiasis-induced hepatic fibrosis via the miR21/smad7/ERK pathway in this animal model.     

Target identification,lead optimization and antitumor evaluation of some new 1,2,4-triazines as c-Met kinase inhibitors

In silico target fishing approach using PharmMapper server identified c-Met kinase as the selective target for our previously synthesized compound NCI 748494/1. This approach was validated by in vitro kinase assay which showed that NCI 748494/1 possessed promising inhibitory activity against c-Met kinase (IC₅₀ = 31.70 μM). Assessment of ADMET profiling, drug-likeness, drug score as well as docking simulation for the binding pose of that compound in the active site of c-Met kinase domain revealed that NCI 748494/1 could be considered as a promising drug lead. Based on target identification and validation, it was observed that there is structure similarity between NCI 748494/1 and the reported type II c-Met kinase inhibitor BMS-777607. Optimization of our lead NCI 748494/1 furnished newly synthesized 1,2,4-triazine derivatives based on well-established structure-activity relationships, whereas three compounds namely; 4d, 7a and 8c displayed excellent in vitro cytotoxicity against three c-Met addicted cancer cell lines; A549 (lung adenocarcinoma), HT-29 (colon cancer) and MKN-45 (gastric carcinoma); with IC₅₀ values in the range 0.01–1.86 µM. In vitro c-Met kinase assay showed 8c to possess the highest c-Met kinase inhibition profile (IC₅₀ = 4.31 µM). Docking of the active compounds in c-Met kinase active site revealed strong binding interactions comparable to the lead NCI 748494/1 and BMS-777607, suggesting that c-Met inhibition is very likely to be the mechanism of the antitumor effect of these derivatives.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

A targeted high-efficiency angiogenesis strategy as therapy for myocardial infarction

AimsThe aim of this study was to prove that an intramyocardial injection of a mixture of low-dose human growth factor (HGF) plasmid and microbubbles (MB) in combination with insonation was an effective therapy for myocardial infarction.Main methodsTwenty dogs with myocardial infarction were divided into 4 groups: (1) HGF, MB and ultrasound (HGF-US/MB), (2) HGF and US (HGF-US), (3) HGF alone and (4) surgery alone (control). In the HGF-US/MB group, HGF plasmid DNA (500 μg) mixed with 0.5 ml of MB solution was injected 5 min after coronary occlusion followed by insonation. With the exception of the control group, the other dogs were divided into two groups, one treated with the HGF gene and insonation and the other with the HGF gene only.Key findingsCompared to the HGF group, infarct size decreased from 32% ± 7% (control) to 23% ± 5% in the HGF-US/MB group 28 d later (P < 0.05). Capillary density increased from 21.7 ± 4.2/mm² (control) to 114.3 ± 28.9/mm² in the HGF-US/MB group (P < 0.01). Compared to the HGF group, there was a 14% decrease in the ratio of left ventricle weight/body weight and a 25% decrease in hydroxyproline content. We also observed a 29% and 20% decrease in collagen volume fraction of type I and type III collagen, respectively in the HGF-US/MB group.SignificanceIntramyocardial injection of HGF and MB in combination with insonation enhances neovascularization and reduces ventricular remodeling and infarct size.     

M1 muscarinic receptors modify oxidative stress response to acetaminophen-induced acute liver injury

The role of muscarinic receptor subtypes in modulating acute liver injury is unknown. We detected M1 muscarinic receptor (M1R) expression in human and murine hepatocytes, and investigated the consequences of M1R deficiency on acute liver injury in vivo and inhibiting M1R activation on hepatocyte injury in vitro. Age-matched wild-type (WT) and M1R-deficient (Chrm1^−/−) male mice were injected intraperitoneally with 200 mg/kg acetaminophen (APAP) and euthanized 0, 2, 4, 16, 24, and 36 h later. Biochemical and histological parameters indicated that liver injury peaked within 16 h after APAP treatment and resolved by 24 h. Compared to WT, M1R-deficient mice had reduced intrahepatic hemorrhage and hepatocyte necrosis, reflected by an attenuated rise in serum alanine aminotransferase levels. Livers of M1R-deficient mice showed reduced hepatocyte DNA fragmentation and attenuated expression of injury cytokines (Il-1α, Il-1β, Il-6, and Fasl). In all mice hepatic glutathione levels decreased after APAP injection, but they recovered more quickly in M1R-deficient mice. During the course of APAP-induced liver injury in M1R-deficient compared to WT mice, hepatic Nrf-2, Gclc, and Nqo1 expressions increased and nitrotyrosine generation decreased. APAP metabolic pathways were not altered by M1R deficiency; expression of hepatic Cyp2e1, Cyp1a2, Cyp3a11, Cyp3a13, Car, and Pxr was similar in Chrm1^−/− and WT mice. Finally, treatment of murine AML12 hepatocytes with a novel M1R antagonist, VU0255035, attenuated H₂O₂-induced oxidative stress, prevented GSH depletion, and enhanced viability. We conclude that M1R modify hepatocyte responses to oxidative stress and that targeting M1R has therapeutic potential for toxic liver injury.     

Evaluation of transient elastography in assessing liver fibrosis in patients with advanced schistosomiasis japonica

Schistosomiasis remains an important public health issue. The presence and extent of liver fibrosis are associated with disease progression and prognosis. The study is aimed at exploring the value of liver stiffness measurement (LSM) by transient elastography in assessing liver fibrosis in patients with advanced schistosomiasis japonica. Seventy-three patients were consecutively recruited for the purpose of this study. The correlation between noninvasive parameters and histological fibrosis stages was analyzed and an area under receiver operating characteristic curve (AUROC) was used to assess diagnostic efficacy. Our results demonstrated that there are significant differences between LSM values of patients with different stages of fibrosis (F1 vs. F2, F2 vs. F3 and F3 vs. F4, P < 0.01). The AUROC values of LSM in detecting significant fibrosis (F ≥ 2), advanced fibrosis (F ≥ 3) and cirrhosis (F = 4) were 0.96, 0.90, and 0.92 respectively. The optimal cut-off LSM values were 8.0 kPa, 9.5 kPa, and 18.0 kPa for significant fibrosis, advanced fibrosis and cirrhosis. Based on differences between AUROC values, LSM was proven to be superior to several serum models in detecting advanced fibrosis and cirrhosis. In conclusion, our study demonstrates that LSM is a reliable parameter for assessing risk of liver fibrosis in patients with advanced schistosomiasis japonica.     

Investigation of switch from ATM to ATR signaling at the sites of DNA damage induced by low and high LET radiation

Upon induction of DNA damage by ionizing radiation (IR), members of the phosphatidylinositol 3-kinase-like kinase family of proteins namely ataxia-telangiectasia mutated (ATM), DNA-PKcs, and ATM- and Rad3-related (ATR) maintain genomic integrity by mounting DNA damage response (DDR). Recent reports suggest that activation of ATM and ATR are oppositely regulated by the length of single stranded overhangs generated during end processing by nucleases at the break sites. These stretches of single stranded overhangs hold the clue for the transition from ATM to ATR signaling at broken DNA ends. We investigated whether differential processing of breaks induced by low and high LET radiation augments the phenomenon of switching from ATM to ATR kinase and hence a concomitant NHEJ to HR transition at the sites of DNA damage. 82-6 human fibroblasts were irradiated with 1 or 2 Gy of γ-rays and particle radiation of increasing LET in order to increase the complexity and variability of DNA double strand breaks (DSB) structures. The activation kinetics of ATM and ATR kinases along with their downstream substrates were determined utilizing Western blotting and immunofluorescence techniques. Our data provide evidence of a potential switch from ATM to ATR kinase signaling in cells treated with γ-rays at approximately 2 h post irradiation, with induction and completion of resection denoted by Rad51 foci resolution kinetics and observed with a significant decline of phosphorylated ATR kinase 8 h after IR. On the other hand, irradiation with high LET 600 MeV/u ⁵⁶Fe (180 keV/μm) and 170 MeV/u ²⁸Si (99 keV/μm) particles show a similar Rad51 foci decay kinetics, however, exhibiting prolonged resection, evident by the persistent phosphorylated ATM and ATR kinase until 24 h post irradiation. This residual effect, however, was significantly reduced for 250 MeV/u ¹⁶O particles of moderate LET (25 keV/μm) and absent for γ-rays. Hence, our results support the hypothesis that the transition from ATM to ATR signaling at DNA break sites is extended for longer periods of time, indicated by sustained resection due to the complex type of damage induced, a hallmark of high LET radiation, which may contribute to its increased biological effectiveness.