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Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor (TGF)-β superfamily. The rejuvenative effect of GDF11 has been called into question recently, and its role in liver regeneration is unclear. Here, we investigated the pathophysiologic role of GDF11, as well as its plausible signaling mechanisms in a mouse model of partial hepatectomy (PH). We demonstrated that both serum and hepatic GDF11 protein expression increased following PH. Treatment with adeno-associated viruses-GDF11 and recombinant GDF11 protein severely impaired liver regeneration, whereas inhibition of GDF11 activity with neutralizing antibodies significantly improved liver regeneration after PH. In vitro, GDF11 treatment significantly delayed cell proliferation and induced cell-cycle arrest in α mouse liver 12 (AML12) cells. Moreover, GDF11 activated TGF-β-SMAD2/3 signaling pathway. Inhibition of GDF11-induced SMAD2/3 activity significantly blocked GDF11-mediated reduction in cell proliferation both in vivo and in vitro. In the clinical setting, GDF11 levels were significantly elevated in patients after hepatectomy. Collectively, these results indicate that rather than a ‘rejuvenating’ agent, GDF11 impairs liver regeneration after PH. Suppression of cell-cycle progression via TGF-β-SMAD2/3 signaling pathway may be a key mechanism by which GDF11 inhibits liver regeneration.

Carbon monoxide (CO) exerts protective effects on hepatic ischemia/reperfusion injury (IRI), but the underlying molecular mechanisms are not fully understood. High-mobility group box 1 (HMGB1) is an important mediator of injury and inflammation in hepatic IRI. Here, we investigated whether CO could attenuate hepatic IRI via inhibition of HMGB1 release, particularly through sirtuin 1 (SIRT1). CO was released by treatment with carbon monoxide-releasing molecule (CORM)-2. CORM-2-delivered CO ameliorated hepatic IRI, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory responses, and less severe ischemia/reperfusion-associated histopathologic changes. Treatment with CORM-2 significantly inhibited IRI-induced HMGB1 translocation and release. SIRT1 expression was increased by CORM-2 pretreatment. When CORM-2-induced SIRT1 expression was inhibited using EX527, HMGB1 translocation and release were increased and hepatic IRI was worsened, whereas SIRT1 activation by resveratrol reversed this trend. In vitro, CORM-2 reduced hypoxia/reoxygenation-induced HMGB1 translocation and release, these inhibitions were blocked by SIRT1 inhibition using EX527 or SIRT1 small interfering RNA both in alpha mouse liver 12 cells and RAW264.7 macrophages. Moreover, SIRT1 directly interacted with and deacetylated HMGB1. IRI increased HMGB1 acetylation, which was abolished by CORM-2 treatment via SIRT1. In conclusion, these results suggest that CO may increase SIRT1 expression, which may decrease HMGB1 acetylation and subsequently reduce its translocation and release, thereby protecting against hepatic IRI. Liver Transplantation 23 510-526 2017 AASLD.

Objective 1,25(OH)2D3 has been reported to attenuate liver steatosis; however, its exact mechanism of action remains poorly understood. This study aimed to determine whether 1,25(OH)2D3 can attenuate hepatic steatosis by inducing autophagy. Methods Male C57BL/6 mice fed a high-fat diet (HFD) were injected with 1,25(OH)2D3 for 4 weeks. These mice were given 3-methyladenine (3-MA) to inhibit autophagy. HepG2 cells were preincubated with a free fatty acid (FFA) and then treated with 1,25(OH)2D3. Vitamin D receptor (VDR) shRNA and autophagy-related 16-like 1 (ATG16L1) siRNA were used for VDR knockdown or ATG16L1 silencing, respectively. Results 1,25(OH)2D3 diminished HFD-induced liver damage and steatosis, changes accompanied by autophagy and ATG16L1 expression upregulation. Inhibition of 1,25(OH)2D3-induced autophagy mediated by 3-MA blocked the protective effects of 1,25(OH)2D3 on hepatic steatosis. Additionally, 1,25(OH)2D3-induced autophagy appeared to play a role in anti-inflammation and lipid metabolism modulation in the liver. In HepG2 cells, 1,25(OH)2D3 reduced lipid accumulation and increased autophagy and ATG16L1 expression; however, this effect was abrogated after VDR knockdown. The protective effects of 1,25(OH)2D3-mediated autophagy against lipid accumulation were abolished by 3-MA. Furthermore, siRNA-mediated ATG16L1 knockdown prevented 1,25(OH)2D3-induced autophagy, resulting in increased fat accumulation. Conclusions The data suggest that 1,25(OH)2D3 may ameliorate hepatic steatosis by inducing autophagy by upregulating ATG16L1.

// Anding Liu 1,2 , Enshuang Guo 3 , Jiankun Yang 1 , Renlong Li 3 , Yan Yang 1 , Shenpei Liu 1 , Jifa Hu 1 , Xiaojing Jiang 3 , Olaf Dirsch 2 , Uta Dahmen 2 , Jian Sun 4 and Mingwen Ouyang 5 1 Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Experimental Transplantation Surgery, Department of General, Visceral and Vascular Surgery, Friedrich-Schiller-University Jena, Jena, Germany 3 Department of Infectious Diseases, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China 4 Department of Biliopancreatic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 5 Department of Anesthesiology, The Fifth Affiliated Hospital, Southern Medical University, Guangzhou, China Correspondence to: Mingwen Ouyang, email: // Keywords : ischemic preconditioning; steatosis; liver ischemia/reperfusion injury; autophagy; heme oxygenase-1, Pathology Section Received : April 25, 2016 Accepted : November 02, 2016 Published : November 10, 2016 Abstract Steatotic livers are more susceptible to ischemia/reperfusion (I/R) injury, which is ameliorated by ischemic preconditioning (IPC). Autophagy possesses protective action on liver I/R injury and declines in steatotic livers. The aim of this study was to test the hypothesis that the increased susceptibility of steatotic livers to I/R injury was associated with defective hepatic autophagy, which could be restored by IPC via heme oxygenase-1 (HO-1) signaling. Obesity and hepatic steatosis was induced using a high fat diet. Obesity impaired hepatic autophagy activity and decreased hepatic HO-1 expression. Induction of HO-1 restored autophagy activity and inhibited calpain 2 activity. Additionally, suppression of calpain 2 activity also restored autophagy activity. Mitochondrial dysfunction and hepatocellular injury were significantly increased in steatotic livers compared to lean livers in response to I/R injury. This increase in sensitivity to I/R injury was associated with defective hepatic autophagy activity in steatotic livers. IPC increased autophagy and reduced mitochondrial dysfunction and hepatocellular damage in steatotic livers following I/R injury. Furthermore, IPC increased HO-1 expression. Inhibition of HO-1 decreased the IPC-induced autophagy, increased calpain 2 activity and diminished the protective effect of IPC against I/R injury. Inhibition of calpain 2 restored autophagic defect and attenuated mitochondrial dysfunction in steatotic livers after I/R. Collectively, IPC might ameliorate steatotic liver damage and restore mitochondrial function via HO-1-mediated autophagy.

Liver dysfunction has been known to occur frequently in cases of sepsis. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in endotoxic shock. However, the role of baicalein in polymicrobial sepsis-induced liver injury and its regulatory mechanisms remain unclear. In this study, we aimed to investigate the protective effects of baicalein on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in C57BL/6 mice. Mice were treated with baicalein (100mg/kg, i.p) at 1h, 6h and 12h following CLP. Baicalein significantly improved the survival of septic mice. Treatment with baicalein ameliorated the CLP-induced liver injury, as indicated by the lower serum aminotransferase levels and the fewer histopathologic abnormalities. Baicalein reduced the neutrophil infiltration and the hepatic inflammatory cytokine expression and release. It also decreased the hepatic and the serum high-mobility group box 1 and macrophage migration inhibitory factor levels in septic mice. Moreover, baicalein significantly inhibited the mitogen-activated protein kinases (MAPKs) activation and suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB). In conclusion, these results suggest that baicalein treatment could protect against the sepsis-induced liver injury, and improve the survival of mice with polymicrobial sepsis. The mechanism of the protective action of baicalein seems to involve its ability to reduce inflammatory response, to inhibit hepatic apoptosis, and to suppress MAPKs and NF-κB activation.

FK866 exhibits a protective effect on D-galactosamine (GaIN)/lipopolysaccharide (LPS) and concanavalin A (ConA)-induced acute liver failure (ALF), but the mechanism by which FK866 affords this benefit has not yet been elucidated. Autophagy has a protective effect on acute liver injury. However, the contribution of autophagy to FK866-conferred hepatoprotection is still unclear. This study aimed to investigate whether FK866 could attenuate GaIN/LPS and ConA-induced ALF through c-jun-N-terminal kinase (JNK)-dependent autophagy. In vivo, Mice were pretreated with FK866 at 24, 12, and 0.5 h before treatment with GaIN/LPS and ConA. 3-methyladenine (3MA) or rapamycin were used to determine the role of autophagy in FK866-conferred hepatoprotection. In primary hepatocytes, autophagy was inhibited by 3MA or autophagy-related protein 7 (Atg7) small interfering RNA (siRNA). JNK was suppressed by SP600125 or Jnk siRNA. FK866 alleviated hepatotoxicity and increased autophagy while decreased JNK activation. Suppression of autophagy abolished the FK866-conferred protection. Inhibition of JNK increased autophagy and exhibited strongly protective effect. Collectively, FK866 could ameliorate GaIN/LPS and ConA-induced ALF through induction of autophagy while suppression of JNK. These findings suggest that FK866 acts as a simple and applicable preconditioning intervention to protect against ALF; autophagy and JNK may also provide therapeutic targets for ALF treatment.

1,25(OH)Male C57BL/6 mice fed a high-fat diet (HFD) were injected with 1,25(OH)1,25(OH)The data suggest that 1,25(OH)

We previously demonstrated that baicalein could protect against liver ischemia/reperfusion (I/R) injury in mice. The exact mechanism of baicalein remains poorly understood. Autophagy plays an important role in protecting against I/R injury. This study was designed to determine whether baicalein could protect against liver I/R injury via induction of autophagy in rats. Baicalein was intraperitoneally injected 1 h before warm ischemia. Pretreatment with baicalein prior to I/R insult significantly blunted I/R-induced elevations of serum aminotransferase levels and significantly improved the histological status of livers. Electron microscopy and expression of the autophagic marker LC3B-II suggested induction of autophagy after baicalein treatment. Moreover, inhibition of the baicalein-induced autophagy using 3-methyladenine (3-MA) worsened liver injury. Furthermore, baicalein treatment increased heme oxygenase (HO)-1 expression and pharmacological inhibition of HO-1 with tin protoporphyrin IX (SnPP) abolished the baicalein-mediated autophagy and the hepatocellular protection. In primary rat hepatocytes, baicalein-induced autophagy also protected hepatocytes from hypoxia/reoxygenation injury in vitro and the beneficial effect was abrogated by 3-MA or Atg7 siRNA, respectively. Suppression of HO-1 activity by SnPP or HO-1 siRNA prevented the baicalein-mediated autophagy and resulted in increased hepatocellular injury. Collectively, these results suggest that baicalein prevents hepatocellular injury via induction of HO-1-mediated autophagy.

To explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism.The hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA.DAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs.0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs.0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg.Down-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

microRNAs (miRNAs) have regulated the expression and function of genes implicated in many pathological settings, but their impact on the pathoetiological characteristics of overactive bladder (OAB) largely remains unknown. We have generated a mouse model in which adult mice can be induced for detrusor deletion of Dicer, an enzyme essential for miRNA processing. Targeted deletion of Dicer did not lead to a significant change for detrusor functionality under physiological conditions; however, loss of Dicer exacerbated cyclophosphamide-induced OAB, manifested by the higher severity of altered detrusor contractile force and sensitivity, abnormal urodynamics, and enhanced macrophage infiltration. Mechanistic studies revealed that loss of Dicer may impair the expression of miRNAs that are capable of targeting P2x mRNAs. As a result, mice deficient in Dicer manifest enhanced P2X expression in the detrusor on cyclophosphamide treatment, predisposing to the increased risk for OAB development. More important, studies using bladder biopsy samples of patients with OAB also demonstrated similar results as those found in animals. Taken together, our results suggest that miRNAs modulate OAB susceptibility by regulating purinergic signaling, in which the pathogenic insult induces the expression of miRNAs capable of targeting P2X mRNAs to suppress OAB symptoms.

Summary Recent studies showing the therapeutic effect of young blood on aging-associated deterioration of organs point to young blood as the solution for clinical problems related to old age. Given that defective autophagy has been implicated in aging and aging-associated organ injuries, this study was designed to determine the effect of young blood on aging-induced alterations in hepatic function and underlying mechanisms, with a focus on autophagy. Aged rats (22 months) were treated with pooled plasma (1 ml, intravenously) collected from young (3 months) or aged rats three times per week for 4 weeks, and 3-methyladenine or wortmannin was used to inhibit young blood-induced autophagy. Aging was associated with elevated levels of alanine transaminase and aspartate aminotransferase, lipofuscin accumulation, steatosis, fibrosis, and defective liver regeneration after partial hepatectomy, which were significantly attenuated by young plasma injections. Young plasma could also restore aging-impaired autophagy activity. Inhibition of the young plasma-restored autophagic activity abrogated the beneficial effect of young plasma against hepatic injury with aging. In vitro, young serum could protect old hepatocytes from senescence, and the antisenescence effect of young serum was abrogated by 3-methyladenine, wortmannin, or small interfering RNA to autophagy-related protein 7. Collectively, our data indicate that young plasma could ameliorate age-dependent alterations in hepatic function partially via the restoration of autophagy.

Ischemia/reperfusion (I/R) is a pathophysiologic process that occurs during hemorrhagic shock, liver resection and liver transplantation. Baicalein, the main active ingredient of the Scutellaria root, exerts anti-inflammatory and anti-apoptotic properties in the setting of I/R injury in the heart and brain. However, the role of baicalein in liver I/R injury and its regulatory mechanisms remain poorly understood. This study was designed to evaluate the effects of baicalein in a model of liver I/R in mice and to explore the possible mechanisms. Baicalein (100mg/kg) was intraperitoneally injected 1h before warm ischemia. Pretreatment with baicalein protected against liver I/R injury, as indicated by the decreased serum aminotransferase levels and the reduced histopathologic abnormalities. Baicalein also significantly reduced cellular hepatic apoptosis in response to I/R injury. Moreover, pretreatment with baicalein significantly inhibited nuclear factor-kappa B (NF-κB) activation and the subsequent proinflammatory cytokine production, and decreased leukocyte infiltration. In vitro studies, baicalein treatment inhibited the proinflammatory cytokine production via the modulation of NF-κB signaling pathway in lipopolysaccharide-stimulated macrophages. Taken together, these results suggest that baicalein could protect against liver I/R injury via inhibition of inflammation by down-regulating NF-κB activity, and suppression of cellular hepatic apoptosis.

Liver dysfunction has been known to occur frequently in cases of sepsis. Excessive inflammation and apoptosis are pathological features of acute liver failure. Recent studies suggest that activation of glycogen synthase kinase- (GSK-) 3βis involved in inflammation and apoptosis. We aimed to investigate the protective effects of GSK-3βinhibition on polymicrobial sepsis-induced liver injury and to explore the possible mechanisms. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and SB216763 was used to inhibit GSK-3βin C57BL/6 mice. GSK-3βwas activated following CLP. Administration of SB216763 decreased mortality, ameliorated liver injury, and reduced hepatic apoptosis. The inhibition of GSK-3βalso reduced leukocyte infiltration and hepatic inflammatory cytokine expression and release. Moreover, GSK-3βinhibition suppressed the transcriptional activity of nuclear factor-kappa B (NF-κB) but enhanced the transcriptional activity of cAMP response element binding protein (CREB) in the liver. In in vitro studies, GSK-3βinhibition reduced inflammatory cytokine production via modulation of NF-κB and CREB signaling pathways in lipopolysaccharide-stimulated macrophages. In conclusion, these findings suggest that GSK-3βblockade protects against CLP-induced liver via inhibition of inflammation by modulating NF-κB and CREB activity and suppression of hepatic apoptosis.

The inflammatory response after liver ischemia/reperfusion (I/R) contributes to increased risk of liver failure after liver surgery. Strategies aimed to preventing inflammation could be beneficial in reducing liver I/R injury. Recent studies have demonstrated that peptide B"15-42 is able to decrease the injury of I/R in heart and kidney by inhibition of leukocyte migration and preserving endothelial barrier function. Prompted by these results, we hypothesized that B"15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury. Therefore, in this study, we aimed to evaluate the effects of B"15-42 in a model of liver I/R injury in rats. Rats were treated with B"15-42 at initiation of reperfusion and 2 h thereafter. Rats were killed at 0.5, 6, 24, and 48 h after reperfusion. Hepatic mRNA levels of fibrinogen-! (Fg!), Fg" ,F g+ were significantly increased after I/R. Treatment with Fg-derived B"15-42 ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels and fewer I/R-associated histopathologic changes. B"15-42 treatment decreased leukocyte infiltration and expression of hepatic inflammatory cytokines. Moreover, B"15-42 significantly reduced high-mobility group box 1 release and altered mitogen-activated protein kinase activation. In conclusion, B"15-42 treatment protected against liver warm I/R injury. The mechanism of protective action of B"15-42 seemed to involve its ability to reduce hepatic inflammatory response through preventing high-mobility group box 1 release and altering mitogen-activated protein kinase activation. KEYWORDS—Liver ischemia/reperfusion, fibrinogen, B"15-42, inflammatory response, HMGB1, MAPK