Alcoholic liver disease and the gut-liver axis

Alcoholic liver disease (ALD) is one of the leading causes of liver diseases and liver-related death worldwide. Of the many factors that contribute to the pathogenesis of ALD, gut-derived lipopolysaccharide (LPS) plays a central role in induction of steatosis, inflammation, and fi brosis in the liver. In this review, we discuss the mechanisms by which alcohol contributes to increased gut permeability, the activation of Kupffer cells, and the infl ammatory cascade by LPS. The role of the Toll-like receptor 4 (TLR4) complex in LPS recognition and the importance of the TLR4-induced signaling pathways are evaluated in ALD.

Emerging role of microRNAs in liver diseases

MicroRNAs are a class of small non-coding RNAs that are found in plants, animals, and some viruses. They modulate the gene function at the post-transcriptional level and act as a fine tuner of various processes, such as development, proliferation, cell signaling, and apopto-sis. They are associated with different types and stages of cancer. Recent studies have shown the involvement of microRNAs in liver diseases caused by various factors, such as Hepatitis C, Hepatitis B, metabolic disorders, and by drug abuse. This review highlights the role of microRNAs in liver diseases and their potential use as therapeutic molecules.

Lipopolysaccharide induces and activates the Nalp3 inflammasome in the liver

AIM:To examine the activation of the Nalp3 inflammasome and its downstream targets following lipopolysaccharide(LPS) -induced stimulation in the liver. METHODS:Six-to-eight-week-old C57BL/6 chow fed mice were injected intraperitoneally with 0.5μg/g bodyweight LPS and sacrificed 2,4,6,18 or 24 h later. LPS-induced liver damage was confirmed by a biochemical assay to detect alanine aminotransferase(ALT) levels.To determine if LPS stimulation in the liver led to activation of the inflammasome,real-time quantitative polymerase chain reaction was used to evaluate the mRNA expression of components of the Nalp3 inflammasome.Enzyme-linked immunosorbent assays were used to determine the protein expression levels of several downstream targets of the Nalp3 inflammasome,including caspase-1 and two cytokine targets of caspase-1,interleukin(IL) -1βand IL-18. RESULTS:We found that LPS injection resulted in liver damage as indicated by elevated ALT levels.This was associated with a significant increase in both mRNA and protein levels of the proinflammatory cy-tokine tumor necrosis factor(TNF) -αin the liver,as well as increased levels of TNFs in serum.We showed that LPS stimulation led to upregulation of mRNA levels in the liver for all the receptor components of the inflammasome,including Nalp3,Nalp1,pannexin-1 and the adaptor molecule apoptosis-associated specklike,caspase recruitment domain-domain containing protein.We also found increased levels of mRNA and protein for caspase-1,a downstream target of the inflammasome.In addition,LPS challenge led to increased levels of both mRNA and protein in the liver for two cytokine targets of caspase-1,IL-1βand IL-18. Interestingly,substantial baseline expression of pre-IL1βand pre-IL-18 was found in the liver.Inflammasome and caspase-1 activation was indicated by the significant increase in the active forms of IL-1βand IL-18 after LPS stimulation. CONCLUSION:Our results show that the Nalp3 inflammasome is upregulated and activated in the liver in response to LPS stimulation.

In vitro and in vivo models of acute alcohol exposure

Alcohol abuse is a global problem due to the financial burden on society and the healthcare system. While the harmful health effects of chronic alcohol abuse are well established, more recent data suggest that acute alcohol consumption also affects human wellbeing. Thus, there is a need for research models in order to fully understand the effect of acute alcohol abuse on different body systems and organs. The present manuscript summarizes the interdisciplinary advantages and disadvantages of currently available human and non-human models of acute alcohol abuse, and identifi es their suitability for biomedical research.

High fat diet feeding results in gender specific steatohepatitis and inflammasome activation

AIM:To develop an animal model that encompasses the different facets of non-alcoholic steatohepatitis(NASH),which has been a challenge.METHODS:In this study,we used a high fat diet(HFD)feeding supplemented with fructose and sucrose in the water mimicking the high-fructose corn syrup that is abundant in the diet in the United States.We used C57Bl/6 wild-type mice for short and long-term feedings of 6 and 16 wk respectively,and evaluated the extent of liver damage,steatosis,and inflammasome activation.Our methods included histopathological analysis to assess liver damage and steatosis,which involved H and E and oil-red-o staining;biochemical studies to look at ALT and triglyceride levels;RNA analysis using quantitative polymerase chain reaction;and cytokine analysis,which included the enzyme-linked immunosorbent assay method to look at interleukin(IL)-1βand tumor necrosis factor-α(TNFα)levels.Furthermore,at each length of feeding we also looked at insulin resistance and glucose tolerance using insulin tolerance tests(ITT)and glucose tolerance tests.RESULTS:There was no insulin resistance,steatosis,or inflammasome activation at 6 wk.In contrast,at16 wk we found significant insulin resistance demonstrated by impaired glucose and ITT in male,but not female mice.In males,elevated alanine aminotransferase and triglyceride levels,indicated liver damage and steatosis,respectively.Increased liver TNFαand monocyte chemoattractant protein-1 mRNA and protein,correlated with steatohepatitis.The inflammasome components,adaptor molecule,Aim2,and NOD-like receptor 4,increased at the mRNA level,and functional inflammasome activation was indicated by increased caspase-1 activity and IL-1βprotein levels in male mice fed a long-term HFD.Male mice on HFD had increasedα-smooth muscle actin and pro-collagen-1 mRNA indicating evolving fibrosis.In contrast,female mice displayed only elevated triglyceride levels,steatosis,and no fibrosis.CONCLUSION:Our data indicate gender differences in NASH.Male mice fed a long-term HFD display steatohepatitis and inflammasome activation,whereas female mice have steatosis without inflammation.

Plasma microRNA profiles distinguish lethal injury in acetaminophen toxicity: A research study

AIM: To investigate plasma microRNA (miRNA) profiles indicative of hepatotoxicity in the setting of lethal acetaminophen (APAP) toxicity in mice. METHODS: Using plasma from APAP poisoned mice, either lethally (500 mg/kg) or sublethally (150 mg/kg) dosed, we screened commercially available murine microRNA libraries (SABiosciences, Qiagen Sciences, MD) to evaluate for unique miRNA profiles between these two dosing parameters. RESULTS: We distinguished numerous, unique plasma miRNAs both up- and downregulated in lethally compared to sublethally dosed mice. Of note, many of the greatest up- and downregulated miRNAs, namely 574-5p, 466g, 466f-3p, 375, 29c, and 148a, have been shown to be associated with asthma in prior studies. Interestingly, a relationship between APAP and asthma has been previously well described in the literature, with an as yet unknown mechanism of pathology. There was a statistically significant increase in alanine aminotransferase levels in the lethal compared to sublethal APAP dosing groups at the 12 h time point (P < 0.001). There was 90% mortality in the lethally compared to sublethally dosed mice at the 48 h time point (P = 0.011). CONCLUSION: We identified unique plasma miRNAs both up- and downregulated in APAP poisoning which are correlated to asthma development.

Distinct toll-like receptor expression in monocytes and T cells in chronic HCV infection

瞄准：丙肝病毒经常建立长期的感染。最近的研究建议病毒、细菌的感染在与控制相比的感染 HCV 的病人是更普通的。病原体被像使用费的受体(TLR ) 认出塑造适应、天生的有免疫力的回答。方法：在这研究，估计感染 HCV 的主人的能力认识到入侵病原体，我们调查了像使用费的受体表示在天生(单核白血球) 并且适应(T 房间) 由即时 PCR 的有免疫力的房间。结果：我们决定 RNA 为 TLR 铺平 2， 6。7， 8， 9 和 10 个 mRNA 层次起来与控制相比在感染 HCV 的病人在单核白血球和 T 房间调整了。TLR4 在 T 淋巴细胞在仅仅上面被调整，当 TLR5 有选择地在感染 HCV 的病人的单核白血球被增加时。MD-2， TLR4 合作受体，当 CD14 和 MyD88 仅仅在单核白血球被增加时，在病人的单核白血球和 T 房间被增加。结论：我们的数据在多半在感染 HCV 的个人联系到病原体和有免疫力的防卫的天生的识别的 TLR 表示上披露新奇详情。

Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease

AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma(HCC) that develops in livers with alcoholic liver disease(ALD).METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine(DEN) followed by 7 wk of 4% Lieber-De Carli diet. Serum alanine aminotransferase(ALT), alpha fetoprotein(AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6 G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/i NOS(M1) and Arg-1/IL-10/CD163/CD206(M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen(PCNA).RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed- DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicatingadvanced liver disease. Molecular characterization showed high estlevels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M 2 macrophages were edominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to al l other groups. This correlated with increased serum alphafetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice.CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC.

Proton pump inhibitors increase the severity of hepatic encephalopathy in cirrhotic patients

BACKGROUND Liver cirrhosis is the late stage of hepatic fibrosis and is characterized by portal hypertension that can clinically lead to decompensation in the form of ascites,esophageal/gastric varices or encephalopathy.The most common sequelae associated with liver cirrhosis are neurologic and neuropsychiatric impairments labeled as hepatic encephalopathy(HE).Well established triggers for HE include infection,gastrointestinal bleeding,constipation,and medications.Alterations to the gut microbiome is one of the leading ammonia producers in the body,and therefore may make patients more susceptible to HE.AIM To investigate the relationship between the use of proton pump inhibitors(PPIs)and HE in patients with cirrhosis.METHODS This is a single center,retrospective analysis.Patients were included in the study with an admitting diagnosis of HE.The degree of HE was determined from subjective and objective portions of hospital admission notes using the West Haven Criteria.The primary outcome of the study was to evaluate the grade of HE in PPI users versus non-users at admission to the hospital and throughout their hospital course.Secondary outcomes included rate of infection,gastrointestinal bleeding within the last 12 mo,mean ammonia level,and model for end-stage liver disease scores at admission.RESULTS The HE grade at admission using the West Haven Criteria was 2.3 in the PPI group compared to 1.7 in the PPI nonuser group(P=0.001).The average length of hospital stay in PPI group was 8.3 d compared to 6.5 d in PPI nonusers(P=0.046).Twenty-seven(31.8%)patients in the PPI user group required an Intensive Care Unit admission during their hospital course compared to 6 in the PPI nonuser group(16.7%)(P=0.138).Finally,10(11.8%)patients in the PPI group expired during their hospital stay compared to 1 in the PPI nonuser group(2.8%)(P=0.220).CONCLUSION Chronic PPI use in cirrhotic patients is associated with significantly higher average West Haven Criteria for HE compared to patients that do not use PPIs.

Mechanisms of alcohol-mediated hepatotoxicity in human-immunodeficiency-virus-infected patients

Clinical observations have demonstrated that excessive chronic alcohol use negatively affects human immuno- deficiency virus (HIV) infection and contributes to the liver manifestations of the disease, even in HIV mono- infection. HIV/hepatitis C virus (HCV) co-infection is as- sociated with increased progression of HVC liver disease compared to HCV infection alone, and both of these are negatively affected by alcohol use. Recent data suggest that alcohol use and HIV infection have common targets that contribute to progression of liver disease. Both HIV infection and chronic alcohol use are associated with increased gut permeability and elevated plasma levels of lipopolysaccharide; a central activator of inflammatory responses. Both alcoholic liver disease and HIV infec tionresult in non-specific activation of innate immunity, proinflammatory cytokine cascade upregulation, as well as impaired antigen presenting cell and dendritic cell functions. Finally, alcohol, HIV and antiretroviral therapyaffect hepatocyte functions, which contributes to liver damage. The common targets of alcohol and HIV infection in liver disease are discussed in this minireview.

Immunopathobiology and therapeutic targets related to cytokines in liver diseases

Chronic liver injury with any etiology can progress to fibrosis and the end-stage diseases cirrhosis and hepatocellular carcinoma.The progression of liver disease is controlled by a variety of factors,including liver injury,inflammatory cells,inflammatory mediators,cytokines,and the gut microbiome.In the current review,we discuss recent data on a large number of cytokines that play important roles in regulating liver injury,inflammation,fibrosis,and regeneration,with a focus on interferons and T helper(Th)1,Th2,Th9,Th17,interleukin(IL)-1 family,IL-6 family,and IL-20 family cytokines.Hepatocytes can also produce certain cytokines(such as IL-7,IL-11;and IL-33),and the functions of these cytokines in the liver are briefly summarized.Several cytokines have great therapeutic potential,and some are currently being tested as therapeutic targets in clinical trials for the treatment of liver diseases,which are also described.

IL-1α in acetaminophen toxicity: a sterile danger signal

Much controversy surrounds the pathogenesis of acetaminophen-induced inflammation.Acetaminophen(APAP)is a well-studied xenobiotic that in high doses results in a deadly,yet preventable,form of liver failure.Although its effects are sudden and often irreversible,its mechanism is far from simple.In fact,at first glance,recent studies seem to refute each other.The field has been grappling with seemingly contradictory results driven mainly by often-generalized assumptions and different experimental systems.