Pathogenesis of the hypertriglyceridemia at early stages of alcoholic liver injury in the baboon

To study the mechanism of alcoholic hypertriglyceridemia, baboons were pair-fed liquid diets containing 50% of energy as ethanol or as additional carbohydrate for 5-16 months. Alcohol-fed animals developed hypertriglyceridemia and early stages of alcoholic injury, namely fatty liver with or without perivenular fibrosis. In the fasting state, the triglyceride content was sixfold higher in very low density (VLDL) and intermediate density (IDL) lipoproteins and twofold higher in low density (LDL) and high density (HDL) lipoproteins. The increase in VLDL was markedly exaggerated in the postprandial state. To investigate the source of these increases, we determined net output or removal of serum triglycerides during circulation through either splanchnic or extrasplanchnic (lower extremities) vascular beds. In the splanchnic territory, there was net output of triglycerides in VLDL and net removal from the other lipoproteins. In alcohol-fed baboons, the output of VLDL-triglycerides into the hepatic (but not into the portal) vein tripled. This increase was mainly due to production of VLDL particles that were larger and had a flotation (Sf greater than 400) different from the Sf 20-400 which predominated in controls. This was associated with increased splanchnic removal of labeled chylomicron- or VLDL-triglycerides. In the lower extremities, there was an arteriovenous difference in VLDL-triglyceride concentration and this was increased in the alcohol-fed animals. Thus, the primary mechanism of the hypertriglyceridemia in alcohol-fed baboons was increased production of large, chylomicron-like VLDL by the liver, whereas both the extrasplanchnic extraction of VLDL-triglycerides and the splanchnic extraction of triglycerides from chylomicron- and VLDL-remnants were secondarily enhanced.     

Current concepts in the pathogenesis of alcoholic liver injury.

Alcoholic liver disease (ALD) develops as a consequence of priming and sensitizing mechanisms rendered by cross-interactions of primary mechanistic factors and secondary risk factors. This concept, albeit not novel, is becoming widely accepted by the field, and more research is directed toward identifying and characterizing the interfaces of the cross-interactions to help understand individual predisposition to the disease. Another pivotal development is the beginning of cell type-specific research to elucidate specific contributions not only of hepatocytes, but also of hepatic macrophages, liver-associated lymphocytes, sinusoidal endothelial cells, and hepatic stellate cells to sensitizing and priming mechanisms. In particular, the critical role of hepatic macrophages has been highlighted and the priming mechanisms concerning this paracrine effect have been proposed. Glutathione depletion in hepatocyte mitochondria is considered the most important sensitizing mechanism. One of the contributing factors is decreased methionine metabolism. Remaining key questions include how altered methionine metabolism contribute to the pathogenesis of ALD; how cross-talk among nonparenchymal liver cells or between nonparenchymal cells and hepatocytes leads to ALD; how dysfunctional mitochondria determine the type of cell death in ALD; and what secondary factors are critical for the development of advanced ALD such as alcoholic hepatitis and cirrhosis.     

Role of mitochondria in alcoholic liver injury

Oxidative stress and oxygen-derived free radicals are well known to play an important role in the pathogenesis of ethanol-associated liver injury. Active oxidants produced during ethanol metabolism induce mitochondrial membrane depolarization and permeability changes in cultured hepatocytes. These mitochondrial alterations (loss of DeltaPsim and mitochondrial permeability transition [MPT]) are now recognized as a key step in apoptosis. In recent studies, including ours, the MPT has been identified as a key step for the induction of mitochondrial cytochrome c release and caspase activation by ethanol. In addition, chronic and/or acute ethanol modulates intracellular, especially mitochondrial, antioxidant levels, leading to the increased susceptibility to alcoholic liver injury induced by several apoptotic stimuli. In this review, we address the mechanism of mitochondrial alterations and liver injury induced by ethanol.     

The functional role of microRNAs in alcoholic liver injury

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver‐inflammatory agents such as tumour necrosis factor‐alpha (TNF‐α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram‐negative bacteria, is responsible for TNF‐α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR‐155 and miR‐21, show a positive correlation in up‐regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF‐α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR‐122 and miR‐34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.     

黑果枸杞汁对大鼠酒精性肝损伤的保护作用*

目的: 研究黑果枸杞汁对大鼠酒精性肝损伤的保护作用,探讨其中Toll样受体4(TLR4)/p38 丝裂原活化蛋白激酶(p38 MAPK)信号通路的调节机制。方法: 60只雄性SD大鼠随机分为空白对照组(C)、模型组(M)、低剂量黑果枸杞汁组(LLM)、中剂量黑果枸杞汁组(MLM)、高剂量黑果枸杞汁组(HLM),每组12只。M、LLM、MLM和HLM组每天以20 ml/kg(8 g/(kg·d))剂量分2次灌胃400 g/L酒精,C组于相同时间点灌胃等体积蒸馏水。每日首次酒精灌胃前4 h,黑果枸杞汁各剂量组分别以2.4、4.8、9.6 ml/(kg·d)进行灌胃,其他组于相同时间点灌胃等体积蒸馏水。实验周期4周,末次实验结束后24 h处死大鼠,取血液和肝脏,计算肝脏指数,HE染色观察肝脏组织形态,比色法检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)活性,免疫组化法检测肝脏TLR4、p38 MAPK及磷酸化p38 MAPK(p-p38 MAPK)蛋白质表达,酶联免疫吸附试验检测肝脏肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-10(IL-10)、白细胞介素-18(IL-18)水平。结果: 与C组比较,M组成功建立酒精性肝损伤模型。与M组比较,黑果枸杞汁各剂量组的相关指标出现改善,其中HLM组肝脏组织形态改善最为显著,其肝脏系数、血清ALT和AST、肝脏TLR4蛋白质表达、p-p38 MAPK/p38 MAPK比值、TNF-α、IL-1β和IL-18水平均显著降低(P＜0.05或P＜0.01),肝脏IL-10水平显著升高(P＜0.01)。黑果枸杞汁各剂量组组间比较,肝脏系数、血清AST、肝脏TLR4蛋白质表达、p-p38 MAPK/p38 MAPK比值、TNF-α和IL-18水平,HLM组较LLM组均显著降低(P＜0.05或P＜0.01);肝脏IL-10水平,HLM组较LLM和MLM组均显著升高(P＜0.05或P＜0.01);其余主要指标组间比较均无统计学差异(P＞0.05)。结论: 黑果枸杞汁可以通过调控TLR4/p38 MAPK信号通路,改善炎症应激,缓解大鼠酒精性肝损伤,高剂量组效果优于其他剂量组。     

益生菌通过调整正常菌群缓解酒精性肝损伤的研究进展

许多临床试验表明慢性酒精性肝损伤会引起肠道菌群的失调,主要表现为双歧杆菌、乳杆菌数量减少,革兰氏阴性菌大量繁殖,破坏肠道屏障功能,增加肠道通透性,使细菌来源的内毒素大量释放出来,引起血液内毒素增加,并在肝脏中累积,超出肝脏的清除能力,导致肝损伤。本文主要综述益生菌通过调整正常菌群这一机制来缓解酒精性肝损伤的研究进展,进而深入了解酒精引起肠道菌群变化(酒精的摄入会导致肠道中拟杆菌、厚壁菌数量减少,革兰氏阴性变形菌、革兰氏阳性放线菌数量增加,同时肠道内细菌来源的内毒素水平增加)导致肝损伤的发病机制,以及益生菌如何通过调整肠道正常菌群改善酒精性肝损伤。     

Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease

Alcoholic liver disease (ALD) remains a leading cause of death from liver disease in the United States for which there is no FDA-approved therapy. Abnormal cytokine metabolism is a major feature of ALD. Elevated serum concentration levels of TNF-alpha and TNF-alpha-inducible cytokines/chemokines, such as IL-6, -8, and -18, have been reported in patients with alcoholic hepatitis and/or cirrhosis, and levels correlated with markers of the acute phase response, liver function, and clinical outcome. Studies in animal models support an etiologic role for cytokines in the liver injury of ALD. Cytokines, such as transforming growth factor-beta, play a critical role in the fibrosis of ALD. Multiple new strategies are under investigation to modulate cytokine metabolism as a form of therapy for ALD.     

Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress

Antioxidants are likely potential pharmaceutical agents for the treatment of alcoholic liver disease. Metallothionein (MT) is a cysteine-rich protein and functions as an antioxidant. This study was designed to determine whether MT confers resistance to acute alcohol-induced hepatotoxicity and to explore the mechanistic link between oxidative stress and alcoholic liver injury. MT-overexpressing transgenic and wild-type mice were administrated three gastric doses of alcohol at 5 g/kg. Liver injury, oxidative stress, and ethanol metabolism-associated changes were determined. Acute ethanol administration in the wild-type mice caused prominent microvesicular steatosis, along with necrosis and elevation of serum alanine aminotransferase. Ultrastructural changes of the hepatocytes include glycogen and fat accumulation, organelle abnormality, and focal cytoplasmic degeneration. This acute alcohol hepatotoxicity was significantly inhibited in the MT-transgenic mice. Furthermore, ethanol treatment decreased hepatic-reduced glutathione, but increased oxidized glutathione along with lipid peroxidation, protein oxidation, and superoxide generation in the wild-type mice. This hepatic oxidative stress was significantly suppressed in the MT-transgenic mice. However, MT did not affect the ethanol metabolism-associated decrease in NAD(+)/NADH ratio or increase in cytochrome P450 2E1. In conclusion, MT is an effective agent in cytoprotection against alcohol-induced liver injury, and hepatic protection by MT is likely through inhibition of alcohol-induced oxidative stress.     

Nf-kappab activation is associated with free radical generation and endotoxemia and precedes pathological liver injury in experimental alcoholic liver disease.

Endotoxemia and oxidative stress activate nuclear factor kappa B (NF-kappaB) in alcoholic liver injury. In alcohol-fed rats, activation of NF-kappaB is associated with the development of necro-inflammatory changes in the liver. Whether activation of NF-kappaB occurs prior to development of liver injury is unknown. We determined whether activation of NF-kappaB preceded histopathological liver changes. Male Wistar rats were fed a liquid diet containing ethanol by continuous infusion through permanently implanted gastric tubes. Radical intermediates detected by spin trapping were measured in bile prior to killing. After 2 weeks of treatment, samples of liver tissue were obtained for histopathological examination, for evaluation of NF-kappaB, and determination of messenger RNA levels of cytokines, chemokines and cyclo-oxygenase-2. No pathological changes in liver were seen after 2 weeks of intragastric feeding. However, activation of NF-kappaB was seen in the livers from ethanol-fed rats. In addition, elevated mRNA levels of hepatic pro-inflammatory cytokines (TNF-alpha and IL12), chemokines MIPIalpha and MIP-2) and cyclo-oxygenase-2 were seen in association with activation of NF-kappaB and increased levels of free radicals and endotoxin. Thus, activation of NF-kappaB, associated with elevated mRNA levels of pro-inflammatory stimuli, precedes the histopathological liver changes in experimental alcoholic liver disease in rats.     

Hepatic deletion of Smad7 in mouse leads to spontaneous liver dysfunction and aggravates alcoholic liver injury

Background

TGF-β has been known to play an important role in various liver diseases including fibrosis and alcohol-induced fatty liver. Smad7 is an intracellular negative regulator of TGF-β signaling. It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.

Methodology/Principal Findings

We used Cre/loxP system by crossing Alb-Cre mice with Smad7^loxP/loxP mice to generate liver-specific deletion of Smad7 with loss of the indispensable MH2 domain. Alcoholic liver injury was achieved by feeding mice with a liquid diet containing 5% ethanol for 6 weeks, followed by a single dose of ethanol gavage. Deletion of Smad7 in the liver was associated with increased Smad2/3 phosphorylation in the liver or upon TGF-β treatment in primary hepatocytes. The majority of mice with liver specific deletion of Smad7 (Smad7^liver-KO) were viable and phenotypically normal, accompanied by only slight or no reduction of Smad7 expression in the liver. However, about 30% of Smad7^liver-KO mice with high efficiency of Smad7 deletion had spontaneous liver dysfunction, demonstrated as low body weight, overall deterioration, and increased serum levels of AST and ALT. Degeneration and elevated apoptosis of liver cells were observed with these mice. TGF-β-induced epithelial to mesenchymal transition (EMT) was accelerated in Smad7-deleted primary hepatocytes. In addition, alcohol-induced liver injury and steatosis were profoundly aggravated in Smad7 deficient mice, associated with upregulation of critical genes involved in lipogenesis and inflammation. Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.

Conclusion/Significance

In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.     

Activity of Cassia auriculata leaf extract in rats with alcoholic liver injury

This study was undertaken to investigate the effect of Cassia auriculata leaf extract on tissue lipid peroxidation and antioxidant status in experimental hepatotoxicity. Administering ethanol to rats for 60 days resulted in significantly elevated levels of serum total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) as compared with those of the experimental control rats. Significantly elevated levels of tissue thiobarbituric acid reactive substances (TBARS), hydroperoxides and lowered activities of superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) were also observed on alcohol treatment as compared with those of experimental control rats. Concentration of serum non-enzymic antioxidants such as vitamin E and vitamin C were also significantly lowered on alcohol supplementation. Treatment with Cassia auriculata leaf extract at a dose of 250 mg kg(-1) body weight and 500 mg kg(-1) body weight to rats administered alcohol, lowered the levels of TBARS and hydroperoxides and elevated the activities of SOD and CAT and the levels of reduced GSH in the liver, brain, kidney and intestine significantly compared to unsupplemented alcohol treated rats. Cassia auriculata leaf extract treatment restored the serum vitamin E, and vitamin C levels also to near those of the experimental control animals. Our data indicate that supplementation with Cassia auriculata leaf extract can offer protection against free radical mediated oxidative stress in experimental hepatotoxicity. In addition, histopathological studies of the liver and brain confirmed the beneficial role of Cassia auriculata leaf extract.     

红托竹荪多糖对大鼠酒精性肝损伤的保护作用

研究红托竹荪多糖(Dictyophora rubrovalvata polysaccharide,DRP)对酒精所致大鼠肝损伤的保护作用。采用苯酚-硫酸法测得DRP的含量为74.68%±1.32%,利用傅里叶红外光谱初步分析表明DRP是含有α-糖苷键和β-糖苷键的吡喃环多糖。当DRP浓度达到3.0 mg/m L时,DPPH自由基的清除率达到80.12%,其还原力为0.31,对羟基自由基的清除率达到88.07%。雄性SD大鼠被随机分为6组：空白对照组(NC)、模型对照组(MC)、阳性对照组(PC)、红托竹荪多糖低(LDRP)、中(MDRP)、高(HDRP)剂量干预组,连续灌胃28 d后将其安乐死,测定血清中AST、ALT、TG水平以及肝脏SOD、GSH、MDA、TNF-α、IL-6水平,并根据病理切片分析红托竹荪多糖对大鼠酒精性肝损伤的保护程度。与MC组相比,DRP各剂量组血清AST、ALT、TG水平显著降低(P<0.05),肝脏SOD和GSH水平显著上升(P<0.05),MDA、TNF-α、IL-6含量显著下降(P<0.05),肝脏细胞变性和坏死等病理现象明显改善。D...     

Genetic markers in alcoholic liver cirrhosis.

11 genetic markers were typed in 157 individuals suffering from alcoholic cirrhosis, and compared with a random sample of healthy individuals. No significant differences were found for transferrin, specific group component, orosomucoid, esterase D, phosphogluconate dehydrogenase and adenylate kinase. Strong associations between alcoholic cirrhosis and alpha-1-antitrypsin PI*Z allele, haptoglobin HP*1 allele and acid phosphatase ACP AC phenotype were observed. The biological significance of these associations and their relationships with the development of alcoholic cirrhosis are also discussed.     

Dietary linoleic acid is required for development of experimentally induced alcoholic liver injury

We had previously hypothesized that linoleic acid (LA) was essential for development of alcoholic induced liver injury in our rat model. Male Wistar rats were fed a nutritionally adequate diet (25% calories as fat) with ethanol (8-17 g/kg/day). The source of fat was tallow (0.7% LA), lard (2.5% LA) or tallow supplemented with linoleic acid (2.5%). Liver damage was followed monthly by obtaining blood for alanine aminotransferase assay and liver biopsy for assessment of morphologic changes. Enzyme and histologic changes (fatty liver, necrosis and inflammation) in the tallow-linoleic acid-ethanol fed animals were more severe than in the lard-ethanol group. The tallow ethanol group did not show any evidence of liver injury. Our results strongly support our hypothesis that LA is essential for development of alcoholic liver disease in our rat model.     

茶多酚对慢性酒精中毒大鼠肝损伤的保护作用

目的：建立慢性酒精诱导的成年大鼠肝损伤动物模型,并进行茶多酚的干预,观察茶多酚的干预对慢性酒精诱导的肝损伤大鼠的防护作用及其可能的机制。方法：将36只SD大鼠适应性喂养一周后,随机分为对照组、酒精损伤组和茶多酚干预组（每组12只）。对照组大鼠用0.9%生理盐水按7 g/kg灌胃,酒精组用体积分数56%的红星牌白酒同剂量灌胃,茶多酚干预组在酒精灌胃同时给予0.25 g/kg剂量的茶多酚。每天定时灌胃一次,连续8周。8周后处死大鼠,取内脏脂肪和肝脏组织,以脂体比衡量内脏脂肪含量,以肝体比和油红O染色结果衡量肝脂质沉积,测定超氧化物歧化酶（SOD）活力、丙二醛（MDA）含量、总抗氧化能力（T-AOC）和谷胱甘肽过氧化物酶（GSH-Px）活力等氧化应激指标,测定肝脏组织中脂肪酸转位酶（FAT/CD36）蛋白水平。结果：与对照组相比,酒精损伤组大鼠内脏脂肪含量、SOD/MDA比值、T-AOC和GSH-Px活力显著下降（（P<0.05或P<0.01）,肝体比、FAT/CD36蛋白水平显著提高（P<0.01）,肝细胞中脂滴增加;与酒精损伤组相比,茶多酚干预组大鼠内脏脂肪含量、SOD/MDA比值、T-AOC和GSH-Px活力显著增加（（P<0.05或P<0.01）,肝体比、FAT/CD36蛋白水平显著下降（P<0.01）,肝细胞中脂滴减少。结论：茶多酚干预能改善慢性酒精中毒大鼠肝脏的脂质沉积和氧化应激状态,并伴有肝细胞膜上FAT/CD36表达的减少。