Non‐alcoholic steatohepatitis: Definitions and pathogenesis

Abstract

The term 'non-alcoholic steatohepatitis' or NASH was first used by Ludwig et al. in 1980 to describe 'the pathological and clinical features of non-alcoholic disease of the liver associated with the pathological features most commonly seen in alcoholic liver disease itself'.1 This description remains appropriate because non-alcoholic fatty liver disease (NAFLD) can range from simple steatosis, through NASH and fibrosis to cirrhosis with fat.2 In addition to fat, the histological diagnosis of NASH ideally requires evidence of (i) hepatocyte injury, manifest by swollen or 'ballooned' cells; (ii) an inflammatory infiltrate, predominantly neutrophils, with or without (iii) fibrosis, typically perivenular/pericellular in distribution. Each of these features can be graded to derive a score recently proposed by Brunt et al.3 Rigorous exclusion of alcohol as a cause of the histology is, of course required and this is best achieved by a combination of repeated questioning of patients and ideally friends/relatives, frequent random blood alcohol estimations and measurement of mean cell corpuscular volume (MCV). No pattern of liver blood test abnormalities is either specific or sensitive enough to distinguish between NAFLD and alcohol-related liver disease. Interest in the pathogenesis of NASH has increased markedly in recent years for a variety of reasons. First, NASH is increasingly recognized to be a common condition, second only to viral hepatitis as a reason for referral in one study of urban North American office practise.4 A recent study in the United Kingdom demonstrated that NAFLD, either fatty liver alone or NASH, is the diagnosis in approximately two-thirds of patients presenting with abnormal liver function tests unrelated to viral hepatitis, immune disease or excessive alcohol intake.5 Some of this increase may be artifactual due to the increasing awareness of the condition; however, a 'real' increase seems likely in view of the increased prevalence of the conditions associated with NAFLD. In the various series reported thus far, 50% of patients are obese, particularly with a central or 'male' pattern and up to 40% of patients have type II diabetes mellitus (T2DM) or impaired glucose tolerance.2 Insulin resistance is an extremely common finding and may be a universal finding.6 Hypertension and hyperlipidemia are also common. These associations have led to the suggestion that NASH, or NAFLD in general, is the liver manifestation of the insulin resistance or 'metabolic' syndrome X.6,7 The interest in the pathogenesis of NASH has also increased in view of accumulating evidence that it may progress to advanced liver disease in at least some individuals. A review of the published biopsy studies of NASH reported up to 1998 showed that between 15 and 50% of patients had fibrosis or cirrhosis on their index biopsy.2 This has now been supplemented with a retrospective follow-up study of patients with NAFLD showing that over a median 8-year follow up, 25% of patients with NASH progressed to cirrhosis and 11% died of a liver-related cause. This is in contrast to only 3.4% of patients with simple fatty liver who developed cirrhosis and less than 2% who died a liver-related death.8 Further evidence that NASH may progress to cirrhosis has been provided by two studies suggesting that a large proportion, if not the majority, of patients with cryptogenic cirrhosis have the classical risk factors for NASH (obesity and T2DM), suggesting that NASH is probably the underlying cause of this disease in the majority of patients.9,10 A growing body of evidence suggests that, rather than being an 'innocent bystander', steatosis per se may be a 'guilty party', playing a role in the progression of NAFLD to NASH and fibrosis.11 This evidence has come from studies in NAFLD as well as in alcoholic liver disease and hepatitis C. In all of these conditions, the severity of steatosis predicts either the risk of concomitant steatohepatitis or the risk of progression to fibrosis and cirrhosis.12–15 In addition, studies in alcoholic fatty liver have shown that the severity of fat correlates with the degree of hepatic stellate cell (HSC) activation.16 Hepatic stellate cells are the principal cells in the liver responsible for the production of extracellular matrix proteins and, accordingly, fibrosis. Explaining the association between the severity of steatosis and the risk of necroinflammation and fibrosis first requires an understanding of the mechanisms of hepatocyte injury thought to play a role in NAFLD. In looking for clues as to the nature of these mechanisms, it is pertinent to emphasize that the histological features of NASH are identical to those of alcoholic hepatitis. The similarity between the histology of these conditions suggests that common mechanisms of injury are likely to be involved. Currently at least three mechanistic pathways of liver injury are thought to play a role in the pathogenesis of alcoholic hepatitis: oxidative stress, endotoxin-mediated cytokine release and immunologically mediated mechanisms (reviewed in 17). In heavy drinkers oxidative stress arises as a result of alcohol metabolism either via cytosolic alcohol dehydrogenase or the microsomal cytochrome P450 CYP2E1. This oxidative stress leads to lipid peroxidation, damaging plasma and intracellular membranes, resulting in cell death due to necrosis and/or apoptosis.18 In addition, the end products of lipid peroxida-tion (malondialdehyde (MDA) and hydroxynonenal (HNE)) are capable of inducing all of the classical features of alcoholic hepatitis. Both are chemotactic for neutrophils. They also stimulate the transcription of extracellular matrix-encoding genes in HSC, and, via induction of the transcription factor nuclear factor (NF)κB, they can lead to the increased transcription of cytokines and other pro-inflammatory genes in both hepatocytes and Kupffer cells. The importance of endotoxin-mediated release of cytokines as an important pathway of liver injury in alcoholic liver disease has been stressed by the animal work of Thurman et al (reviewed in 17). In this model, chronic alcohol consumption first leads to increased intestinal permeability to endotoxin. The resulting portal endotoxemia then activates hepatic Kupffer cells to produce the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Tumor necrosis factor-α subsequently induces mitochondrial oxidative stress in hepatocytes, leading to necrosis and apoptosis, and in endothelial cells leads to increased transcription of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), leading to the recruitment of inflammatory cells. Most of the evidence for this pathway has come from the Tsukamoto–French continuous intragastric feeding rodent model of alcoholic liver injury, although there are some supportive data from studies in humans. Alcohol certainly increases gut permeability in humans,19 and the serum level of TNF-α is increased in patients with alcoholic hepatitis and correlates with abnormal liver function and mortality.20 Acetaldehyde, the principal metabolite arising from alcohol metabolism by alcohol dehydrogenase, and MDA and HNE and other products of alcohol metabolism including hydroxyethyl radicals are capable of binding covalently to 'self' proteins to form adducts that are capable of stimulating an immune response. These adducts have been demonstrated in the blood and liver of animals fed alcohol, and humans with alcoholic liver disease. Importantly, the adducts are present on the surface of hepatocytes. Anti-adduct and autoantibodies have been demonstrated in the serum of heavy drinkers, and titers of these alloantibodes and autoantibodies are highest in patients with advanced alcoholic liver disease.21,22 Finally, the anti-adduct antibodies have been shown to be capable of mediating antibody-dependent cytotoxicity (ADCC) in hepatocytes isolated from alcohol-fed rats.23 Given the histological similarity between NASH and alcoholic hepatitis, is there any evidence that these mechanisms of alcoholic liver injury play a role in the liver injury occurring in NASH? First, with respect to oxidative stress, several studies using immunohistochemical methods to look for proteins adducted to MDA and HNE have provided evidence that lipid peroxidation occurs in the livers of patients with NAFLD.11 Moreover, recent work has demonstrated a variety of potential sources of oxidative stress in patients with NAFLD. At least one study has demonstrated evidence of increased iron in the liver of patients with NASH.24 Studies in animal models of NASH and humans with NASH have shown induction of cytochrome P450 enzymes CYP2E1 and CYP4A, both of which are capable of generating reactive oxygen species (ROS) during the metabolism of fatty acids and ketones.25 Most recently, Sanyal et al. have provided evidence that increased mitochondrial β-oxidation of free fatty acids (FFA) due to hepatic as well as peripheral insulin resistance may be an important source of ROS in NASH.26 As regards endotoxin-induced cytokine release, this is considered to be the important mechanism of the NASH occurring in patients following jejuno-ileal bypass surgery for obesity, because liver injury can be prevented by antibiotics aimed at reducing growth of Gram-negative bacteria in the 'blind loop' and the resulting portal endotoxemia.27 Non-alcoholic steatohepatitis has also been reported to occur in patients presenting with small intestinal bacterial overgrowth, and most recently bacterial overgrowth has been demonstrated in patients with apparently 'primary' NASH.28 Further support for a role for endotoxin in NASH has come from the leptin-deficient (Ob/Ob) mouse model of NASH, in which mice with extensive steatosis have been shown to be extremely sensitive to small doses of intraperitoneally administered endotoxin.29 Further support for a role for this pathway in human NASH has come from our recent work demonstrating tha

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