Inhibition of apoptosis protects mice from ethanol-mediated acceleration of early markers of CCl4 -induced fibrosis but not steatosis or inflammation.

Alcoholic liver disease (ALD) typically progresses through a series of pathologies starting with fatty liver or steatosis. Steatosis often proceeds to steatohepatitis, characterized by inflammation and hepatic cell death (Nagata et al., 2007). Steatohepatitis can further proceed to fibrosis, followed by cirrhosis. Ethanol-induced hepatic inflammation is characterized by infiltration of a variety of pro-inflammatory cells, including macrophages, monocytes, lymphocytes and neutrophils, as well as enhanced production of many pro-inflammatory cytokines (Szabo and Bala, 2010). Macrophage-derived TNFαis central to ethanol-induced hepatic inflammation and injury (Roychowdhury et al., 2009a). Apoptosis is implicated in a variety of pathological conditions in the liver, including ALD (Mundt et al., 2005; Zhou et al., 2001) and non-alcoholic steatohepatitis (NASH) (Galle and Krammer, 1998; Mundt et al., 2005). Induction of apoptosis in hepatocytes closely correlates with the severity of alcoholic and non-alcoholic liver diseases (Benedetti et al., 1988; Guicciardi and Gores, 2005; Witek et al., 2009). Detection of apoptotic hepatocytes in close proximity to inflammatory foci in liver biopsies from patients with ALD suggests that apoptosis could be a critical factor in alcohol-induced inflammation in the liver (Guicciardi and Gores, 2005; Jaeschke, 2002; Ziol et al., 2001). Apoptosis is a complex, caspase-dependent cell death mechanism, mediated by the extrinsic (death-receptor-mediated) and the intrinsic (mitochondria-mediated) pathways (Elmore, 2007). Interaction of the death receptors (TNFαreceptor or Fas) with their respective ligands initiates the extrinsic pathway, while mitochondrial dysfunction triggers the intrinsic pathway. Bid is a key pro-apoptotic Bcl-2 family member that is crucial for the induction of apoptotic death and serves as a bridge between these two fundamental cell death pathways (Yin, 2000a; Yin, 2000b). Since pathological activation of apoptosis in the liver is implicated in the progression of liver disease in both acute and chronic liver disorders, attenuation of apoptotic events can be considered as a potential therapeutic strategy against liver damage (Witek et al., 2009). Hepatocytes also undergo a necrosis-like caspase-independent cell death, called programmed necrosis/necroptosis, in conditions such as ischemia (Rosenbaum et al.) or in response to partial hepatectomy (Zorde-Khvalevsky et al., 2009). Necrosis can either be executed in a rapid, uncontrolled manner leading to rupture of cell membranes and leakage of intracellular proteins in the extra-cellular space, known as oncotic necrosis, or it can be orchestrated by a cascade of proteins, termed as programmed necrosis/necroptosis (Vandenabeele et al.2010). Programmed necroptosis is regulated by specific receptor-interacting protein kinases (RIP), including RIP1 and RIP3 (Vandenabeele et al.2010). Upon activation of the necroptotic pathways, damage-associated molecular pattern (DAMP) molecules are released from the dying cells; DAMPs then trigger a variety of pro-inflammatory signals and oxidative stress (Tsung et al., 2007). Genetic or pharmacological inhibition of apoptosis, using specific caspase-deficient mice or drugs that inhibit caspase activities, is protective in a number of models of liver injury, including the methyl-choline deficient model (MCD) of NASH (Witek et al., 2009), bile duct ligation-induced hepatic fibrosis (Canbay et al., 2004; Nalapareddy et al., 2009), hepatitis C (Masuoka et al., 2009) and liver transplantation (Baskin-Bey et al., 2007). Inhibiting caspases, the central mediators of apoptosis, ameliorates inflammation and fibrosis in the MCD model of NASH, but fails to attenuate liver injury, assessed by increased circulating ALT/AST (Witek et al., 2009). Although correlative evidence indicates that apoptosis is associated with inflammation and hepatic injury during progression of ALD (Malhi et al., 2006), very little is known about the direct role of apoptosis in mediating ethanol-induced liver injury. In a rat model, treatment with a pan-caspase inhibitor ameliorated ethanol-induced sensitization to challenge with LPS (Deaciuc et al., 2001); however, no studies have been carried out to directly link apoptosis to progression of alcohol-induced liver damage. We hypothesized that chronic ethanol feeding induces apoptosis in the liver in a Bid-dependent manner, which subsequently contributes to liver injury. Making use of Bid-deficient mice and the pan-caspase inhibitor VX166, we investigated whether apoptosis contributes to steatosis, inflammation, hepatocyte injury and fibrosis in response to ethanol using both a model of chronic, heavy ethanol feeding to generate steatohepatitis, as well as moderate ethanol feeding combined with carbon tetrachloride (CCl4) to model the acceleration of fibrosis by ethanol. Chronic, heavy ethanol feeding resulted in Bid-dependent hepatocyte apoptosis. While Bid-deficiency or treatment with the pan-caspase inhibitor VX166 had no effect on ethanol-induced hepatocyte injury, steatosis or expression of mRNA for some inflammatory cytokines, inhibition of caspase activity attenuated the expression of early markers of fibrosis in mouse liver in response to moderate ethanol and CCl4.