Alcoholic liver fibrosis is characterized by severe liver inflammatory response and fibrosis owing to augmented oxidative stress as well as generation of cell-toxic and profibrogenic ethanol metabolites, such as acetaldehyde and lipid oxidation products, which cause hepatocellular injury and activation of HSCs. A representative animal model of alcoholic liver fibrosis should reflect the characteristic metabolic changes and typical histological lesions of progressive fibrosing steatohepatitis, enabling ascertainment of pathogenesis and evaluation of drug therapy. In the present study, we established an experimental model of alcoholic liver fibrosis by feeding C57BL/6J mice with 4% ethanol-containing Lieber-DeCarli liquid diet for 8 weeks and combined with 5% CCl4 intraperitoneal injection for the last 4 weeks. After the combined administration, the mice rapidly and consistently developed alcoholic liver fibrosis manifested histologically by pronounced inflammatory infiltration, piecemeal hepatocellular necrosis, perisinusoidal and bridging fibrosis, together with enhanced hepatic expression of α-SMA, as well as significantly elevated serum ALT and AST levels. Conversely, ethanol treatment caused moderate macrosteatosis and mild inflammatory infiltration in the mice liver. These findings indicated that an alcoholic liver fibrosis model could be established rapidly and successfully by feeding mice ethanol liquid diet combined with a small amount of CCl4 intraperitoneal injection. Ethanol was the key mediator to induce liver injury, whilst CCl4 accelerated the progression of liver injury by increasing lipid accumulation [6, 7] and enhancing oxidative stress [8, 9].
With the progression of liver fibrosis, hepatic PPARα expression was reduced, suggesting that abnormal expression and/or dysfunction of PPARα might be involved in the development of ethanol plus CCl4 induced liver injury. We further demonstrated that induction of PPARα by specific agonist WY14643 administration for two weeks prominently attenuated liver injury, as evidenced by decreased serum ALT and AST levels, diminished inflammatory response, reduced collagen deposition, as well as suppressed activation of HSCs. These results indicated that PPARα played an important protective role in the progression of alcoholic liver fibrosis.
Oxidative stress and release of inflammatory cytokines induced by ethanol metabolism can evoke the activation of HSCs. Once activated, HSCs migrate to the site of liver injury and secrete excessive ECM , which is the pivotal event triggering the process of liver fibrogenesis. A variety of proinflammatory and profibrogenic factors are involved in controlling the activation and proliferation of HSCs. TNF-α, a pivotal inflammatory cytokine, exerts a profibrogenic function by inducing HSCs activation and inhibiting HSCs apoptosis . We found that hepatic expression of TNF-α was increased in mice fed with ethanol liquid diet and intraperitoneally injected with CCl4, which was significantly reduced by WY14643 treatment. In addition, we demonstrated that hepatic expression of OPN and TGF-β1 was up-regulated by ethanol with or without CCl4 treatment and restored by WY14643 administration. OPN is a chemoattractant molecule engaged in the hepatic inflammatory response by promoting neutrophil infiltration in the liver . As a biomarker of fibrosis , OPN can bind to integrin on the surface of HSCs to drive the fibrogenic response by regulating collagen I deposition [14, 15]. TGF-β1 is the most critical profibrogenic factor involved in the initiation and maintenance of liver fibrogenesis [4, 16]. It was reported that TGF-β1 increased expression of OPN , which in turn up-regulated TGF-β1 expression and activated HSCs through TGF-β1/Smad pathway , forming a vicious circle promoting liver fibrogenesis. Thus, the role of PPARα induction in alleviating alcoholic fibrotic hepatitis was possibly related to the down-regulation of TNF-α, OPN and TGF-β1, as well as the subsequent suppression of inflammatory response and fibrogenesis.
To further clarify the mechanism by which PPARα alleviated ethanol mediated liver fibrosis in mice, hepatic expression of visfatin, a novel identified adipocytokine, was assessed. It was considered as a proinflammatory cytokine and could be up-regulated by TNF-α [19, 20]. In the livers of ethanol plus CCl4 treated mice, visfatin expression was markedly up-regulated, accompanied with increased hepatic expression of PI3K, MMP-2 and MMP-9. In accord with our results, one study reported that visfatin induced MMP-2 and MMP-9 production in endothelial cells, which was mediated by the PI3K signaling pathway . MMP-2, secreted by activated HSCs, is considered a profibrotic mediator exerting proliferation and migration of HSCs [22, 23], and can be up-regulated by TGF-β and/or ROS stimulation [24, 25]. Meanwhile, MMP-9 can stimulate HSCs activation and up-regulate TGF-β1 expression [26, 27], which in turn induces MMP-9 expression through PI3K/Akt/nuclear factor-kappa B signaling pathway . We demonstrated that PPARα agonist significantly repressed hepatic expression of visfatin, and consequently reduced PI3K, MMP-2 and MMP-9 expression, thus suppressing the progression of ethanol mediated liver fibrosis.
We considered whether protective cytokines mediated the effect of PPARα in alleviating ethanol induced liver injury. Indeed, we found that reduced hepatic expression of adiponectin in alcoholic liver fibrosis mice was restored by WY14643 treatment. Adiponectin exerts its anti-inflammatory property through suppressing TNF-α production and secretion , and alleviates hepatic fibrosis by maintaining quiescence of HSCs and inducting apoptosis of activated HSCs [30–32]. Adiponectin induces expression of several other protective mediators, and an IL-10/HO-1 pathway is involved in the anti-inflammatory effects of adiponectin . IL-10 inhibits intrahepatic fibrogenesis by suppressing production of collagen I [34, 35], down-regulating expression of profibrogenic factors TGF-β1, MMP-9 and TNF-α [36–38], and promoting apoptosis of activated HSCs . In addition, IL-10 up-regulates HO-1 expression through the p38 mitogen-activated protein kinase pathway [40, 41]. HO-1 acts as an anti-oxidant and anti-fibrogenic protein in the liver. It was reported that induction of HO-1 suppressed oxidative stress and HSCs activation, thus inhibiting liver fibrogenesis in nutritional fibrotic steatohepatitis in mice . In keeping with this observation, we found that IL-10 and HO-1 expression was up-regulated by WY14643, together with the improved liver injury induced by ethanol plus CCl4 administration. Therefore, the protective role of PPARα induction against liver inflammation and fibrosis was mediated by up-regulating anti-inflammatory and anti-fibrogenic cytokines.