Mechanisms in the Acceleration of the Regression of Liver Injury in Chronic Liver Fibrosis in Mice

Abstract

Background: Sustained chronic liver injury originating from several factors including viral infection, alcohol consumption, and hepatitis may lead to hepatic fibrosis characterized by an imbalance between extracellular matrix protein synthesis and degradation. Liver fibrosis may lead to liver failure in advanced stages. Regression of liver injury occurs upon cessation of the cause of injury and thus the acceleration of the regression of liver injury might play a beneficial role in the resolution of liver function. Scar-associated macrophage with anti-inflammatory activity and a high phagocytic activity has been shown to play a role in accelerating fibrosis regression. Monoacylglycerol lipase (MAGL) is a pro-inflammatory enzyme and the rate-limiting enzyme in the degradation of monoacylglycerols, responsible for metabolizing 2-arachidonoylglycerol into arachidonic acid. Inhibition of MAGL protects against liver fibrosis. Statins and Rho-Kinase inhibitors were also described to lower inflammation in liver injury through previous studies. The inhibition of monoacylglycerol lipase, the mevalonate pathway and the Rho-Kinase pathway have shown an acceleration of the liver fibrosis resolution.

Aims: In this study, the objective was to explore various mechanisms in the acceleration of regression of liver fibrosis. Thus, we aimed to investigate the involvement of PPAR-γ-mediated pathway and the role of macrophages in the PPAR-γ-dependent acceleration of regression by MAGL inhibition, and the role of autophagy in the acceleration of the regression of liver fibrosis in mice by ROCK inhibition.

Methods: In-vivo, 12-13 weeks old C57BL/6J male mice were used and chronic liver fibrosis was induced by repetitive injections of carbon tetrachloride (0.6 ml/kg, injected intraperitoneally) twice a week for 6 weeks, and the control group was given mineral oil (vehicle). Three treatment protocols have been studied in the present thesis work. In the first, a group of mice was treated with MJN110, a potent selective MAGL inhibitor, along with GW9662, an antagonist of PPAR-γ, and acceleration of liver fibrosis was investigated after macrophage deletion using clodronate liposomes. In the second, Pioglitazone, an agonism of PPAR-γ, was used to assess the involvement of PPAR-γ-mediated pathway in the acceleration of regression of liver fibrosis. In the third treatment protocol, mice were treated with Fasudil (HA-1077), a ROCK inhibitor, and Chloroquine, an autophagy inhibitor. Fibrosis was assessed by Sirius red staining of collagen deposition in liver, and hepatic gene expression was evaluated using RT-PCR.

Results: MJN110 did not show a significant inhibition of liver fibrosis as illustrated in the quantification of collagen deposition as well as the expression of the inflammatory markers. Depletion of macrophages by clodronate liposomes, during inhibition of MAGL by MJN110 and inhibition of PPAR-ɣ pathway by GW9662, showed a significant decrease in fibrotic and inflammatory genes (TGF-β, CCL3, CCL4). Moreover, MMP13 expression significantly increased upon MAGL inhibition whereas expression of MMP13 significantly decreased after macrophage depletion upon MAGL inhibition and in the presence or absence of GW9662. Activation of PPAR-ɣ pathway by pioglitazone showed no acceleration in the regression of liver fibrosis. A significant increase in the mRNA expression of TGF-β was observed as well as that of MMP2, whereas there was no significant change in the expression of inflammatory markers (CCL4, IL-1β, IL-6) and of MMP13. Mice treated with Fasudil (HA-1077), and Chloroquine (CQ) showed no acceleration in the regression of liver fibrosis as evidenced by no significant change in the accumulation of collagen and in the mRNA expression of α-SMA.

Conclusion: MJN110 did not show an acceleration in the regression of liver fibrosis. Both PPAR-ɣ–dependent and independent macrophages are involved in the MJN110 effect on regression of liver fibrosis and can have both anti- and pro-fibrotic effect. MMP13 can come in part from macrophages. The involvement of PPAR-ɣ pathway and autophagy was not clearly understood in this study. Further evaluations using selective and targeted knockout models shall be used to evaluate their effect in the acceleration of the regression of liver fibrosis.