Hepatoprotective Effects of a Natural Flavanol 3,3'-Diindolylmethane against CCl4-Induced Chronic Liver Injury in Mice and TGFß1-Induced EMT in Mouse Hepatocytes via Activation of Nrf2 Cascade.

ABSTRACT

Hepatic fibrosis is a form of irregular wound-healing response with acute and chronic injury triggered by the deposition of excessive extracellular matrix. Epithelial-mesenchymal transition (EMT) is a dynamic process that plays a crucial role in the fibrogenic response and pathogenesis of liver fibrosis. In the present study, we postulated a protective role of 3,3'-diindolylmethane (DIM) against TGF-ß1 mediated epithelial-mesenchymal transition (EMT) in vitro and carbon tetrachloride (CCl4)-induced liver fibrosis in mice. TGF-ß1-induced AML-12 hepatocyte injury was evaluated by monitoring cell morphology, measuring reactive oxygen species (ROS) and mitochondrial membrane potential, and quantifying apoptosis, inflammatory, and EMT-related proteins. Furthermore, CCl4-induced liver fibrosis in mice was evaluated by performing liver function tests, including serum ALT and AST, total bilirubin, and albumin to assess liver injury and by performing H&E and Sirius red staining to determine the degree of liver fibrosis. Immunoblotting was performed to determine the expression levels of inflammation, apoptosis, and Nrf2/HO-1 signaling-related proteins. DIM treatment significantly restored TGF-ß1-induced morphological changes, inhibited the expression of mesenchymal markers by activating E-cadherin, decreased mitochondrial membrane potential, reduced ROS intensity, and upregulated levels of Nrf2-responsive antioxidant genes. In the mouse model of CCl4-induced liver fibrosis, DIM remarkably attenuated liver injury and liver fibrosis, as reflected by the reduced ALT and AST parameters with increased serum Alb activity and fewer lesions in H&E staining. It also mitigated the fibrosis area in Sirius red and Masson staining. Taken together, our results suggest a possible molecular mechanism of DIM by suppressing TGF-ß1-induced EMT in mouse hepatocytes and CCl4-induced liver fibrosis in mice.