Synergistic Effects of Vitis vinifera L. and Centella asiatica against CCl4-Induced Liver Injury in Mice.

Liver injury can be acute or chronic, resulting from a variety of factors, including viral hepatitis, drug overdose, idiosyncratic drug reaction, or toxins, while the progression of pathogenesis in the liver rises due to the involvement of numerous cytokines and growth factor mediators. Thus, the identification of more effective biomarker-based active phytochemicals isolated from medicinal plants is a promising strategy to protect against CCl4-induced liver injury. Vitis vinifera L. (VE) and Centella asiatica (CE) are well-known medicinal plants that possess anti-inflammatory and antioxidant properties. However, synergism between the two has not previously been studied. Here, we investigated the synergistic effects of a V. vinifera L. (VE) leaf, C. asiatica (CE) extract combination (VCEC) against CCl4-induced liver injury. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 mL/kg). VCEC was administered orally for three consecutive days at various concentrations (100 and 200 mg/kg) prior to CCl4 injection. The extent of liver injury and the protective effects of VCEC were evaluated by biochemical analysis and histopathological studies. Oxidative stress was evaluated by measuring malondialdehyde (MDA) and glutathione (GSH) levels and Western blotting. VCEC treatment significantly reduced serum transaminase levels (AST and ALT), tumor necrosis factor-α (TNF-α), and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by VCEC treatment by reducing cleaved caspase-3 and Bcl2-associated X protein (Bax). VCEC-treated mice significantly restored cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) expression in CCl4-treated mice. In addition, VCEC downregulated overexpression of proinflammatory cytokines and hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4-mediated apoptosis. Collectively, VCEC exhibited synergistic protective effects against liver injury through its antioxidant, anti-inflammatory, and antiapoptotic ability against oxidative stress, inflammation, and apoptosis. Therefore, VCEC appears promising as a potential therapeutic agent for CCl4-induced acute liver injury in mice.

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.

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.

3,3'-Diindolylmethane Suppresses the Growth of Hepatocellular Carcinoma by Regulating Its Invasion, Migration, and ER Stress-Mediated Mitochondrial Apoptosis.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide with limited treatment options. Biomarker-based active phenolic flavonoids isolated from medicinal plants might shed some light on potential therapeutics for treating HCC. 3,3'-diindolylmethane (DIM) is a unique biologically active dimer of indole-3-carbinol (I3C), a phytochemical compound derived from Brassica species of cruciferous vegetables-such as broccoli, kale, cabbage, and cauliflower. It has anti-cancer effects on various cancers such as breast cancer, prostate cancer, endometrial cancer, and colon cancer. However, the molecular mechanism of DIM involved in reducing cancer risk and/or enhancing therapy remains unknown. The aim of the present study was to evaluate anti-cancer and therapeutic effects of DIM in human hepatoma cell lines Hep3B and HuhCell proliferation was measured with MTT and trypan blue colony formation assays. Migration, invasion, and apoptosis were measured with Transwell assays and flow cytometry analyses. Reactive oxygen species (ROS) intensity and the loss in mitochondrial membrane potential of Hep3B and Huh7 cells were determined using dihydroethidium (DHE) staining and tetramethylrhodamine ethyl ester dye. Results showed that DIM significantly suppressed HCC cell growth, proliferation, migration, and invasion in a concentration-dependent manner. Furthermore, DIM treatment activated caspase-dependent apoptotic pathway and suppressed epithelial-mesenchymal transition (EMT) via ER stress and unfolded protein response (UPR). Taken together, our results suggest that DIM is a potential anticancer drug for HCC therapy by targeting ER-stress/UPR.

Anticancer effects of Poncirus fructus on hepatocellular carcinoma through regulation of apoptosis, migration, and invasion.

Poncirus fructus (PF) is a phytochemical compound extracted from the dry, immature fruits of Poncirus trifoliate. PF is traditionally used to treat gastrointestinal disorders, allergies, and inflammatory disease. In East Asia, PF is also known for its anticancer properties. There are numerous reports on the anticancer and anti­inflammatory effects of PF in a wide range of cancers and gastrointestinal diseases, respectively. However, the role of PF in inducing apoptosis and suppressing the invasiveness of hepatocellular carcinoma (HCC) remains unclear. This study investigated the ability of PF to induce apoptosis and inhibit the invasiveness and migratory ability of HCC cell lines (Hep3B and Huh7). Wound healing, Transwell migration and invasion, and colony­formation assays, as well as flow cytometry, were used to analyze cell proliferation, migration, invasion, and apoptosis. Epithelial­mesenchymal transition (EMT)­related and apoptotic proteins were assessed by western blotting. The mitochondrial membrane potential of the Hep3B and Huh7 cells was observed with tetramethylrhodamine ethyl ester. The reactive oxygen species (ROS) level was determined by dihydroethidium (DHE) staining. PF treatment significantly decreased the proliferation of Hep3B and Huh7 cells in a dose­dependent manner, reduced the mitochondrial membrane potential, increased ROS levels, decreased the protein levels of Bcl­2, and increased the protein levels of Bax and cleaved caspase­3 and 9, suggesting that PF mediated HCC apoptosis via a mitochondrial pathway. Our findings showed that PF prevented HCC cell migration and invasion by inhibiting the EMT process and downregulating MMP­2 and MMP­9 activities. The results suggest the potential anticancer effects of PF by inhibiting proliferation, inducing apoptosis, and reducing the invasion and migration of HCC cells.

Indole-3-Carbinol Derivative DIM Mitigates Carbon Tetrachloride-Induced Acute Liver Injury in Mice by Inhibiting Inflammatory Response, Apoptosis and Regulating Oxidative Stress.

3,3'-Diindolylmethane (DIM), a metabolic product of indole-3-carbinol extracted from cruciferous vegetables exhibits anti-inflammatory and anti-cancer properties. Earlier, the product has been demonstrated to possess anti-fibrotic properties; however, its protective effects on liver injury have not been clearly elucidated. In this study, we postulated the effects and molecular mechanisms of action of DIM on carbon tetrachloride (CCl4)-induced liver injury in mice. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 ml/kg) into mice. DIM was injected via subcutaneous route for three days at various doses (2.5, 5 and 10 mg/kg) before CCl4 injection. Mice were sacrificed and serum was collected for quantification of serum transaminases. The liver was collected and weighed. Treatment with DIM significantly reduced serum transaminases levels (AST and ALT), tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by DIM treatment by the reduction in the levels of cleaved caspase-3 and Bcl2 associated X protein (Bax). DIM treated mice significantly restored Cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in CCl4 treated mice. In addition, DIM downregulated overexpression of hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4 mediated apoptosis. Our results suggest that the protective effects of DIM against CCl4- induced liver injury are due to the inhibition of ROS, reduction of pro-inflammatory mediators and apoptosis.