Thymoquinone And Betulin Inhibit Liver Fibrosis And Alcoholic Liver Steatosis By Activating LKB1-AMPK Signaling Pathway

Aim:Liver fibrosis is a common consequence of chronic liver injury that is induced by a variety of etiological factors that lead to liver cirrhosis. This progressive pathological process is characterized by the accumulation of extracellular matrix (ECM) proteins. Prolonged liver injury results in hepatocyte damage, which triggers activation of hepatic stellate cells (HSCs). HSCs are recognized as the primary cellular source of matrix components in chronic liver disease, and play a critical role in the development and maintenance of liver fibrosis. Furthermore, chronic alcohol use can result in fibrosis, which refers to the extracellular accumulation of collagen and other matrix proteins. Alcohol consumption is a leading cause of global morbidity and mortality, with much of the burden resulting from alcoholic liver disease (ALD). Excessive alcohol intake can lead to liver damage through its direct action as a hepatotoxin as well as potentiation of other liver diseases including chronic viral hepatitis and nonalcoholic fatty liver disease. Therefore, a greater understanding of molecular mechanisms regulating the hepatic fibrosis in liver is needed for the identification of novel targets for successful antifibrotic therapies. We applied in vivo model of thioacetamide (TAA) induced liver fibrosis and chronic-binge ethanol induced alcoholic liver steatosis to ascertain the protective effect and the underlying mechanism. We conducted in vitro and in vivo investigation to reveal the hepatoprotective mechanism of thymoquinone (TQ), the main active ingredient from the seeds of Nigella sativa Linn, and betulin (BT), an abundant naturally occurring triterpene, available from the bark of Betula platyphylla Suk.Methods:(1) TQ attenuates liver fibrosis via TLR4 and LKB1-AMPK signaling pathways. In vitro, we investigated the anti-fibrotic mechanism of TQ in lipopolysaccharide (LPS)-activated rat hepatic stellate cells line, T-HSC/CI-6. T-HSC/CI-6 cells were treated with TQ (3.125,6.25 and 12.5 μM) prior to LPS (1 μg/ml). In vivo, liver fibrosis was induced in male Kunming mice by intraperitoneal injections of TAA (200mg/kg) three weekly for 5 weeks. Mice were treated concurrently with TAA alone or TAA plus TQ (20 mg/kg or 40 mg/kg) given daily by oral gavage. The administration of TQ was started following TAA injections and was continued for 5 weeks to evaluate the protective effects by hematoxylin and eosin (H&E) staining, Masson staining and immunohistochemistry examination. The expression of collagen-I, a-smooth muscle actin (a-SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), toll-like receptor 4 (TLR4), CD14, phosphatidylinositol 3-kinase (PI3K), p-PI3K, serine/threonine kinase-protein kinase B (Akt), p-Akt, c-FLIPL, XIAP, adenosine monophosphate-activated protein kinase (AMPK), p-AMPK, liver kinase B (LKB)1 and p-LKB1 were measured by Western blot and Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR). (2) BT alleviates alcoholic liver steatosis via modulation of LKB1-AMPK signaling pathways. In vitro, human hepatic stellate cell line, LX-2 cells were treated with BT (6.25,12.5 and 25 μM) prior to ethanol (50 mM) for 24 h. In vivo, we induced alcoholic liver steatosis in male C57BL/6 mice, placing them on Lieber-DeCarli ethanol-containing diets for 10 days and then administering a single dose of ethanol (5 g/kg body weight) via gavage. Animals were divided into normal, ethanol, BT 20, BT 50, ethanol+BT 20 and ethanol+BT 50 groups. BT (20 and 50 mg/kg) were given by gavage every day to evaluate the protective effects by H&E staining and immunohistochemistry examination. The expression of collagen-1, α-SMA, sterol regulatory element-binding protein-1 (SREBP-1), NF-κB p65, AMPK, p-AMPK, LKB1, p-LKB1 and sirtuin 1 (SIRT1) were measured by Western blot and immunofluorescence imaging assay.Results:(1) Our in vitro data demonstrated that TQ effectively decreased activated T-HSC/CI-6 cell viability and the expression of CD14 and TLR4. TQ also significantly inhibited PI3K and Akt phosphorylation. The expression of α-SMA and collagen-l were significantly decreased by TQ. In vivo, TQ treatment obviously reversed liver tissue damage compared with TAA alone group, characterized by less inflammatory infiltration and accumulation of ECM proteins. TQ significantly attenuated TAA-induced liver fibrosis, accompanied by reduced protein and mRNA expression of α-SMA, collagen-I and TIMP-1. TQ downregulated the expression of TLR4 and remarkably decreased proinflammatory cytokines levels as well. TQ also significantly inhibited PI3K phosphorylation, which was in line with in vitro results. Furthermore, TQ enhanced the phosphorylation AMPK and LKB1. (2) Our data demonstrated that BT effectively decreased LX-2 cell viability and the expression of SREBP-1. BT also significantly activated LKB1-AMPK phosphorylation. In vivo, BT administration significantly protected liver from injury by reducing serum aminotransferase and triglyceride levels and improving the histological architecture and hepatic steatosis caused by ethanol. BT also significantly inhibited SREBP-1 expression and activated LKB1-AMPK phosphorylation, which were in line with in vitro results. In addition, the expression of SIRT1 was significantly increased by BT in vitro and in vivo.Conclusions:The potential anti-fibrosis mechanism of TQ might be associated with the depression of α-SMA and collagen-I relating with hepatic fibrosis, down-regulating the accompanying inflammatory response in hepatic fibrosis. TQ suppressed TLR4-PI3K signaling and activated LKB1-AMPK signaling in TAA-induced mouse hepatic fibrosis. BT alleviates alcoholic liver steatosis possibly through blocking SREBP-1 regulated fatty acid synthesis and activating LKB1-AMPK signaling pathway. Further, enhanced hepatic SIRT1 signaling contributes to the protective action of BT against alcoholic liver steatosis. Thus, both of TQ and BT may be useful as a potential pharmacological therapy for the prevention of liver fibrosis and alcoholic liver steatosis. But further investigation is required to determine the exact protective mechanism and to discover the active candidates.