| Objective:As a frequent event following chronic insult, liver fibrosis triggers wound healing reactions, with extracellular matrix components accumulated in the liver. It is the common pathological process of various chronic liver diseases. Currently there still have been no effective drugs for prevention and treatment of liver fibrosis. During liver fibrogenesis, activation of hepatic stellate cells (HSCs) and secretion of large amounts of ECM are the pivotal event. Excessive ECM deposition in liver can lead to severe hepatic dysfunctions. If not treated effectively, it can lead to life-threatening conditions such as liver cirrhosis and hepatocarcinoma. Currently, there is no drug approved by the FDA for the treatment of liver fibrosis, while it had many problems like taking long time and big input to develop new antifibrotic drugs. Our purpose is to research the antifibrotic effects of drugs, which are of clear clinical safety and have been widely applied in clinic.Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. by chineses chemists. There are many derivatives of artemisinin, such as artemether, artesunate and dihydroartemisinin (DHA). Accumulating evidence indicates that Artemisinin, artesunate and artemether exhibited an inhibitory effect on hepatic fibrosis, however, the role of DHA in liver fibrosis remains unclear. But not yet reported about this in existing studies. Giving that DHA is the major active metabolite of artemisinin. In this study, we selected DHA to evaluate the in vivo protective effects of DHA on liver injury and fibrogenesis caused by bile duct ligation (BDL) in a rat model and and further explored the underlying mechanisms.Methods:1) The in vivo study:Fifty-six rats were randomly divided into seven groups (eight rats per group). Group 1 was the vehicle control in which rats did not receive BDL or DHA treatment. Group 2 was the sham group without DHA treatment. Group 3 was the BDL group without DHA treatment. Groups 4,5, and 6 were treatment groups in which rats received BDL and intraperitoneal injection of DHA at 3.5,7, and 14mg/kg, respectively. Group 7 was the positive control in which rats received BDL and colchicine treatment. Groups3-7 were bile duct ligated for 6 weeks. After BDL for 14 days, groups 4-6 were intraperitoneally injected with DHA five times a week for 3-6 weeks. At the end of the experiment, the rats were sacrificed after being anesthetized by i.p. pentobarbital (50mg/kg). Blood was collected, and livers were isolated to calculate liver/bodyweight ratios. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase(ALP) and Bilirubin were evaluated in serum samples. Representative sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome stain and sirius red collagen staining. Immunofluorescence staining and Western blotting analysis were used to evaluate the indicators related with HSC activation such as a-SMA, al(I)Procollagen and Fibronectin. In addition, HA, LN and PCIII in liver tissue were detected to examine the antifibrotic capacity of DHA. Immunofluorescence stainingand Western blotting analysis were used to evaluate the effects of DHA on the apoptosis of HSCs.2) The in vitro study:MTT assay, LDH assay, AST, ALT kits were used to evaluate the effects of DHA on LO2 liver cells in vitro. MTT assay and LDH assay were used to detect the effect of DHA on activation of HSC. Futhermore, we detected the effects of DHA on cell cyce and apotosis by flow cytometry、Hoechst33258 staining assays and Western blot. In addition, we further explored the underlying mechanisms.Results:1. Gross examination showed that compared to the normal liver, the rat liver with BDL underwent pathological morphological changes which, however, were relieved by DHA treatment. Meanwhile, colchicine, as a positive control, also effectively protected the rat liver from BDL-induced injury. HE staining further showed that DHA treatment remarkably improved the liver histology, as evidenced by the alleviated hepatic steatosis, necrosis, and centrilobular congestion of inflammatory cells in the liver. Since BDL triggered liver damage through biliary fibrosis, we analyzed biochemical markers after BDL. Serum AST and ALT levels, which were significantly increased in the model group (groups 3), were reduced dose-dependently by DHA in BDL-treated rats. Moreover, DHA at high dose reduced serum ALT and AST levels more significantly than colchicine did. BDL rats were significantly more prone to cholestasis than sham-operated animals, as characterized by hyperbilirubinemia with yellowish skin and ALP. As shown in ELISA assay, DHA decreased the liver and serum levels of TNF-α and IL-6. Hence, the liver may be protected by DHA treatment after BDL due to suppressed injury and inflammation.2. To assess the impact of DHA on hepatic fibrogenesis caused by BDL, liver sections were stained with Masson’s reagent and picro-sirius red to examine collagen expressions in the rat liver. Collagens were severely deposited in the BDL-injured liver, accompanied by nodular formation, but were reduced by DHA dose-dependently in the liver of rats treated with DHA. Measurement of hepatic hydroxyproline further indicated that collagen production was reduced by DHA in rats with liver fibrosis. Furthermore, we examined the liver and serum levels of PCIII, HA and LN, the indicators of liver fibrosis. In the rat receiving DHA treatment, each of these markers decreased compared with those in the model group, indicating reduced collagen accumulation in the fibrotic liver. We examined the protein abundances of a-SMA, a 1(1) procollagen and fibronectin, three key markers of liver fibrosis. Treatment with DHA diminished their protein expressions dose-dependently, and colchicine also significantly inhibited their expressions in the fibrotic rat liver. BDL caused a significant increase in the number of cells recognized by the antibody against a-SMA. Merging the images demonstrated that these labeled cells by the two different antibodies were the same cells, suggesting the colocalization of cleaved Caspase-3 and a-SMA in these cells. DHA treatment significantly reduced the number of cells labeled with a-SMA antibody and increased the number of cleaved Caspase-3-positive cells, suggesting that DHA might suppress HSCs activation by stimulated apoptosis of HSCs in the rat model.3. MTT assay, LDH assay, AST, ALT kits showed that DHA at a wide range of doses (<40μM) had no cytotoxic effect on hepatocytes in vitro, providing an appropriate dose range for evaluating the DHA effects on HSC. Immunofluorescence staining and Western blotting analysis demonstrated that DHA inhibited HSC proliferation and activation. Furthermore, flow cytometry、Hoechst staining assays and Western blot showed that DHA induced cell cycle arrest at the S checkpoint and activated the mitochondrial apoptosis pathway and Caspase cascades in activated HSCs.4. We next investigated the signaling pathway by which DHA induced mitochondrial apoptosis in HSC. In this study, the PI3K pathway was, as demonstrated by the dose-dependently reduced phosphorylation of PI3K and Akt, blocked by DHA in HSCs. We further used LY294002, a specific inhibitor of PI3K, to test the molecular link between disruption of the PI3K/Akt pathway and DHA-induced apoptosis in activated HSCs. LY294002 inhibited Akt phosphorylation, which was significantly enhanced when combined with DHA. To further strengthen the conclusion, EGF was used to activate PI3K/Akt signaling. Western blot showed that DHA at the selected doses significantly abolished EGF-increased PI3K phosphorylation similarly to LY294002 did. Taken together, inhibition of PI3K/Akt signaling indeed mediated DHA-induced HSC apoptosis.Conclusion:In vivo study, our work demonstrated that DHA improved liver histological architecture, decreased hepatic enzyme levels, and inhibited HSCs activation in the fibrotic rat liver. DHA also induced apoptosis of HSCs in such liver. In addition, in vitro experiments showed that DHA significantly inhibited HSC proliferation and led to dramatic morphological alterations in HSCs. we found that DHA disrupted mitochondrial functions and led to activation of Caspase cascades in HSCs. Mechanistic investigations revealed that DHA induced HSC apoptosis through disrupting PI3K/Akt pathway and that PI3K specific inhibitor LY294002 mimicked the pro-apoptotic effect of DHA. DHA is a promising candidate for the prevention and treatment of liver fibrosis. |
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