Jiangjuan Shao

Activation of PPARγ/P53 signaling is required for curcumin to induce hepatic stellate cell senescence.

Activation of quiescent hepatic stellate cells (HSCs) is the major event in hepatic fibrogenesis, along with enhancement of cell proliferation and overproduction of extracellular matrix. Although inhibition of cell proliferation and induction of apoptosis are potential strategies to block the activation of HSCs, a better understanding of the senescence of activated HSCs can provide a new therapeutic strategy for prevention and treatment of liver fibrosis. The antioxidant curcumin, a phytochemical from turmeric, has been shown to suppress HSC activation in vitro and in vivo. The current work was aimed to evaluate the effect of curcumin on senescence of activated HSCs and to elucidate the underlying mechanisms. In this study, curcumin promoted the expression of senescence marker Hmga1 in rat fibrotic liver. In addition, curcumin increased the number of senescence-associated β-galactosidase-positive HSCs in vitro. At the same time, curcumin induced HSC senescence by elevating the expression of senescence markers P16, P21 and Hmga1, concomitant with reduced abundance of HSC activation markers α-smooth muscle actin and α1(I)-procollagen in cultured HSCs. Moreover, curcumin affected the cell cycle and telomerase activity. We further demonstrated that P53 pharmacological inhibitor pifithrin-α (PFT-α) or transfection with P53 siRNA abrogated the curcumin-induced HSC senescence in vitro. Meanwhile, curcumin disruption of P53 leading to increased senescence of activated HSCs was further verified in vivo. Further studies indicated that curcumin promoted the expression of P53 through a PPARγ activation-dependent mechanism. Moreover, promoting PPARγ transactivating activity by a PPARγ agonist 15d-PGJ2 markedly enhanced curcumin induction of senescence of activated HSCs. However, the PPARγ antagonist PD68235 eliminated curcumin induction of HSC senescence. Taken together, our results provided a novel insight into the mechanisms underlying curcumin inhibition of HSC activation through inducing senescence.

Autophagy regulates turnover of lipid droplets via ROS-dependent Rab25 activation in hepatic stellate cell

Activation of hepatic stellate cells (HSCs) is a pivotal event in liver fibrosis, characterized by dramatic disappearance of lipid droplets (LDs). Although LD disappearance has long been considered one of the hallmarks of HSC activation, the underlying molecular mechanisms are largely unknown. In this study, we sought to investigate the role of autophagy in the process of LD disappearance, and to further examine the underlying mechanisms in this molecular context. We found that LD disappearance during HSC activation was associated with a coordinate increase in autophagy. Inhibition or depletion of autophagy by Atg5 siRNA impaired LD disappearance of quiescent HSCs, and also restored lipocyte phenotype of activated HSCs. In contrast, induction of autophagy by Atg5 plasmid accelerated LD loss of quiescent HSCs. Importantly, our study also identified a crucial role for reactive oxygen species (ROS) in the facilitation of autophagy activation. Antioxidants, such as glutathione and N-acetyl cysteine, significantly abrogated ROS production, and in turn, prevented autophagosome generation and autophagic flux during HSC activation. Besides, we found that HSC activation triggered Rab25 overexpression, and promoted the combination of Rab25 and PI3KCIII, which direct autophagy to recognize, wrap and degrade LDs. Down-regulation of Rab25 activity, using Rab25 siRNA, blocked the target recognition of autophagy on LDs, and inhibited LD disappearance of quiescent HSCs. Moreover, the scavenging of excessive ROS could disrupt the interaction between autophagy and Rab25, and increase intracellular lipid content. Overall, these results provide novel implications to reveal the molecular mechanism of LD disappearance during HSC activation, and also identify ROS-Rab25-dependent autophagy as a potential target for the treatment of liver fibrosis.

ROS-JNK1/2-dependent activation of autophagy is required for the induction of anti-inflammatory effect of dihydroartemisinin in liver fibrosis

Accumulating evidence identifies autophagy as an inflammation-related defensive mechanism against diseases including liver fibrosis. Therefore, autophagy may represent a new pharmacologic target for drug development to treat liver fibrosis. In this study, we sought to investigate the effect of dihydroartemisinin (DHA) on autophagy, and to further examine the molecular mechanisms of DHA-induced anti-inflammatory effects. We found that DHA appeared to play an essential role in controlling excessive inflammation. DHA suppressed inflammation in rat liver fibrosis model and inhibited the expression of proinflammatory cytokines in activated hepatic stellate cells (HSCs). Interestingly, DHA increased the autophagosome generation and autophagic flux in activated HSCs, which is underlying mechanism for the anti-inflammatory activity of DHA. Autophagy depletion impaired the induction of anti-inflammatory effect of DHA, while autophagy induction showed a synergistic effect with DHA. Importantly, our study also identified a crucial role for reactive oxygen species (ROS) in the facilitation of DHA-induced autophagy. Antioxidants, such as glutathione and N-acetyl cysteine, significantly abrogated ROS production, and in turn, prevented DHA-induced autophagosome generation and autophagic flux. Besides, we found that c-Jun N-terminal kinase1/2 (JNK1/2) was a downstream signaling molecule of ROS that mediated the induction of autophagy by DHA. Down-regulation of JNK1/2 activity, using selective JNK1/2 inhibitor (SP600125) or siJNK1/2, led to an inhibition of DHA-induced autophagy. Overall, these results provide novel implications to reveal the molecular mechanism of DHA-induced anti-inflammatory effects, by which points to the possibility of using DHA based proautophagic drugs for the treatment of inflammatory diseases.

Diallyl trisulfide protects against ethanol-induced oxidative stress and apoptosis via a hydrogen sulfide-mediated mechanism.

Garlic is one natural source of organic sulfur containing compounds and has shown promise in the treatment of chronic liver disease. Dietary garlic consumption is inversely correlated with the progression of alcoholic fatty liver (AFL), although the exact underlying mechanisms are not clear. Our previous studies also have shown that diallyl trisulfide (DATS), the primary organosulfur compound from Allium sativum L, displayed anti-lipid deposition and antioxidant properties in AFL. The aim of the present study was to clarify the underlying mechanisms. In the present study, we used the intragastric infusion model of alcohol administration and human normal liver cell line LO2 cultured with suitable ethanol to mimic the pathological condition of AFL. We showed that accumulation of intracellular reactive oxygen species (ROS) was lowered significantly by the administration of DATS, but antioxidant capacity was increased by DATS. Additionally, DATS inhibited hepatocyte apoptosis via down-regulating Bax expression and up-regulating Bcl-2 expression, and attenuated alcohol-induced caspase-dependent apoptosis. More importantly, using iodoacetamide (IAM) to block hydrogen sulfide (H2S) production from DATS, we noted that IAM abolished all the above effects of DATS in ethanol-treated LO2 cells. Lastly, we found DATS could increase the expressions of cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS), the major H2S-producing enzymes. These results demonstrate that DATS protect against alcohol-induced fatty liver via a H2S-mediated mechanism. Therefore, targeting H2S may play a therapeutic role for AFL.

Tetramethylpyrazine prevents ethanol-induced hepatocyte injury via activation of nuclear factor erythroid 2-related factor 2

AIMS Inhibiting the major features of alcoholic liver disease (ALD) such as lipid accumulation and oxidative stress is a promising strategy of treating ALD. Tetramethylpyrazine (TMP) has curative effects on various diseases. However, the effects of TMP on ethanol-induced hepatocyte injury and the related mechanisms remain unclear. The aim of this study is to elucidate the effects of TMP and the potential mechanisms in vitro. MAIN METHODS Ethanol-stimulated LO2 cells were used as an in vitro model of ALD. Several biomarkers related to cell injury, lipid accumulation, and oxidative stress were determined to evaluate the effects of TMP. Nuclear factor erythroid 2-related factor 2 (Nrf2) expression plasmids and Nrf2 small interfering RNA (siRNA) were used to establish the role of Nrf2. KEY FINDINGS TMP increased Nrf2 expression and nuclear translocation. TMP prevented ethanol-induced hepatocyte injury, as indicated by the enhanced cell viability, reduced activities of aspartate transaminase and alanine aminotransferase in the culture medium, and inhibition of cell apoptosis. Furthermore, TMP reduced the levels of lipid droplets, triglyceride, and total cholesterol, probably by regulating genes related to lipid metabolism. Besides, TMP alleviated ethanol-induced oxidative stress by increasing superoxide dismutase activity and the glutathione level and by reducing the levels of reactive oxygen species and malondialdehyde. In addition, overexpression of Nrf2 enhanced the effects of TMP on cell injury, lipid accumulation, and oxidative stress, whereas Nrf2 siRNA eliminated the effects of TMP. SIGNIFICANCE TMP prevents ethanol-induced hepatocyte injury by inhibiting lipid accumulation and oxidative stress, and via an Nrf2 activation-dependent mechanism.

Ligustrazine prevents alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress.

Alcoholic liver disease (ALD) is a major etiology of liver diseases, causing heavy health burdens personally and socially. Ligustrazine has been widely used in China due to its extensive pharmacological activities. However, the role of ligustrazine in ALD treatment remains unclear. Thus, this study is aimed to make up this gap and further uncover the potential mechanisms. The present work demonstrated that compared with the alcohol feeding group, ligustrazine-treated groups showed a clear decrease in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase activities in serum, and a great improvement in liver histology. Additionally, ligustrazine reduced the number of foci containing CD45 positive cells and the expression of proteins associated with hepatic inflammation, apoptosis, and fibrosis. Further, ligustrazine obviously abolished alcohol-induced hepatic steatosis and hyperlipidemia. In addition, ligustrazine reversed alcohol-induced overexpression of sterol regulatory element-binding protein-1c and fatty acid synthase, and inhibition of peroxisome proliferator-activated receptor-alpha and carnitine palmitoyltransferase 1 in liver. Ligustrazine also ameliorated alcohol-induced increases in reactive oxygen species and malondialdehyde levels, and decreases in glutathione, superoxide dismutase, catalase, and glutathione reductase content in liver. Finally, chronic alcohol feeding inhibited the hepatic expression of nuclear factor erythroid 2-related factor 2 (Nrf2) at both mRNA and protein levels. Ligustrazine promoted Nrf2 expression and nuclear translocation in a dose-dependent manner. Collectively, for the first time, the present study demonstrated that ligustrazine remarkably improved chronic alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress. Further, Nrf2 activation might be requisite for ligustrazine to exert its protective effects.

Interaction between autophagy and senescence is required for dihydroartemisinin to alleviate liver fibrosis

Autophagy and cellular senescence are stress responses essential for homeostasis. Therefore, they may represent new pharmacologic targets for drug development to treat diseases. In this study, we sought to evaluate the effect of dihydroartemisinin (DHA) on senescence of activated hepatic stellate cells (HSCs), and to further elucidate the underlying mechanisms. We found that DHA treatment induced the accumulation of senescent activated HSCs in rat fibrotic liver, and promoted the expression of senescence markers p53, p16, p21 and Hmga1 in cell model. Importantly, our study identified the transcription factor GATA6 as an upstream molecule in the facilitation of DHA-induced HSC senescence. GATA6 accumulation promoted DHA-induced p53 and p16 upregulation, and contributed to HSC senescence. By contrast, siRNA-mediated knockdown of GATA6 dramatically abolished DHA-induced upregulation of p53 and p16, and in turn inhibited HSC senescence. Interestingly, DHA also appeared to increase autophagosome generation and autophagic flux in activated HSCs, which was underlying mechanism for DHA-induced GATA6 accumulation. Autophagy depletion impaired GATA6 accumulation, while autophagy induction showed a synergistic effect with DHA. Attractively, p62 was found to act as a negative regulator of GATA6 accumulation. Treatment of cultured HSCs with various autophagy inhibitors, led to an inhibition of DHA-induced p62 degradation, and in turn, prevented DHA-induced GATA6 accumulation and HSC senescence. Overall, these results provide novel implications to reveal the molecular mechanism of DHA-induced senescence, by which points to the possibility of using DHA based proautophagic drugs for the treatment of liver fibrosis.

Dihydroartemisinin prevents liver fibrosis in bile duct ligated rats by inducing hepatic stellate cell apoptosis through modulating the PI3K/Akt pathway.

As a frequent event following chronic insult, liver fibrosis triggers wound healing reactions, with extracellular matrix components accumulated in the liver. During liver fibrogenesis, activation of hepatic stellate cells (HSCs) is the pivotal event. Fibrosis regression can feasibly be treated through pharmacological induction of HSC apoptosis. Herein we showed that dihydroartemisinin (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, as demonstrated by reduced distribution of α‐SMA‐positive cells and the presence of high number of cleaved‐caspase‐3‐positive cells in vivo, as well as by down‐regulation of Bcl‐2 and up‐regulation of Bax. 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 the phosphoinositide 3‐kinase (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.

Curcumin inhibits cobalt chloride-induced epithelial-to-mesenchymal transition associated with interference with TGF-β/Smad signaling in hepatocytes.

Epithelial-mesenchymal transition (EMT) occurs during adult tissue remodeling responses including carcinogenesis and fibrosis. Existing evidence reveals that hepatocytes can undergo EMT in adult liver, which is critically involved in chronic liver injury. We herein established a hypoxia-induced EMT model in human LO2 hepatocytes treated with cobalt chloride (CoCl2) in vitro, and evaluated the effects of curcumin, a natural antifibrotic compound, on hepatocyte EMT and explored the underlying molecular mechanisms. We found that CoCl2 at non-toxic doses induced a mesenchymal cell phenotype in hepatocytes and upregulated several mesenchymal markers including α-smooth muscle actin, vimentin, N-cadherin, fibronectin and Snail (an EMT-related transcription factor), but downregulated the epithelial marker E-cadherin in hepatocytes. However, curcumin reversed the morphological changes, abrogated the increased expression of mesenchymal markers, and rescued E-cadherin expression in CoCl2-treated hepatocytes, suggesting the inhibition of hepatocyte EMT in vitro. We further found that curcumin interfered with the transforming growth factor-β (TGF-β) signaling by reducing the expression of TGF-β receptor I and inhibiting the expression and phosphorylation of Smad2 and Smad3. Use of SB431542, a specific inhibitor of TGF-β receptor I, demonstrated that interference with the TGF-β/Smad pathway was associated with curcumin suppression of hepatocyte EMT. Our in vivo data showed that curcumin affected hepatic EMT in rat fibrotic liver caused by carbon tetrachloride, which was associated with the inhibition of TGF-β/Smad signaling. These findings characterized a novel mechanism by which curcumin modulated hepatocyte EMT implicated in treatment of liver fibrosis.

Dihydroartemisinin restricts hepatic stellate cell contraction via an FXR‐S1PR2‐dependent mechanism

Hepatic stellate cells (HSCs) are universally acknowledged to play a stimulative role in the pathogenesis of hepatic fibrosis and portal hypertension. HSCs when activated in response to liver injury are characterized with many changes, with HSC contraction being the most common cause of portal hypertension. Previous studies have shown that dihydroartemisinine (DHA) is a potential antifibrotic natural product by inducing HSC apoptosis, whereas the role of DHA in regulating HSC contraction and the mechanisms involved remain a riddle. Recent studies have emphasized on the importance of farnesoid X receptor (FXR) and sphingosine‐1‐phosphate receptor 2 (S1PR2) in controlling cell contractility. This study showed that DHA strongly induced the mRNA and protein expression of FXR in LX‐2 cells in a dose‐ and time‐dependent manner and inhibited HSC activation, implying a conceivable impact of DHA on HSC contraction. The gel contraction assays and fluorescence staining of actin cytoskeleton verified that DHA dose‐dependently limited contraction of collagen lattices and reorganization of actin stress fibers in LX‐2 cells. DHA also decreased the phosphorylation of myosin light chain that is responsible for the contractile force of HSCs. Furthermore, gain‐ or loss‐of‐function analyses exhibited a FXR‐ and S1PR2‐dependent mechanism of inhibiting HSC contraction by DHA, and DHA decreased S1PR2 expression by modulating FXR activation. Subsequent work revealed that inhibition of both Ca2+‐dependent and Ca2+‐sensitization signaling transductions contributed to DHA‐induced HSC relaxation. In summary, these findings suggest that DHA could restrict HSC contraction through modulating FXR/S1PR2 pathway‐mediated Ca2+‐dependent and Ca2+‐sensitization signaling. Our discoveries make DHA a potential candidate for portal hypertension.