Chunfeng Lu

Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells

Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver‐specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet‐derived growth factor‐β receptor (PDGF‐βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF‐βR/focal adhesion kinase/RhoA cascade. Gain‐ or loss‐of‐function analyses revealed that activation of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR‐γ activation‐dependent mechanism. PPAR‐γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.

Tetramethylpyrazine reduces inflammation in liver fibrosis and inhibits inflammatory cytokine expression in hepatic stellate cells by modulating NLRP3 inflammasome pathway

Hepatic fibrosis is concomitant with liver inflammation, which has been highlighted as significant treatment of chronic liver disease. We previously demonstrated that tetramethylpyrazine (TMP), the effective component of Ligusticum chuanxiong Hort, can inhibit the activation of HSCs and consequential anti‐hepatic fibrosis. In this study, our work demonstrated that TMP improved liver histological architecture, decreased hepatic enzyme levels and attenuated collagen deposition in the rat fibrotic liver. In addition, TMP significantly protected the liver from CCl4‐caused injury and fibrogenesis by suppressing inflammation with reducing levels of inflammatory cytokines, including tumor necrosis factor‐α (TNF‐α), NLRP3, nuclear factor‐kappa B (NF‐κB) and interleukin‐1β (IL‐1β). Experiments in vitro showed that TMP inhibited inflammatory cytokine expression in HSCs associated with disrupting platelet‐derived growth factor‐b receptor (PDGF‐βR)/NLRP3/caspase1 pathway. These data collectively indicate that TMP can attenuate liver inflammation in liver fibrosis and possibly by targeting HSCs via PDGF‐βR/NLRP3/caspase1 pathway. It provides novel mechanistic insights into TMP as a potential therapeutic remedy for hepatic fibrosis.

Curcumin regulates cell fate and metabolism by inhibiting hedgehog signaling in hepatic stellate cells

Accumulating evidence indicates that Hedgehog (Hh) signaling becomes activated in chronic liver injury and plays a role in the pathogenesis of hepatic fibrosis. Hepatic stellate cells (HSCs) are Hh-responsive cells and activation of the Hh pathway promotes transdifferentiation of HSCs into myofibroblasts. Targeting Hh signaling may be a novel therapeutic strategy for treatment of liver fibrosis. We previously reported that curcumin has potent antifibrotic effects in vivo and in vitro, but the underlying mechanisms are not fully elucidated. This study shows that curcumin downregulated Patched and Smoothened, two key elements in Hh signaling, but restored Hhip expression in rat liver with carbon tetrachloride-induced fibrosis and in cultured HSCs. Curcumin also halted the nuclear translocation, DNA binding, and transcription activity of Gli1. Moreover, the Hh signaling inhibitor cyclopamine, like curcumin, arrested the cell cycle, induced mitochondrial apoptosis, reduced fibrotic gene expression, restored lipid accumulation, and inhibited invasion and migration in HSCs. However, curcumin’s effects on cell fate and fibrogenic properties of HSCs were abolished by the Hh pathway agonist SAG. Furthermore, curcumin and cyclopamine decreased intracellular levels of adenosine triphosphate and lactate, and inhibited the expression and/or function of several key molecules controlling glycolysis. However, SAG abrogated the curcumin effects on these parameters of glycolysis. Animal data also showed that curcumin downregulated glycolysis-regulatory proteins in rat fibrotic liver. These aggregated data therefore indicate that curcumin modulated cell fate and metabolism by disrupting the Hh pathway in HSCs, providing novel molecular insights into curcumin reduction of HSC activation.

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.

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.

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.

Dihydroartemisinin protects against alcoholic liver injury through alleviating hepatocyte steatosis in a farnesoid X receptor-dependent manner.

ABSTRACT Alcoholic liver disease (ALD) is a common etiology of liver diseases, characterized by hepatic steatosis. We previously identified farnesoid X receptor (FXR) as a potential therapeutic target for ALD. Dihydroartemisinin (DHA) has been recently identified to possess potent pharmacological activities on liver diseases. This study was aimed to explore the impact of DHA on ALD and further elaborate the underlying mechanisms. Gain‐ or loss‐of‐function analyses of FXR were applied in both in vivo and in vitro studies. Results demonstrated that DHA rescued FXR expression and activity in alcoholic rat livers. DHA also reduced serodiagnostic markers of liver injury, including aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase. DHA improved alcohol‐induced liver histological lesions, expression of inflammation genes, and inflammatory cell infiltration. In addition, DHA not only attenuated hyperlipidemia but also reduced hepatic steatosis through regulating lipogenesis and lipolysis genes. In vitro experiments further consolidated the concept that DHA ameliorated ethanol‐caused hepatocyte injury and steatosis. Noteworthily, DHA effects were reinforced by FXR agonist obeticholic acid or FXR expression plasmids but abrogated by FXR antagonist Z‐guggulsterone or FXR siRNA. In summary, DHA significantly improved alcoholic liver injury by inhibiting hepatic steatosis, which was dependent on its activation of FXR in hepatocytes. HIGHLIGHTSDHA rescues FXR expression in alcoholic livers.DHA improves alcoholic liver inflammation and steatosis in a FXR‐dependent way.DHA alleviates ethanol‐induced hepatocyte steatosis by activation of FXR.

Nrf2 Activation Is Required for Ligustrazine to Inhibit Hepatic Steatosis in Alcohol-Preferring Mice and Hepatocytes

Hepatic steatosis is the most distinctive feature of alcoholic liver disease (ALD). Our previous in vivo study showed that ligustrazine, a major active alkaloid isolated from Ligusticum chuanxiong Hort, attenuated alcohol-induced hepatic steatosis and in vitro study revealed that nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation might be a prerequisite. This study was aimed to explore the in vivo function of Nrf2 in the protective effect of ligustrazine and illustrate downstream mechanisms. Nrf2 shRNA lentivirus was introduced to establish Nrf2-knockdown mice. Results showed that Nrf2 knockdown aggravated alcoholic liver injury and abolished the protective effect of ligustrazine, evidenced by elevated serum biomarkers and severe liver inflammation. Ligustrazine impressively ameliorated hepatic steatosis through inhibiting hepatic sterol regulatory element-binding protein-1c and inducing peroxisome proliferator-activated receptor-alpha, which was abrogated by Nrf2 knockdown. Noteworthily, Nrf2 knockdown apparently reinforced the inductive effect of alcohol on hypoxia-inducible factor 1-alpha (HIF-1&agr;). Ligustrazine weakened the stimulation of alcohol on HIF-1&agr; expression, which was abrogated by Nrf2 shRNA lentivirus. Consistent results were obtained from in vitro study, implying that Nrf2/HIF-1&agr; pathway participated in the modulation of ligustrazine. Gain- or loss-of-function analyses further revealed that Nrf2 siRNA and dimethyloxalylglycine, a HIF-1&agr; agonist, abolished the inhibitory effect of ligustrazine, whereas HIF-1&agr; siRNA mimicked the role of ligustrazine and even rescued the inhibitory effect of ligustrazine on ethanol-induced lipid accumulation in Nrf2-knockdown hepatocytes. Taken together, we concluded that ligustrazine attenuated alcohol-induced hepatic steatosis via a Nrf2/HIF-1&agr; pathway-dependent mechanism.

Nrf2 knockdown attenuates the ameliorative effects of ligustrazine on hepatic fibrosis by targeting hepatic stellate cell transdifferentiation.

Hepatic fibrosis is a frequent reparative process in response to chronic liver injury and inflammation, which is mainly attributed to hepatic stellate cell (HSC) activation. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was recently highlighted for its negative regulation of HSC behaviors. Our previous studies have revealed the potent antifibrotic effects of ligutrazine without elaborating potential molecular mechanisms. In this work, our in vitro results showed that ligustrazine significantly enhanced Nrf2 expression and nuclear translocation in HSC. Mechanistic investigations using RNAi technology demonstrated that Nrf2 knockdown abolished the ameliorative effects of ligustrazine on serum enzyme activities, hepatic histological architecture, levels of proinflammatory cytokines in serum and liver, intrahepatic inflammatory cell infiltration. Nrf2 shRNA also abrogated the antifibrotic effects of ligustrazine evidenced by increased serum fibrotic biomarkers, hepatic hydroxyproline, profibrogenetic factors in serum and liver, and intrahepatic collagen deposition. Ligustrazine inhibited the induction of CCl4 on β-catenin in HSC, which was cancelled by Nrf2 shRNA lentivirus. In vitro experiments also showed that Nrf2 siRNA abrogated the inhibition of ligustrazine on β-catenin expression. Nrf2 siRNA and IWR-1-endo (a specific antagonist of β-catenin) were applied to investigate the correlation between Nrf2 and β-catenin in mediating the effects of ligustrazine. Results suggested that ligustrazine not only suppressed the viability, contraction, and migration of human HSC but also alleviated lipid droplet loss and extracellular matrix production. Nrf2 siRNA yet weakened the inhibitory action of ligustrazine on HSC behaviors while IWR-1-endo further impaired the suppression of Nrf2 siRNA and restored the capacity of ligustrazine. Collectively, we drew a conclusion that the favorable antifibrotic effects of ligustrazine were attributed to its negative modulation on HSC behaviors by interrupting Nrf2/β-catenin pathway. The findings broaden the width and depth of molecular mechanisms involved in the ligustrazine action, facilitating the development of ligustrazine in antifibrotic therapies.