Huanhuan Jin

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.

Activation of Fas death receptor pathway and Bid in hepatocytes is involved in saikosaponin D induction of hepatotoxicity.

Drug-induced liver injury can lead to acute liver failure. Saikosaponin D (SSD) is a major component isolated from the medicinal herb Bupleurum falcatum, which has been linked to hepatotoxicity. We previously reported that SSD disrupted PDGF-βR pathway leading to mitochondrial apoptosis in human LO2 hepatocytes. The present study was aimed at further exploring the underlying mechanisms in vitro and in vivo. We initially determined the concentration range of SSD at up to 2μM for subsequent apoptosis examinations. SSD significantly upregulated Fas expression, promoted caspase-8 cleavage and activated the pro-apoptotic protein Bid in LO2 cells. Moreover, SSD reduced the abundance of cytochrome c in mitochondria and increased the cleaved-caspase-3 in LO2 cells, but did not apparently affect PI3K/AKT, ERK and STAT3 pathways that are involved in cell fate regulation. Experiments in vivo showed that one-week treatment with SSD at 300 mg/kg significantly elevated the liver/body weight ratio and caused histological injury in mouse liver. Furthermore, SSD treatment induced massive hepatocyte apoptosis, and significantly downregulated Bcl-2 but upregulated Bax in mouse liver. Taken together, these results revealed a specific mechanism of activation of extrinsic apoptosis pathway and Bid by SSD, which was involved in SSD-induced mitochondrial apoptosis in hepatocytes and potential hepatotoxicity.

Diallyl trisulfide attenuates ethanol-induced hepatic steatosis by inhibiting oxidative stress and apoptosis

Inhibiting the major characteristics of alcoholic fatty liver (AFL) such as lipid accumulation, oxidative stress and apoptosis is a promising strategy of treating AFL. Diallyl trisulfide (DATS) is the major constituent isolated from garlic, which shows promise in the treatment of chronic liver disease. However, the effects of DATS on ethanol-induced liver injury and the related mechanisms remain unclear. The aim of this study was to evaluate the potential protective effects of DATS on AFL and the potential mechanisms. A single intragastric dose of ethanol was given to rats in vivo, while ethanol-stimulated LO2 cells were used as an in vitro model. Our results demonstrated that DATS prevented ethanol-induced injury, as indicated by the reduced activities of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum and culture medium, and inhibition of cell apoptosis. Furthermore, DATS reduced hepatic steatosis by up-regulating the expression of peroxisome proliferator-activated receptor-alpha (PPAR-α) and down-regulating the expression of sterolregulatory element binding protein 1c(SREBP-1c). In addition, DATS alleviated ethanol-induced oxidative stress by enhancing non-enzymatic antioxidant and enzymatic antioxidants contents and by reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). These data collectively revealed that DATS protected ethanol-induced liver injury by inhibiting lipid accumulation and oxidative stress.

Canonical hedgehog signalling regulates hepatic stellate cell‐mediated angiogenesis in liver fibrosis

Hepatic stellate cells (HSCs) are liver‐specific pericytes regulating angiogenesis during liver fibrosis. We aimed to elucidate the mechanisms by which hedgehog signalling regulated HSC angiogenic properties and to validate the therapeutic implications.

Curcumin inhibits aerobic glycolysis in hepatic stellate cells associated with activation of adenosine monophosphate-activated protein kinase

Activation of hepatic stellate cells (HSCs) is characterized by expression of extracellular matrix and loss of adipogenic phenotype during liver fibrogenesis. Emerging evidence suggests that HSCs adopt aerobic glycolysis during activation. The present work aimed at investigating whether the anti‐fibrogenic effects of curcumin was associated with interfering with glycolysis in HSCs. Primary rat HSCs were cultured in vitro. We demonstrated that inhibition of glycolysis by 2‐deoxyglucose or galloflavin reduced the expression of α‐smooth muscle actin (α‐SMA) and α1(I)procollagen at both mRNA and protein levels, and increased the intracellular lipid contents and upregulated the gene and protein expression of adipogenic transcription factors C/EBPα and PPAR‐γ in HSCs. Curcumin at 20 μM produced similar effects. Moreover, curcumin decreased the expression of hexokinase (HK), phosphofructokinase‐2 (PFK2), and glucose transporter 4 (glut4), three key glycolytic parameters, at both mRNA and protein levels. Curcumin also reduced lactate production concentration‐dependently in HSCs. Furthermore, curcumin increased the phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK), but AMPK inhibitor BML‐275 significantly abolished the curcumin downregulation of HK, PFK2, and glut4. In addition, curcumin inhibition of α‐SMA and α1(I)procollagen was rescued by BML‐275, and curcumin upregulation of C/EBPα and PPAR‐γ was abrogated by BML‐275. These results collectively indicated that curcumin inhibited glycolysis in an AMPK activation‐dependent manner in HSCs. We revealed a novel mechanism for curcumin suppression of HSC activation implicated in antifibrotic therapy.

Ligustrazine disrupts lipopolysaccharide-activated NLRP3 inflammasome pathway associated with inhibition of Toll-like receptor 4 in hepatocytes

Intestine microbial products may translocate into the liver via portal vein and trigger or exacerbate hepatocyte inflammatory responses during liver injury. The NLRP3 inflammasome pathway plays a key role in regulation of inflammatory cytokines in response to bacterial products. The present study was aimed to investigate the effects of ligustrazine, a natural alkaloid compound, on the NLRP3 inflammasome pathway activation and interleukin-1β (IL-1β) generation in hepatocytes. We cultured human LO2 hepatocytes and treated them with lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, for mimicking hepatic exposure to microbial products in vitro. The results demonstrated that LPS upregulated NLRP3 and cleaved-caspase-1, and promoted the expression and secretion of IL-1β in LO2 cells. Ligustrazine was found to reduce NLRP3 and cleaved-caspase-1, prevented IL-1β cleavage, and decreased IL-1β secretion into extracellular environment. Further examinations showed that LPS upregulated the expression of Toll-like receptor 4 (TLR4), but ligustrazine repressed TLR4 expression in LPS-treated hepatocytes. Moreover, pharmacological inhibition of TLR4 by its specific inhibitor TAK-242 downregulated NLRP3 and cleaved-caspase-1, and combination treatment with TAK-242 and ligustrazine led to more significant inhibitory effects on the NLRP3 pathway. TAK-242 also reduced cleaved-IL-1β, and this reducing effect was enhanced by ligustrazine. Collectively, the current results revealed that ligustrazine interrupted LPS-activated NLRP3 inflammasome signaling and reduced generation of IL-1β in hepatocytes, which was associated with inhibition of TLR4. This study uncovered a novel mechanism for ligustrazine as a potential hepatoprotective agent.

Inhibition of YAP signaling contributes to senescence of hepatic stellate cells induced by tetramethylpyrazine.

&NA; Accumulating evidence indicates that hepatic stellate cells (HSCs) are the central mediators and major effectors in the development of hepatic fibrosis. It is well‐known that regulation of cell proliferation and apoptosis are potential strategies to block the activation of HSCs. Recently, several studies have revealed that induction of HSC senescence could prevent and cure the liver fibrosis. In our previous work, we have demonstrated that the natural product tetramethylpyrazine (TMP) could inhibit the activation of HSCs and ameliorate hepatic fibrosis. The aim of this study was to identify a new role of TMP in the regulation of activated HSC senescence and to elucidate the underlying mechanisms. In this study, our data showed that TMP could promote HSC senescence in vivo and in vitro. Moreover, TMP affected the cell cycle and telomerase activity. We further demonstrated that P53 siRNA or P53 pharmacological inhibitor PFT‐&agr; abrogated the TMP‐induced HSC senescence in vitro. Meanwhile, similar results were obtained in vivo. Further studies indicated that TMP promoted the expression of P53 through a YAP inhibition‐dependent mechanism. Moreover, silencing YAP enhanced TMP induction of activated HSC senescence. Collectively, our results suggested that TMP inhibited the activation of HSCs by inducing senescence and had therapeutic implication for the treatment of liver fibrosis. Graphical abstract Figure. No caption available.