The herbal medicine inchin-ko-to (TJ-135) induces apoptosis in cultured rat hepatic stellate cells

Use of herbal remedies in the treatment of various diseases has a long tradition in Eastern medicine and the liver diseases are not an exception. In their use, lack of elucidation of mechanism(s) as well as randomized, placebo-controlled clinical trials has been a problem. Recently, we and others reported that inchin-ko-to (TJ-135), one of herbal remedies, suppressed hepatic fibrosis in animal models. In the course of clarifying the mechanism, we directed our focus on hepatic stellate cells (HSCs), playing a pivotal role in hepatic fibrosis, and found that rat HSCs cultured with TJ-135 changed their morphology to star-like configuration with thin, slender and dendritic processes with fewer stress fibers, which might be the features in apoptosis. In fact, TJ-135 induced HSC apoptosis in a time- and concentration-dependent manner as judged by the nuclear morphology, quantitation of cytoplasmic histone-associated DNA oligonucleosome fragments and caspase 3 activity. In HSCs treated with TJ-135, increased expression of p53 and decreased expression of Bcl-2 and phosphorylated Akt and Bad were determined. HSC apoptosis is shown to be involved in the mechanisms of spontaneous resolution of rat hepatic fibrosis and the agent which induces HSC apoptosis has been shown to reduce experimental hepatic fibrosis in rats. Thus, the induction of HSC apoptosis could be the mechanism how TJ-135 works on the resolution of hepatic fibrosis. Our current data may shed light on the novel effect of the herbal remedy.     

Tetramethylpyrazine induces G0/G1 cell cycle arrest and stimulates mitochondrial-mediated and caspase-dependent apoptosis through modulating ERK/p53 signaling in hepatic stellate cells in vitro

Activation of hepatic stellate cells (HSCs) is a pivotal event in the pathogenesis of liver fibrosis. Pharmacological induction of HSC apoptosis could be a promising strategy for fibrosis regression. Natural product tetramethylpyrazine (TMP) exhibits potent antifibrotic activities in vivo. However, the molecular mechanisms remain to be defined. The present study aimed at investigating the anti-proliferative and pro-apoptotic effects of TMP on HSCs and elucidating the underlying mechanisms. Our results demonstrated that TMP had no apparent cytotoxic effects on hepatocytes, but significantly inhibited HSC proliferation and induced cell cycle arrest at the G0/G1 checkpoint. These effects were associated with TMP regulation of cyclin D1, p21, p27 and p53. Furthermore, we found that TMP disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that TMP selectively blocked the extracellular signal-regulated kinase (ERK) signaling and activated p53, which was required for TMP induction of caspase-dependent mitochondrial apoptosis in HSCs. Autodock simulations predicted that TMP could directly bind to ERK2 with two hydrogen bonds and low energy score, indicating that ERK2 could be a direct target molecule for TMP within HSCs. Moreover, TMP altered expression of some marker proteins relevant to HSC activation. These data collectively revealed that TMP modulation of ERK/p53 signaling led to mitochondrial-mediated and caspase-dependent apoptosis in HSCs in vitro. These studies provided mechanistic insights into the antifibrotic properties of TMP that may be exploited as a potential option for hepatic fibrosis.     

Wnt signaling enhances the activation and survival of human hepatic stellate cells

Wnt signaling was implicated in pulmonary and renal fibrosis. Since Wnt activity is enhanced in liver cirrhosis, Wnt signaling may also participate in hepatic fibrogenesis. Thus, we determined if Wnt signaling modulates hepatic stellate cell (HSC) activation and survival. Wnt3A treatment significantly activated human HSCs, while this was inhibited in secreted frizzled-related protein 1 (sFRP1) overexpressing cells. Wnt3A treatment significantly suppressed TRAIL-induced apoptosis in control HSCs versus sFRP1 over-expressing cells. Particularly, caspase 3 was more activated in sFRP1 over-expressing cells following TRAIL and Wnt3A treatment. These observations imply that Wnt signaling promotes hepatic fibrosis by enhancing HSC activation and survival.     

Loss of expression of miR-335 is implicated in hepatic stellate cell migration and activation

Activation and migration of resident stellate cells (HSCs) within the hepatic space of Disse play an important role in hepatic fibrosis, which accounts for the increased numbers of activated HSCs in areas of inflammation during hepatic fibrosis. Currently, microRNAs have been found to play essential roles in HSC differentiation, proliferation, apoptosis, fat accumulation and collagen production. However, little is known about microRNA mediated HSC activation and migration. In this study, the miRNA expression profiles of quiescent HSCs, partially activated HSCs and fully activated HSCs were compared in pairs. Gene ontology (GO) and GO-Map network analysis indicated that the activation of HSCs was regulated by microRNAs. Among them miR-335 was confirmed to be significantly reduced during HSC activation by qRT-PCR, and restoring expression of miR-335 inhibited HSC migration and reduced α-SMA and collagen type I. Previous study revealed that tenascin-C (TNC), an extracellular matrix glycoprotein involved in cell migration, might be a target of miR-335. Therefore, we further studied the TNC expression in miR-335 over-expressed HSCs. Our data showed that exogenous TNC could enhance HSC migration in vitro and miR-335 restoration resulted in a significant inhibition of TNC expression. These results demonstrated that miR-335 restoration inhibited HSC migration, at least in part, via downregulating the TNC expression.     

Cytotoxic effects of the conjugated linoleic acid isomers t10c12, c9t11-CLA and mixed form on rat hepatic stellate cells and CCl4-induced hepatic fibrosis

Rat hepatic stellate cells (HSC-T6) were incubated for 24 h with 10-180 microM of t10c12 (98%), c9t11 (96%) and a mixed form (c9,t11:t10,c12; 41%:44%) of conjugated linoleic acid (CLA). The MTS dye reduction was measured to verify cell viability in a dose-dependent manner. Among the three CLAs, c9,t11-CLA exhibited the most intense cytotoxic effect on HSCs, the survival rate of which was reduced to 60% under 80 microM of treatment, while cell survival was slightly affected by the mixed form. Three CLA-induced cell deaths were determined by measuring DNA fragmentation using 4',6-diamidino-2-phenylindole staining. The degrees of DNA fragmentation were the most severe in HSC treated with 80 microM of c9,t11-CLA. The mitogen-activated protein kinase/extracellular signal-regulated kinase-kinase and mitogen-activated or extracellular signal-regulated protein kinase (MEK) 1 and 2 were not activated in the t10,c12-CLA treatment. This suggests that the MEK-dependent apoptosis signal is crucial in HSC, which is induced by c9,t11 and mixed CLA. In order to evaluate the protective effect of CLA on carbon tetrachloride (CCl4)-induced hepatic fibrosis in vivo, animals were treated with 10% CCl4 to induce hepatic fibrosis during all experimental periods. Rats were divided into two treatment groups: (1) control diet with tap water ad libitum (n=15) and (2) 1% CLA diet with tap water ad libitum (n=15). In the CLA-supplemented rat livers, alpha-smooth muscle actin-positive cells were significantly reduced around the portal vein. In addition, collagen fibers were not detected in the CLA-treated group. These results suggest that 9c,11t-CLA influences cytotoxic effect on HSC in an MEK-dependent manner and preserving liver from fibrosis.     

Transforming growth factor-beta1 downregulation of Smad1 gene expression in rat hepatic stellate cells

Smads are intracellular signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily that play an important role in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis. Excepting the regulation of Smad7, receptor-regulated Smad gene expression is still unclear. We employed rat HSCs to investigate the expression and regulation of the Smad1 gene, which is a bone morphogenetic protein (BMP) receptor-regulated Smad. We found that the expression and phosphorylation of Smad1 are increased during the activation of HSCs. Moreover, TGF-beta significantly inhibits Smad1 gene expression in HSCs in a time- and dose-dependent manner. Furthermore, although both TGF-beta1 and BMP2 stimulate the activation of HSCs, they have different effects on HSC proliferation. In conclusion, Smad1 expression and phosphorylation are increased during the activation of HSCs and TGF-beta1 significantly inhibits the expression of the Smad1 gene.     

Rho kinase promotes alloimmune responses by regulating the proliferation and structure of T cells

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling.     

Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway

The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT(1)R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension.     

Hepatic stellate cell damage may lead to decreased plasma ADAMTS13 activity in rats

ADAMTS13 is gaining attention, because its deficiency causes thrombotic thrombocytopenic purpura. Although its regulatory mechanism is not fully understood, we wondered if hepatic stellate cells (HSCs) play a role, because ADAMTS13 mRNA is exclusively expressed in the liver and primarily in HSCs. Plasma ADAMTS13 activity was markedly reduced in dimethylnitrosamine-treated rats, where HSC apoptosis is an essential event, but not in carbon tetrachloride- or thioacetamide-treated rats without HSC apoptosis. Furthermore, plasma ADAMTS13 activity was also reduced in 70% hepatectomized rats, where HSC loss occurs. These results suggest that HSC may be involved in the regulation of plasma ADAMTS13 activity.     

The ATM-BID pathway regulates quiescence and survival of haematopoietic stem cells

BID, a BH3-only BCL2 family member, functions in apoptosis as well as the DNA-damage response. Our previous data demonstrated that BID is an ATM effector acting to induce cell-cycle arrest and inhibition of apoptosis following DNA damage. Here we show that ATM-mediated BID phosphorylation plays an unexpected role in maintaining the quiescence of haematopoietic stem cells (HSCs). Loss of BID phosphorylation leads to escape from quiescence of HSCs, resulting in exhaustion of the HSC pool and a marked reduction of HSC repopulating potential in vivo. We also demonstrate that BID phosphorylation plays a role in protecting HSCs from irradiation, and that regulating both quiescence and survival of HSCs depends on BID's ability to regulate oxidative stress. Moreover, loss of BID phosphorylation, ATM knockout or exposing mice to irradiation leads to an increase in mitochondrial BID, which correlates with an increase in mitochondrial oxidative stress. These results show that the ATM-BID pathway serves as a critical checkpoint for coupling HSC homeostasis and the DNA-damage stress response to enable long-term regenerative capacity.     

Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration

Activated hepatic stellate cells (HSCs) are the main producers of extracellular matrix in the fibrotic liver and are involved in the regulation of hepatic inflammation. The aim of this study was to characterize the role of regulated on activation, normal T-cell expressed, and presumably secreted (RANTES) in activated HSCs. RANTES mRNA and protein secretion were strongly induced after stimulating HSCs with TNF-alpha, IL-1beta, or CD40L. RANTES production was NF-kappaB dependent, because inhibitor-kappaB (IkappaB) superrepressor and dominant-negative IkappaB kinase-2 almost completely blocked RANTES expression. NF-kappaB activation was sufficient to drive RANTES expression as demonstrated by the strong induction of RANTES in HSCs expressing NF-kappaB-inducing kinase. The JNK/activator protein-1 pathway also contributed to RANTES expression as demonstrated by the blocking effects of the JNK inhibitor SP600125. HSCs responded to stimulation with recombinant human (rh)RANTES with an increase in intracellular calcium concentration and a rapid increase in free radical formation. Furthermore, rhRANTES induced ERK phosphorylation, ERK-dependent [3H]thymidine incorporation, and HSC proliferation. Additionally, rhRANTES induced focal adhesion kinase phosphorylation and a substantial increase in HSC migration. HSCs functionally expressed chemokine receptor-5 (CCR5), as shown by flow-cytometric analysis and RT-PCR, and the inhibitory effects of a blocking CCR5 antibody on rhRANTES-induced ERK activation, proliferation, and migration. Diphenylene iodonium and N-acetylcysteine inhibited rhRANTES-induced ERK activation and HSC proliferation, indicating that NADPH oxidase-dependent production of reactive oxygen species was required. In conclusion, RANTES and CCR5 represent potential mediators of 1) HSC migration and proliferation and 2) a cross-talk between HSCs and leukocytes during fibrogenesis.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

Increased contractility of hepatic stellate cells in cirrhosis is mediated by enhanced Ca2+-dependent and Ca2+-sensitization pathways

Activation of hepatic stellate cells (HSCs) results in cirrhosis and portal hypertension due to intrahepatic resistance. Activated HSCs increase their contraction after receptor agonist stimulation; however, the signaling pathways for the regulation of contraction are not fully understood. The aim of this study was to elucidate the change in contractile mechanisms of HSCs after cirrhotic activation. The expression pattern of contractile regulatory proteins was analyzed with quantitative RT-PCR and Western blotting. The phosphorylation levels of myosin light chain (MLC), 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), and MLC phosphatase targeting subunit 1 (MYPT1) after endothelin-1 (ET-1) stimulation in culture-activated HSCs were measured using phosphorylation-specific antibodies. In vivo-activated HSCs were isolated from rats subjected to bile duct ligation and repeated dimethylnitrosoamine injections. HSCs showed increased expression of not only α-smooth muscle actin, but also the contractile regulatory proteins MLC kinase (MLCK), Rho kinase 2 (ROCK2), and CPI-17 during HSC activation in vitro. In culture-activated HSCs, ET-1 increased phosphorylation of CPI-17 at Thr18, which was markedly inhibited by the PKC inhibitor Ro-31-8425. ET-1 induced phosphorylation of MYPT1 at Thr853, which was suppressed by the ROCK inhibitor Y-27632. ET-1 induced sustained phosphorylation of MLC at Thr18/Ser19, which was inhibited by both Ro-31-8425 and Y-27632. Consistent with the data obtained from the in vitro study, HSCs isolated from cirrhotic rats showed increased expression of α-smooth muscle actin, MLCK, CPI-17, and ROCK2 compared with HSCs from nontreated rats. Furthermore, MLC phosphorylation in in vivo-activated HSCs was increased, according to enhanced phosphorylation of CPI-17 and MYPT1 in the presence of ET-1. These results suggest that activated HSCs may participate in constriction of hepatic sinusoids in the cirrhotic liver through both Ca(2+)-dependent (MLCK pathway) and Ca(2+)-sensitization mechanism (CPI-17 and MYPT1 pathways).     

NP603, a novel and potent inhibitor of FGFR1 tyrosine kinase, inhibits hepatic stellate cell proliferation and ameliorates hepatic fibrosis in rats

Fibroblast growth factor 2 (FGF-2) and its main receptor FGFR1 have been shown to promote hepatic stellate cell (HSC) activation and proliferation. However, scant information is available on the anti-fibrogenic activity of FGFR1 inhibitors. The aim of this study was to assess the impact of a selective FGFR1 tyrosine kinase inhibitor NP603 on HSC proliferation and hepatic fibrosis. We demonstrated that rat primary HSCs secreted significant amounts of FGF-2, and its tyrosine phosphorylation of FGFR1 was attenuated by NP603. NP603 inhibited HSC activaton by measuring the expression of α-smooth muscle actin (α-SMA) and the production of type I collagen using ELISA. Furthermore, NP603 (25 μM) in vitro strongly suppressed HSC growth induced by FGF-2 (10 ng/ml) and FCS. This effect correlated with the suppression of extracellular-regulated kinase (ERK) activity and its downstream targets cyclin D1 and p21. In addition, PO NP603 (20 mg·kg(-1)·day(-1)) administration significantly decreased hepatic collagen deposition and α-SMA expression in CCl(4)-treated rats. Collectively, these studies suggest that selective blocking of the FGFR1-mediated pathway could be a promising therapeutic approach for the treatment of hepatic fibrosis.