Proliferation Of HSC-T6 Cells Research Articles

Quercetin induces hepatic stellate cell apoptosis by inhibiting the PI3K/Akt signaling pathway via upregulating miR-146

To investigate whether quercetin induces apoptosis of hepatic stellate cells by regulating miR-146 to inhibit the PI3K/Akt signaling pathway. Rat hepatic stellate cells (HSC-T6) were treated with TGF-β and different concentrations (40, 60 and 80 μmol/L) of quercetin, and the changes in cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry. RT-qPCR was used to detect the expression of miR-146 and mRNA expressions of α-SMA, collagenⅠ, TRAF6, PI3K and Akt in the treated cells, and the protein expressions of α-SMA, collagenⅠ, TRAF6, PI3K, Akt and p-Akt were detected using Western blotting. Immunofluorescence assay was used to detect the protein expression of α-SMA and collagenⅠ. The effects of transfection with miR-146 mimic and inhibitor on the protein expressions of the cells were also examined using Western blotting. Treatment with quercetin dose- and time-dependently inhibited the proliferation of HSC-T6 cells and significantly increased the total cell apoptosis rate (P<0.01). TGF-β-stimulated HSC-T6 cells showed significantly increased mRNA and protein expression levels of α-SMA, collagenⅠ, TRAF6, PI3K and Akt (P<0.05), which were significantly down-regulated by quercetin treatment (P<0.05). Quercetin significantly upregulated the expression of miR-146 in HSC-T6 cells (P<0.01), Transfection of the cells with miR-146 mimic significantly decreased the mRNA and protein expression levels of α-SMA, collagen Ⅰ, TRAF6, PI3K and Akt (P<0.05), and miR- 146 inhibitor produced the opposite effects (P<0.05). Quercetin inhibits proliferation and promotes apoptosis of HSCs by upregulating miR-146 to inhibit the PI3K/Akt signaling pathway.

Herpetetrone nanosuspensions enhance drug solubility and bioavailability to improve anti-hepatic fibrosis effects

The aim of this study was to enhance the dissolution rate and oral bioavailability of herpetetrone (HPT) by preparing nanosuspensions (NSs) and evaluate the changes in its anti-hepatic fibrosis effect. Herpetetrone nanosuspension (HPT-NS) was prepared using the ultrasound-precipitation technique, and characterised on the basis of mean diameter, zeta potential (ZP), encapsulation efficiency percent (EE%), scanning electron microscopy (SEM), and X-ray powder diffraction (XRPD). In addition, the pharmacokinetics and anti-hepatic fibrosis activity were evaluated. HPT-NS prepared with the optimised formulation was found to be spherical with mean diameter of 177.48 ± 6.13 nm, polydispersity index (PDI) of 0.108 ± 0.002 and ZP of −17.28 ± 2.02 mV. The EE (m/m, %) was 83.25 ± 0.27. XRPD analyses confirmed that the amorphous state of HPT in HPT-NS remained unchanged. The dissolution rate of HPT-NS was significantly higher than that of HPT coarse suspensions (HPT-CSs). Following oral administration, C max and AUC0– t of HPT-NS showed a significant increase (p < 0.05). In vitro, HPT inhibited the proliferation of HSC-T6 cells and induced apoptosis by up-regulating the expression of Bax proteins and down-regulating the expression of Bcl-2 and TGF-β1 proteins. Compared with HPT-CS, HPT-NS exhibited a more pronounced anti-fibrotic effect. HPT-NS, as a new drug formulation designed to improve the solubility and bioavailability of the drug, shows promising potential in enhancing the anti-liver fibrosis effect.

Effect of ADHI on hepatic stellate cell activation and liver fibrosis in mice

The relationship between alcohol dehydrogenase (ADH) and liver fibrosis has been studied, but the mechanism of ADH involvement in liver fibrosis remains unclear. The aim of the present study was to explore the role of ADHI, the classical liver ADH, in hepatic stellate cell (HSC) activation and the effect of 4-methylpyrazole (4-MP), an ADH inhibitor, on liver fibrosis induced by carbon tetrachloride (CCl4) in mice. The results showed that overexpression of ADHI significantly increases proliferation, migration, adhesion and invasion rates of HSC-T6 cells as compared with controls. When HSC-T6 cells were activated by ethanol, TGF-β1 or LPS, the expression of ADHI was elevated significantly (P < 0.05). Overexpression of ADHI significantly increased the levels of COL1A1 and α-SMA, markers of HSC activation. Moreover, the expression of COL1A1 and α-SMA was decreased significantly by transfection of ADHI siRNA (P < 0.01). In a liver fibrosis mouse model ADH activity increased significantly and was highest in the 3rd week. The activity of ADH in the liver was correlated with its activity in the serum (P < 0.05). 4-MP significantly decreased ADH activity and ameliorated liver injury, and ADH activity was positively correlated with the Ishak score of liver fibrosis. In conclusion, ADHI plays an important role in the activation of HSC, and inhibition of ADH ameliorates liver fibrosis in mice.

One-Way Intestinal Perfusion of PVP/VA-Poloxamer 188-Curcuma longa L. Extract Solid Dispersion in Rats In Vivo and Its Effect on HSC-T6 Cell Proliferation.

Turmeric was the dried rhizome of Curcuma longa L., and its extract had important pharmacological effects such as anti-tumor, cholagogic, and antioxidant. However, curcuma extract had poor water solubility and low bioavailability, which had become the main limiting factor for its clinical application. The purpose of this study was to prepare PVP/VA-Poloxamer-188-curcuma extract solid dispersion (PAP-CSD) to improve the solubility and bioavailability of the curcuma extract. The intestinal absorption mechanism of solid dispersion of this extract was studied by one-way intestinal perfusion in rats. PAP-CSD,PVP/VA-curcuma extract solid dispersion (PA-CSD) and Poloxamer-188-curcuma extract solid dispersion (P-CSD) was able to improve the intestinal absorption of the curcuma extract (P < 0.05), and PAP-CSD (combined use of two carriers) was better than that of PA-CSD and P-CSD. CCK8 method was used to investigate the effects of the curcuma extract and PAP-CSD on the proliferation of hepatic stellate cells (HSC)-T6 cells. The inhibitory effect of PAP-CSD on the proliferation of HSC-T6 cells, related to the p38 MAPK pathway, was better than that of the curcuma extract.

Study on mechanism of coix seed oil intervening hepatic fibrosis in immune injury rats

This study aims to investigate the effect and specific mechanism of Coix Seed Oil (CSO) on immune hepatic fibrosis in immune injury rats. In this study, healthy Wister rats were injected with porcine serum in order to establish hepatic fibrosis model. We choose the compound Sophora flavescens injection as the positive control drug. HE staining was used to observe the morphological and histological changes. HSC-T6 cells were cultured in vitro. MTT and AO/EB staining were used to detect the effect of CSO on the proliferation and apoptosis of HSC-T6 cells. The results shown that Coix seed oil significantly improved hepatic fibrosis of immune injury rats, and the degree of hepatic fibrosis in CSO group was significantly reduced. Intraperitoneal injection of 15 mg/kg, 30 mg/kg and 40 mg/kg significantly reduced the levels of serum ALT, AST, ALP, HA, LN, PIIINP and C-IV in rats (P < 0.01).), MTT assay showed that the cells proliferated well. The results of HE staining showed that Coix seed oil had a good therapeutic effect on immune-induced hepatic fibrosis in rats. AO/EB staining showed that 0.5 mg/mL CSO for 24 h and 0.1 mg/mL CSO for 48 h both significantly promoted the apoptosis of HSC-T6 cells. In conclusion, Coix seed oil has a significant therapeutic effect on immune hepatic fibrosis, and its molecular mechanism may be related to antagonizing cytokine TGF-β1, inhibiting the expression of TIMP-1, promoting collagen degradation, inhibiting the activation of hepatocyte and promoting the apoptosis of activated hepatic stellate cells.

CD39-mediated ATP-adenosine signalling promotes hepatic stellate cell activation and alcoholic liver disease

CD39 is associated with diverse physiological and pathological processes, including cell proliferation and differentiation. Adenosine triphosphate (ATP) is hydrolysed to adenosine by different enzymes including ecto-nucleoside triphosphate diphosphohydrolase-1/ENTPD1 (CD39) and ecto-5′-nucleotidase (CD73), regulating many physiological and pathological processes in various diseases, but these changes and functions in alcoholic liver disease are generally unknown. In this study, an alcoholic liver disease model in vivo was induced by ethanol plus carbon tetrachloride(CCl4) administered to C57BL/6 mice, who were the intraperitoneally injected with the CD39 inhibitor sodium polyoxotungstate (POM1) or colchicine from the 5th week to the 8th week. Meanwhile, hepatic stellate cells were stimulated by acetaldehyde to replicate alcoholic liver fibrosis models in vitro. Exogenous ATP and POM1 were added in turn to the culture system. Pharmacological blockade of CD39 largely prevents liver damage and collagen deposition. We found that blockade or silencing of CD39 prevented acetaldehyde-induced proliferation of HSC-T6 cells and the expression of fibrogenic factors. Moreover, blockade or silencing of CD39 could block the activation of the adenosine A2A and adenosine A2B receptors and the TGF-β/Smad3 pathway, which are essential events in HSC activation. Thus, blockade of CD39 to inhibit the transduction of ATP to adenosine may prevent HSC activation, alleviating alcoholic hepatic fibrosis. The findings from this study suggest ATP-adenosine signalling is a novel therapeutic and preventive target for alcoholic liver disease.

Carvacrol alleviates liver fibrosis by inhibiting TRPM7 and modulating the MAPK signaling pathway

Liver fibrosis is a compensatory response to the tissue repair process. The activation and proliferation of hepatic stellate cells (HSCs) are thought to be related to the occurrence of hepatic fibrosis. Therefore, inhibiting the activation and proliferation of HSCs is a key step in alleviating liver fibrosis. As a non-specific inhibitor of transient receptor potential melastatin 7 (TRPM7), carvacrol has anti-tumor, anti-inflammatory and anti-hepatic fibrosis activities. This study aimed to explore the protective effect of carvacrol on liver fibrosis and related molecular mechanisms. A CCl4-induced liver fibrosis mouse model and platelet-derived growth factor (PDGF-BB)-activated HSC-T6 cells (a rat hepatic stellate cell line) were employed for in vivo and in vitro experiments. C57BL/6J mice were orally administered different concentrations of carvacrol every day for 6 weeks during the development of CCl4-induced liver fibrosis. The results show that carvacrol could effectively reduce liver damage and the progression of liver fibrosis in mice, which are expressed as fibrotic markers levels were reduced and histopathological characteristics were improved. Moreover, carvacrol inhibited the proliferation and activation of HSC-T6 cells induced by PDGF-BB. In addition, it was found that carvacrol inhibits the expression of TRPM7 and mediated through mitogen-activated protein kinases (MAPK). Collectively, our study shows that carvacrol can reduce liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and the MAPK signaling pathway might be involved in this process.

MicroRNA-92b-3p promotes the progression of liver fibrosis by targeting CREB3L2 through the JAK/STAT signaling pathway

Liver fibrosis is a common feature of almost all chronic liver diseases, which eventually leads to cirrhosis and even hepatocellular carcinoma (HCC). The current study showed that miR-92b plays an important role in the progression of HCC but its role in liver fibrosis is still unclear. Here we aimed to explore the role and underlying molecular mechanism of miR-92b-3p in the activated hepatic stellate cells (HSCs) and the pathological process of hepatic fibrosis. We found that miR-92b-3p was highly expressed both in fibrotic liver tissues from patients and model mice and in activated LX-2 cells stimulated with TGF-β1. However, the expression of miR-92b-3p was downregulated in inactivated LX-2 cells treated with adipogenic differentiation mixture (MDI). In addition, we found that miR-92b-3p mimic could promote the activation, proliferation, and migration of LX-2 and HSC-T6 cells, while miR-92b-3p inhibitor could reverse this process. From the TargetScan databases, we found that CREB3L2 is a potential target of miR-92b-3p and the luciferase assay revealed the suppressed CREB3L2 expression by miR-92b-3p. Mechanistically, we found that miR-92b-3p promotes the activation of HSCs and thereby the progression of liver fibrosis by activating JAK/STAT pathway via targeting CREB3L2, providing a new target for the diagnosis and treatment of liver fibrosis.

Effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice

To investigate the effects of persistent Echinococcus multilocularis infections on hepatic fibrosis in mice, so as to provide insights into the understanding of liver fibrogenesis induced by E. multilocularis infections and the treatment of alveolar echinococcosis. Hepatic stellate HSC-T6 and LX-2 cells were exposed to the sera (25, 50 and 100 μL) from Meriones unguiculatus infected with E. multilocularis, and E. multilocularis, germinal layer cells (GCs) and protoscoleces (PSCs) for 48 hours, respectively. The cell proliferation was measured using a CCK-8 assay, and the levels of collagen 1 (Col1) and α-smooth muscle actin (α-SMA) were measured in the culture supernatant of HSC-T6 cells using ELISA. In addition, the serum and liver samples were collected 1, 2, 4, 6, 8 months post-infection with E. multilocularis, respectively. The serum Col1 and α-SMA concentrations were measured using enzyme-linked immunosorbent assay (ELISA), and the deposition of collagen fibers was examined in mice livers using Sirius red staining. The sera of E. multilocularis-infected gerbils promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences seen in the proliferative rate of HSC-T6 (FHSC-T6 = 126.50, P < 0.05) and LX-2 cells (FLX-2 = 201.50, P < 0.05) among different serum groups, with the highest proliferative rate of HSC-T6 (573.36% ± 206.34%) and LX-2 cells (940.38% ± 61.65%) found following exposure to 100 μL mouse sera. Exposure to serum from E. multilocularis-infected gerbils resulted in an increase in the Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, with the greatest Col1 (20.99 ng/mL ± 2.01 ng/mL) and α-SMA levels (305.52 pg/mL ± 16.67 pg/mL) measured following exposure to 100 μL sera. The metacestodes (142.65% ± 9.17% and 189.99% ± 7.75%), GCs (118.55% ± 8.96% and 122.54% ± 0.21%) and PSCs of E. multilocularis (156.34% ± 17.45% and 160.59% ± 31.41%) all promoted the proliferation of HSC-T6 and LX-2 cells in vitro, and there were significant differences in the proliferative rates of HSC-T6 (FHSC-T6 = 11.24, P < 0.05) and LX-2 cells among groups (FLX-2 = 47.72, P < 0.05). Exposure to E. multilocularis resulted in an increase in Col1 and α-SMA levels in the culture supernatant of HSC-T6 cells, and the highest Col1 (4.43 ng/mL ± 2.23 ng/mL) and α-SMA levels (285.20 pg/mL ± 90.67 pg/mL) were detected following treatment with E. multilocularis metacestodes. In addition, a persistent increase was seen in the deposition of collagen fibers in mice livers 1 to 8 months post-infection with E. multilocularis, with the greatest Col1 level (280.26 ng/mL ± 23.04 ng/mL) seen 6 months post-infection and the highest α-SMA level (33.68 ng/mL ± 4.45 ng/mL) detected 8 months post-infection, respectively. Persistent E. multilocularis infections promote hepatic stellate cell proliferation, induce an increase in mouse serum Col1 and α-SMA levels, and cause elevated deposition of collagen fibers in mice livers. The infective stage of E. multilocularis is a critical period for inducing hepatic fibrosis of alveolar echinococcosis.

Chrysanthemum indicum ethanol extract attenuates hepatic stellate cell activation in vitro and thioacetamide-induced hepatofibrosis in rats

Objective: To investigate the antifibrotic effects of Chrysanthemum indicum ethanol extract (CIEE) against activated hepatic stellate cells (HSC) and thioacetamide (TAA)-induced hepatofibrosis in rats. Methods: Cell viability and proliferation of HSC-T6 cells were measured using MTT assay. Primary HSCs were used to study morphology. TAA (200 mg/kg) was used to induced hepatic fibrosis in rats. CIEE (100 and 500 mg/kg) and silymarin (50 mg/kg) were administered orally. Liver functions including alanine transaminase, aspartate transaminase, glutathione, and hydroxyproline levels were measured using commercial kits. Liver sections and fibrotic biomarker expression were measured using hematoxylin and eosin staining and real-time polymerase chain reaction. Results: In vitro study revealed that CIEE (0.1, 0.25, and 0.5 mg/mL) inhibited the proliferation of activated HSCs exposed to transforming growth factor (TGF)-β and restored the activated primary HSC morphology. In in vivo studies, TAA-induced increase in liver/body weight ratio (5.46 ± 0.26) was significantly reduced (4.13 ± 0.22) by CIEE (P Conclusions: CIEE significantly protects against TAA-induced liver damage in rats and can be used in the treatment of liver fibrosis.

Preparation and Characterization of PEG-PLA Genistein Micelles Using a Modified Emulsion-Evaporation Method

The objective of this study is to improve the bioavailability of genistein by encapsulation with polyethylene glycol-polylactic acid (PEG-PLA) copolymers. Genistein micelles (GMs) prepared using a modified emulsion-evaporation method were more stable than those made with the original method. The effect of polyvinyl alcohol, Tween 80, sonication time, PEG-PLA/genistein ratio, and organic phase (acetone)/H2O ratio on the size, polydispersity index, encapsulation efficiency, and drug loading efficiency of GMs was investigated. GMs were obtained and characterized under optimal experimental conditions. For long-term storage, GMs were lyophilized by freeze drying with trehalose to produce genistein lyophilized powder (GLP). The analysis of GLP by Fourier-transform infrared spectroscopy and differential scanning calorimetry showed that genistein was successfully incorporated into the micellar structure. In vitro release experiments revealed that the incorporation of genistein into PEG-PLA copolymers significantly improved its solubility and bioavailability. GLP was more potent in inhibiting the proliferation of HSC-T6 cells than genistein. Treatment with GLP at 10–20 μg/mL for 48 h significantly inhibited the protein expression of α-smooth muscle actin and collagen I in HSC-T6 cells compared with the control. These data demonstrated that the improved solubility and bioavailability of genistein in the form of GLP enhanced its antifibrotic effect in vitro.

CD73 regulates hepatic stellate cells activation and proliferation through Wnt/β-catenin signaling pathway

Alcoholic liver fibrosis (ALF) is commonly associated with long-term alcohol consumption and the activation of hepatic stellate cells (HSCs). Inhibiting the activation and proliferation of HSCs is a critical step to alleviate liver fibrosis. Increasing evidence indicates that ecto-5′-nucleotidase (CD73) plays a vital role in liver disease as a critical component of extracellular adenosine pathway. However, the regulatory role of CD73 in ALF has not been elucidated. In this study, both ethanol plus CCl4-induced liver fibrosis mice model and acetaldehyde-activated HSC-T6 cell model were employed and the expression of CD73 was consistently elevated in vivo and in vitro. C57BL/6 J mice were intraperitoneally injected with CD73 inhibitor Adenosine 5′-(α, β-methylene) diphosphate sodium salt (APCP) from 5th week to the 8th week in the development of ALF. The results showed APCP could inhibit the activation of HSCs, reduce fibrogenesis marker expression and thus alleviate ALF. Silencing of CD73 inhibited the activation of HSC-T6 cells and promoted apoptosis of activated HSC-T6 cells. What's more, the proliferation of HSC-T6 cells was inhibited, which was characterized by decreased cell viability and cycle arrest. Mechanistically, Wnt/β-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/β-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/β-catenin signaling pathway might be involved in this progression.

Effects of cannabinoid receptor 2 agonist AM1241 on proliferation, activation and apoptosis ofHSC-T6 cells induced by TGF-β1

Effects of cannabinoid receptor 2 agonist AM1241 on proliferation, activation and apoptosis ofHSC-T6 cells induced by TGF-β1

Schisandrin B suppresses liver fibrosis in rats by targeting miR-101-5p through the TGF-β signaling pathway

Schisandrin B (Sch B) and miR-101 family members play critical roles in the pathogenesis of liver fibrosis. However, the relationship between them has not been reported yet. Thus, this study aims to fill this research gap. Results showed that Sch B significantly upregulated the expression of miR-101-5p in HSC-T6 cells. Sch B also increased the expression of miR-101-5p by combined administration of TGF-β1 and Sch B. Using miR-101-5p inhibitor, we demonstrated that Sch B can target miR-101-5p through the TGF-β signalling pathway to regulate the proliferation and activation of HSC-T6 cells. A rat model of carbon tetrachloride-induced liver fibrosis was established, and results indicated that Sch B can attenuate liver fibrosis by upregulating the expression of miR-101-5p. In conclusion, Sch B can directly target miR-101 to suppress liver fibrosis. Sch B or miR-101-5p may be used as a therapeutic approach for the prevention and treatment of liver fibrosis.

Mixed micelles loaded with the 5-benzylidenethiazolidine-2,4-dione derivative SKLB023 for efficient treatment of non-alcoholic steatohepatitis.

Background: SKLB023, a novel 5-benzylidenethiazolidine-2,4-dione based-derivative, specifically inhibits inducible nitric oxide synthase and shows promise for treating non-alcoholic steatohepatitis (NASH). However, its poor water solubility and low bioavailability limits its clinical use. Here the drug was loaded into phosphatidylcholine-bile salt-mixed micelles (PBMM/SKLB023) to overcome these limitations.Methods: PBMM/SKLB023 was developed using a simple co-precipitation method, and formulation parameters were optimized. The pharmacokinetics of PBMM/SKLB023 were investigated in Wistar rats, and therapeutic efficacy was assessed in a mouse model of NASH induced by a diet deficient in methionine- and choline.Results: PBMM/SKLB023 particles were 11.36±2.08 nm based on dynamic light scattering, and loading the drug into micelles improved its water solubility 300-fold. PBMM/SKLB023 inhibited proliferation and activation of HSC-T6 cells more strongly than free SKLB023. PBMM/SKLB023 showed longer mean retention time and higher bioavailability than the free drug after intravenous injection in Wistar rats. In the mouse model of NASH, PBMM/SKLB023 alleviated hepatic lipid accumulation, inflammation, and fibrosis to a significantly greater extent than free SKLB023.Conclusion: PBMM/SKLB023 shows therapeutic potential for treating NASH and liver fibrosis.

Geniposide, a sonic hedgehog signaling inhibitor, inhibits the activation of hepatic stellate cell

Liver fibrosis is a continuous wound-healing process, which is due to excessive deposition of extracellular matrix (ECM) caused by activated Hepatic Stellate Cells (HSCs). Geniposide (GP) is a naturally occurring iridoid glucoside and is extracted from Gardenia jasminoides Ellis. GP has long been speculated to play a vital role in the treatment of hyperlipidemia and fatty liver. Emerging evidence has demonstrated that GP may be importantly associated with the pathophysiology of liver fibrosis. Nevertheless, the fundamental mechanism is yet uncertain. This studywas designed to explore the possible mechanism for the inhibitory effect of GP on CCl4-induced mice liver fibrosis. In line with several clinical reports, GP significantly reduced the levels of hyaluronic acid (HA), laminin (LN), hydroxyproline (HYP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Meanwhile, liver fibrosis was significantly alleviated by GP as indicated by decreased α-smooth muscle actin (α-SMA) expression and type I collagen alpha-1 (Col I α1) deposition. In addition, GP could reduce the cell viability (IC50 = 77.11 and 42.88 μM at 24 and 48 h respectively) and cause G2/M cell arrest of the activated HSC-T6 cells. Noteworthy, GP prominently suppressed the Sonic hedgehog (Shh) signaling pathway and it might inhibit the activation and proliferation of HSC-T6 cells through Shh signaling pathway. Taken together, the current research indicate that GP has a promising antifibrotic effect which could be partially attributed to its suppression of Shh signaling pathway. Hence GP could be employed as a promising therapeutic strategy for the treatment of clinical hepatic injury.

Blockade of YAP alleviates hepatic fibrosis through accelerating apoptosis and reversion of activated hepatic stellate cells

Yes-associated protein (YAP) is a significant downstream protein in the Hippo signaling pathway with important functions in cell proliferation, apoptosis, invasion and migration. YAP also plays a role in the progression and development of various liver diseases. In hepatic fibrosis development and reversion, the proliferation and apoptosis of activated hepatic stellate cells (HSCs) play a critical role. However, the contribution of YAP to hepatic fibrosis progression and reversion and the underlying mechanism have not been investigated. Here we investigated the expression and function of YAP in the proliferation and apoptosis of activated HSCs. We found that YAP expression was increased in liver fibrosis tissues from CCl4-induced model mice and restored to normal level after stopping CCl4 injection and 6 weeks of spontaneously recovery. YAP expression was elevated in HSC-T6 cells treated with TGF-β1 and recovered after MDI treatment. Silencing of YAP inhibited the activation and proliferation of HSC-T6 cells stimulated by TGF-β1. In addition, the apoptosis of activated HSC-T6 cells silenced for YAP was slightly enhanced. Furthermore, over-expression of YAP repressed the reversion of activated HSC-T6 cells mediated by MDI reversal. We found that HSC-T6 cells activated by TGF-β1 showed higher levels of nuclear YAP compared with MDI-treated cells, indicating that YAP was activated in HSC-T6 cells treated by TGF-β1. We also found that loss of YAP attenuated Wnt/β-catenin pathway activity in activated HSC-T6 cells. Treatment of VP, an inhibitor of the YAP-TEAD complex, reduced both activation and proliferation of HSC-T6 cells and increased apoptosis. Together these results indicated that reduced expression of YAP contributes to acquisition of the quiescent phenotype in HSCs. Our results suggest that YAP may be a useful target in HSCs activation and reversion.

Xiaochaihutang Inhibits the Activation of Hepatic Stellate Cell Line T6 Through the Nrf2 Pathway

Xiaochaihutang (XCHT) is one of classic prescriptions in Treatise on Febrile Diseases in China which was reported to have the effect of anti-hepatic fibrosis in vivo. Activation of hepatic stellate cells (HSCs) is now well established as a central driver of fibrosis in liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important element for anti-oxidative damage which is one of the key factors responsible for occurrence. This study was to investigate the effect of XCHT compound serum on HSCs activation and focus on the Nrf2 pathway. Rats in treatment groups were given the appropriate doses of XCHT granules (5 g/kg) and Silybin (50 mg/kg) for 6 days, and the serum were obtained. The compound serum was used to intervene HSCs. The results found that XCHT compound serum significantly inhibited the proliferation of HSCT6 cells. The number of α-SMA positive stained cells in HSCT6 cells and the content of Collagen type I (collagen-I) in supernatant were significantly decreased indicating suppression of activated HSCs. Compared with the control group, the nuclear transcription of Nrf2 and the expressions of Nqo1, GCLC, and GCLM were significantly increased in XCHT group. However, the effects of XCHT were inhibited in Nrf2-siRNA transfected HSCT6 cells. These studies demonstrated that XCHT could inhibit HSCT6 cell proliferation and activation. The mechanism might be related to up-regulation of the Nrf2 pathway against oxidative stress.

Repression of liver cirrhosis achieved by inhibitory effect of miR-454 on hepatic stellate cells activation and proliferation via Wnt10a.

As is known, hepatic stellate cells (HSCs) activation contributes to liver cirrhosis. This study aims to find out the acting mechanisms of miR-454 inhibiting the activation and proliferation of hepatic stellate cells. The expression of Col1A1, α-smooth muscle actin (α-SMA) and Wnt10a were determined by western blot, and the miR-454 level was determined by quantitative real-time PCR in this study. We took two objects as experiment subjects, one was liver cirrhosis rats, and the other was transforming growth factor (TGF)-β1-stimulated HSC-T6 cells. After activated with TGF-β1 and transfected with microRNA-454 mimic, separately or successively, the changes on the Col1A1 and α-SMA expression, HSC proliferation, miR-454 level and Wnt10a expression were examined in HSC-T6 cells, respectively. Interaction between miR-454 and Wnt10a was evaluated with dual luciferase reporter assay. MiR-454 expression was down-regulated in tissues of liver cirrhosis rats. TGF-β1 caused the down-regulation of the miR-454 in HSC-T6 cells. MiR-454 inhibited the activation and proliferation of HSC-T6 cells. Wnt10a had a targeting relationship with miR-454. TGF-β1 promoted HSC-T6 activation and proliferation via down-regulating miR-454 expression, which further up-regulated Wnt10a expression. MiR-454 mimic inhibited cirrhosis progression in liver cirrhosis rats. MiR-454 can inhibit the activation and proliferation of HSCs via suppressing the expression of Wnt10a, to restrain liver cirrhosis.

Effects of angiotensin II on protein kinase Cε and Cα expression in hepatic stellate cells

Objective To investigate the effect of angiotensinⅡon protein kinase Ce (PKCe) and protein kinase Cα (PKCα) expression in hepatic stellate cells. Methods Hepatic stellate cell (HSC)-T6 cells were treated with different concentrations of angiotensinⅡand the proliferation of HSC-T6 cells was detected by methyl thiazolyl tetrazolium (MTT) assay. The expression of PKCe and PKCα was detected by immunofluorescence staining. PKCe and PKCα mRNA levels was detected by real time polymerase chain reaction (PCR). Results AngiotensinⅡconcentrated the proliferation of HSC-T6 cells and the level of hydroxyproline (F=25.321, 13.283, P<0.001) and showed a dose-dependent effect. With the increase of angiotensin Ⅱ concentration, PKCe significantly increased and translocated in the cell membrane; PKCα increased significantly, especially in transplanted membrane and cytoplasm (F=21.387, 19.431, P<0.01), and showed obvious dose effect. Meanwhile, Angiotensin Ⅱ increased the expression of PKCe and PKCα, and induced cell proliferation by up-regulating PKCe and PKCα mRNA levels (F=13.279, 15.174, P<0.05). Conclusions AngiotensinⅡcan up-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner, increase the expression of protein kinase Ce and Cα, and promote the proliferation of hepatic stellate cells. Key words: Angiotensin Ⅱ/PD; Hepatic stellate cells/DE/EN; Protein kinase C-alpha/ME; Protein kinase C-epsilon/ME; Cell proliferation