Effect of lipid on proliferation and activation of rat hepatic stellate cells(Ⅰ)

AIM To study the effect of lipid (triglyceride and very low density lipoprotein, VLDL) on proliferation and activation of rat hepatic stellate cells (HSC). METHODS HSC were isolated and cultured from liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz. HSC proliferation was examined with MTT colorimetric assay. RESULT Triglyceride of 12.5mg/L had a promoting effect on proliferation of HSC ( P <0 05), 25, 50, 100 and 200mg/L had no effects ( P >0 05), but 400mg/L had an inhibiting effect ( P <0 01). VLDL of 6 25 and 12 5mg/L had no effect on proliferation of HSC ( P >0 05), but increased concentration of VLDL could promote the HSC proliferation ( P <0 05). CONCLUSION Lipid had an effect on proliferation of HSC. Triglyceride and VLDL may promote HSC proliferation and may be associated with fatty liver and hepatic fibrogenesis.

Hepatitis B virus X protein promotes proliferation and upregulates TGF-β1 and CTGF in human hepatic stellate cell line,LX-2

BACKGROUND:Chronic hepatitis B virus(HBV)infection is a major cause of liver fibrosis,but the mechanisms underlying HBV-related fibrogenesis are still unknown. Although the roles of HBV X protein(HBx)remain poorly understood,it is thought to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to determine the role of HBx in liver fibrogenesis by studying the effect of HBx on the proliferation and expression of fibrosis-related molecules in the human hepatic stellate cell line,LX-2. METHODS:We established an in vitro co-culture system with LX-2 cells and a stable QSG7701-HBx cell line which had been transfected with the HBx gene. 3 H-TdR incorporation and flow cytometry were used to determine the effects of HBx on the proliferation of LX-2 cells. α-smooth muscle actin(α-SMA),transforming growth factor-β1(TGF-β1),transforming growth factor-βreceptor Ⅱ(TGF-βRⅡ),and connective tissue growth factor (CTGF)in LX-2 cells were analyzed by Western blotting.In addition,the expression levels of collagen typeⅠ(ColⅠ) from the co-cultured media were measured by ELISA. RESULTS: 3 H-TdR incorporation increased significantly in LX-2 cells co-cultured with QSG7701-HBx cells compared to those cultured with QSG7701-pcDNA3 and QSG7701 (non-tumorigenic human liver cell line).Cell cycle results revealed that HBx accelerated the progression of G1 to S in LX-2 cells.The expressions ofα-SMA,TGF-β1,TGF-βR Ⅱ,CTGF and ColⅠwere significantly increased in the co- cultures of LX-2 cells with stable QSG7701-HBx cells. CONCLUSION:These results suggest that HBx may facilitate liver fibrosis by promoting hepatic stellate cell proliferation and upregulating the expression of fibrosis- related molecules.

Role of free fatty acids in proliferation of rat hepatic stellate cells(Ⅱ)

AIM To study the role of free fatty acids (arachidonic acid and linoleic acid) in proliferation of rat hepatic stellate cells (HSC). METHODS HSC were isolated and cultured from liver of Wistar rats by in situ perfusion with pronase and collagenase, and density gradient centrifugation with Nycodenz. MTT colorimetric assay was detected for HSC proliferation. RESULTS Arachidonic acid and linoleic acid had an effect on proliferation of HSC. Arachidonic acid of 25mg/L promoted HSC proliferation ( P <0 01), but 50 and 100mg/L had an inhibitory effect on HSC ( P <0 01), and showed cytotoxity on HSC under inverted microscope; 6 25, 12 5 and 25mg/L of linoleic acid had no effect on HSC proliferation ( P >0 05), but with concentration increasing, 50 and 100mg/L of linoleic acid might promote HSC proliferation ( P <0 05 or 0 01). CONCLUSION Arachidonic acid and linoleic acid may promote HSC proliferation, but increased concentration can have cytotoxity on HSC. Arachidonic acid and linoleic acid might be associated with fatty liver and hepatic fibrogenesis by lipid peroxidation.

In vivo effects of Chinese herbal recipe, Danshaohuaxian, on apoptosis and proliferation of hepatic stellate cells in hepatic fibrotic rats

AIM: To investigate the effects of Danshaohuaxian (DSHX), a Chinese herbal recipe, on the apoptosis and cell cycles of hepatic stellate cells (HSCs) in rat hepatic fibrosis and its possible mechanisms. METHODS: Seventy-six male Wistar rats were randomly divided into normal control group, hepatic fibrosis group, non-DSHX-treated group and DSHX-treated group. Except for the normal control group, rat hepatic fibrotic models were induced by subcutaneous injection of carbon tetrachloride (CCl4), drinking alcohol, giving diet of hyperlipid and hypoprotein for 8 wk. When the hepatic fibrotic models were produced, 12 rats of hepatic fibrosis group (15 rats survived, others died during the 8 wk) were sacrificed to collect blood and livers. HSCs were isolated from the other 3 rats to detect the apoptotic index (AI) and cell cycles by flow cytometry. DSHX was then given to the DSHX-treated group (1.0 g/kg, PO, daily) for 8 wk. At the same time, normal control group and non-DSHX-treated group were given normal saline for 8 wk. At end of the experiment, some rats in these three groups were sacrificed to collect blood and livers, the other rats were used for HSC isolation to detect the apoptotic index (AI) and cell cycles. Then the liver index, serum hyaluronic acid (HA) and alanine aminotransferase (ALT), degree of hepatic fibrosis, urinary excretion of hydroxyproline (Hyp) and expression of collagen types Ⅰ and Ⅲ (COL Ⅰ and Ⅲ) in these four groups were detected respectively. RESULTS: Compared with the indexes of the hepatic fibrosis group and non-DSHX-treated group, the DSHX-treated group revealed a liver index of (0.0267±0.0017 vs 0.0423±0.0044, 0.0295±0.0019, P<0.05), levels of serum HA (200.78±31.71 vs 316.17±78.48, 300.86±72.73, P<0.05) and ALT(93.13±5.79 vs 174.5±6.02, 104.75±6.54, P<0.01), and stage of hepatic fibrosis (1.30 vs 4.25, 2.60, P<0.01) all reduced. The urinary excretion of Hyp increased (541.09±73.39 vs 62.00±6.40, 182.44±30.83, P<0.01), the COL Ⅰ and Ⅲ expression decreased (COL I: 1.07±0.96 vs 4.18±2.26, 3.22±1.44, P<0.01; COL Ⅲ: 1.09±0.58 vs 3.04±0.62, 2.23±0.58, P<0.01), the HSCs apoptotic index of HSCs (7.81±0.47 vs 1.63±0.25, 1.78±0.4, P<0.05) and the ratio of G0-G1 phase cells increased (94.30±1.33 vs 62.27±17.96, 50.53±2.25, P<0.05). The ratios of S-phase cells (3.11±1.27 vs 9.83±1.81, 11.87±1.9, P<0.05) and G2-M phase cells (2.58±0.73 vs 23.26±10.95, 13.60±1.15, P<0.01) declined. CONCLUSION: DSHX capsule shows certain therapeutic effects on hepatic fibrosis in rats and inhibits abnormal deposition of COL I and III in rat livers by promoting the apoptosis of HSCs and preventing their proliferation.

Rosmarinic acid attenuates hepatic fibrogenesis via suppression of hepatic stellate cell activation/proliferation and induction of apoptosis

Objective:To investigate the antifibrotic role of rosmarinic acid(RA),a natural polyphenolic compound,on HSCs activation/proliferation and apoptosis in vitro and in vivo. Methods:The impact of RA on stellate cell line(HSC-T6) proliferation,activation and apoptosis was assessed along with its safety on primary hepatocytes. In vivo,rats were divided into:(i) normal;(ii) thioacetamide(TAA)-intoxicated rats for 12 weeks;(iii) TAA+silymarin or(iv) TAA+RA. At the end of experiment,liver functions,oxidative stress,inflammatory and profibrogenic markers,tissue inhibitor metalloproteinases type-1(TIMP-1) and hydroxyproline(HP) levels were evaluated. Additionally,liver histopathology and immunohistochemical examinations of alpha-smooth muscle actin(α-SMA),caspase-3 and proliferation cellular nuclear antigen(PCNA) were determined. Results:RA exhibited anti-proliferative effects on cultured HSCs in a time and concentration dependent manner showing an IC50 of 276 μg/mL and 171 μg/mL for 24 h and 48 h,respectively,with morphological reversion of activated stellate cell morphology to quiescent form. It significantly improved ALT,AST,oxidative stress markers and reduced TIMP-1,HP levels,inflammatory markers and fibrosis score(S1 vs S4). Furthermore,reduction in α-SMA plus elevation in caspase-3 expressions of HSCs in vitro and in vivo associated with an inhibition in proliferation of damaged hepatocytes were recorded. Conclusions:RA impeded the progression of liver fibrosis through inhibition of HSCs activation/proliferation and induction of apoptosis with preservation of hepatic architecture.

Laparoscopic hepatic left lateral lobectomy combined with fiber choledochoscopic exploration of the common bile duct and traditional open operation

AIM: To investigate the possibilities and advantages of laparoscopic hepatic left lateral lobectomy combined with fiber choledochoscopic exploration of the common bile duct compaired with traditional open operation.METHODS: Laparoscopic hepatic left lateral lobectomy combined with fiber choledochoscopic exploration of the common bile duct and traditional open operation were performed in two groups of patients who had gallstones in the left lobe of liver and in the common bile duct. The hospitalization time, hospitalization costs, operation time, operative complications and post-operative liver functions of the two groups of patients were studied.RESULTS: The operation time and post-operative liver functions of the two groups of patients had no significant differences, while the hospitalization time, hospitalization costs and operative complications of the laparoscopic hepatic left lateral lobectomy combined with fiber choledochoscopic exploration in the common bile duct group were significantly lower than those in the traditional open operation group.CONCLUSION: For patients with gallstones in the left lobe of liver and in the common bile duct, laparoscopic hepatic left lateral lobectomy combined with fiber choledochoscopic exploration of the common bile duct can significantly shorten the hospitalization time, reduce the hospitalization costs and the post-operative complications,without prolonging the operation time and bringing about more liver function damages compared with traditional open operation. This kind of operation has more advantages than traditional open operation.

KN-93,a specific inhibitor of CaMKⅡ inhibits human hepatic stellate cell proliferation in vitro

AIM： To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate cells. METHODS： Human hepatic stellate cells （LX-2） were incubated with various concentrations （0-50 μmol/L） of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting. RESULTS： KN-93 （5-50 μmol/L） decreased the proliferation of human hepatic stellate cells in a dosedependent manner from 81.76% （81.76% ± 2.58% vs 96.63% ± 2.69%, P 〈 0.05） to 27.15% （27.15% ± 2.86% vs 96.59% ± 2.44%, P 〈 0.01） after 24 h treatment. Incubation of 10 μmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21. CONCLUSION： KN-93 has potent inhibitory effect on proliferation of LX-2 cells by modulating the expression of two special cell cycle regulators, p53 and p21.

BMP-4 induced proliferation and oriented differentiation of rat hepatic oval cells into hepatocytes

Objective:To explore the role of bone morphogenetic protein 4(BMP-4) in hepatic progenitor cells(HPCs).Methods:The effect of BMP-4 on rat hepatic oval cells was examined by using the WB-F344 rat hepatocytic epithelial stem-cell-like cell line.This hepatocytic cell line could exert various hepatocytc functions including the secretion of albumin and urea.Immunohistochemistry was used to examine the effects of BMP-4 and its antagonist,Noggin,on the proliferation and differentiation of these cells,cellular uptake and excretion of indocyanine green,the periodic acid-schiff(PAS) assay for glycogen storage and the expression of hepatic markers.Results:Our results showed for the first time that BMP-4 may acted as a potential inducer of hepatic differentiation in rat hepatic oval cells.Conclusions:This cell source offers a much-needed attractive and expandable source for future investigations of drug screening,stem cell technologies and cellular transplantation,in a society with increasing levels of liver disease and damage.

Effects of Guangxi Hepu Pearl Hydrolysate on Proliferation Activity and Apoptosis of Human Hepatic Stellate Cells

[Objective] The paper was to observe the effects of Guangxi Hepu pearl hydrolysate on proliferation and apoptosis of human hepatic stellate cells. [Method] The inhibition rate against the proliferation of human hepatic stellate cells was determined by MTT colorimetry after co-incubation with pearl hydrolysate for 48 h. The percentage of early and late apoptotic cells was determined by flow cytometry(Annexin V-FITC/PI staining). [Result] Low, medium and high concentrations of pearl hydrolysate could inhibit the proliferation of human hepatic stellate cells, and the inhibition rates at 48 h were 12.4%, 27.4% and 37.8%, respectively. The low, medium and high concentrations of pearl hydrolysate could induce the apoptosis of cells. The percentages of total apoptotic cells(early apoptotic + late apoptotic) at 48 h were 8.97%, 16.09% and 26.98%, respec-tively. [Conclusion] Pearl hydrolysate can inhibit the proliferation of human hepatic stellate cells and induce the apoptosis of these cells, which may be the mechanism of anti-hepatic fibrosis effect of pearl hydrolysate. These effects are dose-dependent. It is also found that the above cellular effects of high dose pearl hydrolysate are stronger than those of colchicine and compound Biejiaruangan tablets.

Trichloroethylene Induces Biphasic Concentration-dependent Changes in Cell Proliferation and the Expression of SET-Associated Proteins in Human Hepatic L-02 Cells

Trichloroethylene （TCE） is a major pollutant that affects both occupational and general environments. The liver is an important target organ of TCEE. Substantial efforts and remarkable progress into understanding TCE cytotoxicity have been made in cultured liver cells. However, the molecular mechanisms by which TCE induces hepatotoxicity are not well understood. SET （also known as protein phosphatase 2A inhibitor, 12PP2A, or template-activating factor-I, TAF-D is a nuclear protein that regulates histone modification, gene transcription, DNA replication, nucleosome assembly,

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background:Activated hepatic stellate cells(HSCs)are closely involved in the initiation,perpetuation,and resolution of liver fibrosis.Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis.Methods:In this study,we investigated the effect of the pro-inflammatory cytokine interleukin(IL)-1βon the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells,an HSC cell line,using western blotting and cell proliferation assays.Results:IL-1βincreased the proliferation rate andα-smooth muscle actin(SMA)expression of LX-2 cells in a dose-dependent manner.Within 1 h after IL-1βtreatment,c-Jun N-terminal kinase(JNK),p38,and nuclear factor-κB(NF-κB)signaling was activated in LX-2 cells.Subsequently,protein kinase B(AKT)phosphorylation and an increase inα-SMA expression were observed in LX-2 cells.Each inhibitor of JNK,p38,or NF-κB decreased cell proliferation,AKT phosphorylation,andα-SMA expression in IL-1β-treated LX-2 cells.Conclusion:These results indicate that JNK,p38,and NF-κB signals converge at AKT phosphorylation,leading to LX-2 activation by IL-1β.Therefore,the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.

Hollow Fiber Module Applied for Effective Proliferation and Harvest of Cultured Chondrocytes

Steady and useful culture for chondrocytes is essential for cartilage regenerative medicine. However, in conventional plate culture, the chondrocytes become dedifferentiated and lose their ability to make cartilage matrices. Three-dimensional culture mimicking the physiological environment in native chondrocytes is useful to maintain the chondrocyte properties during the proliferation culture. However, the three-dimensional culture is practically a hard task due to difficult harvest of the cells. Thus, we attempted to apply porous materials, hollow fibers for the three-dimensional culture, and developed their module to realize the effective harvest of the cells. Polyethersulfone-based hollow fibers, whose safety and cell affinity were confirmed by the experiment of the coculture with human chondrocytes, were collected to fabricate a module. The hollow fiber module was installed with screw ends, and enabled the easy removal of chondrocytes from the inner unit. Cultured human chondrocytes embedded within collagen hydrogel were put into the outer lumen of the hollow fiber module, while chondrocyte prolfieration medium was perfused through the inner lumen at 0 to 30 mL/min. After 2 weeks’ culture, the flow rate of 3 to 10 mL/min effectively supported the chondrocyte proliferation. Then, long-term culture using the hollow fiber module at flow rate of 5 mL/min was performed, revealing that the cell growth in this module at 3 weeks was approximately twice larger than that in static culture. The numbers of viable cells could be maintained by week 7. The hollow fiber module installed with screw ends can effectively culture and harvest the chondrocytes.

Anti-proliferative and pro-apoptotic effects of tectorigenin on hepatic stellate cells

AIM: To investigate the effect of tectorigenin on proliferation and apoptosis of hepatic stellate cells (HSC)-T6 cells. METHODS: HSC-T6 cells were incubated with tectorigenin at different concentrations, and their proliferation was assessed by bromodeoxyuridine incorporation assay. Apoptosis was detected by flow cytometry assay with Hoechst 33342 staining. Also, generation of reactive oxygen species (ROS), intracellular [Ca2+]i, potential of mitochondrial membrane, activities of cytochrome c and caspase-9 and-3 were investigated to explore a conceivable apoptotic pathway. RESULTS: Tectorigenin suppressed the proliferation of HSC-T6 cells and induced apoptosis of HSC-T6 cells in a time-and dose-dependent manner. Tectorigenin at the concentration of 100 μg/mL greatly inhibited the viability of HSC-T6 cells and induced the condensation of chromatin and fragmentation of nuclei. When treated for 48 h, the percentage of cell growth and apoptosis reached 46.3% ± 2.37% (P = 0.004) and 50.67% ± 3.24% (P = 0.003), respectively. Furthermore, tectorigenin-induced apoptosis of HSC-T6 cells was associated with the generation of ROS, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3. CONCLUSION: Tectorigenin inhibits proliferation of HSC-T6 cells and induces apoptosis of HSC-T6 cells.

Effect of hepatitis C virus core protein on modulation of cellular proliferation and apoptosis in hilar cholangiocarcinoma

BACKGROUND: Hepatitis C virus (HCV) is believed to be an important human pathogen causing carcinoma. But the effect of HCV infection on the alteration of cellular pro- liferation and apoptosis and the relationship between the effect and the development of hilar cholangiocarcinoma are largely unknown. The aim of this study was to assess the effect of HCV core protein on proliferation and apoptosis of hilar cholangiocarcinoma. METHODS: HCV core protein (HCV C protein) was de- tected by peroxidase-antiperoxidase assay in surgical speci- mens from 48 patients with hilar cholangiocarcinoma. The apoptosis index ( AI) and PCNA index ( PI) in hilar cholangiocarcinoma were detected by in situ end labeling assay and streptavidin-biotin assay respectively. RESULTS: The expression of HCV C protein was observed in 32 (67.7%) of the 48 specimens of hilar cholangiocarci- noma. The mean ± standard deviation for AI and PI was 3.52%±0.64% and 46.24%±11.46% respectively. The AI of hilar cholangiocarcinoma specimens with HCV C protein expression was significantly lower than that of HCV C pro- tein negative specimens (P<0.01), whereas the PI of HCV C protein positive specimens was significantly higher than that of HCV C protein negative specimens (P<0.01). CONCLUSION: HCV C protein may promote the cellular proliferation of hilar cholangiocarcinoma and inhibit its cel- lular apoptosis.

Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression

BACKGROUND： Proliferation of hepatic stellate cells （HSCs） plays a pivotal role in the progression of liver fibrosis conse- quent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. METHODS： The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin （10, 50 and 100 μmol/L） for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. RESULTS： Silibinin inhibits LX-2 cell proliferation in doseand time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin- inhibited proliferation of LX-2 cells. CONCLUSION： The anti-proliferative effect of silibinin on LX-2 human steUate cells is via the inhibition of the expres- sions of various cell cycle targets including p27, Akt and sir- tuin signaling.

Dynamic expression of extracellular signal-regulated kinase in rat liver tissue during hepatic fibrogenesis

AIM： To investigate whether extracellular signal-regulated kinase 1 （ERK1） is activated and associated with hepatic stellate cell （HSC） proliferation in fibrotic rat liver tissue.METHODS： Rat hepatic fibrosis was induced by bile duct ligation （BDL）. Histopathological changes were evalo uated by hernatoxylin and eosin staining, and Masson＇ s trichrorne method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polyrnerase chain reaction, while the distribution of ERK1 was assessed by irnrnunohistochernistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin （α-MA） staining.RESULTS： With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber sepia and perisinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct （12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively） were significantly larger than those in the control group （5.88% ± 1.46%, P 〈 0.01）. With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber sepia around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK1 protein was up-regulated during the model course, and its level was the highest 4 wk after operation, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days after BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-MA expression （r = 0.958, P 〈 0.05）.CONCLUSION： The expression of ERK1 protein and mRNA is greatly increased in fibrotic rat liver tissues, which may play a key role in HSC proliferation and hepatic fibrogenesis.

Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2

Hepatitis B virus X protein （HBx） plays a crucial role in the development of hepatocellular carcinoma. Here, we sought to identify the mechanisms by which HBx mediates liver cell proliferation. We found that HBx upregulated the levels of cyclooxygenase-2 （COX-2）, 5-1ipoxygenase （5-LOX） and phosphorylated extracellular signal-regulated protein kinases 1/2 （p-ERK1/2） in liver cells. HBx-induced p-ERK1/2 was abolished by inhibition of Gi/o proteins, COX or LOX. In addition, HBx increased the amounts of prostaglandin E2 （PGE2） and leukotriene B4 （LTB4） released from cell lines derived from hepatocytes. Moreover, these released arachidonic acid metabolites were able to activate ERK1/2. Interestingly, activated ERK1/2 could upregulate the expression of COX-2 and 5-LOX in a positive feedback manner. In conclusion, HBx enhances and maintains liver cell proliferation via a positive feedback loop involving COX-2, 5-LOX, released arachidonic acid metabolites, Gi/o proteins and p-ERK1/2.

Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors

The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor(EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGFinduced receptor down-regulation. Furthermore, the overexpression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma sMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/timedependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction.

Ezrin Promotes the Proliferation, Migration, and Invasion of Ovarian Cancer Cells

Objective The underlying mechanism of Ezrin in ovarian cancer(OVCA) is far from being understood.Therefore, this study aimed to assess the role of Ezrin in OVCA cells(SKOV3 and Ca OV3) and investigate the associated molecular mechanisms.Methods We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, Rho A and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells.Results The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial–mesenchymal transition(EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of Rho A rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation.Conclusion Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.