Involvement of 90-kuD ribosomal S6 kinase in collagen type I expression in rat hepatic fibrosis

AIM： To investigate the relationship between 90-kuD ribosomal $6 kinase （pg0RSK） and collagen type I expression during the development of hepatic fibrosis in vivo and in vitro.METHODS： Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type I were determined by co-immunofluoresence and confocal microscopy.Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell （HSC） line. The expression of collagen type I in HSC-T6 cells was assessed by Western blotting and real-time polymerase chainreaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, thencollagen type I promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated withor without platelet-derived growth factor （PDGF）-BB at a final concentration of 20μg/L and the cell growthwas determined by MTS conversion.RESULTS： In fibrotic liver tissues, p90RSK was over-expressed in activated HSCs and had a significantpositive correlation with collagen type I levels.In HSC-T6 cells transfected with RNAi targeted top90RSK, the expression of collagen type I was down-regulated （61.8% in mRNA, P 〈 0.01, 89.1% inprotein, P 〈 0.01）. However, collagen type ] promoteractivity was not increased with over-expression of p90RSK and not decreased with low expression either,compared with controls in the same cell line （P = 0.076）.Furthermore, p90RSK siRNA exerted the inhibitionof HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation.CONCLUSION： p90RSK is over-expressed in activatedHSCs and involved in regulating the abnormalexpression of collagen type I through initiating theproliferation of HSCs.     

Effects of c-myb antisense RNA on TGF-β1 and α1-Ⅰ collagen expression in cultured hepatic stellate cells

AIM: To investigate the effects of c-myb antisense RNA on cell proliferation and the expression of c-myb, TGF-β1 and α1-Ⅰ collagen in cultured hepatic stellate cells (HSC) from rats.METHODS: Recombinant retroviral vector of c-myb antisense gene (pDOR-myb) was constructed, and then transfected into retroviral package cell line PA317 by means of DOTAP.The pseudoviruses produced from the resistant PA317 cells were selected with G418 to infect HSCs isolated from rat livers. The cell proliferation was measured by 3-[4,5-Dimethylthiazolzyl]-2, 5-diphenyl tetrazo-dium bromide (MTT) method.The expression of c-myb, α1-Ⅰ collagen and TGF-β1 rnRNA, and c-myb protein in HSCs was detected with semi-quantitive reverse transeription-polymerase chain reaction (RT-PCR) and Western-blot respectively.RESULTS: HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, the cmyb protein expression, cell proliferation,and α1-Ⅰ collagen and TGF-β1 mRNA expression were repressed significantly compared with their corresponding control groups (P<0.01).CONCLUSION: c-myb plays a key role in activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation, α1-Ⅰ collagen and TGF-β1 mRNA expression,suggesting that inhibition of c-myb gene expression might be a potential way for the treatment of liver fibrosis.     

Specific shRNA targeting of FAK influenced collagen metabolism in rat hepatic stellate cells

AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of ...     

Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro

AIM: To investigate the role of nuclear factor-κB (NF-κB)inhibitor caffeic acid phenethy1 ester (CAPE) in the proliferation, collagen synthesis and apoptosis of hepatic stellate cells (HSCs) of rats. METHODS: The HSCs from rats were isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with CAPE. The proliferation and collagen synthesis of HSCs were determined by 3H-TdR and 3H-proline incorporation respectively, and the expression of type Ⅰ, Ⅲ procollagen genes was further explored byin situ hybridization. Apoptosis cell indices (AIs) were examined using terminal deoxynucleotidyl transferase- mediated DIG-dUTP nick end labeling (TUNEL). RESULTS: Tn activated HSC in culture, CAPE significantly inhibited 3H-TdR and 3H-proline incorporation by HSCs at concentrations of 5 μmol/L and 10 μmol/L respectively. CAPE also reduced the type I procollagen gene expression (P＜0.05)at higher concentration. Apoptosis of HSC was induced by CAPE and the AIs were time-and dose-dependently increased from 2.82+0.73 % to 7.66±1.25 % at 12 h (P＜0.01) and from 3.15±0.88 % to 10.6L±2.88 % at 24 h (P＜0.01). CONCLUSION: CAPE inhibits proliferation and collagen synthesis of HSC at lower concentration and induces HSC apoptosis at higher concentration.     

Effect of ligand of peroxisome proliferator-activated receptor γ on the biological characters of hepatic stellate cells

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARγ), on the expression of PPARγ in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 μmol/L rosiglitazone group, and 10 μmol/L rosiglitazone group. The expression of PPARγ, α-smooth muscle actin (α-SMA), and type Ⅰ and Ⅲ collagen was detected by RT-PCR, Western blot and immunocytochemiccal staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARy at mRNA and protein level markedly increased in HSCs of 10 umol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 μmol/L rosiglitazone group decreased significantly (t=5.542, P<0.01), α-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0,01 in both). The apoptotic rate of HSCs significantly increased in 10 μmol/L rosiglitazone group vs control (X2=16.682, P<0,01). CONCLUSION: By increasing expression of PPARγ in activated HSCs, rosiglitazone, an agonist of PPARγ, decreases α-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.     

Effects of c-myb antisense RNA on TGF-β1 and α1-I collagen expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA oncell proliferation and the expression of c-myb,TGF-131 andα1-I collagen in cultured hepatic stellate cells (HSC) from rats.METHODS:Recombinant retroviral vector of c-myb antisensegene (pDOR-myb) was constructed,and then transfectedinto retroviral package cell line PA317 by means of DOTARThe pseudoviruses produced from the resistant PA317 cellswere selected with G418 to infect HSCs isolated from ratlivers.The cell proliferation was measured by 3-[4,5-Dimethylthiazolzyl]-2,5-diphenyl tetrazo-dium bromide(MIT) method.The expression of c-myb,α_1-I collagen andTGF-β1 mRNA,and c-myb protein in HSCs was detectedwith semi-quantitive reverse transeription-polymerase chainreaction (RT-PCR) and Western-blot respectively.RESULTS:HSCs from rats were isolated successfully withthe viability>98%.In the pDOR-myb infected HSCs,the c-myb protein expression,cell proliferation,and α_1-I collagenand TGF-β1 mRNA expression were repressed significantlycompared with their corresponding control groups (P<0.01).CONCLUSION:c-myb plays a key role in activation andproliferation of HSC.c-myb antisense RNA can inhibit cellproliferation,α_1-I collagen and TGF-β1 mRNA expression,suggesting that inhibition of c-myb gene expression mightbe a potential way for the treatment of liver fibrosis.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, a smooth muscle actin expression and typeⅠcollagen synthesis in vitro

AIM: To study the effect of oxymatrine-baicalin combination (OB) against HBV replication in 2.2.15 cells and a smooth muscle actin (αSMA) expression, typeⅠ, collagen synthesis in HSC-T6 cells. METHODS: The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to p actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by p actin. RESULTS: In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group (HBsAg, P=0.043; HBeAg, P=0.026; respectively); HBV DNA level in the OB group was significantly lower than that in the oxymatrine group (P = 0.041). In HSC-T6 cells the mRNA and protein expression levels of a SMA in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P = 0.013; protein, P-0.042; respectively); The mRNA and protein expression levels of typeⅠcollagen in the OB group were significantly lower as compared with those in the oxymatrine group (mRNA, P<0.01; protein, P<0.01; respectively). CONCLUSION: OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against a SMA expression and typeⅠcollagen synthesis in HSC-T6 cells than oxymatrine in vitro.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

丹参素对大鼠肝星状细胞氨基末端蛋白激酶信号转导的影响

Objective To investigate the inhibitory effect of danshensu on the activation of JNK signaling in rat hepatic stellate cells (HSCs) induced by IL-1 β. Methods The proliferation of primary rat HSCs treated with different concentration of Danshensu was checked by MTT colorimetric assay. The expres-sion and phosphorylation of JNK and P-JNK was detected by western blotting. Synthesis and secretion of collagen Ⅰ were detected by the quantitative immunocytochemical assay and ELISA. Results Danshensu inhibited the proliferation of HSCs in a dose-dependent manner. At the concentration of 0.0625 to 0.25 mmol/L, Danshensu significantly repressed the proliferation of HSC induced by IL-115 (P < 0.05). Synthesis and secretion of Type β collagen was significantly decreased 24 hours after 0.25 mmol/L Danshensu treatment (P < 0.01). The phosphorylation of JNK induced by IL-1 β was significantly inhibited by Danshensu treatment (P < 0.05), however, the expression of JNK was not regulated by Danshensu. Conelutions Danshensu represses the activation and proliferation of HSCs, and inhibits the synthesis and secretion of Type β collagen, possibly via the repression of the JNK signal transduction.     

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 consequent 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 silibinininhibited proliferation of LX-2 cells. CONCLUSION: The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.