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.     

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 S6 kinase (p90RSK) 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 chain reaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, then collagen type Ⅰ promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated with or without platelet-derived growth factor (PDGF)-BB at a final concentration of 20 μg/L and the cell growth was determined by MTS conversion. RESULTS: In fibrotic liver tissues, p90RSK was overexpressed in activated HSCs and had a significant positive correlation with collagen type Ⅰ levels. In HSC-T6 cells transfected with RNAi targeted to p90RSK, the expression of collagen type Ⅰ was downregulated (61.8% in mRNA, P < 0.01, 89.1% in protein, P < 0.01). However, collagen type Ⅰ promoter activity 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 inhibition of HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation. CONCLUSION: p90RSK is over-expressed in activated HSCs and involved in regulating the abnormal expression of collagen type Ⅰ through initiating the proliferation of HSCs.     

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.     

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.     

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM： To investigate the role of insulin-like growth factor binding protein-7 （IGFBP-7） in the activation and transdifferentiation of hepatic stellate cells （HSC） in vitro.
METHODS： Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor （TGF）-β1, IGFBP-7 or anti- IGFBP-7 antibody for 24 h. The supernatant or a cytoplasm suspension was obtained from cultured HSC, followed by transfer of cells to form cell-coated dishes. Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin, collagen Ⅰ and α-smooth muscle actin （SMA）. The pro-apoptotic effect of anti- IGFBP-7 antibody was determined by flow cytometry.
RESULTS： Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group. In addition, fibronectin, collagen Ⅰ and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent. Moreover, flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC, which is responsible for the development of liver fibrosis, and may represent a novel pathway and target for therapeutic intervention.
CONCLUSION： IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC. Anti- IGFBP-7 antibody may ameliorate liver fibrogenesis.     

Resistin mediates the hepatic stellate cell phenotype

AIM: To describe the role of resistin in liver fibrosis. METHODS: For the in vivo animal study, Sprague Dawley rats were subjected to bile duct ligation (BDL) for 4 wk. Rat liver, adipose tissue (epididymal fat) and serum were analyzed for resistin expression. For the in vitro experiment, rat primary hepatic stellate cells (HSCs) and Kupffer cells (KCs) were used. HSCs were exposed to recombinant resistin, and collagenⅠ, transforming growth factor β1, α smooth muscle actin, tissue inhibitor of metalloproteinase 1 and connective tissue growth factor expression were analyzed. Resistin gene and protein expression was quantified as was the expression of pro-inflammatory cytokines including tumor necrosis factor α (TNFα), interleukin (IL)-1, IL-6, IL-8 and monocyte chemotactic protein-1 (MCP-1). The effects of resistin on HSC proliferation, migration and apoptosis were determined. The effects of resistin on KCs were also investigated. RESULTS: Following BDL, rat epididymal fat and serum rather than liver showed higher resistin expression compared to control rats. In liver, resistin was expressed in quiescent HSCs and KCs. Resistin treatment resulted in enhancement of TNFα , IL-6 , IL-8 and MCP-1 gene expression and increased IL-6 and MCP-1 protein in HSCs. Resistin activated HSC phospho-MAPK/p38, and p38 inhibition diminished IL-6 and MCP-1 expression. Furthermore, resistin facilitated HSC proliferation and migration, but decreased apoptosis which was via an IL-6 and MCP-1 mechanism. Finally, resistin-induced transforming growth factor β1 from KCs enhanced HSC collagenⅠexpression. CONCLUSION: Resistin directly and indirectly modulates HSC behavior towards a more pro-fibrogenic phenotype.     

TGF-β1 promotes differentiation of hiPSC into functional smooth-muscle-like cells

Background Cell source is one of the most important constructions for tissue engineered blood vessels(TEBV). As human adult vascular cells are limited by the replicative life spans and poor collagen secretion, stem cell has become a promising cell source. Hence, we investigated the differentiation of human induced pluripotent stem cells(hiPSC) into functional smooth-muscle-like cells(SMLCs) by embryoid bodies method and explored whether transforming growth factor-β1(TGF-β1) can promote the differentiation. Methods HiPSCs were cultured in smooth muscle cell medium with or without TGF-β1 after forming embryoid bodies. The cell morphology, cell characteristics and contractility were compared after 7 days of differentiation. Real-time PCR and Western blot were used to assess the mRNA and protein expression levels of α-SMA, Calponin, SM22α, Collagen I and Collagen III. Functional contraction study was performed using carbachol. Results HiPSC could successfully differentiate into cells that were similar to typical smooth muscle cells in morphology. The expression of α-SMA, Calponin and SM22α up-regulated after induction. TGF-β1 could further up-regulated α-SMA expression.Immunofluorescence images showed that more than 80% of the hiPSC-derived SMLCs by TGF-β1 stained with smooth muscle cell markers α-SMA, SMMHC, SM22α and Calponin. Analyses of expression in collagen showed that hiPSC-derived SMLCs exhibited higher levels of Collagen I and Collagen III after induction by TGF-β1. Conclusion The hiPSC could successfully differentiate into smooth-muscle-like cells using embryoid bodies method. TGF-β1 can promote the differentiation and enhance collagen synthesis[.S Chin J Cardiol 2019;20(1):44-53]     

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 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 mRNA, 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.     

The role of Smad3 in mediating mouse hepatic stellate cell activation.

Transforming growth factor beta (TGF-beta) is the most potent profibrogenic mediator in liver fibrosis. Although Smad proteins have been identified as intracellular mediators in the TGF-beta signaling pathway, the function of individual Smad proteins remains poorly understood. The aim of this study was to explore the contribution of Smad3 in mediating TGF-beta responses in a model of acute liver injury in vivo and in culture-activated hepatic stellate cells (HSCs). Wild-type, Smad3 heterozygous or Smad3 homozygous knockout mice were treated with a single intragastric administration of CCl(4). After 72 hours, the induction of hepatic collagen alpha1(I) and alpha2(I) messenger RNA (mRNA) levels in Smad3 knockout mice was only 42% and 64%, respectively, of the levels induced in wild-type mice. However, smooth muscle alpha-actin (alpha-SMA) was expressed at a slightly higher level in livers from knockout mice compared with wild-type mice. In culture-activated HSCs from Smad3 knockout mice, collagen alpha1(I) mRNA was 73% of wild-type HSCs, but alpha-SMA expression was the same. HSCs from knockout mice showed a higher proliferation rate than wild-type HSCs. Smad3-deficient HSCs did not form TGF-beta1-induced Smad-containing DNA-binding complexes. In conclusion, (1) maximal expression of collagen type I in activated HSCs requires Smad3 in vivo and in culture; (2) Smad3 is not necessary for HSC activation as assessed by alpha-SMA expression; (3) Smad3 is necessary for inhibition of proliferation of HSCs, which might be TGF-beta-dependent; and (4) Smad3 is required for TGF-beta1-mediated Smad-containing DNA-binding complex formation in cultured HSCs.     

Increased 9,13-di-cis-retinoic acid in rat hepatic fibrosis: implication for a potential link between retinoid loss and TGF-beta mediated fibrogenesis in vivo.

BACKGROUND/AIMS: During hepatic fibrosis, hepatic stellate cells (HSCs) transform into myofibroblastic cells and lose their intracellular droplets of retinyl esters, the storage form of vitamin A. Recently, we have demonstrated that 9,13-di-cis-retinoic acid (RA), a geometric isomer identified as a stable and major metabolite of vitamin A in circulation, stimulates the synthesis of plasminogen activator (PA) and induces PA/plasmin-dependent latent transforming growth factor (TGF)-beta activation in HSC cultures, probably via induction and activation of RA receptor (RAR) alpha. The aim of the present study was to address a potential link between the loss of retinyl esters to increased formation of RA(s), which might play a role in facilitating TGF-beta-mediated liver fibrogenesis in vivo. METHODS: We examined the effect of 9,13-di-cis-RA on transactivating activity of RARalpha in HeLa cells as well as its effect on PA- and TGF-beta-dependent collagen synthesis in rat and human HSC cultures. We measured the changes in 9,13-di-cis-RA levels both during activation of rat HSCs in vitro and during porcine serum-induced rat hepatic fibrosis in vivo and correlated this with RAR alpha/beta, PA, TGF-beta and type I procollagen mRNA expression in the fibrotic liver. RESULTS: 9,13-di-cis-RA transactivated RARalpha, and provoked PA/plasmin and TGF-beta-dependent procollagen synthesis in HSCs. 9,13-di-cis-RA levels were increased both in activated HSCs in vitro and in fibrotic liver accompanying the enhanced expression of RAR alpha/beta, PA, TGF-beta and procollagen in vivo. CONCLUSIONS: These findings suggest a potential link between 9,13-di-cis RA formation and hepatic fibrosis via formation of TGF-beta in vivo, and thus provide further insight into the biologic role of retinoids during hepatic fibrogenesis.     

TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.

After liver injury, hepatic stellate cells (HSCs) undergo a process of activation with expression of smooth muscle alpha-actin (alpha-SMA), an increased proliferation rate, and a dramatic increase in synthesis of type I collagen. The intracellular signaling mechanisms of activation and perpetuation of the activated phenotype in HSCs are largely unknown. In this study the role of the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, were evaluated in primary cultures of rat HSCs. The effect of JNK was assessed by using an adenovirus expressing a dominant negative form of transforming growth factor beta (TGF-beta)-activated kinase 1 (TAK1) (Ad5dnTAK1) and a new selective pharmacologic inhibitor SP600125. The effect of p38 was assessed with the selective pharmacologic inhibitor SB203580. These kinases were inhibited starting either in quiescent HSCs (culture day 1) or in activated HSCs (culture day 5). Although blocking TAK1/JNK and p38 decreased the expression of alpha-SMA protein in early stages of HSC activation, no effect was observed when TAK1/JNK or p38 were inhibited in activated HSCs. JNK inhibition increased and p38 inhibition decreased collagen alpha1(I) mRNA level as measured by RNase protection assays, with maximal effects observed in early stages of HSC activation. Furthermore, TAK1/JNK inhibition decreased HSC proliferation, whereas p38 inhibition led to an increased proliferation rate of HSCs, independently of its activation status. These results show novel roles for the TAK1/JNK pathway and p38 during HSC activation in culture. Despite similar activators of TAK1/JNK and p38, their functions in HSCs are distinct and opposed.     

Prostaglandin E2 inhibits transforming growth factor beta 1-mediated induction of collagen alpha 1(I) in hepatic stellate cells

BACKGROUND/AIMS: Cyclooxygenase-2 (COX-2) has been implicated in a number of hepatic stellate cell (HSC) functions but its relationship to transforming growth factor-beta 1 (TGF-beta 1)-mediated fibrogenesis is unknown. We assessed the impact of COX-2 inhibition and PGE(2) on the regulation of TGF-beta 1-stimulated matrix synthesis in an immortalized human HSC line, LX-1 and corroborated these findings in primary stellate cells. METHODS: Expression of COX-2 was assessed by Western blotting and real time quantitative PCR. The effect of NS398, a selective COX-2 inhibitor, and PGE(2) on TGF-beta 1-mediated fibrogenesis was examined by measuring mRNA levels of collagen alpha1(I). PGE(2) receptor expression was analyzed by RT-PCR. RESULTS: Under basal conditions, NS398 suppressed PGE(2) synthesis and induced collagen alpha 1(I) whereas exogenous PGE(2) suppressed expression of collagen alpha1(I). TGF-beta 1 induced COX-2 mRNA, COX-2 protein and PGE(2) biosynthesis. Importantly, TGF-beta 1-mediated induction of collagen alpha 1(I) was markedly suppressed by the addition of exogenous PGE(2). All four major PGE(2) receptors were expressed in LX-1 cells. CONCLUSIONS: These results suggest that COX-2-derived PGE(2) inhibits both basal and TGF-beta 1-mediated induction of collagen synthesis by HSC. Based on these findings, it will be important to determine whether inhibiting COX-derived PGE(2) synthesis alters the progression of liver fibrosis in vivo.     

转化生长因子β1对不同活化状态肝星状细胞增殖与Ⅰ型胶原表达的影响

目的 观察不同活化状态肝星状细胞(HSC)对外源性转化生长因子-β_1(TGF-β_1)旁分泌刺激的生物学效应作用。方法 原代分离培养大鼠HSC,无包被塑料培养皿上分别培养1、4、7d,细胞处于静止、中间活化与完全活化状态,继以10～500 pmol/L TGF-β_1温育细胞24h,~3H—TdR掺入法测定细胞增殖,western blot法检测细胞α-平滑肌肌动蛋白(α-SMA)与Ⅰ型胶原蛋白表达沉积,~3H-脯氨酸掺入与胶原酶消化法测定细胞总胶原的分泌量。100pmol/L TGF-β_1温育细胞15～90min,northern blot法检测细胞Ⅰ型前胶原mRNA的表达水平。结果 TGF-β_1浓度依赖性抑制培养1d HSC的细胞增殖,10～500 pmol/L TGF-β_1浓度组细胞内~3H—TdR掺入率分别为对照组的52.8％～16.8％,与对照组比较,q值为5.44～10.37,P<0.01。但TGF-β_1对培养4d与7d的细胞增殖无影响。随细胞活化,HSC基础性α-SMA、Ⅰ型胶原蛋白与mRNA水平明显增加,而TGF-β_1刺激各培养时间HSC以上蛋白与基因的表达。培养1、4、7d HSC基础水平与TGF-β_1刺激的总胶原分泌量分别为(804±274)dpm/孔与(1 200±708)dpm/孔;(2 966±1 701)dpm/孔与(6 160±1 123)dpm/孔;(2 580±767)dpm/孔与(4 583±1 467)dpm/孔,后2组组内比较,t值分别为3.84与2.96,P<0.01或P<0.05。以培养4d HSC     

转录因子c-myb及肝脏激活蛋白对α1(Ⅰ)胶原基因在肝星状细胞中表达的调控作用

目的 了解激活的ＨＳＣ中ｃ－ｍｙｂ及肝脏组织特异的转录因子－肝脏激活蛋白（ｌｉｖｅｒ ａｃｔｉｖａｔｏｒｐｒｏｔｅｉｎ，ＬＡＰ）在α１（Ｉ）基因表达调控中的作用。方法 采用链霉蛋白酶，胶原酶原位灌注、Ｎｙｃｏｄｅｎｚ密度梯度离心分离大鼠ＨＳＣ，并进行体外培养使之激活，构建含人α１（Ｉ）胶原基因启动子片段（－８０４～＋１４５或－８０４～＋２２２碱基）的荧光素酶报告基因质粒，用构建的报告基因质粒与ｃ－