AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-β1 (TGFβ1)/ activin receptor-like kinase 1 (ALK1, type Ⅰ receptor) signaling-pathway-related gene expression in the L...AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-β1 (TGFβ1)/ activin receptor-like kinase 1 (ALK1, type Ⅰ receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells. METHODS: LX-2 cells were treated with TGFβ1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGFβ1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA (α-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA. RESULTS: In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h. CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.展开更多
Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in ...Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in a wide array of physiological and pathological processes.This study aimed to investigate the effect of KLF4 on the proliferation,apoptosis and phenotype of quiescent HSCs Methods We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector,to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection.Cell proliferation was assessed using the CCK-8 assay.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.Western blotting was used to determine the levels of some quiescence and activation markers of HSCs Results Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1,which are quiescent HSC markers,while significantly decreased the levels of N-cadherin and a-SMA,known activated HSC markers.In contrast,cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced Conclusion KLF4 inhibits the proliferation and activation of human LX-2 HSCs.It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.展开更多
目的研究IL-32γ可否诱导肝星状细胞LX-2表达Ⅰ型胶原(CollagenⅠ)。方法不同浓度的IL-32γ与LX-2共培养,分别收集培养上清液、提取细胞总RNA,real-time PCR检测CollagenⅠmRNA表达水平,ELISA法检测CollagenⅠ蛋白质表达水平。用不同浓...目的研究IL-32γ可否诱导肝星状细胞LX-2表达Ⅰ型胶原(CollagenⅠ)。方法不同浓度的IL-32γ与LX-2共培养,分别收集培养上清液、提取细胞总RNA,real-time PCR检测CollagenⅠmRNA表达水平,ELISA法检测CollagenⅠ蛋白质表达水平。用不同浓度的人重组磷酸化p38α蛋白(recombinant human active p38α蛋白)与LX-2细胞共培养,48 h后收集细胞培养上清液,ELISA法检测CollagenⅠ蛋白质表达水平。再用不同浓度的p38MAPK抑制剂SB203580加入LX-2细胞与IL-32γ共培养的培养液中,分别收集培养上清液,采用ELISA检测CollagenⅠ蛋白质表达水平,同时提取细胞总RNA采用real-time PCR检测CollagenⅠmRNA表达水平。结果 IL-32γ诱导人LX-2细胞表达CollagenⅠ,且其表达水平随IL-32γ浓度的增加而增强;人重组磷酸化p38α蛋白可诱导LX-2细胞表达CollagenⅠ增高,阻断p38MAPK可降低IL-32γ诱导的CollagenⅠ表达。结论 IL-32γ通过活化p38MAPK通路诱导LX-2细胞表达CollagenⅠ,IL-32γ可能通过诱导肝星状细胞表达CollagenⅠ而参与肝纤维化的形成。展开更多
[目的]建立人肝星状细胞(LX-2)与人肾小管上皮细胞(HK-2)共培养体系的理论模型,研究LX-2对HK-2转分化的影响。[方法]以Transwell小室建立体外LX-2细胞和HK-2细胞间接共培养体系。应用细胞免疫组织化学法检测HK-2细胞平滑肌肌动蛋白(α-S...[目的]建立人肝星状细胞(LX-2)与人肾小管上皮细胞(HK-2)共培养体系的理论模型,研究LX-2对HK-2转分化的影响。[方法]以Transwell小室建立体外LX-2细胞和HK-2细胞间接共培养体系。应用细胞免疫组织化学法检测HK-2细胞平滑肌肌动蛋白(α-SMA)表达;用酶联免疫吸附法(ELISA法)检测各组细胞培养液中转化生长因子-β1(TGF-β1)的含量;逆转录-聚合酶链反应法(RT-PCR法)检测HK-2细胞TβRⅠm RNA、TβRⅡm RNA、Smad2 m RNA、Smad3 m RNA、Smad7 m RNA的表达。[结果]除了空白组,形态学上其他两组均可看到HK-2细胞胞浆染棕黄色,不同数量细胞由立方铺路石样转变为梭形长条状,细胞肥大;HK-2细胞高表达α-SMA(P<0.01);HK-2细胞上清液中TGF-β1含量明显高于空白组(P<0.01);HK-2细胞TβRⅠ、Smad2、Smad3基因表达明显上调,而TβRⅡ、Smad7基因表达明显下调。[结论]Transwell共培养法可诱导HK-2细胞转分化,其机制可能与TGF-β1/Smad信号通路有关。展开更多
文摘AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-β1 (TGFβ1)/ activin receptor-like kinase 1 (ALK1, type Ⅰ receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells. METHODS: LX-2 cells were treated with TGFβ1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGFβ1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA (α-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA. RESULTS: In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h. CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.
基金supported by the National Natural Science Foundation of China(No.81071541).
文摘Objective Hepatic stellate cells(HSCs)play a crucial role in liver fibrosis.Early-stage liver fibrosis is reversible and intimately associated with the state of HSCs.Kruppel-like factor 4(KLF4)plays a pivotal role in a wide array of physiological and pathological processes.This study aimed to investigate the effect of KLF4 on the proliferation,apoptosis and phenotype of quiescent HSCs Methods We designed a KLF4 lentiviral vector and a KLF4 siRNA lentiviral vector,to upregulate and silence KLF4 expression in human HSC LX-2 cells via transfection.Cell proliferation was assessed using the CCK-8 assay.Flow cytometry was used to detect the cell cycle distribution and apoptosis rate.Western blotting was used to determine the levels of some quiescence and activation markers of HSCs Results Overexpression of KLF4 significantly increased the levels of E-cadherin and ZO-1,which are quiescent HSC markers,while significantly decreased the levels of N-cadherin and a-SMA,known activated HSC markers.In contrast,cell proliferation and apoptosis rates were elevated in LX-2 cells in which KLF4 expression was silenced Conclusion KLF4 inhibits the proliferation and activation of human LX-2 HSCs.It might be a key regulatory protein in the maintenance of HSC quiescence and may serve as a target for the inhibition of hepatic fibrosis.
文摘目的研究IL-32γ可否诱导肝星状细胞LX-2表达Ⅰ型胶原(CollagenⅠ)。方法不同浓度的IL-32γ与LX-2共培养,分别收集培养上清液、提取细胞总RNA,real-time PCR检测CollagenⅠmRNA表达水平,ELISA法检测CollagenⅠ蛋白质表达水平。用不同浓度的人重组磷酸化p38α蛋白(recombinant human active p38α蛋白)与LX-2细胞共培养,48 h后收集细胞培养上清液,ELISA法检测CollagenⅠ蛋白质表达水平。再用不同浓度的p38MAPK抑制剂SB203580加入LX-2细胞与IL-32γ共培养的培养液中,分别收集培养上清液,采用ELISA检测CollagenⅠ蛋白质表达水平,同时提取细胞总RNA采用real-time PCR检测CollagenⅠmRNA表达水平。结果 IL-32γ诱导人LX-2细胞表达CollagenⅠ,且其表达水平随IL-32γ浓度的增加而增强;人重组磷酸化p38α蛋白可诱导LX-2细胞表达CollagenⅠ增高,阻断p38MAPK可降低IL-32γ诱导的CollagenⅠ表达。结论 IL-32γ通过活化p38MAPK通路诱导LX-2细胞表达CollagenⅠ,IL-32γ可能通过诱导肝星状细胞表达CollagenⅠ而参与肝纤维化的形成。
文摘[目的]建立人肝星状细胞(LX-2)与人肾小管上皮细胞(HK-2)共培养体系的理论模型,研究LX-2对HK-2转分化的影响。[方法]以Transwell小室建立体外LX-2细胞和HK-2细胞间接共培养体系。应用细胞免疫组织化学法检测HK-2细胞平滑肌肌动蛋白(α-SMA)表达;用酶联免疫吸附法(ELISA法)检测各组细胞培养液中转化生长因子-β1(TGF-β1)的含量;逆转录-聚合酶链反应法(RT-PCR法)检测HK-2细胞TβRⅠm RNA、TβRⅡm RNA、Smad2 m RNA、Smad3 m RNA、Smad7 m RNA的表达。[结果]除了空白组,形态学上其他两组均可看到HK-2细胞胞浆染棕黄色,不同数量细胞由立方铺路石样转变为梭形长条状,细胞肥大;HK-2细胞高表达α-SMA(P<0.01);HK-2细胞上清液中TGF-β1含量明显高于空白组(P<0.01);HK-2细胞TβRⅠ、Smad2、Smad3基因表达明显上调,而TβRⅡ、Smad7基因表达明显下调。[结论]Transwell共培养法可诱导HK-2细胞转分化,其机制可能与TGF-β1/Smad信号通路有关。
