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
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） w...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.展开更多
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.
