The role and mechanism of NADPH oxidase in leptin-induced reactive oxygen species production in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether or not NADPH oxidase (NOX) participates in leptin-induced reactive oxygen species (ROS) production in hepatic stellate cells (HSC) and to explore the possible mechanism.</p><p><b>METHODS</b>HSC-T6 cells (rat hepatic stellate cells line) were divided into nine groups: Group1: leptin (100 ng/ml) treated; Group2-6: leptin treated together with inhibitors that block different ROS-producing systems: diphenylene-iodonium (DPI) (20 micromol/L), Rotenone (20 micromol/L), Metyrapone (250 micromol/L), Allopurinol (100 micromol/L) and Indomethacin(100 micromol/L); Group7: leptin treated together with Janus kinase (JAK) inhibitor AG490 50 micromol/L; Group8: normal control group (treated DMEM with 0.1% DMSO); Group9: negative control group (untreated). Intracellular ROS levels were measured with dichlorodihydrofluorescein diacetate (DCFH-DA) dye assay by Fluorescence microscope and/or flow cytometry. NOX activity was analyzed by using spectrophotometer to calculate the absorbance of NADPH. The mRNA levels of Rac1 and p22Phox were evaluated by RT-PCR.</p><p><b>RESULTS</b>(1) Leptin increased significantly the ROS production as compared to normal control group (92.91+/-4.19 vs.27.56+/-6.27, P<0.01) in HSC-T6 cells. Both the NADPH oxidase inhibitor DPI and AG490 (50 micromol/L) blocked the ROS production, inhibitors of other ROS producing systems had no significant effect on ROS production induced by lepin (P is more than 0.05). (2) Leptin treated HSC-T6 cells for 1 hour up-regulated the NOX activity significantly compared with that in normal control group [(1.90+/-0.22) pmol.min(-1).mg(-1) vs. (0.76+/-0.06) pmol.min(-1).mg(-1), P<0.05]. Furthermore, the NOX activity increased after being treated with leptin for 12 hours and 24 hours than being treated for 1 hour. Leptin-induced up-regulation of NOX activity was inhibited by pretreatment with DPI or AG490. (3) The RT-PCR results indicated that mRNA expressions of Rac1 and p22Phox in HSC-T6 cells with 12 hours of leptin stimulation increased significantly as compared with normal control group (0.41+/-0.13 vs 0.14+/-0.08, 0.45+/-0.12 vs 0.20+/-0.08, all P<0.05), while the DPI and AG490 had no effect on the mRNA expressions of Rac1 and p22Phox.</p><p><b>CONCLUSION</b>NOX is the main cellular source of the reactive oxygen species (ROS) generated by HSCs in response to leptin stimulation. The mechanism is probably that leptin can directly activate NOX through JAK signal transduction and hence induce the expression of NOX subunit to promote the activity of NOX which generates considerable ROS in HSC.</p>

Effect of ursolic acid on proliferation and apoptosis of hepatic stellate cells in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of ursolic acid on proliferation and apoptosis of hepatic stellate cells (HSC) in vitro and explore the mechanisms of apoptosis of HSC induced by ursolic acid by studying the expressions of apoptosis-regulating proteins Bcl-2, Bax and Caspase 3 in HSC.</p><p><b>METHODS</b>Hepatic stellate cells HSC-T6 and hepatocytes L02 were incubated with different concentrations of ursolic acid (25, 50, 75, 100, 125 and 150 micromol/L) for 24 h, 48 h and 72 h. The effect of ursolic acid on the cell proliferation was studied by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The rate of HSC-T6 apoptosis was identified by flow cytometry (FCM) and the morphological change of apoptosis was observed with light microscopy. The expressions of apoptosis-regulating protein Bcl-2, Bax and Caspase 3 in HSC-T6 after apoptosis induced by ursolic acid were examined by immunocytochemical staining assay.</p><p><b>RESULTS</b>MTT analysis indicated administration of 25-150 micromol/L ursolic acid incubated with HSC-T6 for 24 h, 48 h and 72 h significantly inhibited HSC-T6 proliferation in a dose-dependent and time-dependent manner compared with the control group. Promotive effect of ursolic acid on proliferation of hepatocyte L02 was observed in the 25, 50, 75 micromol/L concentration groups. Ursolic acid inhibited L02 proliferation when its concentration was higher than 100 micromol/L and for 72 hours or longer. HE stained histological slides demonstrated morphologic changes of HSC-T6, including karyorrhexis and cytoplasm vacuolization, when they were treated with ursolic acid at 75 micromol/L concentrations for 48 h. FCM showed the apoptosis ratios of HSC-T6 were 10.30%+/-3.85%, 21.87%+/-4.46% and 31.33%+/-6.18% after treating HSC-T6 with ursolic acid at concentrations of 25, 50 and 75 micromol/L for 48 h. They were significantly higher than that of the control group 2.93%+/-1.60%. Immunocytochemistry also indicated the expressions of Bax and caspase 3 protein in HSC-T6 cells were up-regulated in a dose-dependent manner, but expressions of Bcl-2 protein were not significantly different from that of the blank control group (P more than 0.05).</p><p><b>CONCLUSIONS</b>Ursolic acid could significantly inhibit HSC proliferation and induce apoptosis in a dose-dependent and time-dependent manner. Ursolic acid in low concentration promotes proliferation of L02 cells, but in high concentrations (more than 100 micromol/L) it inhibits the growth of hepatocytes. Expressions of Bax and Caspase 3 in apoptotic HSC were increased; expressions of Bcl-2 protein were not significantly different from that of the control group, while Bcl-2/Bax ratio was reduced. Our results suggest that HSC-T6 cell apoptosis induced by ursolic acid occurs through mechanisms involving mitochondrial pathways and Bcl-2 family proteins.</p>

Methodology for Aptamer Selection / 中国生物工程杂志

The methods for aptamer selection have been modified and developed in recent years. Selection efficiency has been increased due to the improvement in separation of bound and unbound nucleic acids; applicability of aptamers has been improved due to the development of primer-free SELEX and the utilization of short oligonucleotide library; more research requirements have been satisfied due to the functional modifications of aptamer selection methods. All the methodological advances enhance the potential value of aptamers in basic researches and clinical applications.