The role and mechanism of NADPH oxidase in leptin-induced reactive oxygen species production in hepatic stellate cells

Abstract

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. 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. (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. 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.