Fig. 6: NPM downregulated ROS and reduced the apoptosis of HSCs via the Akt pathway.

CellROX Orange reagent and dihydroethidium were used to label intracellular hydrogen peroxide and superoxide anion. The ROS level and apoptosis of HSCs were detected by flow cytometry. A Flow cytometry results showed that NPM knockdown remarkably reduced hydrogen peroxide and superoxide anion in human LX-2 cells and primary mouse HSCs. B Flow cytometry showed NPM knockdown significantly induced the apoptosis of LX-2 and primary mouse HSCs. C Flow cytometry analysis of primary HSCS isolated from liver tissue of fibrotic mice showed that when VA-lip-siNPM specifically down-regulated the NPM expression of HSC in liver tissue, ROS was up-regulated and apoptosis was increased. When the up-regulated ROS in liver tissue was down-regulated by NAC, the apoptosis of HSC was reduced (n = 3). D Flow cytometry results showed that the ROS inhibitor NAC dose-dependently inhibited CIGB-induced hydrogen peroxide and apoptosis in LX-2 cells. E NAC inhibited CIGB-induced hydrogen peroxide and apoptosis in primary mouse HSCs. F Flow cytometry results showed that NAC reduced MK2206-induced hydrogen peroxide and reduced apoptosis in LX-2 cells. G NAC reduced MK2206-induced hydrogen peroxide and reduced apoptosis in primary mouse HSCs. The data shown are representative of three independent experiments and quantification data were presented as mean ± SEM; MFI mean fluorescence intensity, WT untreated cells, NC negative control, ns non-significant difference, **p < 0.01, ***p < 0.001; T test.