Research Paper Volume 13, Issue 14 pp 18606—18619

Obesity increases neuropathic pain via the AMPK-ERK-NOX4 pathway in rats

Overexpression of NOX4 abolishes the protective effect of an ERK inhibitor on DRG cells under high-fat environment. DRG neurons isolated from rats were cultured. PA was applied to simulate a high-fat environment and U0126 to inhibit ERK. NOX4 cDNA was transfected into cells to overexpress NOX4. (A) The transfection effect of NOX4 was indicated by western blot. (B–D) The supernatant of cultured cells was collected and the secretion of IL-1β, IL-6, and TNF-α was determined by ELISA. (E) The ROS content was detected by oxidant-sensitive fluorescence probe DHE. (F, G) The expression of cleaved caspase3, bcl-2 and bax in DRG neurons were detected by western blot. (H) Cell apoptosis level was detected by TUNEL method. N = 5 per group, *P P

Figure 5. Overexpression of NOX4 abolishes the protective effect of an ERK inhibitor on DRG cells under high-fat environment. DRG neurons isolated from rats were cultured. PA was applied to simulate a high-fat environment and U0126 to inhibit ERK. NOX4 cDNA was transfected into cells to overexpress NOX4. (A) The transfection effect of NOX4 was indicated by western blot. (BD) The supernatant of cultured cells was collected and the secretion of IL-1β, IL-6, and TNF-α was determined by ELISA. (E) The ROS content was detected by oxidant-sensitive fluorescence probe DHE. (F, G) The expression of cleaved caspase3, bcl-2 and bax in DRG neurons were detected by western blot. (H) Cell apoptosis level was detected by TUNEL method. N = 5 per group, *P < 0.05 vs. U0126+Vector group, **P < 0.01 vs. U0126+Vector group. U0126, ERK inhibitor; ERK, extracellular-regulated kinase; NOX4, NAD(P)H oxidase 4; PA, palmitic acid; DRG, dorsal root ganglia; IL-1β, interleukin-1 beta; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha; ELISA, enzyme-linked immunosorbent assay; DHE, dihydroethidium; ROS, reactive oxygen species.