ObjectiveTo investigate the role and molecular mechanism of necrostatin-1 (Nec-1), a specific programmed cell necrosis inhibitor, in promoting the oxidative stress response of macrophages under high glucose (HG) environment.
MethodsMacrophages were cultured in control (5.5 mmol·L-1glucose) or HG (25 mmol·L-1glucose) medium for 72 h. The HG+Nec-1 group was given HG and 5 μmol·L-1Nec-1. Reactive oxygen species (ROS) level, malondialdehyde (MDA) activity, and superoxide dismutase (SOD) activity were measured by 2’-7’dichlorofluorescin diacetate, MDA, and SOD enzyme linked immunosorbent assay kits, respectively. Moreover, receptor interacting protein 1 (RIP1) expression was assessed through real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB). Finally, after the expression of RIP1 in macrophages was silenced, the effect of HG environment on oxidative stress response was evaluated in the gene-deficient cells.
ResultsThe HG group had increased ROS level and MDA activity (P<0 .000 1) and decreased sod activity (P<0 .000 1) compared with the control group. the hg+nec-1 group had higher ros level and mda activity (P<0 .000 1) and lower sod activity (P<0 .01) than the hg group. the qrt-pcr and wb results showed that rip1 mrna level (P<0 .001) and protein expression level (P<0 .000 1) in the hg group were significantly higher than those in the control group, and rip1 mrna and protein expression levels in the hg+nec-1 group were significantly lower than those in the hg group (P<0 .000 1). after rip1 was silenced effectively (P<0 .001) with si-rna, the ros level and mda activity of the hg+si-rip1 group decreased compared with those of the hg+si-negative control (si-nc) group (P<0 .001), and sod activity in the hg+si-rip1 group increased than that in the hg+si-nc group (P<0 .000 1).
ConclusionHG promotes oxidative stress on macrophages by upregulating RIP1 expression.
