CORM-2 attenuates red blood cell-mediated inflammation by upregulating phagocytic capacity of cerebral microglial cells of the neonatal rat in vitro

• Effectively inhibiting inflammatory response in peri -hematoma area may reduce ICH-mediated secondary ischemic brain injury. • We established microglia and RBCs co-culture in vitro, to investigate the effect of HO-1/CO on promoting microglia phagocytosis. • CO, an endogenous antioxidant gas molecule produced by HO-1-mediated heme decomposition, exerts strong antioxidant effects. • CORM-2 can inhibit RBCs-induced inflammatory reactions in vitro by promoting microglial phagocytosis to RBCs and down regulating IL-1β and NF-κB p65.x'c'c To explore the mechanism of CORM-2 on attenuating hemorrhage-related inflammation. Microglia were isolated from neonatal rats (1–2 days old) and identified using CD11b/c antibody. Subsequently, some of the microglia were co-cultured with red blood cells (RBCs) marked with PKH26 fluorescent dye, and then treated with CORM-2. Microglial cells were divided into three cultures: microglia, microglia+ PKH26+ RBCs and microglia + PKH26+ RBCs + CORM-2. Microglial phagocytosis of PKH26+ RBCs was observed under an inverted fluorescence microscope and were determined by cell counting plates under microscopy. Moreover, the fluorescence intensity of microglia that phagocytized PKH26+ RBCs was detected through immunofluorescence. The expression levels of HO-1, NF-κB p65, and IL-1β were detected using RT-qPCR, western blotting, and immunofluorescence, respectively. The levels of carbon monoxide hemoglobin (HbCO) in the cell supernatant in each group were measured using enzyme-linked immunoassay (ELISA). After 1-day of co-culturing, the reduction in the number of residual PKH26+ RBCs in the microglia+ PKH26+ RBCs + CORM-2 group was significantly greater than that in the microglia+ PKH26+ RBCs, which revealed that microglia phagocytosis was enhanced in CORM-2 treated group. Moreover, compared with microglia + PKH26+ RBCs group, the microglia+ PKH26+ RBCs + CORM-2 group showed higher levels of HO-1 mRNA and protein expression at day-3 and day-5 after co-culturing. Furthermore, CORM-2 significantly inhibited the expression of mRNA and proteins of NF‐κB p65 and IL-1β after 3 days of co-culturing. Meanwhile, CORM-2 did not increase the level of HbCO in the cell supernatant. CORM-2 inhibits RBCs-induced inflammation in vitro by promoting microglial phagocytosis of RBCs and decreases IL-1β and NF-κB p65; the mechanism may implicate the HO-1/CO system.