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

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.

This study was supported by the National Science & Technology Fundamental Resource Investigation Program of China (2018FY100903 to XPY), and Jiangxi Provincial Health Commission Science and Technology Plan project(202311506 to ZYC), Jiangxi Provincial Administration of Traditional Chinese Medicine science and technology plan project (2022A322 to ZYC), Key Projects of Jiangxi Provincial Department of Education (GJJ2201902 to ZYC), Youth Foundation of Natural Science Foundation of Jiangxi Province (20224BAB216045 to ZYC).