OBJECTIVE: To investigate whether the HMGB1 DAMP signaling pathway is involved in resveratrol anti-oxygen glucose deprivation (OGD)-induced microglial inflammatory processes and explore its underlying mechanisms.
METHODS: Cell viability was tested by MTT assay to determine the appropriate resveratrol and EX527 concentration and OGD time, and the cells were divided into four groups: Control, OGD+DMSO, OGD+RES and OGD+RES+EX527, ELISA. Rt-PCR and western blot were used to detect inflammatory factors and HMGB1 signaling pathway-related protein changes. WB and immunofluorescence were used to demonstrate the localization of HMGB1 in cells, the acetylation level of HMGB1 and the interaction between HMGB1 and Sirt1 were detected by CoIP. Different groups BV2 cells were co-cultured with primary mouse neurons, and the release of HMGB1 was observed and the level of LDH in the supernatant was detected.
RESULTS: We determined that RES (100umol), EX527 (100umol) and OGD3h were the optimal treatment conditions. RES inhibited the increase of inflammatory mediators and HMGB1 signaling pathway-related proteins and reduce the increase of HMGB1 level in cell supernatant after OGD, and EX-527 reversed this effect; immunofluorescence indicated that RES can limit HMGB1 in cells, however, different from the change of HMGB1 in the culture medium, there was no significant difference in the mRNA level of HMGB1 in each group, suggesting that the increase of HMGB1 level in supernatant after hypoxia is mainly due to the increase of active secretion rather than synthesis. CoIP results showed that the binding of HMGB1 to deacetylase SIRT1 was decreased and the level of acetylation was decreased after OGD. RES could increase the interaction between HMGB1 and sirt1 and increase the acetylation level of HMGB1 but EX527 reduced this interaction. In the neuron co-culture system, the extracellular release of HMGB1 and LDH was increased in the supernatant of the OGD+DMSO group, while this change in the RES group was attenuated, and the OGD+RES+EX527 group restored the increase of LDH and HMGB1.
CONCLUSIONS: The activation of downstream signaling pathway by active release of HMGB1 is partially involved in OGD induced BV2 inflammatory process. Resveratrol reduces inflammation by inhibiting HMGB1 release, a role associated with its ability to activate SIRT1-mediated acetylation of HMGB1.