Abstract
OBJECTIVE: To investigate whether the HMGB1 DAMP signaling pathway is involved inresveratrol anti-oxygen glucose deprivation (OGD)-induced microglialinflammatory processes and explore its underlying mechanisms.
METHODS: Cell viability was tested by MTT assay to determine the appropriateresveratrol and EX527 concentration and OGD time, and the cells were dividedinto four groups: Control, OGD+DMSO, OGD+RES and OGD+RES+EX527, ELISA. Rt-PCRand western blot were used to detect inflammatory factors and HMGB1 signalingpathway-related protein changes. WB and immunofluorescence were used todemonstrate the localization of HMGB1 in cells, the acetylation level of HMGB1and the interaction between HMGB1 and Sirt1 were detected by CoIP. Differentgroups BV2 cells were co-cultured with primary mouse neurons, and the release ofHMGB1 was observed and the level of LDH in the supernatant was detected.
RESULTS: We determined that RES (100umol), EX527 (100umol) and OGD3h were theoptimal treatment conditions. RES inhibited the increase of inflammatorymediators and HMGB1 signaling pathway-related proteins and reduce the increaseof 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 nosignificant difference in the mRNA level of HMGB1 in each group, suggesting thatthe increase of HMGB1 level in supernatant after hypoxia is mainly due to theincrease of active secretion rather than synthesis. CoIP results showed that thebinding of HMGB1 to deacetylase SIRT1 was decreased and the level of acetylationwas decreased after OGD. RES could increase the interaction between HMGB1 andsirt1 and increase the acetylation level of HMGB1 but EX527 reduced thisinteraction. In the neuron co-culture system, the extracellular release of HMGB1and LDH was increased in the supernatant of the OGD+DMSO group, while thischange in the RES group was attenuated, and the OGD+RES+EX527 group restored theincrease of LDH and HMGB1.
CONCLUSIONS: The activation of downstream signaling pathway by active release ofHMGB1 is partially involved in OGD induced BV2 inflammatory process. Resveratrolreduces inflammation by inhibiting HMGB1 release, a role associated with itsability to activate SIRT1-mediated acetylation of HMGB1.
