High mobility group box 1, a novel serotonin receptor-7 negative modulator, contributes to M2 microglial ferroptosis and neuroinflammation in post-stroke depression.

Emerging evidence points to a pivotal role of microglial polarization in the neuroinflammatory processes linked to post-stroke depression (PSD), with both high mobility group box 1 (HMGB1) and serotonin receptor-7 (5-HT7R) being implicated in the microglial inflammatory response. However, the underlying mechanisms remain unclear. This study investigated the potential roles of HMGB1 and 5-HT7R in PSD. We first examined the interaction between HMGB1 and 5-HT7R in HEK293 cells using bioluminescence resonance energy transfer (BRET) technology. The expression of 5-HT7R was most significantly increased in microglia in vitro upon HMGB1 stimulation and in a transient middle cerebral artery occlusion (tMCAO) model, although it is also enhanced in astrocytes and neurons to some extent. Subsequently, the interaction between HMGB1 and 5-HT7R in primary microglia was examined using co-immunoprecipitation (Co-IP) and pull-down assays. Meanwhile, we found that HMGB1 upregulated the expression of 5-HT7R and downregulated the accumulation of second messenger cyclic adenosine monophosphate (cAMP) in HEK293 cells and primary microglia. Additionally, employing a 4-week tMCAO mice model, we found that 5-HT7R deletion or HMGB1 inhibition alleviated ischemic brain injury, depressive-like behaviors, M2 microglial ferroptosis, and neuroinflammation, meanwhile upregulating the cAMP/PKA and Nrf2/xCT/GPX4 pathways. Moreover, in vitro, recombinant HMGB1 (rHMGB1) effectively induced M2 microglial ferroptosis and neuroinflammation and suppressed the cAMP/PKA and Nrf2/xCT/GPX4 pathways in primary microglia, which were markedly attenuated by 5-HT7R deletion. Finally, cAMP analog dibutyryl cAMP (db-cAMP) or Nrf2 activator sulforaphane (SFN) attenuates ferroptosis in M2-polarized BV2 microglial cells and neuroinflammation induced by rHMGB1. Altogether, these results suggest that HMGB1 functions as a novel negative regulator of 5-HT7R, contributing to ischemic brain injury and PSD by promoting M2 microglial ferroptosis and neuroinflammation.