Hydrogen Sulfide Modulates Microglial Polarization Via AMPK Following Cerebral Ischemia

Objective: H2 S suppresses stroke-mediated neuroinflammation. However, the underlying mechanisms are poorly understood, although AMPK signal path way has been implicated in H2 S inhibition on neuroinflammation induced by LPS.We investigated if the H2 S donor(ADT-OH) polarized microglia to an anti-inflammatory(M2) phenotype by activating AMP-activated kinase(AMPK) and promote the angiogenesis following cerebral ischemia.Methods: Conditioned media were collected from primary neurons subjected to oxygen glucose deprivation(OGD neuronal conditioned medium) as well as control neurons without OGD treatment(Neuron Condition Medium). OGD neuronal conditioned medium was used to stimulate BV2 and primary microglia and neuron conditioned medium served as the control to mimic in vivo stroke.Mouse middle cerebral artery occlusion(MCAO) was applied as the in vivo stroke model. To investigate the mechanisms underlying H2 S inhibition on stroke-induced neuroinflammation, we used the H2 S donor(ADT-OH) and over-expressed H2 S synthase cystathionine-beta-synthase(CBS) in BV2 microglia. The expression levels of p-AMPK, CBS and cystathionine-γ-lyase(CSE) were assayed by Western Blot analysis. The expression levels of M1/M2 signature genes were assessed by Q-PCR or ELISA. AMPK/Ca MMKβ pharmacological inhibitors and AMPK si RNAs were used to explore whether H2 S suppression on neuroinflammation was depended on AMPK and Ca MMKβ.To investigate if ADT-OH promoted post-ischemic angiogenesis through microglia via the M2 polarization-depedent manner we used an in vitro angiogenesis model,in which the brain microvessel endothelial cells(b END3) formed vascular structures on matrigels.Finally,we examined the effect of ADT-OH on cerebral AMPK activation and M1/M2 polarization in the brain cortex in the mouse MCAO model.Result: Compared to neuron condition medium, OGD neuron conditioned medium reduced AMPK activation and enhanced M1 signature genes expression in BV2 and primary microlial cells. When microglia were co-treated with ADT-OH and OGD neuron condition medium at the same time, the AMPK activation was enhanced and ADT-OH cotreatment promoted M2 polarization, as evidenced by decreaed expression of M1 signature genes and enhanced expression of M2 signature genes.The promoting effects of ADT-OH on M2 polarization were attenuated by the AMPK or the Ca MMKβ inhibitor or AMPK si RNA. The results suggested that H2 S promoted M2 polarization via AMPK activation. Compared to neuron condition medium collected from neurons without OGD treatment, OGD-conditioned neuronal medium reduced CBS genes expression in BV2. Upon stimulation with OGD neuron conditioned medium, BV2 microglia also displayed a reduction in the expression of the endogenous H2 S synthase cystathionine-beta-synthase(CBS). Consistently, compared with the vector transfection, over-expression of CBS in BV2 cells elevated AMPK activation promoted M2 polarization in BV2 microglia treated with OGD neuron condition medium.To investigate if ADT-OH promoted angiogenesis via microglial polarization, the conditioned medium was collected from BV2 microglia co-treated with ADT-OH and OGD neuron condition medium, and then was added to b End3 cells seeded on the matrigels. Media collected from BV2 cells treated with neuron condition medium alone, OGD neuron condition medium alone or control BV2 cells without theatment. Only conditioned medium collected from BV2 microglia co-treated with ADT-OH and OGD neuron condition medium promoted in vitro angiogenesis, as evidenced by the vascular structures formed by b End3 on matrigels. Moreover, AMPK knockdown in BV2 microglia by si RNA aboragted the promoting effects of ADT-OH on angiogenesis. The results indicated the AMPK signal pathway was indespensiable for promoting effects of ADT-OH on angiogenesis through the microglial polarization-dependent mechanim.In the in vivo mouse MCAO model, ADT-OH enhanced the activation of AMPK, promoted M2 polarization and suppressed M1 polarization in the ischemic cortex.Conclusion: For the first time, our results indicated that H2 S donor suppressed post-ischemic neuroinflammation and thus enhanced angiogenesis via polarizing microglia to the M2 phenotype in an AMPK-dependent manner.