CaMKKβ-dependent Activation Of AMP-activated Kinase Is Critical To Suppressive Effects Of Hydrogen Sulfide On Neuroinflammation

Objective: Hydrogen sulfide (H2S) suppresses microglia-mediatedneuroinflammation. However, the underlying mechanisms are poorly understood, althoughnuclear factor-kappa B (NF-κB) and MAPK signaling cascades have been implicated inH2S inhibition on neuroinflammation. We investigated if H2S polarized microglia to ananti-inflammatory (M2) phenotype by activating AMP-activated kinase (AMPK).Methods: Lipopolysaccharide (LPS)-treated BV-2microglial cells and primarymicroglial cells were used as the in vitro microglia-mediated neuroinflammation model. Toinvestigate the mechanisms underlying H2S inhibition on neuroinflammation, threestructurally unrelated exogenous H2S donors (NaSH/GYY4137/ADT-OH) as well as theapproach to over-express the H2S synthase cystathionine-beta-synthase in microglia wereused. The concentrations at which the H2S donors inhibited microglia-mediatedneuroinflammation were determined by assessing LPS-provoked the production of thepro-inflammtion mediator nitric oxide (NO) with Griess reagent method. The expressionlevels of p-AMPK, CBS and Ca2+/calmodulin dependent protein kinase kinase beta(CaMKKβ) were assayed by Western Blot analysis. The expression levels of M1/M2signature genes were assessed by Q-PCR or ELISA. Both AMPK/CaMKKβpharmacological inhibitors and AMPK/CaMKKβ siRNAs were used to explore whetherH2S suppression on neuroinflammation depended on AMPK and CaMKKβ. Finally, in anin vivo neuroinflammation model induced by intracerebroventricular (ICV) injection ofLPS, we examined the effects of H2S donors on microglial AMPK activation and M1/M2polarization in the brain lateral septal complex area where microglia were over-activatedupon ICV injection of LPS.Results: Compared to vehicle or vector transfection, both H2S donors(ADT-OH/GYY4137/NaHS) and over-expression of the CBS plasmids enhanced AMPKactivation (phosphorylation) in BV-2microglial cells either in the presence or absence oflipopolysaccharide (LPS). Upon LPS stimulation, both ADT-OH and CBS over-expression promoted M2polarization of BV-2cells, as evidenced by reduced M1and elevated M2signature gene expression. The promoting effects of ADT-OH on M2polarization wereattenuated by an AMPK inhibitor or AMPK knockdown by siRNA. The results suggestedthat H2S suppressed neuroinflammation via AMPK activation.Furthermore, ADT-OH activated AMPK in Hela cells lacking1iver-kinase B1(LKB1), while both the CaMKKβ inhibitor and siRNA abolished ADT-OH activation ofAMPK and blunted ADT-OH suppression on M1gene expression and enhancement of M2gene expression in LPS-stimulated BV-2cells. These data suggested that H2S activatedAMPK to inhibit neuroinflammation in a CaMKKβ-dependent manner.Moreover, in primary microglia, ADT-OH promoted M2polarization ofLPS-stimulated primary microglia, which was blocked by the AMPK inhibitor andCaMKKβ inhibitor.In the LPS-induced in vivo neuroinflammation model, both ADT-OH and NaHSenhanced AMPK activation in the lateral septal complex area where microglial cells wereover-activated upon LPS stimulation. Furthermore, ADT-OH suppressed M1and promotedM2gene expression in the area. Immunohistochemistry (ICH) results showed that cellspositive for Iba1were co-stained with p-AMPK in the lateral septal complex area of themice co-treated with ADT-OH plus LPS.