The Impact Of Cerebral Ischemia On The Cystathionine ?-synthase/hydrogen Sulfide Pathway And The Mechanism Underlying Modulating Effects Of H₂S On Post-ischemic Neuroinflammation

Objective: The mechanisms underlying neuroinflammation following cerebral ischemia remain unclear.Hydrogen sulfide(H2S),a newly identified gasotransmitter,has been reported to regulate inflammation.However,the role that the endogenous H2 S production pathway plays in post-ischemic neuroinflammation is not clear.Cystathionine ?-synthase(CBS)is an endogenous H2 S synthase that is responsible for cerebral H2 S production and is preferentially expressed by glial cells.In the current study,we investigated whether the endogenous H2 S production pathway(CBS/H2 S pathway)contributed to microglia-mediated neuroinflammation following stroke.AMPK signal pathway has been implicated in H2 S inhibition on neuroinflammation induced by LPS.We investigated whether H2 S reduced acute infarction and promote the functional recovery following cerebral ischemia by activating AMPK to polarize microglia to an anti-inflammatory(M2)phenotype.To increase the translatability of our study,we further examined whether ADT,a clinical hepatoprotective and antixerostomia drug with a high safety profile,displayed similar effects as ADT-OH on microglial polarization in the stroke models.Indeed,ADT is a methylated derivative of ADT-OH and is rapidly and almost completely metabolized to ADT-OH in vivo.We have shown that ADT acts as an H2 S donor both in vivo and in vitro.Methods: Conditioned media were collected from primary neurons subjected to oxygen glucose deprivation(OGD neuronal conditioned medium)as well as from the control neurons without OGD treatment(Neuron Condition Medium).To mimic microglial activation in vitro,OGD neuronal conditioned medium was used to stimulate BV2 or primary microglia and neuron conditioned medium served as the control.This in vitro microglial activation model as well as mouse middle cerebral artery occlusion(MCAO)was used to investigate the effects of cerebral ischemia on microglial CBS/H2 S pathway,the mechanisms by which H2 S inhibited stroke-induced neuroinflammation.To investigate whether H2 S promoted functional recovery following stroke,mice were subjected to behavioral tests following photothrombotic stroke.The behavioral tests included left paw placement test,foot-fault test,cylinder test and reach for food test.The expression levels of CBS,p-AMPK,and cystathionine-?-lyase(CSE)were assayed by Western Blot analysis.The expression levels of M1/M2 signature genes were assessed by Q-PCR or ELISA.H2 S concentrations in BV2 and primary microglia media and H2 S synthesizing activity in cortical homogenate were measured by absorbance at 670 nm with infinite 2000 PRO.Microglial polarization was also characterized with immunohistochemistrical examinantion of the morphology of macrophages/microglia co-localization of the M1 marker CD16/32 or the M2 marker CD206 with the microglial/macrophage marker Iba1.Microglial phagocytosis was futher assessed to indicate microglia/macrophage polarization using aqueous red fluorescent latex beads of 1 ?M diameter.Result: in the cellular model:1.Western Blot and Q-PCR results suggested thatboth m RNA and protein expression of the H2 S synthase CBS in microglia were rapidly reduced following the treatment with OGD neuronal conditioned medium.However,CSE expression remained unchanged.2.H2 S synthesizing activity was also rapidly and dramatically decreased in microglia under the ischemic condition,as indicated by markably reduced sulfide levels in the medium collected from microglia treated with OGD neuronal conditioned medium.3.Q-PCR results suggested that the H2 S donor ADT remarkably attenuated the expression of M1 signature genes(IL-1?,IL-6,i NOS,TNF-a and CD32)and enhanced the expression of M2 signature genes(ARG and CD206)in microglia treated with OGD neuronal conditioned medium.4.Western Blot results suggested that the H2 S donor ADT enhanced AMPK activation in microglia treated with OGD neuronal conditioned medium.5.Immunofluorescence results suggested thatmost microglia adopted round shapes after the treatment with OGD neuron CM,whereas almost all microglia co-treated with H2 S donor(ADT/ADT-OH)and OGD neuron CM re-assumed elongated bipolar morphology,indicating H2 S skewed ischemia induced M1 polarization of microglia/macrophages toward M2 polarization6.H2 S donor(ADT)enahenced the phagocytosis capacity of microglia treated with OGD neuronal conditioned medium.7.Western Blot hat theexpression of CBS,but not that of CSE,was rapidly reduced in mixed glial cultures as well as in astrocyte-enriched cultures.however,as indicated by ELISA results,The expression of the proinflammatory mediator(IL-1?)was not induced in astrocytes upon treatment with OGD Neuron CM.Moreover supplementation of H2 S donor ADT-OH or ADT also did not affect astrocytic expression of the M1 signature gene.These results suggested that H2 S mainly acted through microglia rather than astrocytes to inhibit post-ischemic neuroinflammation.In the mouse MCAO model:1.Western Blot results suggested thatprotein expression of the H2 S synthase CBS were rapidly reduced in the mouse ischemic brain following MCAO.However,CSE unchanged in the ischemic cortex.2.H2 S synthesizing activity in the ischemic cortexl was rapidly and dramatically decreased following cerebral ischemia.3.Q-PCR and ELISA results suggested thatthe H2 S donor(ADT)inhibited cerebral ischemia-evoked expression of M1 signature genes and enhanced M2 signature gene expression in the mouse ischemic cortex following MCAO.4.The H2 S donor ADT decreased acute infarction and promoted functional recovery after in the mice subjected to MCAO or photothrombotic stroke.5.Western Blot results suggested that: AMPK activation was enhanced in the ischemic ipsilateral cortex,which was further enhanced by the H2 S donor ADT following MCAO.6.Co-localization results suggested that the H2 S donor ADT significantly reduced the number of CD16/32 + Iba1 + double positive cells while enhanced the number of CD206 + Iba1 + double positive cells in the mouse ischemic cortex following MCAO.Conclusion: These data suggested that cerebral ischemia inhibited CBS-mediated production of endogenous H2 S in microglia,while supplementing exogenous H2 S following cerebral ischemia ameliorated acute infarct damage and promoted the functional recovery by activating AMPK to polarize microglia to an anti-inflammatory(M2)phenotype.