H₂S Donors Modulate Post-ischemic Neuron-inflammation Via SQR

Objective:Our previous results showed that the endogenous gaseous transmitter hydrogen sulfide(H2S)selectively induced microglia/macrophage polarization from a pro-inflammatory(M1)status to an anti-inflammatory(M2)status by activating AMP-activated protein kinase(AMPK)in LPS stimulated microglia.However,the mechanisms underlying AMPK activation by H2S remain to be investigated.Here,by using a variety of H2S donor,including inorganic and organic slow-release H2S donor,we explored how AMPK was activated by H2S to induce M2 polarization of microglia following cerebral ischemia and whether H2S acted through this mechanism to inhibit post-ischemia neuron-inflaumation.Methods:1)Mitochondrial complex IV activity was assayed to investigate whether H2S donors activate AMPK by inhibiting the activity of mitochondrial complex IV;2)To explore whether the effects of H2S donors were mediated by the released H2S,ZnCl2 was used to remove free H2S released from H2S donors;3)By measuring cell oxygen consumption(OCR)and the mitochondrial membrane potential with the fluorescence probes(JC-1 and TMRM),we investigated whether H2S donors activated AMPK by uncoupling mitochondria;4)By Western blot and Q-PCR were performed to detect cellular expression pattern of the sulfide quinone oxidoreductase(SQR)expression in microglia,astrocytes and neurons;5)To investigate whether SQR mediated mitochondria uncoupling and AMPK activation conferred by H2S donors,lentivirus mediated technique was used to over-expressing SQR in primary neurons and to knock down SQR in primary microglia;6)siRNA technique was further used to confirm the role of SQR in AMPK activation induced by H2S donors via the mechanism of mitochondrial uncoupling;7)By using mitochondrial complex I specific inhibitor rotenone,we investigated whether mitochondrial uncoupling induced by H2S slow-release donor ADT-OH depends on the reverse electron transport(RET)at the mitochondrial complex I driven by H2S oxidation;8)Rotenone was used to explore whether AMPK activation induced by H2S donor with mitochondrial uncoupling effects was dependent on the mitochondrial complex I RET;9)primary neurons were subjected to oxygen-glucose deprivation(OGD)and conditioned medium was collected following OGD and termed as OGD Neuron CM to mimic microglia activation following cerebral ischemia,OGD Neuron CM was used to treat primary cultured microglia and BV2 microglia cell line in vitro,we investigated whether the AMPK activation induced of H2S donor ADT-OH in the cellular model was dependent on SQR.Furthermore,in this cellular model,the expression of inflanmmatory factors was examined with real-time quantitative PCR and ELISA(enzyme-linked immunosorbent assay)and AMPK activation was estimated with Western blot.Results:1)Inorganic H2S donor NaHS(20-200?M)and slow-releasing H2S donor ADT-OH(50-100?M)and 5a(50-150?M)reduced the levels of intracellular ATP and elevated AMPK activation.In the presence of ZnCl2,the effects on intracellular levels of ATP and the activation of AMPK conferred by the above H2S donors were abolished.NaHS(20-200?M)inhibited mitochondrial complex IV activity while the organic slow-releasing donors ADT-OH(50-100?M),5a(50?M)activated AMPK but did not inhibited mitochondrial complex IV activity.These results suggested that NaHS likely activated AMPK via inhibiting mitochondrial complex IV,while ADT-OH and 5a(50?M)may activate AMPK through other mechanisms.2)In the presence of ATP synthase inhibitor oligomycin,H2S donors ADT-OH(50-100?M)and 5a(50?M)elevated cellular oxygen consumption(OCR)in microglia.ADT-OH(50-100?M)and 5a(50?M)also reduced the mitochondrial membrane potential as assayed with the fluorescence probes TMRM and dyeing JC-1.These results suggested that organic slow-releasing H2S donors ADT-OH and 5a induced mitochondrial uncoupling in microglia.3)Western blot and Q-PCR results showed that SQR lRNA and protein were abundantly expressed in primary microglia and primary astrocytes but not in primary neurons.4)In primary neurons,ADT-OH(50-1000?M)neither enhanced OCR in the presence of oligomycin nor reduced cellular ATP levels or mitochondrial membrane potential.5a at 50?M did notreduce cellular ATP levels and mitochondrial membrane potential in primary neurons either.In contrast,NaHS(20 100 200?M)and 5a at 100?M reduced cellular ATP levels and mitochondrial membrane potential in primary neurons.But in the primary neurons over-expressing SQR,ADT-OH(50-100?M)enhanced OCR in the presence of oligomyein and reduced mitochondrial membrane potential and cellular ATP levels.In the primary microglia,SQR knockdown abolished the effects of ADT-OH on OCR in the presence of oligomycin,ATP content and mitochondrial membrane potential after.These results suggest that the slow-releasing H2S donors acted through SQR to induce mitochondrial uncoupling.5)Compared to vehicle,ADT-OH or rotenone alone increased mitochondrial NADH content and decreased NAD+/NADH ratio.However,in ADT-OH and rotenone co-treated microglia,rotenone inhibited the effects of ADT-OH on mitochondrial NADH and NAD+/NADH ratio.The results suggest that ADT-OH caused reverse electron transport(RET)at the mitochondrial complex I.Furthermore,in microglia,rotenone attenuated the enhancing effects of ADT-OH on OCR in the presence of oligomycin and inhibiting effects on intracellular ATP and mitochondrial membrane potential.These results suggested that organic slow-releasing H2S donor ADT-OH induced mitochondrial uncoupling dependent on RET at mitochondrial complex I.6)In primary neurons without endogenous SQR expression,ADT-OH did not increase AMPK activation.Consistently,SQR knockdown blunted ADT-OH enhanced AMPK activation blunted in microglia.In contrast,in primary neurons over-expressing SQR,the H2S donor ADT-OH significantly activated AMPK.Knockdown of SQR in microglia also inhibited AMPK activation by 5a at 50?M in primary microglia.However,5a at 100?M-150?M and NaHS at 20-200?M can activated AMPK in primary neurons.These results suggest that 5a at 100?M-150?M and NaHS at 20-200 ?M activated AMPK independent of SQR mediated mitochondrial uncoupling.The H2S donors ADT-OH and 5a at 50?M activated AMPK through SQR mediated mitochondrial uncoupling.In further investigation,we found that rotenone(the inhibitor of mitochondrial complex I)inhibited ADT-OH induced AMPK activation.These results suggest that ADT-OH activation of AMPK depended on mitochondrial complex I RET.7)Compared to vehicle,OGD Neuron CM decreased microglia of AMPK,which was significantly enhanced by ADT-OH.Knockdown of microglia SQR abolished the promoting effects of ADT-OH on AMPK activation.Moreover,knockdown of microglia SQR attenuated the promoting effects of ADT-OH on M2 polarization of microglia treated with OGD Neuron CM.These results suggest that the promoting effects of ADT-OH on AMPK activation and M2 polarization of microglia following cerebral ischemia were mediated by SQR in this in vitro cellular model.Conclusion:H2S signals in part via SQR mediated mitochondrial uncoupling and the signaling cascade also mediates the inhibiting effects on post-ischemic neuron-inflammation conferred by some organic slow-releasing H2S donors.