Short-term Dietary Restriction Protects Against Cerebral Ischemia

Research purposes:Dietary restriction(DR)extends the lifespan of most organisms and contributes to metabolic health.Studies have shown that DR contributes to the prevention and treatment of various diseases,possibly through the restriction of sulfur amino acid(SAA).However,the impacts of DR on cerebral ischemia as well as the underlying mechanisms remain to be explored.Our previous studies show that the endogenous gasotransmitter hydrogen sulfide(H2S)inhibits microglia-mediated neuroinflammation via the activation of AMP-activated protein kinase(AMPK).Here we investigated whether short-term DR acted through endogenously produced H2S to inhibit neuroinflammation and protect against ischemic brain injury following cerebral ischemia.Method:In vivo studies:(1)To explore whether(?)term DR led to(?)production of endogenous H2S,DR treatment was performed on male ICR mice for one week.Western blot was used to detect the protein level of the H2S synthases cystathionine β-synthase(CBS)and cystathionine-γ-lyase(CSE)in animal tissues.The synthetic activity of H2S in the brain tissues was also examined following DR.(2)DR treatment was performed on male ICR mice for one week.Then cerebral ischemia was induced by the photothrombotic approach.And the cerebral infarctin volumes were assessed with Nissl staining.The protein expression level of pro-inflammatory cytokine in cerebral tissues after ischemia was detected by enzyme-linked immunosorbent assay(ELISA).(3)In order to explore whether DR reduced ischemic brain injury and inhibited neuroinflammation through the CBS of microglia/macrophagcs,one week of DR was performed on the mice with deletion of cbs in microglia/macrophages.Then,cerebral ischemia was induced by the photothrombotic approach.The cerebral infarction volumes were assessed with Nissl staining.And the protein expression level of pro-inflammatory cytokine in cerebral tissues after ischemia was detected by ELISA(4)To further explore whether short-term DR reduced inflammation through H2S,wild type mice were supplemented with exogenous H2S donor ADT for 7 consecutive days before cerebral ischemia.Next,cerebral ischemia was induced by the photothrombotic approach and the infarction volumes were assessed with Nissl staining.The protein expression level of pro-inflammatory cytokine in cerebral tissues after ischemia was detected by ELISAIn vitro research:(1)SAA restriction was performed on BV-2 microglial cells to mimic DR.After SAA restriction,the protein levels of CBS and CSE were detected with western blot,and H2S level in culture medium were assayed with a well-established chemical method.Moreover,BV-2 microglial cells were treated with lipopolysaccharide(LPS)after SAA restriction.Then the protein expression level of pro-inflammatory cytokine was detected by ELISA.(2)In order to investigate whether the inhibition or microglial inflammation by SAA restriction after LPS treatment was mediated by CBS,the primary microglia with cbs deletion or control microglia were subjected to SAA restriction following by LPS stimulation.Then the protein expression level of pro-inflammatory cytokine of the primary microglia was assessed by ELISA.(3)BV-2 microglial dells wvere subjected to SAA restriction followed by LPS treatment.Then western blot was used to detect the activation of AMPK.To knock down the expression of endogenous AMPK,BV-2 microglial cells were transfectced with small interfering RNA against AMPK(AMPK-siRNA)for 2 days.Then,BV-2 microglial cells were subjected to SAA restriction followed by LPS treatment.ELISA was performed to explore whethcr SAA restriction inhibited the inflammation of microglia via AMPK activation.Results:(1)Compare to the ad libitum group,the protein level of CBS and H2S synthesizing activity in the brain tissues were enhanced in the DR group.Moreover,DR decreased infarction volumes and inhibited neuroinflamnation after cerebral ischemia(2)Deleting cbs in microglia/macrophages abolished the protective effects of short-term DR on cerebral ischemia injury as well as the inhibitory effects of DR on post-ischemic neuroinflammation in mice(3)The supplementation of exogenous H2S before cerebral ischemia mimicked the effect of short-term DR on cerebral ischemia injury and post-ischemic neuroinflammation in mice(4)SAA restriction increased the protein expression level of CBS and H2S synthesizing activity in microglia.Moreover,SAA restriction inhibited the up-regulation of pro-inflammatory cytokine expression induced by LPS in microglia(5)AMPK knock-down in microglia blocked the inhibitory effect of SAA restriction on the neuroinflammation following LPS stimulationConclusion:Short-term DR exerts protective effects on cerebral ischemia injury by inhibling neuroinflammation,which can be mediated by CBS-H2S signaling pathway.Endogenously produced H2S(?)AMPK and inhibits neuroinflamatory in microglia.