The Protective Effects Of Sulforaphane On Cerebral Ischemia-reperfusion Injury In Rats And Its Anti-inflammatory Mechanism

Background :The brain’s metabolism and physiological functions depends on good blood supply.Focal cerebral ischemia often makes the brain cells injury,and after reperfusion,cellular functions,metabolic disorders and structural damage will further aggravate.This phenomenon was known as ischemia-reperfusion injury.Cerebral infarction,is a life-threatening sentinel event with substantial morbidity and mortality across all populations.Sulforaphane(SFN),a member of the isothiocyanate family,is found in cruciferous vegetables such as broccoli,cabbages and Brussels sprouts.Sulforaphane has been known to have a variety of beneficial properties including anticarcinogenic,antiapoptosis,antioxidant and anti-inflammatory.Sulforaphane can pass the blood brain barrier,which makes it has the potential on neuroprotection.In addition,recent studies have suggested that Sulforaphane has protective effects against cerebral ischemia-reperfusion injury.The molecular and cellular mechanisms underlying cerebral ischemia are complex and poorly understood,but involve bioenergetic failure,intracellular calcium overloading,increased release of excitatory amion acids,acidosis,apoptosis-related genes activation,oxidative stress,inflammation and so on,all of which result in cell death.Oxidative stress damage was a research focus,but in recent years,a lot of experimental evidence indicate that inflammation plays an important role in cerebral ischemia reperfusion injury.Many factors cause inflammatory factors activated in the cerebral ischemia phase and induce inflammatory cascade amplification effect in the reperfusion phase which leading to ischemiareperfusion injury.Studies have showed that the activation of inflammasome plays an important role in the inflammation.The NLRP3 inflammasome are multiprotein complexes that activated in the presence of pathogenic microorganism and other danger signals.NLRP3 is able to identify the corresponding ligand and to collect and bind the pro-Caspase-1 and trigger the release of IL-1β and IL-18 via Caspase-1 activation.In addition,inflammasome can mediate cell death relied on Caspase-1,which is also called "pyroptosis",promote ischemic brain injury.Janus kinase is a kind of soluble protein kinase in the cytoplasmic size,There are four members in the family(JAK1-3,TKY2).JAK2 is widely existed in brain tissue.STATs(Signal Transducers and Activators of Transcription)is a kind of DNA binding protein,which is JAKs substrate directly.The STAT family has seven members(STAT1-4、STAT5a、STAT5b and STAT6),STAT3 is widely existed the central nervous system.Some research showed that JAK-STAT as important cytokine signal transduction pathways,involved in cell proliferation,differentiation,apoptosis and necrosis,stress,inflammatory reaction,immune regulation,and so on.Recent research shows that JAK2-STAT3 signal transduction pathway is an important reason of tissue damage after cerebral ischemia,which is a key pathway to regulate cytokine reaction and induce cell’s survival or death after cerebral ischemia.AG490 is a specific JAK2 inhibitor,which can inhibit tyrosine phosphorylation of JAK2,thus inhibiting JAK2-STAT3 signaling pathway activation.Now,the research about JAK2-STAT3 pathway in cerebral ischemia-reperfusion inflammatory reaction is less and the results are different.There are two conclusions,Someone thought that JAK2-STAT3 pathway could promote damage in cerebral ischemia-reperfusion and others thought that JAK2-STAT3 pathway has neuroprotective effect.JAK2-STAT3 is a key pathway in cerebral ischemia reperfusion upstream,but the mechanism is not clear.So,we plan to build a stable and reliable rat focal cerebral ischemia reperfusion and investigate sulforaphane’s neuroprotective effect in cerebral ischemia-reperfusion.Find the drug concentration with which the protection effect is better.Then,we will observe the expression of NLRP3 and other inflammatory mediators with SFN treatment.To indicate whether Sulforaphane could protect nerve cells through anti-inflammatory effects.In addition,we plan to compare effects of sulforaphane and AG490 on JAK2-STAT3 signaling pathway,and explore the mechanism of JAK2-STAT3 signaling pathway in cerebral ischemia-reperfusion.Part one:Sulforaphane protecte against cerebral ischemia-reperfusion injury in rat Objective:Build a stable and reliable rat focal cerebral ischemia reperfusion model and investigate sulforaphane’s neuroprotective effect.Methods:1.Model: Focal cerebral ischemia-reperfusion model in rats was established by middle cerebral artery occlusion using modified suture occlusion technique.2.Groups: The rats were randomly divided into five groups.Sham group,MCAO group,MCAO +DMSO group,MCAO +SFN(5mg/kg)group,MCAO +SFN(10mg/kg)group。3.Neuroscores were evaluated.Infarct volume was measured by TTC staining.The degree of neutrophil infiltration was measured by myeloperoxidase(MPO)level detection.Results:1.The Neuroscores in MCAO group is higher than in sham group.The Neuroscores in Sulforaphane group is lower than in MCAO group.2.Infarct volume in Sulforaphane group is smaller than in MCAO group.3.MPO activity in MCAO group is higher than in sham group.MPO activity in Sulforaphane group is lower than in MCAO group.Conclusion:1.Middle cerebral artery occlusion(MCAO)by using modified suture occlusion technique is a good model for researching on cerebral ischemia-reperfusion injury.2.Sulforaphane improved nerve function,reduced cerebral infarction volume,and alleviated inflammation after ischemia-reperfusion,so it has neuroprotective effect.Part two : The role of NLRP3 inflammasome in the process of sulforaphane neuroprotection Objective:To investigate whether Sulforaphane protects against cerebral ischemia-reperfusion injury by inhibiting NLRP3 inflammasome activation.Methods:1.Groups: The rats were randomly divided into four groups: Sham group,MCAO group,MCAO +SFN(5mg/kg)group。2.The expression of IL-1β and IL-18 was measured by ELISA,RT-PCR was used to measure the level of NLRP3 mRNA and Western blot was used to measure the expression of NLRP3,caspase-1,IL-1β and IL-18.Results:1.The mRNA and protein expression of NLRP3 in MCAO group were higher than in sham group.The mRNA and protein expression of NLRP3 in Sulforaphane group were lower than in MCAO group.2.The expression of Caspase-1,IL-1β and IL-18 in MCAO group was higher than in sham group.The expression of Caspase-1,IL-1β and IL-18 in Sulforaphane group was lower than in MCAO group.Conclusion:1.Sulforaphane treatment inhibited NLRP3 inflammasome activation and the downregulation of cleaved Caspase-1,while reducing IL-1β and IL-18 expression.2.Sulforaphane treatment improved outcomes after focal cerebral ischemia.This neuroprotective effect was likely exerted by Sulforaphane inhibiting NLRP3 inflammasome activation and inflammatory response.Part Three: The role of JAK2-STAT3 signaling pathway in the process of sulforaphane neuroprotection Objective:To investigate the role of JAK2-STAT3 signaling pathway in the process of sulforaphane neuroprotection.Methods:1.Groups: The rats were randomly divided into five groups,Sham group,MCAO group,MCAO +SFNgroup,MCAO +AG490 group,MCAO +SFN+AG490 group。2.Neuroscores were evaluated.Infarct volume was measured by TTC staining.The degree of neutrophil infiltration measured by myeloperoxidase(MPO)level detection.Western blot was used to measure the level of JAK2,P-JAK2,STAT3,P-STAT3,NLRP3,Caspase-1,IL-1β and IL-18.Results:1.The Neuroscores in MCAO group is higher than in sham group,and the Neuroscores increased in MCAO+AG490 group when compared with MCAO group.The Neuroscores in MCAO+SFN group is lower than in MCAO group.The Neuroscores in MCAO+SFN+ AG490 group is higher than in MCAO+SFN group.2.The infarct volume in MCAO group is bigger than in sham group,and the infarct volume in MCAO+AG490 group is bigger than in MCAO group.The infarct volume in MCAO+SFN group is smaller than in MCAO group.The infarct volume in MCAO++SFN AG490 group is bigger than in MCAO+SFN group.3.The MPO activity in MCAO group is higher than in sham group,and the MPO activity increased in MCAO+AG490 group when compared with MCAO group.The MPO activity in MCAO+SFN group is lower than in MCAO group.The MPO activity in MCAO+SFN +AG490 group is higher than in MCAO+SFN group.4.The level of P-JAK2 and P-STAT3 in MCAO group is higher than in sham group,and the level of P-JAK2 and P-STAT3 decreased in MCAO+AG490 group when compared with MCAO group.The level of P-JAK2 and P-STAT3 in MCAO+SFN group is higher than in MCAO group.The level of P-JAK2 and P-STAT3 in MCAO+SFN +AG490 group is lower than in MCAO+SFN group.5.The expression of NLRP3,Caspase-1,IL-1β and IL-18 in MCAO group is higher than in sham group,and the expression of NLRP3,Caspase-1,IL-1β and IL-18 increased in MCAO+AG490 group when compared with MCAO group.The expression of NLRP3,Caspase-1,IL-1β and IL-18 in MCAO+SFN group is lower than in MCAO group.But,the expression of NLRP3,Caspase-1,IL-1β and IL-18 in MCAO +SFN+AG490 group is higher than in MCAO+SFN group Conclusion:1.Sulforaphane improved nerve function,reduced cerebral infarction volume,and alleviated inflammation after ischemia-reperfusion,so it has neuroprotective effect.But AG490 weakened this neuroprotective effect of sulforaphane2.Sulforaphane activated the JAK2 and STAT3,increased the level of P-JAK2 and P-STAT3,at the same time suppressed the activation of NLRP3 inflammasome,decreased expression of the NLRP3,Caspase-1,IL-1β and IL-18.AG490 reversed these effects of sulforaphane.3.Sulforaphane protected against cerebral ischemia-reperfusion injury may be through the activation of JAK2-STAT3 signaling pathway and the inhibition of NLRP3 inflammasome and inflammatory response.