Effects And Mechanisms Of L-Cys On Hypoxic-ischemic Brain Damage In Neonatal Mice

BackgroundHypoxic-ischemic encephalopathy(HIE)is a hypoxic-ischemic(HI)injury caused by perinatal asphyxia,which is a common disease leading to neonatal death and long-term neurological dysfunction of survivors.The condition of HIE is very serious and the incidence rate is very high.The incidence rate of full-term newborns is 30‰(pregnancy>36 weeks),and that of premature newborns is 70‰(pregnancy?36 weeks).About 40%of HIE patients are unable to survive in the neonatal period,and 30%of HIE patients suffer from long-term neurological diseases,such as cerebral palsy,learning disabilities and neurosensory defects,which bring serious economic burden to families and society.However,at present,the clinical treatment for this disease is mainly comprehensive treatment,combined with mild hypothermia,hyperbaric oxygen,magnesium sulfate,gangli-oside and other methods.However,the treatment effect is not significant,and it is easy to cause adverse consequences such as tissue damage and infection.The decrease of blood flow and energy depletion after HI brain injury induce apoptosis and necrosis of neurons,which rapidly release many pro-inflammatory factors,such as oxygen free radicals and excitatory amino acids.These pro-inflammatory factors induce microglia to move,proliferate,infiltrate and activate into damaged brain regions,causing secondary brain edema and acute inflammation.In addition,microglia release many inflamematory mediators after activation,further aggravating neuronal damage and eventually leading to cerebral infarction.Therefore,terminating the vicious cycle between neurons and microglia is beneficial to the recovery of neurological function after HI brain injury.Hydrogen sulfide(H2S)is the third endogenous gas signal molecule discovered by human beings and widely distributed in the body,such as heart,blood,and central nervous system(CNS).H2S in the CNS is produced by catalyzing respective substrates by Cystathionine-?-synthas-synthases(CBS),3-mercaptopy-rouvate sulfurtransfer(3?MST)and D-amino acid oxidase(DAO).H2S plays an important physiological and pathological role in the body.Under normal circumstances,H2S participates in N-methy1-D-aspartic acid receptor signaling,Na+-K+-ATP enzyme signaling,glutathione formation,and maintenance and regulation of neuronal function.Under pathological conditions,H2S can play anti-inflammatory and antioxidant roles,protecting damaged neurons and synapses.ObjectiveRice-Vannucci method was used to establish HI model to simulate HI of human newborn,and to explore whether L-Cysteine(L-Cys),a donor of H2S,can reduce acute brain injury and improve HI-induced neurological dysfunction in newborn mice,providing reference for clinical drug therapy.Methods1.Establishment of model:C57BL/6J suckling mice were selected for postnatal day(P7)to establish HI model.After mice were anesthetized with 2%isoflurane and fixed the right common carotid artery was separated and ligated with 4-0 nylon monofilament suture.After 60 min rest,and then treating mice in hypoxia(8%O2+92%N2)environment for 90 min could produce physiological and pathological changes like human newborn HI.If there is obvious edema 3 days after HI,the model is successfully established.The Sham operation group(Sham group)was anesthetized and exposed the right common carotid artery,but did not ligature the right common carotid artery and did not undergo hypoxia treatment.2.Experimental grouping:In most of the experiments,P7 suckling mice were randomly divided into the following four groups:Sham group,HI group,HI+L-Cys intervention group(HI + L-Cys group),HI+ L-Cys + aminooxyacetic acid(AOAA)intervention group(HI + L-Cys + AOAA group).In the experiment to detect the autophagy effect of L-Cys on HI,P7 suckling mice were randomly divided into the following five groups:Sham group,HI group,HI+ L-Cys group,HI + chloroquine(Cloroquine,CQ)intervention group(HI+CQ group),HI + L-Cys + CQ intervention group(HI + L-Cys + CQ group).3.Intervention method:L-Cys,AOAA(which is a non-specific inhibitor of CBS,but the inhibitory effect is strong and no better alternative has been found,so it is widely used in in vivo and in vitro experiments)and CQ(Which is an inhibitor of lysosome)were dissolved in 1 × phosphate buffered saline(PBS)at PH=7.0.Drugs were injected intraperitoneally 1,2 and 3 days after HI,respectively.Sham and HI groups were injected with the same volume of PBS solution.In HI+ L-Cys group,L-Cys(5 mg/kg)solution was injected.In HI+ L-Cys + AOAA group,AOAA(5 mg/kg)solution was given first,and then L-Cys(5 mg/kg)solution was given first,and 30 min later.In HI+ CQ group,CQ(50 mg/kg)solution was injected.In HI + L-Cys + CQ group,CQ(50 mg/kg)solution was given first,and then L-Cys(5 mg/kg)solution was given 30 min later.4.Detection method:(1)Western blot,reverse transcription-polymerase chain reaction(RT-PCR),Flow Cytometry(FCM),immunohistochemistry and immunofluorescence were used to detect the effects of L-Cys on inflammatory factors,oxidative stress,glial cells,autophagy and synapse in the cortex of injured side after HI.(2)Transmission electron microscope(TEM)was used to observe the effect of L-Cys on autophagy and synapse after HI.(3)Behavioral tests:Negative geotaxis,Gait reflex and Front-limb suspension expe-riments were performed 3 days,5 days and 7 days after HI to detect their motor coor-dination,forelimb movement ability and forelimb grasping ability.28 days after HI,a Novel object recognition task(NORT)was performed to test the learning and memory abilities.Experimental results1.At 3 days after HI,obvious edema appeared in the rnght hemisphere of HI group mice.However,compared with HI group,cerebral edema in HI + L-Cys group was significantly reduced.Brain edema was obvious in HI + L-Cys + AOAA group.In addition,the water content in the right hemisphere of HI group increased significantly(/P<0.001).After L-Cys treatment,the water content of right hemisphere brain decreased(P<0.05).When L-Cys and AOAA are combined,the effect of L-Cys on improving cerebral edema is blocked by AOAA.2.At 3 days after HI,the EB content in the right hemisphere of HI group increased significantly(P<0.001).After L-Cys administration,EB content in HI+ L-Cys group decreased significantly(P<0.001).When L-Cys and AOAA were combined,EB content in the right hemisphere of mice in HI + L-Cys + AOAA group increased(P<0.05).3.At 3 days after HI,cerebral cortex,hippocampus and striatum cells in HI group showed obvious damage and mass cells were loss(P<0.001).L-Cys treatment reduced cell degeneration and necrosis in the right brain region of mice.Significant damage occurred in the brain regions of the HI + L-Cys + AOAA group.4.At 3 days after HI,the number of Cleaved caspase-3/NeuN double positive neuron cells in injured cortex of HI group mice increased significantly(P<0.001).Compared with HI group,L-Cys treatment decreased the number of Cleaved caspase-3/NeuN double positive neurons(P<0.001).AOAA pretreatment significantly inhibited the role of L-Cys in reducing HI-induced caspase-3 activation.5.At 3 days after HI,the expression of tumor necrosis factor ?(TNF-?),Interleukin-1?(IL-1?)and cluster of differentiation 86(CD86)in the injured cortex of HI group increased significantly(P<0.001).After administration of L-Cys,the expression of TNF-?,IL-1? and CD86 decreased(P<0.001,P<0.01,P<0.001,respectively).After AOAA pretreatment,the expression of TNF-??IL-1? and CD86 increased again(P<0.05,P<0.05,P<0.01,respectively).6.At 3 days after HI in newborn mice,microglia and astrocytes in the infarct core area in the injured cortex of HI group were activated,the number of ionized calcium binding adaptor molecule-1(Iba-1)positive cells and collagen fibrillary acidic protein(GFAP)positive cells increased significantly(P<0.001);After L-Cys was administered,the number of Iba-l+cells and GFAP+cells decreased significantly(P<0.01);AOAA pretreatment significantly inhibited the effect of L-Cys on astrocytes(P<0.01).After HI injury,astrocytes around the infarct area of the injured cortex were activated,and the number of GFAP+cells increased(P<0.001),while the number of Iba-l+cells did not change(P>0.05).CD11b+/CD45rh'gh cells were significantly increased in the cortex of HI group(P<0.001).After administration of L-Cys,the number of CD11b+/CD45highcells decreased(P<0.001),and the effect of L-Cys was reversed after AOAA pretreatment.In addition,Western blot results showed that the expression of GFAP in injured cortex of HI group increased(P<0.01),and AOAA pretreatment blocked the effect of L-Cys.The expression of GFAP decreased after L-Cys administration(P<0.05).When L-Cys and AOAA were combined,the expression of GFAP increased significantly(P<0.01).7.At 3 days after HI,the expressions of reactive oxygen species(ROS)and malondia-ldehyde(MDA)in the cortex of HI group were increased significantly.After adminis-tration of L-Cys,the levels of ROS and MDA were decreased,while AOAA pretreatment reversed the effect of L-Cys.8.At 3 days after HI,microtubule associated protein 1 light chain 3 ?(LC3-?),p62 and phosphorylated signal transduction and activator of transcription 3(p-Stat3)in cortex of HI group were increased(P<0.05);After administration of L-Cys,the expressions of p62,phosphorylated mammalian rapamycin target of rapamycin(p-mTOR)and p-Stat3 were decreased(P<0.05,P<0.01,P<0.01,respectively),and the expression of LC3-? and Beclinlwere increased(P<0.05).9.At 3,14,and 28 days after HI,synaptic morphology in cortex of injured side of HI group was destroyed and synaptic number was significantly reduced(P<0.015 P<0.001,P<0.01,respectively);After administration of L-Cys,the abnormal synaptic morphology was significantly improved and the number of synapses increased(P<0.01,P<0.01,P<0.05,respectively).AOAA pretreatment reversed the effect of L-Cys(P<0.01,P<0.05,P<0.05 respectively).In HI group,the expression of Synapto-physin(Syn)and postsynaptic density protein 95(PSD95)decreased significantly;After administration of L-Cys,the expression of Syn and PSD95 significantly upregulated.10.The results of Negative geotaxis test showed that the latency of HI group was signify-cantly prolonged in P10 test compared with Sham group(P<0.05),and the effect of HI on chemotaxis could be significantly reversed after administration of L-Cys.Compared with Sham group,the latency of HI group did not change significantly during P12 and P14 days of testing.Gait reflex test results showed that the latency of P10 and P12 in HI group was significantly longer than that in Sham group(P<0.01).The latency of HI group did not change significantly in P14 day test.Compared with the HI group,the latency of P10 and P12 in HI + L-Cys group mice was significantly shortened(P<0.05).The results of forelimb suspension experiment showed that compared with Sham group,the suspension time of HI group did not change significantly in P10 and P12 tests,while the suspension time of HI group decreased significantly in P14 tests(P<0.05).However,L-Cys treatment had no effect on the forelimb suspension duration of these mice.NORT test results showed that compared with Sham group,HI group mice have significantly reduced ability to distinguish old and new things.After administration of L-Cys,the discrimination ability of mice in HI+ L-Cys group was significantly improved.AOAA pretreatment reverses the neur-oprotective effect of L-Cys.11.At 3 days after HI,the expressions of CBS,3-MST and DAO in the injured cortex of HI group decreased significantly(P<0.01).The expression of CBS,3-MST and DAO increased in HI + L-Cys group(p<0.001,P<0.01,P<0.01 respectively).At the same time,H2S content in HI+ L-Cys group increased significantly(P<0.01).The expression of CBS and DAO decreased in HI + L-Cys + AOAA group(P<0.01).In addition,AOAA pretreatment reversed the effect of L-Cys on H2S content.Conclusion and significanceL-Cys inhibits activation of glial cells,alleviates synaptic damage and promotes autophagy,and effectively alleviates HI brain injury and improves behavioral deficits.Therefore,L-Cys can provide a new treatment method for HIE by forming H2S.