NGR1 Protects Against Hypoxic-ischemic Injury In Rats And Its Mechanism

Hypoxic-ischemic encephalopathy(HIE)is a major reason that can cause acute neonatal death,chronic injury of the nervous systemand brain retardation during neonatal period.Neuronal death is the most direct and mostserious consequence inhypoxic-ischemic brain injury.Therefore,protecting normal neuronal survival and reducing mortality becomes one of the most important strategies to solve neurological diseases.Recent studies have reported that estrogen has neuroprotective effect and can affect the process of cerebral hypoxic-ischemic injury,which may be related to the regulation of neuronal apoptosis signaling pathway,including alleviate damage from free radicals,reduce calcium influxandinhibit excitatory amino acids toxicity.Neuroprotective effect of estrogen ismainlyrelated with estrogen receptor(Estrogen receptor,ER).NGR1(Notoginsenoside R1,NGR1)as a natural plant estrogen receptor agonisthas anti-inflammatory,antioxidant,anti-apoptotic and neuro-protection,and plays a protective effect in multiple organ injury.Thus,it is worthy to explore whether NGR1 has protective effect on neonatal rats with HIE injury.Although numerous signaling pathways,which regulating neuronal survival and death,are activated after hypoxic-ischemic brain injury,PI3K-Akt-m TOR pathway is the central focus,that mainly regulates cell survival,proliferation,differentiation and inhibition of apoptosis,and then exertsneuroprotective effect on hypoxic-ischemic brain injury.JNK pathway cancause apoptosisby activatingthe downstream pro-apoptotic genes c-Jun.The balance between PI3K-Akt and JNK determine whether the neuron survival or apoptosis after neuronal brain injury.Akt activated by PI3 K can activate downstream m TOR signaling and promote cell survival and proliferation;on the other hand,AKT can inhibit the activation of JNKby direct or indirect ways and reduce neuronal apoptosis.Thus,the level of PI3 K activityplays a key role.Many scholars have reported that ER and PI3 K may act on directly or indirectly,which activatethe downstream Akt family and cause a series of signaling pathway cascade.We researchwhether NGR1 can act on ER and protect neurons by regulating the activity of PI3K-Akt and JNK pathways.In summary,we plan to explore NGR1 effect and its mechanism as NGR1,ER,PI3K-Akt-m TOR and JNK signaling pathway for the axis clues.Part one NGR1 protects against hypoxic-ischemic injury in ratsObjective:Investigate the neuroprotective effect NGR1 on hypoxic-ischemic injury in vivo and in vitro.Methods: 1.Model:(1)rat primary cortical neuron cultures(from 17-day-old embryonic SD rats),establish oxygen-glucose deprivation(Oxygen-Glucose Deprivation,OGD)rat cortical neurons OGD/R model;(2)Establish HIE model with newborn 7 days SD ratsin vivo.2.Groups: control group(CON or sham group),OGD/R(HIE)model group,model + NGR1 treatment group,model + DMSO(dimethyl sulfoxide,Dimethyl sulfoxide,the solvent control vehicle)group.3.Detection Indicator:(1)In vitro: neuronal cell viability tested by MTT assay;LDH membrane integrity tests to assess cortical neurons injury andthe protective effect of NGR1.(2)In vivo: detected water content of rat brain tissue,infarct size,light microscope pathological brain damage,laser Doppler flowery to detect cerebral blood flow,nerve behavioral tests(balance beam experiment,the corner test and water maze experiments),TUNEL apoptosis detection to assess the HIE damage and the neuroprotective effects of NGR1 in rats.Results: 1.In vitro we found thatneurons survival rate of NGR1 treatment group increased significantly and LDH leakage rate decreased compared to OGD/R model group.2.In vivo we found that compared with HIE model group,NGR1 therapy group brain edemaalleviated;Infarct size was significantly reduced;cerebral blood flow increased;neurobehavioral test including body movement and coordination counterbalance learning significantly improved 5 weeks after model;reduced the number of apoptotic cells by TUNEL.Conclusion NGR1 has the neuroprotective effects on OGD/R injury and HIE damage in neonatal rats(P<0.05 or P<0.01).PART TWOTO INVESTIGATE THE MECHANISM OF NGR1'S PROTECTIVE EFFECTS ON HYPOXIC-ISCHEMIC INJURY IN NEONATAL RATS SECTION ONE NGR1 NEUROPROTECTIVE EFFECTS RELATED WITH ESTROGEN RECEPTOR.Objective To discuss whether NGR1'sneuroprotective effects related with ER.Methods 1.Groups: control group(CON or sham group),OGD/R(HIE)model group,model + NGR1 treatment group,model + NGR1 + ICI182780(ER receptor blocker)group,model +ICI182780 group2.Detection Indicator:(1)ER expression changes at each timepoint after OGD / HIE injury by Western Blot.(2)The neuronal cell survival by MTT,LDH cell membrane integrity testing.(3)To detect the rat brain tissue water content,infarct size,pathological lesions of brain tissue,cerebral blood flow by laser Doppler flowery,nerve behavioral tests(balance beam experiment,the corner test and water maze test),apoptotic cell count by TUNEL.Results 1 In vitro and in vivo we found that after OGD/R or HIE injury,neuronal ER protein expression decreased,and reached the minimum at 24 h,continued until 48 h,which hinted that ER participate in hypoxic-ischemic injury.2.In vivo and in vitro we used the ER blocker ICI18780,the results was as follows:(1)ICI182780 can block the NGR1's protective effect on cortical neurons in OGD/R injury,which induced neuronal survival rate decreased and LDH leakage rate increased.(2)ICI182780 can block the NGR1's protective effect on neonatal rat model of brain injury.Compared with NGR1 treatment group,cerebral edema aggravated,infarct size was significantly increased,cerebral blood flowdecreased in blocker group.Neurobehavioral tests prompt damage increased,the number of TUNEL apoptotic cells increased 5weeks after modelConclusion NGR1 plays a protective effect on hypoxic ischemic brain injury in neonatal rats by ER and ER's blocker ICI182780 could inhibit theeffect(P<0.05 or P<0.01).SECTION TWONGR1 HAS THE NEUROPROTECTIVE EFFECTS RELATEDWITHER REGULATED PI3K-AKT-MTOR AND JNK SIGNALING PATHWAYS.Objective :To investigate whether the neuroprotective effects of NGR1 is related with ER regulated PI3K-Akt-m TOR and JNK signaling pathway.Methods: 1.Groups:(1)NGR1 regulation of PI3K-Akt-m TOR and JNK signaling pathway through ER: control group(CON or sham),OGD/R(HIE)24h group,OGD/R(HIE)24h+NGR1treatmentgroup,OGD/R(HIE)24h+NGR1+ICI182780 group(2)The NGR1 regulation of PI3 K as a target to control the downstream pathway activity: the control group(CON or sham);OGD/R24 h group;OGD/R24h+NGR1group;OGD24h+NGR1+ICI182780+740Y-P(PI3K agonist)group;OGD24h+NGR1+ LY294002(PI3K inhibitor)group 2.Detection index: Expression changes of PI3 K in different time points after OGD/HIE damage by Western Blot.Expression changes in protein expression of PI3K-Akt-m TOR and JNK signaling pathway24 hafter OGD/HIE by Western Blot.3.MTT method was used to detect the survival rate of the neurons in each group,and the integrity of the cell membrane was detected by LDH.Results 1 In vivo and in vitro we have found that 24 hafter OGD/R or HIE,theexpression of PI3 K in model group decreased gradually,24 h down to the minimum,the effect continued to 48 hafter OGD/R or HIE,which suggested that PI3 K signaling pathway is involved in hypoxic ischemic injury.2 In vitro and in vivo,the results showed that compared with the model group,PI3 K expression increased significantly in NGR1 treatment group and its downstream Akt-m TOR-4EBP-1(p70S6K)phosphorylation was increased,the expression of phosphorylated JNK and c-Jun decreased significantly.ER antagonist ICI182780 can block the protective effect of NGR1 compared with the NGR1 treatment group.PI3K-Aktm TOR-4EBP-1 inhibitor group(p70S6K)pathway is inhibited,while theJNK and c-Jun pathway is activated.3 In order to make clear which is the target of NGR1,we used the PI3 K blocking agent LY294002 and agonist 740Y-P for the experiment.The results showed that in NGR1 treatment group LY294002 can cause the cell survival rate decreased,the leakage rate of LDH increased,Akt expression decreased,JNK expression increased;while the PI3 K agonist 740Y-P can make the blocking effects of ICI182780 NGR1 reversed,the survival rate was increased,the leakage rate of LDH decreased,Akt expression increased and JNKgene expression decreased.Conclusion: 1.NGR1 may regulate the PI3K-Akt-m TOR signaling pathway and the JNK signaling pathway by ER(P<0.05 or P<0.01).2.PI3 K may be the target of NGR1 through ER.