Neuroprotective Effects Of Oxymatrine On PI3K/Akt/mTOR Pathway After Hypoxic-ischemic Brain Damage In Neonatal Rats

Objective: To investigated in depth the protective effect of Oxymatrine(OMT)and explore the possible relationship between the protective effect of OMT and the PI3K/Akt/mTOR signal pathway.Methods: 1.To study the protective effect of OMT on neonatal rats with HIBDIn vivo: 7-day-old neonatal rats were anesthetized to establish hypoxic-ischemic brain injury model,and were randomly divided into sham group,HI group and OMT(120 mg/kg)group.To observe the protective effect of OMT on HIBD in neonatal rats,neurological development of neonatal rats was evaluated by righting reflex,negative geotaxis reflex and cliff reflex.Dry-wet weight method was detected brain water content.HE and TUNEL staining was used to observe neuronal morphological changes and the degree of necrosis.In vitro: The primary hippocampal neurons were deprived of oxygen and glucose for 2 h and reperfusion 24 h,and subjected to oxygen and glucose deprivation and reperfusion(OGD/R)injury model.They were randomly divided into control group,OGD/R group and OMT(5 ?g/mL)group.The effects of OMT on the primary hippocampal neurons OGD/R injury were observed: MTT method was used to determine the cell viability;Chemiluminescence method was used to detect the opening degree of mPTP.2.To explore the protective mechanism of OMT on neonatal rats with HIBDIn vivo: The formation of autophagosome was observed by transmission electron microscopy.TTC staining was used to observe brain infarction volume and the expression of PI3 K,Akt and mTOR protein were detected by Western blot.In vitro: MDC staining was evaluated the autophagy formation level;Western blot was used to detect the expression levels of PI3K/p-PI3 K and Akt/p-Akt proteins;Immunofluorescence localization of p-mTOR;q-PCR was used to detect the expression of Beclin1,LC3 and P62 mRNA.Results: 1.Protective effect of OMT on HIBD in neonatal ratsIn vivo: compared with Sham group,the time of righting reflex,negative geotaxis reflex and cliff reflex in HI group increased significantly(P<0.01),cerebral water content were increased(P<0.01),histomorphological damage of neurons.After administration of OMT(120mg/kg),the time of each reflex decreased significantly(P<0.01);the brain water content of the ischemic side was decreased(P<0.05),the histopathological changes,injury and death of neurons were alleviated.In vitro: compared with the control group,the viability of hippocampal neurons in OGD/R group decreased significantly(P<0.01),intracellular the openness of mPTP increased significantly(P<0.01).Compared with OGD/R group,the survival rate of neurons increased after OMT(5?g/mL)administration(P < 0.01),which stabilized the openness of mPTP in neurons(P < 0.01).2.Protective mechanism of OMT on HIBD in neonatal ratsIn vivo: compared with Sham group,autophagy level and cerebral infarct volume increased significantly(P<0.01).The expression of PI3 K,Akt and mTOR protein in ischemic brain tissue decreased significantly(P<0.01).After administration of 120mg/kg OMT,autophagy level and cerebral infarct volume decreased significantly(P<0.01),the expression of related proteins was reversed.In vitro: compared with control group,autophagy levels of neurons after OGD/R injury increased,p-PI3 K and p-Akt protein decreased significant ly(P<0.01),p-mTOR fluorescence expression decreased significantly(P<0.01),Beclin-1 and LC3 mRNA expression increased,while P62 mRNA expression decreased(P <0.01).Compared with OGD/R group,OMT reduced the autophagy of neurons.The expression levels of p-PI3 K and p-Akt protein increased significantly(P<0.01)and the fluorescence expression of p-mTOR also changed accordingly(P<0.01).In addition,OMT decreased the relative expression levels of Beclin-1 and LC3 mRNA(P<0.01,P<0.05)and increased the relative expression levels of P62 mRNA(p<0.01).The regulatory effect of OMT on above indicators was abolished by RAPA.Conclusions: It is further confirmed that OMT exerts neuroprotective effects on HIBD in neonatal rats.The protective effect of OMT on brain injury is related to activating PI3K/Akt/mTOR signaling pathway and inhibiting excessive autophagy of primary hippocampal neurons in neonatal rats.