| Objective: Neonatal hypoxic-ischemic brain damage(HIBD)is one of the leading causes of permanent neurological dysfunction and acute death in infants,such as epilepsy,intellectual disability,and cerebral palsy.As the only effective treatment for neonatal HIBD,therapeutic hypothermia(TH)can only moderately improve the prognosis,and developing new and more effective treatments for neonatal HIBD has become the top priority.Icariin(ICA),as a flavonoid glycoside compound isolated from Herba Epimedii of Berberidaceae,has the biological activities of activating autophagy,anti-apoptosis,anti-oxidation,anti-inflammation,and so on.It has a significant neuroprotective effect in such diseases as stroke,Parkinson’s disease,and Alzheimer’s disease.Our previous result showed that ICA could inhibit apoptosis and exert a neuroprotective effect on HIBD in neonatal mice,but the specific mechanism remained unknown.Through reviewing the literature,previous studies indicate that the activation of autophagy may contribute to the outcome of HIBD,and there is a positive regulatory relationship between estrogen receptor(ER)and autophagy.Therefore,this study aimed to explore whether ICA played a neuroprotective role in inhibiting apoptosis by regulating autophagy through the estrogen receptor α(ERα)and estrogen receptor β(ERβ)pathway in neonatal mice with HIBD.Methods:In vivo experiments,the HIBD model of neonatal mice was constructed according to the modified Rice-Vannucci method.The effects of ICA pretreatment on the protein expression levels of autophagy-related proteins(Beclin1,LC3,and p62)as well as ERα and ERβ were detected by tissue immunofluorescence and western blot.At the same time as ICA pretreatment,HIBD newborn mice were injected with the autophagy inhibitor3-methyladenine(3-MA)into the lateral ventricle to test whether ICA pretreatment might exert the neuroprotective effect of HIBD on newborn mice by promoting autophagy and inhibiting HIBD-induced apoptosis.After determining the promoting effect of ICA on autophagy of neonatal mice after HIBD and the effects of ERα and ERβ protein expressions,At the same time as ICA pretreatment,the HIBD newborn mice were injected with ERαinhibitor methylpiperidino pyrazole(MPP)or ERβ inhibitor4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo [1,5-a] pyrimidin-3-yl)phenol(PHTPP),Further,whether ICA pretreatment may promote autophagy by activating ERα or ERβ pathways and inhibit HIBD-induced apoptosis to play a neuroprotective role on neonatal HIBD mice was further investigated.The cerebral infarct volume and the degree of brain injury in the mice were evaluated by TTC staining and brain water content determination.Body weight measurements and neurobehavioral tests(righting reflex,negative geotaxis,and grip tests)were carried out to assess neurological recovery after HIBD in neonatal mice;The expressions of ERα,ERβ,autophagy(Beclin1,LC3,and p62)and apoptosis(p53,PUMA,Bax,Bcl-2,caspase-3,and cleaved caspase-3)related proteins were detected by tissue immunofluorescence and western blot,and the level of apoptosis was further detected by TUNEL and FJC staining.A caspase-3 activity detection kit was used to detect the relative activity level of caspase-3.The oxygen and glucose deprivation(OGD)model of mouse hippocampal neuron line HT22 was constructed in vitro.The effects of ICA pretreatment on the protein expression levels of autophagy-related proteins(Beclin1,LC3,and p62)as well as ERα and ERβ were detected by tissue immunofluorescence and western blot.Subsequently,OGD-injured HT22 cells were treated with 3-MA and ICA at the same time,further validating whether ICA pretreatment might exert neuroprotective effects on neonatal mice HIBD by promoting autophagy and inhibiting HIBD-induced apoptosis.After in vitro verification of ICA’s promoting effect on autophagy and the influence of ERα and ERβ protein expression after HIBD,OGD-injured HT22 cells were treated with MPP or PHTPP and ICA at the same time.To further verify whether ICA pretreatment may promote autophagy by activating ERα or ERβ pathways and thereby inhibit HIBD-induced apoptosis and play a neuroprotective role on neonatal HIBD in mice.The optimal hypoxia time of OGD,the optimal dose of ICA,the optimal inhibitory concentration of MPP and PHTPP as well as the cytotoxicity of ICA,MPP,and PHTPP at different doses were detected by CCK8.Cell immunofluorescence that labeled cytoskeleton protein βIII-Tubulin was conducted,to observe the effect of ICA on the morphology of HT22 cells under the injury of OGD.The expressions of ERα,ERβ,autophagy(Beclin1,LC3,and p62),and apoptosis(p53,PUMA,Bax,Bcl-2,caspase-3,and cleaved caspase-3)related proteins were detected by cellular immunofluorescence and western blot.TUNEL staining and Annexin V-FITC/PI staining were performed to further detect the apoptotic level.A caspase-3 activity detection kit was used to detect the relative activity level of caspase-3.Result:(1)ICA pretreatment significantly promoted the autophagy level of HIBD mice(P < 0.05).(2)Autophagy inhibitor 3-MA significantly inhibited the increase of autophagy induced by ICA pretreatment(P < 0.05),reversed the neuroprotective effect of ICA pretreatment(P < 0.05),promoted the apoptosis of HIBD neonatal mice pretreated by ICA(P < 0.05),and aggravated HIBD in neonatal mice.(3)ICA pretreatment significantly increased the expression levels of ERα and ERβ proteins in HIBD newborn mice(P < 0.05).(4)The treatment with ERα inhibitor MPP and ERβ inhibitor PHTPP both significantly inhibited the expression levels of ERα and ERβ proteins activated by ICA pretreatment(P < 0.05),reversed the neuroprotective effect of ICA pretreatment(P < 0.05),inhibited autophagy of HIBD neonatal mice pretreated by ICA(P < 0.05),promoted apoptosis(P < 0.05),and aggravated HIBD in neonatal mice.Conclusion:ICA pretreatment might play a neuroprotective role in neonatal mice with HIBD by activating the ERα and ERβ pathways to promote autophagy and thus inhibit HIBD-induced apoptosis. |
