Neuroprotective mechanism of icariin on hypoxic- ischemic brain damage in neonatal mice
Authors: Wang Mengxia , Yang Xiaoxia , Zhou Qian , Guo Yingqi , Chen Yingxiu , Song Linyang , Yang Junhua , Li Lixia , Luo Li 
Published: July 2022

Abstract
Objective: Hypoxic-ischemic brain damage (HIBD) is one of the leading causes of permanent neurological dysfunction and acute death in infants and young children. As the only effective treatment for neonatal HIBD, therapeutic hypothermia (TH) can only moderately improve the prognosis. Previous evidence has shown that icariin (ICA) can inhibit apoptosis and exert a neuroprotective effect on neonatal mouse HIBD, but the specific mechanism remains unknown. 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) pathways in neonatal mice with HIBD.Method: In vivo experiments, the HIBD model of neonatal mice were constructed according to the modified Rice-Vannucci method. The effects of ICA on the expression levels of autophagy-related proteins as well as ERα and ERβ proteins were detected. At the same time as ICA pretreatment, HIBD newborn mice were injected with the autophagy inhibitor 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. At the same time as ICA pretreatment, HIBD newborn mice were injected with ERα inhibitor MPP or ERβ inhibitor PHTPP into the lateral ventricle, to further test whether ICA might promote autophagy and inhibit HIBD-induced apoptosis by activating ERα or ERβ pathways, thereby exerting a neuroprotective effect on HIBD in newborn mice. 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 were carried out to assess neurological recovery after HIBD in neonatal mice; The expressions of ERα, ERβ, autophagy and apoptosis-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 glucose and oxygen deprivation (OGD) model of HT22 cells in the mouse hippocampal neuron system was constructed and experimented in vitro.Result: ICA pretreatment significantly promoted the autophagy level of HIBD mice. Treatment with 3-MA significantly inhibited the increase of autophagy induced by ICA pretreatment, reversed the neuroprotective effect of ICA pretreatment, and promoted apoptosis. ICA pretreatment significantly increased the expression levels of ERα and ERβ proteins in HIBD newborn mice. The MPP and PHTPP treatments significantly inhibited the expression levels of ERα and ERβ proteins activated by ICA pretreatment, reversed the neuroprotective effects of ICA pretreatment, inhibited the increase in autophagy induced by ICA pretreatment, and promoted apoptosis.Conclusion: ICA pretreatment may promote autophagy by activating the ERα and ERβ pathway, and thus reduce the apoptosis induced by HIBD, then exert a neuroprotective effect on neonatal mice with HIBD.