The Protective Effect And Mechanism Of Neural Stem Cell Secretome On Damaged Neurons In Intracerebral Hemorrhage

Background Intracerebral hemorrhage(ICH)is a common subtype of stroke with high mortality and morbidity.Patients with ICH have symptoms such as confusion,coma and flaccid paralysis of limbs and have some residual neurological dysfunction.Secondary Brain Injury(SBI)is the main cause of neurological deficit after ICH,and neuronal damage caused by various reasons is an important mechanism of SBI after ICH.At present,there is still no effective cure for ICH.Compared with stem cell transplantation,neural stem cell secretome(NSC-S)has the advantages of low immunogenicity,low tumorigenicity and rich nutrients,which has become the focus of scholars’ research.Previous studies have shown that NSC-S can improve the neurological deficit of central nervous system diseases,inhibit inflammation,resist oxidative stress and protect neurons.However,the anti-ferroptosis effect of NSC-S in ICH and its specific mechanism remain unclear.Objectives To explore the effects of NSC-S on behavior improvement,heme metabolism and neuronal damage in mice model of ICH.The protective effect of NSC-S on neurons in ICH cell model in vitro and its specific mechanism are further studied in vitro.Methods(1)ICH mouse model was established by stereotactic brain injection of collagenase IV;(2)The effects of NSC-S on behavior disorder of ICH mice were observed by modified Neurologic Severity Score(m NSS),beam walking test,adhesive removal test and open field test(OFT);(3)The effects of NSC-S on heme accumulation and neuronal damage in ICH mice were observed by hematoma volume detection,brain edema,Prussian blue staining,Fe2+ detection,RT-q PCR and Western blot; (4)The ICH cell model was established by using hemin to damage BV2 microglia and N2 a neurons co-culture system in vitro;(5)The direct effects of NSC-S on heme metabolism and ferroptosis of N2 a neurons were observed by immunofluorescence staining,heme content detection,Fe2+ assay,TEM,RT-q PCR and Western blot;(6)Whether NSC-S can indirectly protect N2 a neurons by regulating heme metabolism and inflammation of BV2 microglia in co-culture system was observed by heme content,Fe2+ detection,immunofluorescence staining,RT-q PCR and Western blot;(7)The specific mechanism of NSC-S inhibiting ferroptosis of N2 a neurons in coculture system was explored by Western blot,immunofluorescence staining,LCMS/MS and bioinformatics analysis.Results(1)NSC-S treatment significantly improved the behavior disorder of ICH mice(P < 0.05).(2)Compared with the ICH group,NSC-S treatment significantly reduced the hematoma volume,decreased the brain water content(P < 0.01)and heme content of ICH mice(P < 0.001),increased the expression of ferroptosis-related glutathione peroxidase 4(GPX4)gene and protein,and decreased the expression of ferroptosis-related PTGS2 m RNA and corresponding COX2 protein(P < 0.05).(3)Compared with the Hemin group,NSC-S treatment significantly reduced the heme uptake(P < 0.001),alleviated the morphological damage of mitochondria and increased the expression of anti-ferroptosis related protein GPX4(P < 0.05).(4)Compared with the Hemin group,NSC-S treatment significantly increased the heme uptake of BV2 microglia in co-culture system(P < 0.001)and inhibited the expression of inflammatory mediators(ROS)and inflammatory factors(IL-6,IL-1β,TNF-α)in BV2 microglia(P < 0.01).(5)Compared with the Hemin group,NSC-S treatment significantly promoted the nuclear factor-erythroid 2-related factor 2(Nrf-2)nuclear transfer of N2 a neurons and BV2 microglia in co-culture system(P < 0.05),while the Nrf-2 inhibitor ML385 blocked the direct anti-ferroptosis protection of NSC-S(P < 0.05)and the indirect protective effect of NSC-S on N2 a neurons through heme uptake and inflammation inhibition of BV2 microglia in co-culture system(P < 0.05).(6)Bioinformatics analysis showed that the E1 heat shock protein family(HSPE1)was involved in the regulation of Nrf-2 pathway.The results of RT-q PCR and Western blot showed that N2 a neurons treated with exogenous recombinant HSPE1 had nuclear transfer of Nrf-2 protein,and the expressions of KEAP1 protein and anti-ferroptosis related proteins GPX4 and HO-1 increased(P < 0.05).Conclusions(1)NSC-S can improve the neurological deficit,inhibit heme accumulation and ferroptosis in the ICH mouse model.(2)HSPE1 in NSC-S might directly protect injured neurons by promoting Nrf-2 nuclear transfer,inhibiting heme uptake and ferroptosis of N2 a neurons,and indirectly play a protective role in ICH neurons by promoting heme uptake of microglia and inhibiting inflammation of microglia in co-culture system.