ULK1/FUNDC1 Prevents Nerve Cells From Hypoxia-induced Apoptosis By Promoting Cell Autophagy

Cerebrovascular disease has been considered to be one of the most common causes for death and a leading cause for disability all over the world.Ischemic cerebral infarction(ICI),also known as cerebral ischemic stroke,accounts for 60–80% of cerebrovascular disease,which is induced by the insufficiency of oxygen-rich blood in nervous system.Ischemic infarction is a serious threat to human life and health because of its rising incidence,disability rate,mortality and recurrence rate.Therefore,it is particularly important to study the pathogenesis of ischemic stroke and to prevent and treat its pathogenesis and complications in a timely manner,which has been a major disease that has attracted much attention in the field of medical science in the world.In addition,cerebral ischemic stroke is also a very serious perioperative complication.Cerebral perfusion insufficiency or embolism during operation may lead to cerebral blood flow reduction or interruption of ischemic stroke.It not only seriously affects the postoperative rehabilitation of patients,prolongs the length of stay in hospital,increases the financial burden of patients,but also increases the potential risk of medical disputes.Therefore,the study of intraoperative important organ function protection,especially brain protection,is also of great significance to ensure the safety of patients' lives during operation,promote the rapid recovery of patients and improve the prognosis.Autophagy and apoptosis are biological phenomena observed in the process of hypoxic-induced ischemic cerebral infarction(ICI).Autophagy plays an important role in regulating homeostasis in cell survival regulation.There is growing evidence that autophagy is activated in various cell types in the brain after ischemic stroke,such as neuronal cells,glial cells and cerebral microvascular cells.However,the exact role and molecular mechanism of autophagy related to ischemic cerebral infarction have not been fully elucidated.At the same time,the specific mechanism of the transformation between autophagy and apoptosis is not very clear.At present,the research on the interaction and regulation mechanism between autophagy and apoptosis has become one of the hot issues,especially in the occurrence,development and treatment of cerebral ischemia,which still needs to be further studied.This will provide a new idea for the prevention and treatment of cerebral ischemia.And it will also provide new theoretical support fororgan function protection in clinical anesthesia.Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction(ICI).Unc-51 like autophagy activating kinase 1(ULK1)and FUN14 Domain-containing Protein 1(FUNDC1)are both involved in the regulation of cell autophagy.This study aimed to investigate the regulatory effects of ULK1 and FUNDC1 on hypoxia induced nerve cell autophagy and apoptosis,is in order to find a possible molecular target to reduce neuronal apoptosis after cerebral ischemic stroke.Rat pheochromocytoma PC-12 cells were cultured,subcultured,cryopreserved and resuscitated.The hypoxia model was established in a hypoxia chamber containing5%CO2,90% nitrogen and oxygen.The survival rate,apoptosis rate,autophagy and the expression of apoptosis-related proteins in PC-12 cells were detected at 37 ? for0,3,6,24 hours.The full-length ULK1 sequence was constructed into pcDNA3.1 plasmid(GenePharma,Shanghai,China)and labeled as pc-ULK1.Empty vector pcDNA3.1 was used as control to up-regulate the expression of ULK1.The expression of ULK1 was inhibited by transfection of PC-12 cells with a mixture of inhibitor siRNA ULK1 and Lipofectamine 3000 prepared in advance.The expression of ULK1 mRNA,FUNDC1 and p-FUNDC1-related proteins,the apoptosis rate,autophagy and the expression of apoptosis-related proteins in PC-12 cells were detected.3-Methyladenine(3-MA)was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments.SP600125 was used as c-Jun N-terminal kinase(JNK)inhibitor.After adding autophagy inhibitor,JNK inhibitor and autophagy activator respectively,the expression of key molecules related to JNK pathway was detected.Cell viability was measured using cell counting kit-8(CCK-8)assay.Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay.RT-qPCR was used to quantify the mRNA levels of ULK1 and FUNDC1 in PC-12 cells.Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy,apoptosis and JNK pathway.PC-12 cell viability and apoptosis after hypoxia stimulation were detected using CCK-8 assay and Annexin V-PE/7-ADD staining assay,respectively.6 and 24 h hypoxia stimulation remarkably inhibited the viability of PC-12 cells(P < 0.05 or P < 0.01).The rate of cell viability reduced to 46.29% after hypoxia treatment for 24 h.In addition,hypoxia stimulation significantly induced PC-12 cell apoptosis in a time-dependent manner(P < 0.05 or P < 0.01).The rates of apoptotic cells were 3.20%,7.81% and11.76% after 3,6 and 24 h hypoxia treatment,respectively.Western blotting presented that the expression levels of Bax,c/p-caspase 3,and c/p-caspase 9 were all obviously increased and the expression of Bcl-2 was decreased after hypoxia treatment for 24 h(P< 0.05,P < 0.01 or P < 0.001).These results suggested that hypoxia significantly induced PC-12 cell apoptosis.Autophagy of PC-12 cells after hypoxia treatment was analyzed using western blotting.The expression rate of LC3-II/I and the expression level of Beclin-1 were up-regulated after hypoxia treatment(P < 0.05,P < 0.01 or P <0.001).Moreover,the expression level of p62 was down-regulated after hypoxia treatment(P < 0.05).These results indicated that hypoxia remarkably induced PC-12 cell autophagy.The relative mRNA expression of ULK1 and FUNDC1 in PC-12 cells after hypoxia induction was measured using RT-qPCR.Hypoxia treatment obviously up-regulated the mRNA expression of ULK1 in PC-12 cells(P < 0.05 or P <0.01).The mRNA expression of FUNDC1 was not changed after hypoxia induction.However,western blotting showed that the expression level of ULK1 and the expression rate of p/t-FUNDC1 were both increased after hypoxia stimulation in PC-12 cells(P < 0.05 or P < 0.001).These findings indicated that hypoxia promoted the activation of ULK1 and FUNDC1 in PC-12 cells.Pc-ULK1 and si-ULK1 were transfected into PC-12 cells to clarify the effects of ULK1 on FUNDC1 expression in PC-12 cells.Pc-ULK1 transfection significantly increased the mRNA expression level of ULK1 in PC-12 cells(P < 0.05).The protein expression level of ULK1 and the protein expression rate of p/t-FUNDC1 was both raised after transfection with pc-ULK1(P < 0.01 or P <0.001).Moreover,si-ULK1 transfection notably reduced the mRNA expression of ULK1 in PC-12 cells(P < 0.01).Si-ULK1 transfection remarkably down-regulated the protein expression of ULK1 and the protein expression rate of p/t-FUNDC1 in PC-12 cells(P < 0.01 or P < 0.001).These above results suggested that ULK1 positively regulated the expression of FUNDC1 in PC-12 cells.The effects of pc-ULK1 and si-ULK1 on hypoxia-induced PC-12 cell apoptosis and cell autophagy were detected using Annexin V-PE/7-ADD staining and western blotting,respectively.Pc-ULK1 transfection had no significant effects on PC-12 cell apoptosis under normoxia condition.Moreover,the rate of apoptotic cells was notably increased after hypoxia treatment(P < 0.01),but obviously decreased after hypoxia stimulation+pc-ULK1 transfection(P < 0.05).Western blotting displayed that compared to hypoxia group,the expression of Bax,c/p-caspase 3 and c/p-caspase 9 were decreased,while the expression of Bcl-2 was increased,in PC-12 cells after hypoxia stimulation+pc-ULK1 transfection(P < 0.05,P < 0.01 or P < 0.001).On the contrary,si-ULK1 transfection had no effects on PC-12 cell apoptosis and obviously aggravated hypoxia-induced PC-12 cell apoptosis(P <0.05).The results of western blotting indicate that compared to the hypoxia group,the expression of Bax,c/p-caspase 3 and c/p-caspase9 was increased,while the expression of Bcl-2 was decreased,in PC-12 cells after hypoxia stimulation+pc-ULK1 transfection(P < 0.05 or P < 0.01).Furthermore,pc-ULK1 and si-ULK1 also had no significant effects on PC-12 cell autophagy.Compared to hypoxia group,the autophagy of PC-12 cells was enhanced in hypoxia+pc-ULK1 group(P < 0.05 or P < 0.01)and reduced in hypoxia+si-ULK1 group(P < 0.05,P < 0.01 or P < 0.001).These above results indicated that ULK1 participated in the regulation of PC-12 cell apoptosis and cell autophagy.Overexpression of ULK1 decreased PC-12 cell apoptosis and increased cell autophagy.To analyze the effects of JNK pathway on ULK1 overexpression-induced cell autophagy enhancement and cell apoptosis reduction,the protein expression of key factor involved in JNK pathway was measured after relevant treatment.Single hypoxia treatment significantly up-regulated the expression rate of p/t-JNK and p/t-c-Jun in PC-12 cells(P < 0.01),which suggested that hypoxia activated JNK pathway in PC-12 cells.Moreover,compared to hypoxia group,the expression rates of p/t-JNK and p/t-c-Jun in PC-12 cells were enhanced in hypoxia+pc-ULK1 group(P < 0.05),which suggested that pc-ULK1 enhanced JNK pathway activation in PC-12 cells.Furthermore,compared to hypoxia+pc-ULK1 group,the expression rates of p/t-JNK and p/t-c-Jun in PC-12 cells were decreased in hypoxia+pc-ULK1+SP600125(JNK inhibitor)group and hypoxia+pc-ULK1+3-MA(autophagy inhibitor)group(P < 0.01).Compared to hypoxia+pc-ULK1 group,the viability of PC-12 cells was decreased in hypoxia+pc-ULK1+SP600125 group and hypoxia+pc-ULK1+3-MA group(P < 0.05).Compared to hypoxia+pc-ULK1 group,the apoptosis of PC-12 cells was enhanced in hypoxia+pc-ULK1+SP600125 group and hypoxia+pc-ULK1+3-MA group(P<0.05).These results suggested that JNK pathway was involved in the ULK1 overexpression-induced PC-12 cell apoptosis reduction and ULK1/FUNDC1 was not solely factor that involve the protection of cell death via promoting cell autophagy.In addition,compared to single hypoxia group,the apoptosis of PC-12 cells was reduced in hypoxia+rapamycin(autophagy activator)group(P<0.05).Compared to hypoxia+rapamycin+pcDNA3.1group,the apoptosis of PC-12 cells was reduced in hypoxia+ rapamycin+pc-ULK1 group(P<0.05).These findings further indicated that enhancement of PC-12 cell autophagy could reduce cell apoptosis.To conclude,ULK1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis.it can promote autophagy and inhibit apoptosis under hypoxia.Overexpression of ULK1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.ULK1 may be a possible molecular target to reduce neuronal apoptosis after ICI.JNK signaling pathway is involved in the decrease of neuronal apoptosis induced by hypoxia induced by overexpression of ULK1 gene.