| Neural stem cells(NSCs) have the ability to differentiate into astrocytes, neurons and oligodendrocytes, which can self renew and generate a large number of brain cells. In embryonic stage, NSCs are mainly distributed in the cortex, hippocampus, striatum and midbrain of the brain, while in adulthood they are mainly localized in subgranular zone(SGZ) of hippocampal dentate gyrus, subventricular zone(SVZ) of lateral ventricle and striatum. NSCs bring a new hope for the treatment of neurodegenerative diseases by transplantation of neural stem cells which have the potential of self-renewal and multiple-differentiation. So the study on the proliferation and differentiation of NSCs has been becoming hotspotin recent years. The proliferation of NSCs is impacted and regulated by many factors. The internal factors include genes, proteins, transcription factors in NSCs and so on; the external factors include the nutritional factors, the interaction between cells, various physical and chemical factors such as hypoxia, etc. In mammals due to the special structure of the brain region, at the embryonic development stage and the adult stage the oxygen concentration in brain tissue is only 3%-5%, which is called "physiological hypoxia". Therefore, research on the proliferation of neural stem cells under the condition of physiological hypoxia is significant. This study was conducted to explore the proliferation ability of NSCs under physiological hypoxia, furthermore to discuss the effect of hypoxia on the proliferation of NSCs from the perspective of metabolic pathway.Autophagy is a normal metabolic process of intracellular proteins and organelles. It plays an important role in maintaining the stability of the cell itself and protecting the cells from stress injury. In a variety of stress conditions, autophagy is activated and removes the abnormal folding proteins and aging or damaged organelles, and thus promotes cell survival. On the contrary, if the autophagy is over activated, the extent of autophagy is beyond of the level of its regulation, which will accelerate the death of cell. However, the role of autophagy in regulating NSCs under hypoxia is unknown.Wild type p53-induced phosphatase 1(Wip1) is an oncogene, belonging to a member of the serine/threonine family. Studies on Wip1 were mainly focused on identifying target proteins and the phosphorylation sites. At least 7 Wip1 target proteins have been found so far, including p53 and p38 which are closely related to cell proliferation cycle. Wip1 promotes the dephosphorization of p38 and p53 and relieves their inhibitory effect on cell proliferation. So we want to know whether Wip1 participates in regulating proliferation of NSCs under hypoxia? Is this regulation related to autophagy? These issues will be discussed separately in this paper.In this study, we used a 3% O2 hypoxia model(20% O2 as the control group) to study the changes of autophagy in neural stem cells and their effects on cell viability. We examined the expression of Wip1 in hypoxic process and its effect on cell survival and autophagy, and we analyzed the association between Wip1 and autophagy in regulating the proliferation of neural stem cells under hypoxic conditions.The main results are as follows: 1. Effects of hypoxia on the activity of neural stem cellsNormal oxygen(20%) and hypoxia(3%) were used to detect the effects of hypoxia on cell status. By comparing the effects of hypoxia and normoxia on the viability and proliferation of neural stem cells and the changes of reactive oxygen species(ROS) in neural stem cells under different oxygen concentrations, we try to explore the effect of hypoxia on the activity of neural stem cells. 1.1 Isolation and culture of embryonic neural stem cellsThe midbrain derived from the 14.5 Day Embryo of SD rats was seeded in the serumfree medium containing EGF, b FGF and other factors. The proliferation of neural stem cells can be observed in the primary cells after a day of culture. Neural stem cells were suspended in the culture medium, and neural spheres could be observed in 2 to 3 days. Neural spheres should be digested and passaged in a suitable size. 1.2 Identification of neural stem cellsIn order to identify the cells that we have isolated and cultured are neural stem cells, we chose the neural spheres with good growth state to conduct immunostaining of NSCs’ marker nestin. The results showed that most of the neural stem cells were positive for nestin staining. We know from the result that the cultured cells were neural stem cells, and have a higher purity of NSCs. 1.3 Effects of hypoxia on the viability of neural stem cellsWe compared the effects of different oxygen concentrations and different treatment time on the viability of neural stem cells detected by CCK-8. The results demonstrated that the viability of neural stem cells after 24 h, 48 h, 72 h of exposure to hypoxia was significantly increased, respectively. The data revealed that hypoxia can significantly promote the viability of neural stem cells. 1.4 Effects of hypoxia on the proliferation of neural stem cellsIn order to explore the effect of hypoxia on the proliferation of neural stem cells, we further used Brd U and HPI hypoxia probe to examined the proliferation status of NSCs under different oxygen concentrations. The results showed that the neural stem cells exposed to hypoxia displayed more Brd U-positive cells compared with those exposed to normoxia, and the Brd U-positive cells were mainly distributed in the periphery of the neural spheres, which hinted that this area was the main resource of neural stem cell proliferation. 1.5 Effects of hypoxia on the content of ROS in neural stem cellsHypoxia and ROS is closely relative, in view of the fact that hypoxia generates ROS in mitochondria. The normal content of ROS can be used as a second messenger to affect the cell cycle, but excessive ROS lead to cell damage. To measure the level of ROS, we incubated neural stem cells with the ROS fluorescence probe and then detected with FACS. Compared with the normoxia group, the ROS content in hypoxia were significantly more than those in normoxia. And the most obvious increase of ROS appeared at 48 h of hypoxia, about a 2.5-fold increase, and then at 72 h of hypoxia the ROS content decreased but still more than those in normoxia. 1.6 Effects of autophagy(autophagy) inhibitor Mdivi-1 on the content of ROS inneural stem cellsROS is mainly produced in mitochondria. Mitophagy, as a type of autophagy, we further examined the content of ROS in the neural stem cells treated with the inhibitor of mitochondrial autophagy Mdivi-1 for 48 h. Compared with normoxia group, hypoxia group and normoxia Mdivi-1 group ROS increased significantly. Compared with the hypoxia group, hypoxia Mdivi-1 group increased significantly to ROS levels. The results suggested that the ROS content were increased under hypoxia, and the levels of ROS in neural stem cells after inhibition of autophagy(mitochondrial autophagy) were significantly increased. 2. Effects of hypoxia on autophagy of neural stem cellsAutophagy is a metabolic mechanism of cells. If the level of ROS in cells is too high, it can activate autophagy to protect cells from the damage caused by excessive ROS. In order to determine the level of autophagy in neural stem cells under hypoxia and the relationship with ROS and cell survival, We separately carried out the following experiments: 2.1 Effects of hypoxia on the level of autophagy in neural stem cellsThe neural stem cells were treated with 3% O2 at different time points, and then Western Blot and immunofluorescence assays were used to detect the autophagy. The results showed that compared with the normoxia group, the level of autophagy in neural stem cells increased significantly after exposure to hypoxia. Autophagy was inhibited by the autophagy inhibitor 3-MA, which suggest that the neural stem cells occurred autophagy in this hypoxia condition. 2.2 Inhibition of autophagyblocks the proliferation of neural stem cellsTo further verify the effect of autophagy on the viability of neural stem cells, we used autophagy inhibitor 3-MA to treat neural stem cells, and CCK-8 was used to detected cell viability at different time points.The results showed that the viability of the neural stem cells was reduced after the inhibition of autophagy in both hypoxia and normoxia conditions. The decrease was especially significant under hypoxia, which suggests that autophagy played a significant role in the proliferation of neural stem cells. 3. Effects of hypoxia on the expression of Wip1 in neural stem cells and the role of Wip1 in cell proliferationWip1 is a phosphatase closely related to cell cycle, and plays an important role in cell proliferation. In order to explore the effect of Wip1 on the proliferation of neural stem cells under hypoxia conditions, we carried out the following experiments: 3.1 Effect of hypoxia on the expression of Wip1 in neural stem cellsWe examined the expression of Wip1 in neural stem cells at different time points in different oxygen concentrations. The results showed that the protein expression of Wip1 was significantly increased in hypoxia compared with that in normoxia examined by western blot.PCR real-time technique was used to detect the m RNA levels of Wip1 after treatment with different oxygen concentrations at different time points. We found that there was no statistically significant difference between the hypoxia group and the normoxia group at 24 h and 72 h, and the Wip1 m RNA level in hypoxia was obviously lower than that in normoxia at 48 h, which suggests that the transcription activity of Wip1 did not contribute to the increased protein level under this hypoxia condition. 3.2 The impact of Wip1 knockdown on cell survivalNeural stem cells were transfected with Wip1 si RNA, and then cell survival was measured by CCK-8. The results showed that the survival of neural stem cells was reduced with Wip1 si RNA under both normoxia and hypoxia conditions. 3.3 The impact of Wip1 knockout on cell survivalThe survival rate of neural stem cells derived from Wip-/- mice in hypoxia was significantly decreased compared with those in normoxia at different time points, and the inhibitory effect of Wip1 si RNA increased with time, which suggests that Wip1 plays an important role in the process of regulating proliferation of neural stem cells under hypoxia. 3.4 Effects of Wip1 knockdown on autophagy activity of neural stem cellsFrom the studies above, we found that both autophagy and Wip1 are involved in the regulation of NSCs proliferation under hypoxia, but the interaction between them is not clear. In order to explore the relationship between Wip1 and autophagy in the process of hypoxia, we firstly used Wip1 si RNA to treat neural stem cells, and then examined whether the autophagy activity in neural stem cells was changed. The results showed that the activation of autophagy was inhibited in the hypoxia group after Wip1 knockdown, but no significant change was found in the normoxia group.Taken together,this study confirmed the enhanced proliferation of neural stem cells in hypoxia(3%), and furthermore we found that the activity of autophagy was significantly increased by hypoxia at different time points. Inhibition of autophagy clearly reduced the survival of NSCs at different time points under hypoxia, which hints that autophagy was deeply involved in the proliferation of NSCs promoted by hypoxia. Apart from that, we found that the expression level of Wip1 protein in neural stem cells was increased under hypoxia. Knockdown/knockout Wip1 remarkably decreased the survival/proliferation of NSCs under hypoxia, and the autophagy activity was also inhibited by Wip1 knockdown with its si RNA. In conclusion, this study firstly reported that autophagy and Wip1 may be co-associated with the proliferation of neural stem cells under the hypoxia conditions, which provides a new way to explore the molecular mechanism of proliferation of neural stem cells promoted by hypoxia. In the future study we will explore in more depth the interaction between autophagy and Wip1 in regulating the proliferation of NSCs under hypoxia.Stroke is a kind of disease that seriously threatens human health, and even become a major killer of human life. China is the most populous country in the world, and with the improvement of people’s living standards, the number of elderly people in China has increased significantly. The incidence of age-related diseases is increasing year by year, and stroke has a serious negative impact on the patients themselves and their families. Ischemic stroke is the most common type of stroke. It is mainly caused by cerebral artery clog, which leads to severe damage to the brain tissue. Because of the acute and serious harmness resulted from ischemic stroke, only a few patients can get timely treatment, so it is very important to further explore the pathophysiological process of ischemic stroke.Autophagy is an ancient mechanism of self protection in cells. In recent years mitophagy served as the most common type of autophagy is widely studied at home and aboard. The term of mitophagy is first proposed by Lemasters in 2005. The main viewpoint is that in the conditions of reactive oxygen species(ROS), nutrient deprivation, ischemia or hypoxia, mitochondria with damage or aging were recognized by autophagosome. At last, these unwanted mitochondria was degradated in lysosome. Researches have shown recently that many proteins in cells may involve in the process of mitophagy, and among them,BNIP3 attracted more and more attention.Bc1-2/adenovirus E1 B 19 k D protein interacting protein 3, abbreviated as BNIP3, belongs to the Bcl-2 family. BNIP3 is located in the outer mitochondrial membrane, and it can interact with Bcl-2 or E1 B encoded by E1 B gene which was transcribed by adenovirus gene. In the promotor region of BINP3 gene, hypoxia response element(HRE) could contact with hypoxia-inducible factor 1(HIF-1). In the event of cerebral stroke, the tissue ischemia or hypoxia, HIF-1 can up regulate the expression level of BNIP3. The functional study on BNIP3 mainly focuses on two aspects. On the one hand, BNIP3 can promote cell apoptosis or necrosis, plays a role in causing death, on the other hand, BNIP3 can lead to mitophagy, thereby promoting cell survival. Current research is increasingly inclined to study the role of BNIP3 in promoting survival.This study used the p MCAO(permanent Middle Cerebral Artery Occlusion) model to examine the role of BNIP3 in ischemic stroke via increasing mitophagy. We detected the expression of BNIP3 and mitophagy-related proteins at different time points; Analysis of the relationship between the degree of cerebral ischemic injury and the level of BNIP3 expression-is to explore the underlying roles of mitophagy and BNIP3 in the pathogenesis of ischemic stroke. We found that at the early stage of cerebral ischemia, mitophagy is activated, and inhibited at the late stage, which led to the increased brain damage. BNIP3 may play an important regulatory role in this process. The main results are as follows: 1. Changes of cerebral infarction volume at different time points after cerebral ischemia in rats 1.1 TTC staining of brain tissue in ratsIn order to evaluate the brain damage after p MCAO, we used TTC staining to determine the degree of brain damage in rats at different time points. The results showed that the infarct volume increased with the time of ischemia, and the percentage of infarct volume was approximately 50% at 24 h. 2. Changes of mitochondrial morphology during cerebral ischemia 2.1 Changes of mitochondrial autophagy at different time points after p MCAOThe morphology and structure of mitochondria sequestered by autophagosome were observed by a transmission electron microscope at different time points after p MCAO. We observed the normal structure of mitochondria in the brain tissues in the 0 h(sham) group. The typical mitophagy observed via electron transmission microscope presented at 3 h and 9 h, and the severe damage of mitochondria appeared at 24 h, such as mitochondrial swelling, crista disappearing, and membrane breaking down. 3. Expressions of BNIP3 and mitophagy-related proteins in cerebral ischemia 3.1 The dynamic changes of BNIP3 expression at different time points after p MCAOIn order to detect the changes of BNIP3 in protein level at different time points after p MCAO, the brain tissues were collected for Western Blot assay. The results showed that the expression level of BNIP3 was higher in p MCAO 3 h and 9 h groups compared with 0 h group, while the expression of BNIP3 was down-regulated in 24 h group. This demonstrates that the level of BNIP3 is increased first and then decreased, which may play a key role in the early period of cerebral ischemia. 3.2 Changes of mitophagyrelated proteins at different time points after p MCAOWe further examined the expression of mitophagy-related proteins at different time points after p MCAO. Western blot revealed that the expression level of beclin-1 increased, the ratio of LC3-II/I elevated, while P62, Tom20 and Hsp60 levels declined at 3-9 h after p MCAO. On the contrary, the protein level of Beclin-1 was down-regulated, the ratio of LC3-II/I decreased, and P62, TOM20 and HSP60 increased at 24 h of p MCAO. These results suggest that mitophagy is activated at the early stage of cerebral ischemia, and at the late stage, mitophagy is inhibited.In summary, our study demonstrated that mitophagy and BNIP3 are closely related to the degree of cerebral ischemic injury. Mitophagy is activated in the early period of cerebral ischemia, which is inhibited in the late period of cerebral ischemia; higher expression of BNIP3 may contribute to the reduction of ischemic brain injury, howver the lower expression of BNIP3 may aggravate the degree of injury. The results provide direct evidence for further study on the role of BNIP3 against brain damaged induced by ischemic stroke and provide a new target for clinical treatment for ischemic brain injury. |
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