Expression Changes And Significance Of NDRG3 In Massive Cerebral Infarction

Background and PurposeThe mainly mechanisms of neuron death caused by hypoxia are divided into cell necrosis and apoptosis.But,the mechanisms of the death of nerve cells are determined by the rate of hypoxia.As in ischemic stroke,the chief manifestation was neuronal necrosis in the ischemic core,while was the coexistence of neuronal necrosis and apoptosis in ischemic penumbra.Apoptosis is the generated response and sequential death process of the cells to the physiological and pathological stimulation of environment,the changes of environmental condition or mild damages.The mechanism of neuron apoptosis is due to the intracellular and extracellular unbalanced ion concentration gradient of the neuronal cells caused by the lack of glucose or/and hypoxia,which could destroy the ion concentration gradient and could lead to depolarization of the neuronal cells,and then the depolarizing nerve cells will consume much more energy to re-polarize,causing further lack of glucose or oxygen.This process forms a malignant cycle,until the energy of nerve cells is depleted and then died.Many studies have demonstrated that the time of neuron apoptosis ranges from a few hours to several weeks.Ischemic stroke,the most common type of cerebrovascular diseases,refers to the death of nerve cells caused by ischemia and hypoxia due to arterial blood circulation disorder in the local brain.The massive cerebral infarction is one of the most serious types in ischemic stroke with high mortality and disability.N-myc downstream regulated(NDRG)family of proteins contains of 4 members:NDRG1,NDRG2,NDRG3 and NDRG4,and the four members are all well conserved through evolution.NDRGs consist of about 340-394 amino acid residues,with about 53-65%of the common gene sequence.All the family proteins members are characterized by having an NDR-and an ?/? hydrolase-fold region.More and more researches show that NDRGs play an important role in growth and development,immune response,endocrine regulation,tumor gene regulation,lactate-induced hypoxia signaling.A variety of syndromes of neurological and neuro-physiological is related to the lack of NDRG family genes.In the cerebrum,NDRG3 and NDRG4 expression is ubiquitous.NDRG3 is mainly expressed in the nuclei of cerebrum and cerebellum most cells,while NDRG1 and NDRG2 expression is localized in oligodendrocytes and astrocytes,respectively.In cerebellum,NDRG1 and NDRG4 are mainly localized in Purkinje cells and NDRG2 in Bergmann glial cells.NDRG1 is expressed in peripheral nerves and is localized in the cytoplasm of myelinating Schwann cells.Organisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts.Regulation of hypoxic responses via the hypoxiainducible factor(HIF)is well established,but evidence indicates that other,HIF-independent mechanisms are also involved.A recent research have identified a lactate-dependent signaling pathway in hypoxia,mediated by the oxygen-and lactate-regulated protein NDRG family member 3(NDRG3),which involves hypoxic responses' regulation in a HIF-independent manner.Lactate is a metabolite whose production increases inhypoxic conditions.Study found that the NDRG3 protein is degraded and is negatively regulated by oxygen in a PHD2/VHL-dependent manner in normoxia which suggested NDRG3 protein is an bona fide oxygen-regulated substrate of the PHD2/VHL system.However,a large amount of lactate is produced during prolonged hypoxia and NDRG3 protein is protected from destruction by binding to lactate that accumulates under hypoxia.The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway,promoting angiogenesis and cell growth by activating Raf-ERK pathway in hypoxic conditions.Inhibiting cellular lactate production abolishes NDRG3-mediated hypoxia responses.NDRG3 acts as a hypoxia-induced lactate sensor and plays a critical role in promoting hypoxia responses,and the process does not depend on HIF.The NDRG3-Raf-ERK axis provides the genetic basis for lactate-induced hypoxia signaling,which can be exploited for the development of therapies targeting hypoxia-induced diseases in addition to advancing our understanding of the mechanisms of hypoxia-induced diseases.As an oxygen-regulated protein,NDRG3 plays a critical role in promoting hypoxia responses.Therefore,in this paper,we preliminarily explored the expression of NDRG3 protein in lactate-NDRG3-Raf-ERK pathway in patients with massive cerebral infarction.In this study,we detected the expression level of NDRG3 proteins in vitro,rats with cerebral infarction and patients with massive cerebral infarction,respectively.In vitro,the expression level of NDRG3 proteins increased with prolonged hypoxia,but the level gradually decreased in the later period of hypoxia.In rats and patients with cerebral infarction,the results are similar to in vitro.It stands to reason that expression mechanisms of NDRG3 proteins may be that elevated lactate due to hypoxia and upregulation of glycolysis highly induced NDRG3 protein expression in the local tissues.Increased NDRG3 protein provides a self-sufficient mechanism for the cells in local tissues to recover from hypoxia without the need for additional extracellular signals by leading to the activation of the Raf-ERK pathway to promote angiogenesis and hypoxic cell growth.It is suggested that NDRG3 protein may play a protective role in massive cerebral infarction.Therefore,it may be an effective strategy for the further treatment of massive cerebral infarction by regulating the expression of NDRG3.Materials and methods1.Primary culture of neurons from cerebral cortex:cortical neurons were prepared from SD rats born within 24h.The Oxygen-Glucose Deprivation(OGD)model of cerebral cortex neurons:the cell culture medium was replaced with glucose-free DMEM,and the culture plates were placed in an anaerobic chamber filled with 95%N2 and 5%C02 at 37?,then the cells were fixed with 4%paraformaldehyde after 0.5 hours,6 hours,12 hours for QGD.The detection for expression levels of NDRG3 protein in the control group and the test groups:immunofluorescence and western blot were used to detected the level of NDRG3 protein.2.Establishment of cerebral infarction model of SD rats:the cerebral infarction model was established by embolizing the middle cerebral artery in rats.NDRG3 protein expression sites in brain were detected by immunohistochemisty and the level of NDRG3 protein was detected by western blot in 24 hours,48 hours,72 hours and 1 week after cerebral infarction and compared with the control group.3.By using prospective cohort study method,30 patients with massive cerebral infarction were randomly collected in the NICU in a row(brain computed tomography ischemic signs involving>50%of the MCA territory;and a diffusion-weighted imaging(DWI)infarct volume>145 cm3)and 30 normal individuals were collected as the control group.The level of NDRG3 protein was detected in serum of patients by ELISA kits at 24h,48h,72h and 1 week after the first onset and compared with control group.Results1.Compared with the control group,the level of NDRG3 protein increased with prolonged hypoxia time and then decreased at the later phase of hypoxia in cortical neurons of rats.2.The expression of NDRG3 protein increased with prolonged infarction time in cerebral infarction model of SD rats,peaked in 72h and decreased in 1 week,but were higher than that in control group.3.The level of NDRG3 protein increased with prolonged infarction time in patients with massive cerebral infarction,peaked in 72h and decreased in 1 week,but were higher than that in control group.Conclusions1.Hypoxia induces an increase of NDRG3 protein in neurons both in vitro and vivo.2.NDRG3 is a hypoxia-dependent oxygen-regulated protein in neurons.3.The expression of NDRG3 protein is increased in neurons in a hypoxic environment.It is suggested increased NDRG3 protein may play a protective role for hypoxic neurons by the mechanism of activation of the Raf-ERK pathway to promote angiogenesis and hypoxic cell growth.