The Expression Of P53-dependent Gene In Hippocampal Neurons Under Hypoxia

BackgroudThe transcription factor p53 responds to diverse stresses to regulate many target genes that induce cell-cycle arrest,apoptosis,senescence,DNA repair or alter metabolism.Under nomal conditions,the stability of p53 protein is tightly regulated.MDM2—a ubiquitin ligases mediate ubiquitin-dependet proteasomal degradation of p53.when cell exposed different types of cellular stress,p53 is stabilized and activated,for example,DNA damage,oncogenic signalling and hypoxia.The molecular mechanisms that govern the choice between cell-cycle arrest and apoptosis in response to p53 activation are only partially understood.Also,it remains unclear why different cell types display differential response to functional p53.The biological outcome of p53 activation presumably depends on several factors, including cellular context,cell type and type of stress agent.It is plausible that p53 activates different specific and possibly overlapping set of target genes in response to different stress signals.Previous studies have indicated that several hundred genes are potentially regulated by p53,but not necessarily in the same cell in response to the same stress signal.Hypoxia represents one of the most physiologically relevant stresses,having significant roles in both normal development and malignant progression.Exposure to severe hypoxia leads to the accumulation of p53 which can in turn lead to rapid apoptosis.In contrast to the response to DNA-damaging agents,hypoxia-induced p53 has little or no transcriptional transactivation capabilities and instead seems to function primarily as a transrepressor in order to induce apoptosis.This indicates that activation of p53 in response to hypoxia leads to a gene expression pattern that is different from that induced by p53 in response toγ-irradiation,ultraviolet(UV)light and other DNA-damaging agents.In the central nervous system(CNS),hippocampal CA1 neurons are known to be extremely vulnerable to low oxygen concentrations or anoxia.Understanding the mechanisms governing tolerance to oxygen depletion is vital for developing strategies to protect the brain from hypoxic-ischemic insult.Therefore,in this experiment,we investigated the expression of p53-dependent gene in hippocampal neurons under hypoxia treatment,in order to provide important clue for the elucidation of cerebral hypoxia mechanism.ObjectivesInvestigate the molecular mechanism of hypoxia tolerance difference during the different development stage;Studing p53-dependent gene expression pattern in response to hypoxia in hippocampal neurons,in order to provide important clue for the elucidation of cerebral hypoxia mechanism.MethodsTo establish a primary culture technique for rat hippocampal tissue,We divided the neurons into control group,medication administration group and hypoxia group. Inverted microscope and immunohistochemistry of NF was performed to assess the growth and morphology of the cells.Investigate protein expression of UNC5B by immunofluorescence.The neurons were exposed to hypoxia condition(0%O₂, 5%CO₂,and 95%N₂)for 12h or 24h after 1 week seeding.MTT cell assays and AO/EB staining measured cell viability.Medicine was added to the culture medium 2 h before hypoxia condition were introduced and included for the full hypoxia period. Total RNA was isolated using TRIZOL according to the manufacturer's instructions. RT-PCR using RT-PCR kit.Results1.â‘ The growth of hippocampal neurons from 5 days old rat in oxygenation group was more vigorous than in control group.â‘¡The cell viability of neurons from 1 day old rat and 5 days old rat with 12h hypoxia exposure were 89.4±2.197% (n=6)and 81.04±2.25%(n=7)(p＜0.01)respectively,with 24h hypoxia exposure were 85.02±2.242%(n=7)and 74.99±2.751%(n=7)(p＜0.01)respectively.â‘¢The growth of hippocampal neurons from 5 days old rat in oxygenation group was more vigorous than in control group.During hypoxia,the hippocampal neurons disassociated from 5 days old rat were more tolerant than from 1 day old rat.2.â‘ Multipolar neuron ratio of primary cultured hippocampal neurons in hypoxia group was 20.6±2.0%(n=6),in diazoxide and tolbutamide with hypoxia groups were 26.1±1.7%(n=6)and 8.7±1.7%(n=6).Those two kinds of medicine had effect on multipolar neuron ratio of cell under hypoxia condition(p＜0.01).In normoxia,diazoxide and tolbutamide had no significant effect on multipolar neuron ratio of cell compared with those of in control group(p＞0.05,n=6).â‘¡Using MTT analysis,we observed a much lower survival rate(75.7±2.8%)in neurons exposed to severe hypoxia(PO2=0 mmHg)compared with those exposed to normoxia(p＜0.01, n=7).At the same time,tolbutamide(100μM)reduced the survival rate of neurons (55.7±2.5%),however,diazoxide(100μM)increased the survival rate of neurons (81.1±2.4%)(p＜0.01,n=6).In normoxia,neither diazoxide nor tolbutamide had a significant effect on cell viability(p＞0.05,n=6).â‘¢The experiments showed a significant increase in Bax(p＜0.01)and Fas(p＜0.01)mRNA levels in neurons cultured at severe hypoxia condition(PO2=0 mmHg)compared with those cultured at normoxia(PO2=144 mmHg).In hypoxia groups,tolbutamide increased Bax(p＜0.05)and Fas(p＜0.05)mRNA expression,while diazoxide reduced it to levels observed in normoxic condition(n=5 for each),a-tubulin had a different expression pattern.It's mRNA levels decreased in neurons cultured at severe hypoxia condition compared with those cultured at normoxia.In hypoxia groups,tolbutamide and diazoxide didn't change a-tubulin mRNA expression.3.â‘ To confirm the relationship between UNC5B and p53,we measured UNC5B mRNA levels with RT-PCR in neurons that have been treated with p53 inhibitor PFT-a(100nM[5])or p53 activator nutlin-3(2μM)in normoxia or anoxia for 24h.These experiments showed a significant decrease in UNC5B(p＜0.01,n=5)mRNA levels in neurons cultured at severe hypoxia condition(PO2=0 mmHg)compared with those cultured at normoxia(PO2=144 mmHg).In the normoxia group,nutlin-3 increased UNC5B(p＜0.01,n=5)mRNA expression,while PFT-a had no effect on it.In hypoxia groups,PFT-a increased UNC5B(p＜0.01,n=5) mRNA expression,while nutlin-3 reduced it(p＜0.01,n=5).â‘¡MTT cell assays measured cell viability.Using MTT analysis,we observed a much lower survival rate (75.0±2.8%)in neurons exposed to severe hypoxia(PO₂=0mmHg)compared with those exposed to normoxia(p＜0.01,n=7).In normoxia,neither PFT-a nor nutlin-3 had a significant effect on cell viability(p＞0.05,n=5).In anoxia,however,PFT-a increased the survival rate of neurons(85.2±5.6%)(p＜0.01,n=6).At the same time, nutlin-3 reduced the survival rate of neurons(57.1±4.1%)(p＜0.01,n= 7).â‘¢Immunofluorescence of UNC5B was performed to assess its protein expression in all groups.UNC5B was significantly reduced in neurons exposed to severe hypoxia(PO2 =0mmHg)(104.078±19.058,n =98)compared with those exposed to normoxia(129.959±21.633,n= 122)(p=0.000＜0.01,n=7).In normoxia,nutlin-3 (150.014±26.303,n=123)increased the expression of UNC5B(p=0.000＜0.01), while PFT-a(124.072±28.016,n=106)had no effect on it(p=0.709＞0.05).In anoxia,however,nutlin-3(95.244±12.354,n=108)decreased the expression of UNC5B on neurons(p=0.002＜0.01).At the same time, PFT-a(104.541±19.080,n=104)had no effect on it as it acted in normoxia(p=1.000) (F=100.675).Conclusions1.The oxygenation during dissection of hippocampal tissue was beneficial to the growth of neurons,and the hypoxia tolerance of this kind of neuron was better than the one in 1 day old group.2.The result suggested that the activation of K_ATPchannels can protially reduce cell death during hypoxia.And we also proved that K_ATPinfluenced transactivation of p53 tumor suppressor.3.We present the first analysis of p53-dependent gene expression patterns in hypoxia-treated Hippocampal neurons.We have found that hypoxia induces gene Bax that were previously not known to be regulated in a p53-dependent manner.Moreover, we found that the membrane receptor UNC5B which is transactivated by p53 under other stress is downgulated by p53 in response to hypoxia.