| [Background]Hypoxic ischemic encephalopathy refers to a reduced perinatal cerebral oxygen and blood supply by a variety of caurses,resulting in brain energy metabolism disordered caused a variety of clinical manifestations such as convulsions,coma,serious case would result in death.After treatment a lot of cases would with the sequelae of nervous system,it is one of the important causes of disability among children in Chia.With the development of domestic peripheral medicine,the incidence of neonatal asphyxia,especially severe asphyxia,was gradually reduced,but the treatment of this disease stil lacked of progress.Mild hypothermia therapy was used to treat brain injury in foreign countries.Meta analysis showed that mild hypothermia therapy had a significant effect on neonatal encephalopathy.A number of studies have shown the effect of hyperbaric oxygen therapy in the treatment of hypoxic ischemic encephalopathy,the study shows that it can effectively improve the symptoms of the nervous system,reduce the incidence of mortality and sequelae.However,no evidence of hyperbaric oxygen therapy has been shown in other studies.Some other applications was also applied,including the treatment of oxygen free radical scavenger and inhibitor,excitatory amino acid antagonists,neuroprotective agents,which were based on the basic research of oxidative stress pathway.new targets for the treatment of neonatal hypoxic ischemic encephalopathy were insufficient.The etiology Neonatal hypoxic ischemic brain damage(hypoxic ischemic brain damage,HIBD)was involved in brain blood flow and oxygen supply reduced induced result in brain injury,apoptosis plays an important role in it.Mitogen activated protein kinase(mitogen-activated protein,kinases,MAPKs)was important pathway mediated signal transduction from the cell surface to the nucleus,involved in a variety of physiological processes,cell proliferation,differentiation,apoptosis.Among MAPK families,p3 8 MAPK is a more important member.At present,studies have shown that phosphorylated and activated p38 MAPK protein resulting in the expression of apoptosis related genes and proteins.p38 MAPK inhibitors have been used to alleviate renal and intestinal ischemia reperfusion injury in rats.Previous studies have shown that,filling nitrogen into the water can simulate the hypoxic environment,it can induce the apoptosis of neonatal zebrafish brain cells,resulting in abnormal expression of apoptosis related proteins.[Objective]Based on the previous research,the aim of this study was to construct a zebrafish model of hypoxia injury in zebrafish,and to simulate the pathophysiological process of neonatal hypoxic-ischemic brain damage(HIBD).The zebrafish juvenile behavior observation software was used to observe spontaneous movement and the reaction of light stimulation on juvenile zebrafish interved by MAPK inhibitor on p3 8 after hypoxia reoxygenation.In order to reveal the intervention effect of inhibitors from the macroscopic view.TUNEL method was used to detect the apoptosis in brain cells,and B lymphocytes of tumor,brain-2(B-cell lymphoma-2 Bcl-2),Bcl-2(Bcl-2 Associated Protein,Bax)and caspase-3(cysteine-containing aspartate-specific proteases,caspase-3)protein expression was detected by Wetstenblot and rt-qPCR was alos conducted to detect related protein mRNA expression.The molecular mechanism and optimal concentration of inhibitor intervention in hypoxia process would be revealed.[Methods]1.Study animalsThe Southern Medical University in Guangdong province(human disease and drug screening zebrafish model base for international cooperation)provided the wild type AB in zebrafish,male and female zebrafish at 6 O' clock was matched by 1:1,light was turned at 8 O'clock in the next day after the natural spawning,fertilization were checked in microscope,determining the development stage.Normal fertilized eggs were cultivated in a constant temperature box.2.Hypoxia reoxygenation model preparationJuvenile zebrafish hypoxia model prepared refering to the relevant literature,the water was filled with high purity nitrogen to simulate hypoxic environment by reducing water soluble oxygen in the sealed water tank.Dissolved oxygen monitoring was used to monitor dissolved oxygen concentration.hypoxia environment was succefully sitimulated when the oxygen concentration below 0.3 mg/L(normal>6.5mg/L).The end point of hypoxia was the young fish sink to the bottom of the water and remained motionless for 3min.3.Research groupsThe fish of 5 day after fertilization was selected as the experiment object,randomly divided into five groups.the control group were normal fish,however fishes in model group and intervention group were were prepared by hypoxia model,fishes in model group were fed with DMSO water(DMSO concentration of 0.11%)for reoxygenation,and specific water containing SB202190(inhibitor of p38MAPK)was used in intervention group.According to the concentration of inhibitor,the intervention group was divided into 3 subgroups(intervention group 1/2/3),and the concentration was 5,10 and 20 mol/L respectively,and three hour after reoxygenation was selected as the end of observation.4.Observation and experimental methods4.1 Behavior observationThe behavior observation system of ZebraLab was used to record the movement.After 5 minutes in the system,the fish's movement were recorded by the system.After that opening the bottom light for 1 minutes,then quickly closed,observe the changes of behavior.By the software of the system,the motion trajectory is visualized,and the moving parameters such as moving distance and moving speed are calculated.4.2 Apoptosis detectionIn each group,8 young fish were fixed with paraformaldehyde and stored at 4?refrigerator.All fish specimens were dehydrated and paraffin embedded,then the frontal plane eyes serial sections were stained with the whole brain,the Swiss company Roche fluorescent TUNEL kit was used to conduct apoptosis staining.The apoptotic index(AI),the number of apoptotic cells and the total number of apoptotic cells were measured by and Image-Pro 6 software.The apoptotic cells were observed by inverted fluorescence microscope and Plus.4.3 Protein detectionWestern blot was used to detect the expression of phosphorylated p38 MAPK,p38 MAPK,Bcl-2,Bax and activated caspase-3 protein.The total protein was extracted from whole brain tissue and the protein concentration was measured by BCA method.The samples were prepared by gel electrophoresis and SDS-PAGE gel electrophoresis,and then the PVDF films were incubated at 4? using Rabbit anti beta-actin(1:1000)and Rabbit anti protein antibodies.Alpha Innotech software was used to analysis gray value,with-actin as a reference to calculate protein expression.4.4 Gene detectionTotal brain tissue was extracted with RNAiso Plus reagent(Takara,Japan)and total RNA was extracted from the brain tissue,and cDNA was synthesized by reverse transcription of the Prime Script RT reagent Kit reverse transcription Kit(Takara).The beta-actin was selected as reference,primers synthesized by Shanghai Generay biotech company.According to the SYBR Premix TaqTM II(TaKaRa)sample,the Roche Light Cycler Nano instrument on the qPCR reaction,each set of 3 holes.The Ct value of 2-was calculated by software,and the relative expression of gene mRNA was expressed by 2-Ct value.4.5 Statistical analysisStatistical analysis was performed using SAS9.2 software.The measurement data were used Mean + SD,count data was used N(%)for statistical description.Measurement data were compared between grops with t test or analysis of variance,count data were compared by chi-square test,LSD test was used for subgroups comparing,the difference was statistically significant with P<0.05.[Results]1.spontaneous movementThe moving distance and velocity of zebrafishes in the model group and the intervention group were lower than those of the control group(P<0.01).High speed/low speed duration and distance in intervention group two showed higher than those in the model group and intervention group one(P<0.05),and there was no significant difference between the intervention group(P>0.05),these suggested that 10 and 20 mol/L concentration of inhibitors had a significant protective effect on hypoxia injury behavior induced by reoxygenation.2.light stimulation reactionAfter the light stimulation,the zebrafishes showed increased mobility,and the moving speed decreased gradually after the removal of the strong light.After 6 min of removal,the model group and the intervention group one were still more active,and the movement speed was higher than that of other groups(P<0.01).In the control group and the intervention group two/three,the activity of the fishes were restored to the level before the light stimulation,and there was no significant difference between the groups(P>0.05).The results showed that the inhibitor with the concentration of 10 and 20 mol/L had a protective effect on the light sitmulation reaction.3.Apoptosis of brain cellsThe results of TUNEL staining,AI index(%)detected in the model group was higher than that of the control group(P<0.01),it suggested that hypoxia/reoxygenation injury induced apoptosis of brain cells.AI in the intervention group showed lower than model group(P<0.01),intervention group two showed smallest,the intervention group three was in the second level and he intervention group one was biggest(P<0.01),these suggested inhibitors could reduce cell apoptosis of brain after hypoxia/reoxygen injury.4.Apoptosis related protein expression in brain tissueWestern blot results showed that phosphorylation p38/p38 protein ratio in the model group and intervention group of zebrafish brain is higher than that in the control group.This ratio in the intervention group showed lower than the model group(P<0.01),suggesting that SB202190 could inhibit p38 protein phosphorylation induced by hypoxia.The expression of Bcl-2 protein in the intervention group two and three were higher than that in the model group(P<0.01),suggesting that the inhibitor increased the expression of anti-apoptotic protein Bcl-2.Bax protein expression in the intervention group two and three was lower than that in model group and intervention group one(P<0.05),caspase-3 protein expression in the intervention group one and two was lower compared with model group(P<0.05),suggesting that the inhibitor decreased expression of apoptosis related protein.5.Expression of apoptosis related protein mRNA in brain tissueThe mRNA expression of Bcl-2,Bax and Caspase-3 in model group and intervention group was higher than that in control group(P<0.01),suggested that hypoxia could increase the mRNA expression of apoptosis related protein.Bcl-2 mRNA expression in the intervention group two was higher than that in model group and intervention group three(P<0.01).Bax mRNA expression in the intervention group two and three were lower than that in the model group and intervention group one(P<0.01),the expression level of Caspase-3 mRNA in the intervention group two is lower than the model group and intervention group one(P<0.01),suggesting that inhibitors can increase mRNA expression of the anti-apoptotic protein(Bcl-2)and to reduce the apoptosis induced protein(Bax/caspase-3).[Conclusion]1.p38 MAPK pathway plays an important role in hypoxia reoxygenation injury,which may play the protective role by regulating the expression of apoptosis related proteins.2.p38 MAPK inhibitor can reduce the apoptosis of brain cells and the expression of apoptosis induced protein and mRNA after hypoxia,which can be seen an increase in the ability of movement in zebrafish larvae.3.From the observation of movement behavior comparison and the analysis of protein and gene expression,the 10 mol/L of inhilibor was the best concentration for the protective effect. |
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