Effect Of Taurine On The Expression Of GRP78 And GADD34 In Zebrafish Larvae Brain After Hypoxia/reoxygenation Injury

BackgroundNeonatal hypoxic-ischemic encephalopathy (HIE) is the brain hypoxia ischemic damage results from perinatal asphyxia, and appears a series of central nervous system abnormalities of clinical features. HIE is the most important cause of cerebral damage and long-term neurological sequelae in the perinatal period. The pathogenesis of Perinatal HIE is a complex process caused by multiple factors, apoptosis contributes significantly to cerebral damage in the perinatal period. The research of perinatal HIE is mostly used by animal model, via simulating perinatal HIE pathological development process. Despite a considerable research effort, there is still no approved neuroprotective pharmacotherapy for acute neurological conditions. Therefor, it is crucial to find effective treatment strategy for brain injury.The endoplasmic reticulum is the major location in which the synthesis of new proteins and the storage of calcium occur. Abnormal protein conformations are a major cause for disturbed cellular homeostasis; therefore, perturbations in the endoplasmic reticulum are thought to be the origin of many diseases and developmental abnormalities. Many external factors, such as hypoxia, ischemia-reperfusion injury, oxidative stress, ATP depletion, and calcium disturbances and imbalances, can disturb the homeostasis of endoplasmic reticulum function, leading to endoplasmic reticulum stress (ERS). Cells on unstress conditions, glucose regulated protein 78 (GRP78) are binding to protein kinase R-like ER kinase(PERK), inositol requiring enzyme 1(IRE1) and activating transcription factor 6(ATF6) respectively. Under stress conditions, misfold protein make GRP78 dissociates from these endoplasmic reticulum transmembrane proteins, then activate a series of signal transduction cascade to recover the function of endoplasmic reticulum to the physiological state, that was known as unfolded protein response (UPR). However, If the ERS is severe or prolonged, UPR can also initiate pro-apoptotic pathways. Usually, GRP78 is widely considered as a sentinel marker for ERS under pathologic conditions, and the degree of GRP78 can reaction the severity of the ERS. ERS plays an important role in a range of neurological disorders, such as neurodegenation diseases, cerebral ischemia, spinal cord injury, sclerosis, and diabetic neuropathy. Recent developments have revealed that ERS induced apoptosis is an essential signaling event for neuronal injury resulting from ischemia/reperfusion, but less researched in neonatal HIE.Growth arrest and DNA damage-inducible protein 34 (GADD34) is a cell cycle regulating protein which is up-regulated by DNA damage, cell cycle stagnation and endoplasmic reticulum dysfunction, then directly involved in the process of apoptosis. It is also a downstream protein of PERK pathway. After being activated, PERK, in turn phosphorylates eukaryotic translation initiation factor 2a (eIF2a) and elicits various cellular responses such as protein synthesis inhibition and apoptotic signal activation. ATF4 is a stress responsive gene, whose initiation of translation is dependent on phosphorylation of eIF2a, up-regulated in ER stress. The increasing translation of ATF4 induces the expression of downstream gene GADD34 and CCAAT/enhancer binding protein homologous protein (CHOP). GADD34 in turn dephosphorylates eIF2a, which functions as a recovery from a shutoff of protein synthesis, may be important for the maintenance of the homeostasis of cells in the UPR. GADD34-mediated dephosphorylation of eIF2 in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the UPR. This fact indicates that GADD34 may play a role in apoptosis, but its expression and fuction in neonatal HIE is still unclear.Taurine,2-Aminoethanesulfonic acid, is an inhibitory neurotransmitter which has the highest content in neonatal brain, with the brain mature it gradually fell to adult level. Taurine has many physiological functions, it plays a role in neuroprotective through anti-apoptosis, oxidative stress, anti-inflammation, inhibition of excitotoxicity induced by glutamate. Long-term and light supplement of taurine may prevent age-related cognitive decline. A recent experimental study detected protection of taurine against ERS in both middle cerebral artery Occlusion (MCAO) stroke model and in primary neuronal cultures under hypoxia/reoxygenation.Zebrafish is a widely used vertebrate model organism in biomedical fields and evolutionary biology research. Zebrafish has gained great importance in the fields of developmental genetics, functional genomics, aquatic toxicology, neuroscience and many other areas of biomedical research. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of them resemble human clinical disorders. Because of the creation of critical genetic reagents, coupled with the rapid progress of the zebrafish genome initiative directed by the National Institutes of Health, are bringing this model system to its full potential for the study of vertebrate biology, physiology and human disease research fields.In our early research, up-regulation of c-fos was found in the brain tissue of zebrafish embryo after hypoxia/reoxygenation, that may be one of the mechanism which leading to apoptosis in brain tissue at later period of hypoxia. Furthermore, c-Jun amino-terminal kinase (JNK), one of the down-stream protein of UPR, can cause apoptosis by phosphorylation of c-Fos protein. Later treated with taurine (1mmol/L,5mmol/L,10mmol/L) in zebrafish larvae after hypoxia/reoxygenation injury, we found that, hypoxia/reoxygenation induced ERS. Taurine may present neuroprotection against hypoxia/reperfusion by down-regulation of GRP78, CHOP and cysteine-asparate protease (caspase-12),then reduce the nerve cell apoptosis, relieve the zebrafish larvae anoxia/reoxygenation cerebral injury. The research object is zebrafish which is the new model organism. Taurine treatment concentration is 10 mmol/L, observing five different time points. The zebrafish larvae hypoxia/reoxygenation brain injury model was used to simulate neonatal HIE pathological development process. To further explore the neuroprotective effect of taurine and its possible mechanism, our observed the neuronal apoptosis and the dynamic changes of ERS marker protein GRP78 and downstream protein GADD34 in the brain tissue on hypoxia/reoxygenation brain injury zebrafish larvae.Objective and significance1. Establish hypoxia/reoxygenation brain injury model on zebrafish larvae by flushing pure nitrogen (N2) into water to reduce the dissolved oxygen (DO), simulate neonatal HIE pathological development process. It plays an important role in exploring disease mechanism, discovery of new pathways and screening high-throughput for new drugs.2. To explore the pathophysiological role of ERS in zebrafish larvae after hypoxia/reoxygenation brain damage by comparison the dynamic changes of apoptosis index, and the expression of ERS marker protein GRP78 and downstream protein GADD34 between normal zebrafish larvae and zebrafish larvae after hypoxia/ reoxygenation in different time points, that aims to find new therapeutic direction For the treatment of neonatal HIE.3.To investigate whether taurine has neuroprotective effects on hypoxia/ reoxygenation brain damage in zebrafish larvae and explore its mechanisms by observe the dynamic changes of apoptosis index, and the expression of GRP78 and GADD34.Methods1. The 5 day post-fertilization (5dpf) zebrafish larvae were randomly assigned to 3 groups:control group, hypoxia/reoxygenation model group (reoxygenation after hypoxic) and taurine treatment group (lOmmol/L taurine solution after hypoxic). According to the different observed time points, each group was divided into 5 subgroups (1h,3h,6h,24h,48h) with 100 zebrafish larvae each.2. Establish hypoxia/reoxygenation brain injury model on zebrafish larvae by flushing pure N2 into water in sealed tank to reduce the DO to make hypoxia environment. When the DO was below 0.3mg/L,5 dpf zebrafish larvae were quickly put in the tank. The endpoint time of hypoxia was determined as when the zebrafish lay on the bottom of the tank for 1 minute, motionless (except for occasionally opercular movement). When reached the time endpoint, the hypoxic larvae were quickly removed into petri dishes with circulating water or taurine solution to recovery.3. Observe behavior and morphology change of zebrafish larvae after hypoxia. Recorded recovery time (the first time with balanced swimming after the endpoint time in hypoxia) of model group and taurine group, then continuously observed for 30 days after hypoxia/reoxygenation and calculated the median survival time and mortality rates.4. The pathological changes of brain tissue and cell apoptosis in each group were detected by fluorescence terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling(TUNEL) at each time point.5. The expression of GRP78 and GADD34 mRNA in the brain of zebrafish larvae in each group were detected under revers transcription real-time quantitative PCR(RT-qPCR) at each time point. The changes of GRP78 and GADD34 protein were detected by western blot.Result1. Behavior and morphology:after enter the DO< 0.3 mg/L water, zebrafish larvae were initially swimming at the top; About 5-8 min, zebrafish larvae loss of posture, body and water level is not parallel, irregular to move around. After 8 min, larvae began to appear vertical drop down to the bottom of the water gradually, and some motionless(except for occasionally opercular movement), some appear different degree of body bent, and untill to (18.71+2.08) min all drowned in the bottom and motionless (except for occasionally opercular movement).2. Recovery time, median survival time and mortality rates:Recovery time in model and taurine group were (45.69±3.87) min, (22.39±1.55) min, there were significantly difference (all P<0.05). The median survival time and mortality rates in control, model and taurine group were 16d,6d,13d and 57.36%,82.32%,70.26%, respectively, there were significantly difference (all P<0.05).3. Apoptosis index:Fluorescent TUNEL staining showed that apoptosis index was very low in control group, but it was began to increase atlh after hypoxia/reoxygenation, and reached the peak at 3h in model group and then decreased,48 h is still higher than control group, The apoptosis index in taurine group was decreased than that in model group at the same time point, there were significantly difference (all P<0.05).4. GRP78^ GADD34 mRNA:RT-qPCR showed that the expression of GRP78 and GADD34 mRNA was increased at 1h after hypoxia/reoxygenation than that in control group, and reached the peak and then decreased. GRP78 mRNA in model group at 48 h time point was still higher than in control group, but GADD34 mRNA at 48 h time point was approximate in control group. The expression of that in taurine group was decreased than that in model group at 11k 31k 6h、24h time point, the differences were statistically significant (all P<0.05).5. GRP78、GADD34 protein:Western blot showed that the expression of GRP78 and GADD34 protein in control group were extremely low, that was increased at 1h time point, reached the peak at 6h and then decreased, GRP78 mRNA in model group at 48 h time point was still higher than in control group, but GADD34 mRNA at 48 h time point was approximate in control group. The expression of that in taurine group was decreased than that in model group at the same time point, the differences were statistically significant (all P<0.05).6. Correlation analysis:the expression of GRP78 and GADD34 protein was significantly positively related with apoptosis index in model group (r=0.53、0.56, all P<0.05).Conclusion1. This zebrafish larvae model of hypoxia/reoxygenation brain damage could be established to simulate neonatal HIE pathological development process.2. hypoxia/reoxygenation could induced ERS in zebrafish larvae, with neural cell apoptosis index increasing, indicated that hypoxia/reoxygenation brain damage zebrafish larvae may initiate the apoptosis pathway through up-regulating the expression of GRP78, GADD34 mRNA and protein.3. Zebrafish larvae were treated with taurine under hypoxia/reoxygenation, we found that neural cell apoptosis index increased and the expression of GRP78, GADD34 mRNA and protein down-regulated, indicated that one of neuroprotective mechanisms of taurine relieve zebrafish larvae hypoxia/reoxygenation brain injury might by against ER stress.