The Effects Of Hypoxic Stress On Glutathione Antioxidant And Nrf2 Expression Levels In Gansu Zokor (Eospalax Cansus)

Gansu zokor(Eospalax cansus)is subterranean rodents,which lifelong lives in underground tunnel and belongs to the Eospalax family genus of Rodentia,Spalacidae,Myospalacinae.The environment of the underground tunnel(low O2 and high CO2)makes the E.cansus forms a series of adaptive physiological and biochemical mechanisms to deal with environmental stress.Therefore,E.cansus becomes an excellent model to study animals of hypoxic adaptation.Under hypoxia stress,animals produce a large number of reactive oxygen free radicals(ROS),thus lead to cell and tissue damage.Antioxidant enzymes can scavenge free radicals to maintain the body’s oxidation-antioxidant system balance.Glutathione antioxidant enzymes is an important part of antioxidant enzymes,can protect the cell membrane thiol of the protein for the reduction of state,and can remove hydroxyl radicals,unsaturated fatty acid free radicals,alkyl peroxy radicals to prevent free radical damage.Nuclear factor-related factor 2(Nrf2)is an important transcription factor of regulating various antioxidants.It can from Nrf2-ARE(antioxidant responsive element)signaling pathway to start the transcription of the downstream phase Ⅱ detoxification enzymes and antioxidant enzymes by bonding with downstream oxidation reaction components ARE in oxidative stress injury.At present,most studies focus on superoxide dismutase(SOD)and catalase(CAT),glutathione antioxidant enzymes are neglected.About subterranean rodents of hypoxia adaptation,it is not clear results that whether glutathione antioxidant system produces a major antioxidant effect on hypoglycemic adaptation and the effect of upstream regulation factor Nrf2 of antioxidant pathway.The paper discussed the adaptability of glutathione antioxidant systems and Nrf2 under hypoxia stress,and the structural transformation and evolutionary analysis of Nrf2 in E.cansus,and to lay a foundation for the further study of the physiological and molecular mechanisms of hypoxia adaptation in E.cansus.In this study,we used E.cansus as experimental animals,SD rats as control animals,and take animals brains and livers as the material,set three oxygen conditions groups(21%O2;10.5%O2 44 hours;6.5%O2 4 hours).The glutathione-related enzyme(GR,GST)activities were determined by biochemical methods.The glutathione-related enzyme(GR,GST)mRNA expression levels were determined by real-time PCR methods.Bioinformatics analysis of cloned Nrf2 gene was carried out using software.The major experiment results are as follows:1.Effects of oxygen conditions on the enzyme activities and mRNA expression levels of GR and GSTAfter hypoxia treatment,the activity of GR in the brain of E.cansus was increased,though there were no significant differences among groups.The GR activity in the brain of SD rat was increased,and it was significantly increased under 10.5%oxygen condition.In the liver,the GR activities of E.cansus and SD rat were significantly increased under 10.5%oxygen condition;the GR activity of E.cansus was increased under 6.5%oxygen condition,and the GR activity SD rat was significantly increased.The results suggested that GR activity in the brain of E.cansus might lead to a slight decrease under 21%oxygen condition.Meanwhile,10.5%and 6.5%oxygen conditions did not cause stress in the brain of E.cansus.But in the SD rat with oxygen-sensitive animal,the treatment of 10.5%oxygen condition for 44h can increase the GR activity to improve its antioxidant capacity.GR catalytic oxidized glutathione(GSSG)reduced to reduced glutathione(GSH)by NAPH+.The important physiological function of GSH is reducing the generation of hydrogen peroxide(H2O2),is one of the major antioxidants.Enhance of GR enzyme activity may cause GSH to produce a greater antioxidant effect.Under different oxygen conditions,the levels of GR mRNA expression in the brain of both E.cansus and SD rat were consistent with the enzyme activities:mRNA expression of E.cansus had no significantly increased,mRNA expression of SD rat under 10.5%oxygen condition was significantly increased.In the liver,the GR mRNA expression was down-regulated in E.cansus after hypoxia treatment after 10.5%oxygen condition treatment,was significantly down-regulated in SD rat.The GR mRNA expression was down-regulated in E.cansus,and the expression was up-regulated in SD rat after acute hypoxia 6.5%oxygen condition treatment.The changes of GR activity and mRNA expression in E.cansus and SD rat were opposite.On the one hand,this difference should be related to the modification of protein processing(such as ubiquitination,phosphorylation,glycosylation,etc.).On the other hand,it may be related to activation of enzyme activity assist group.After 44 hours of hypoxia(10.5%oxygen condition)treatment,the activities of GST were increased in the brain and liver of E.cansus and SD rat,while the extent was greater in SD rat.Under the condition of 6.5%oxygen,the activities of GST were increased in the brain and liver of E.cansus,and the GST activities of SD rat were decreased.The results suggest that acute hypoxia(6.5%oxygen condition)could stimulate the activities of antioxidant enzymes in E.cansus to respond to hypoxia stress.Acute hypoxia could damage the antioxidant system of SD rat and decrease the enzyme activities.After 10.5%and 6.5%oxygen conditions treatment,the GST mRNA expression levels in the brain of E.cansus were not significantly increased,but the expression levels of SD rat were significantly increased.The results indicated that the GST hypoxia adaptation mechanism was established in E.cansus.SD rats still need to enhance GST transcription to deal with hypoxia.In the liver,the GST mRNA expression levels of E.cansus were higher under 10.5%oxygen condition and lower under 6.5%oxygen condition than those under 21%oxygen condition,but there was no significant difference;the GST mRNA expression levels of E.cansus were significantly loewer under 6.5%oxygen condition than those under 10.5%oxygen condition.The GST mRNA expression levels SD rats were significantly decreased under 10.5%and 6.5%oxygen conditions.This may be due to the body of E.cansus and SD rat cannot respond timely manner when acute hypoxia treatment.Under different oxygen conditions,the enzyme activity and mRNA expression levels of liver in E.cansus and SD rats were higher than those of brain tissue.The liver acts as the main metabolic and detoxification organ of the body,and hypoxia stress accumulates a large amount of reactive oxygen species.There was a difference in the hypoxia response mechanism between E.cansus and SD rats.According to the study of antioxidant enzymes in our laboratory,it is speculated that glutathione antioxidant systems may not play an important role to cope with hypoxia stress,but SD rats should try their best to start the body of all the role of antioxidants.2.Effects of oxygen conditions on the Nrf2 mRNA expressionAfter hypoxia treatment,Nrf2 gene expression in E.cansus was often unaltered or even reduced;the expression level in SD rat was significantly increased.Under different oxygen conditions,the Nrf2 mRNA expression levels were extremely significantly lower in E.cansus than those in SD rat.Based on our cloning and bioinformatics analysis of the Nrf2 gene,it is speculated that the Nrf2 gene not plays a key role to effect Nrf2-ARE pathway.E.cansus Nrf2 reveals unique structural changes,which may be functionally important for this transcription factor and indicate positive selsction.The rise of Nrf2 expression level should be one of the ways to cope with hypoxia in SD rat.3.Structural change of Nrf2 to hypoxic adaption in E.cansusThe full sequence of the E.cansus Nrf2 gene was 1818 bp,encoding a protein of 605 amino acids.Ser is the most amino acids.Ser40 of Nrf2 is the target of PKC,and activation of PKC can lead to an increase in the expression of ARE(antioxidant response element)by phosphorylation of Nrf2.Nrf2 protein molecular weight is 68.19693kDa,molecular formula is C2980H4667N809O981S21,isoelectric point is 4.8.It is unstable protein,fat coefficient is 73.34,and it doesn’t have signal peptide and transmembrane domain.Predicted localization for the Eukarya domain was the nucleus.Nrf2 as an inactive state coupled with Keapl in the cytoplasm,and as active state in the nucleus.The results showed that most of the Nrf2 of Eospalax cansus were active in the nucleus,which were beneficial to the hypoxia tolerance.The Nrf2 protein has two conservative domains,TPX2 and bZIP_NFE2-like domain-containing protein,which are microtubule-binding proteins and DNA binding and dimerization domains,respectively.They can regulate the pathway of Nrf2-ARE.The experience results including 4 N-glycosylation sites;7 Protein kinase C phosphorylation sites;13 Casein kinase Ⅱ phosphorylation sites;3 N-myristoylation sites;1 Basic-leucine zipper(bZIP)domain signature;1 Tyrosine kinase phosphorylation site.The role of phosphorylation can indirectly let Nrf2 dissociate from Keapl,thereby initiating downstream antioxidant gene transcription.A common positive selection site,Gln,was predicted by different methods.Gln is a conditional essential amino acid,is a must for the synthesis of GSH substances,can reduce the oxidation of neutrophils damages.Therefore,this positive adaptation reflects the evolution of the common niche of the subterranean rat.The results showed that the secondary structure of Nrf2 in Eospalax cansus was a mixed protein,and the tertiary structure of Nrf2 found ligand binding sites.The results showed that the Eospalax cansus and Nannospalax galili have very similar sequences(nucleotide,protein).And the genetic distance between them was the closest,stayed in the same branch of the phylogenetic tree.This suggests that the evolution of the Eospalax cansus was the convergence of the common niche.And this convergent evolution was driven by similar hypoxic ecological niches.In conclusion,glutathione antioxidant system is not major antioxidant to resistance to hypoxia stress in E.cansus;the level of Nrf2 mRNA expression was no significant different under hypoxic stress;Nrf2 was regulated the antioxidant pathway Nrf2-ARE by using Nrf2 structure change,functional differentiation and positive selected adaptation.