Antioxidative Research On Antioxidant Enzymes HO-1, NQO-1 And Pathway NRF2-ARE In Gansu Zokor Under Hypoxia

E cansus is a unique underground rodent in China.It lives in low-oxygen and high-carbon dioxide tunnels for life.Therefore,it has evolved a series of adaptive mechanisms in morphology,physiology and molecular.It has become an ideal model for studying hypoxia tolerance.Antioxidant enzyme systems and regulatory mechanisms are important strategies for tolerance to hypoxia.Studies have shown that oxygen is an important environm ental factor for aerobic organisms to sustain life.If the body lacks sufficient oxygen supply,it will cause oxidative stress and generate excess reactive oxygen species(ROS),which will cause biological macromolecules such as nucleic acids,lipids and proteins to be destroyed.Oxidation of ROS eventually leads to pathological damage such as abnormal morphology and structure,and tissue metabolic disorders.Antioxidant enzyme system can remove excess ROS in the body and maintain the dynamic balance of oxidation-antioxidation,mainly including NADPH:quinone oxidoreductase-1(NQO-1)and haem oxygenase-1(HO-1).The NRF2-ARE pathway is the main regulatory mechanism of antioxidant enzyme expression under hypoxic stress.Its core regulatory factors are nuclear factor erythroid 2-related factor-2(NRF2),Kelch-like ECH-associated protein-1(KEAP1)and ?-transducin repeat-containing protein(?-TRCP).Therefore,the response of the underground rodent E cansus to the antioxidant enzymes NQO-1,HO-1 and the regulatory pathway NRF2-ARE under hypoxic conditions may be different from that of ground rodents.It exhibits anti-hypoxic mechanism and prevents oxidative stress from causing body damage,but these need to be verified by experiments.In this experiment,E cansus was used as the research object,and SD rats were used as controls to establish a hypoxia model(normal hypoxia:21%oxygen concentration,chronic hypoxia:10.5%oxygen concentration for 44h,acute hypoxia:6.5%oxygen concentration for 6h).Enzyme-linked immunoassay was used to measure the HO-1,NQO-1 in brain and liver tissues.Real-time quantitative PCR and Western Blot technology were used to detect HO-1,NQO-1 and the pathway NRF2-ARE transcription factors NRF2,KEAP1,?-TRCP mRNA and protein expression.To investigate the role of antioxidant enzymes and its regulatory pathway NRF2-ARE in hypoxic tolerance of E cansus,and to seek a unique antioxidant mechanism that distinguishes E cansus from ground rodents,in order to provide a theoretical basis for the adaptation of organisms to hypoxia.The experimental results are as follows:1.Antioxidant effects of HO-1,NQO-1 in brain and liver of E cansus and SD rats(1)HO-1,NQO-1 enzyme activityUnder different oxygen conditions:21%oxygen concentration was used as a control.At 10.5%oxygen concentration,HO-1 was significantly up-regulated in the brain and liver of E cansus and SD rats;NQO-1 was significantly up-regulated in the E cansus liver and SD rats brain.At 6.5%oxygen concentration,HO-1 was significantly down-regulated in E cansus liver and SD rat brain and liver.NQO-1 was significantly down-regulated in E cansus brain and SD rat liver.Comparison between E cansus and SD rat:HO-1 in the brain was not significantly different between E cansus and SD rats,SD rats were significantly higher than E cansus at 21%,6.5%oxygen concentrations in the liver.NQO-1 in E cansus liver was higher than SD rats at 10.5%oxygen concentration,but it was significantly lower in brains than SD rats.The results suggest that the HO-1 activity in the brain and liver of E cansus and SD rats are basically the same under hypoxia stress,with 10.5%chronic hypoxia up-regulation and 6.5%acute hypoxia down-regulation.However,NQO-1 activity was species-specific and tissue-specific,with the highest activity under 10.5%chronic hypoxia in E cansus liver.(2)Ho-1,Nqo-1 mRNA expressionUnder different oxygen conditions:21%oxygen concentration was used as a control.At 10.5%oxygen concentration,Ho-1 and Nqo-1 were significantly up-regulated in the brain and liver of E cansus;SD rats were unchanged.At 6.5%oxygen concentration,Ho-1 was significantly up-regulated in the E cansus liver,but there was no change in the brain;Nqo-1 was not changed in the brain and liver of E cansus.Ho-1 and Nqo-1 tend to be up-regulated in SD rats.Comparison of E cansus with SD rats:Ho-1 and Nqo-1 were significantly higher than SD rats at three oxygen concentrations.The results suggest that E cansus evolved from hypoxic adaptation characteristics different from SD rats at the transcription level,and Ho-1 and Nqo-1 were significantly higher than SD rats.Chronic hypoxia stress of 10.5%could maximize the transcription of Ho-1 and Nqo-1 genes in E cansus.(3)HO-1,NQO-1 protein expressionUnder different oxygen conditions:21%oxygen concentration was used as a control.At 10.5%oxygen concentration,HO-1 was significantly up-regulated in the E cansus brain,no change in liver;there was no change in brain and liver of SD rats.NQO-1 was significantly up-regulated in the brain and liver of E cansus.There was no change in NQO-1 in the brain of SD rats,and it was significantly down-regulated in liver.At 6.5%oxygen concentration,HO-1 and NQO-1 did not change in the brain and liver of E cansus,and there was no change in the brain of SD rats.Comparison of E cansus with SD rats:Except that the HO-1 in E cansus liver was significantly lower than that of SD rats at 6.5%oxygen concentration,the HO-1 in the brain and liver of E cansus was significantly higher than SD rats.NQO-1 in E cansus brain was significantly higher than SD rats,but liver was significantly lower than SD rats.The results suggest that HO-1 protein is highly expressed in brain and liver tissues of E cansus,but NQO-1 protein is mainly expressed in E cansus brain and SD rat liver in response to hypoxia.And 10.5%chronic hypoxia can stimulate HO-1 and NQO-1 protein translation more than 6.5%of acute hypoxia.2.Antioxidative regulation of Nrf2-ARE pathway in brain and liver of E cansus and SD rats(1)Nrf2,Keap1,?-trcp mRNA expressionUnder different oxygen conditions:21%oxygen concentration was used as a control.At 10.5%oxygen concentration,Nrf2 was significantly up-regulated in the brain and liver of E cansus and SD rat liver;Keap1 and ?-trcp were significantly down-regulated in the brains of E cansus and SD rats.Different expressions in the liver,no significant change in E cansus,and Keapl in SD rats were significantly up-regulated.At 6.5%oxygen concentration,Nrf2 was significantly up-regulated in the brain and liver of E cansus;Keap1 and ?-trcp were significantly down-regulated in the brain of E cansus and SD rats,with no change in the liver.Comparing E cansus with SD rats:Nrf2 and ?-trcp were significantly higher than SD rats at three oxygen concentrations,and Keapl was significantly lower than SD rats at 10.5%oxygen concentration.The results suggest that hypoxia at the transcription level can promote the transcription of the positive regulator Nrf2 gene and inhibit the transcription of the negative regulator Keapl and ?-trcp genes.The level of Nrf2 transcription in the brain and liver of E cansus is significantly higher than that in SD rats,which is more conducive to the activation of downstream antioxidant enzyme expression.(2)NRF2,KEAP1,?-TRCP protein expressionUnder different oxygen conditions:21%oxygen concentration was used as a control.At 10.5%oxygen concentration,NRF2 was significantly up-regulated in the brain of E cansus,while KEAP1 and ?-TRCP were not changed;NRF2 and KEAP1 were not changed in the liver,and ?-TRCP was significantly down-regulated.At 6.5%oxygen concentration,NRF2 was significantly up-regulated in the brain and liver of E cansus.KEAP1 is significantly down-regulated in the brain,and ?-TRCP is significantly down-regulated in the liver.Comparison between E cansus and SD rat:Nrf2 was significantly higher in SD rats at 10.5%oxygen concentration than that in SD rats,and significantly lower in liver than SD rats.KEAP1 was significantly lower than SD rats at 6.5%oxygen concentration in the brain of E cansus and three oxygen concentrations in the liver.Under hypoxia,?-TRCP in E cansus liver was significantly lower than SD rats,and brain was higher than SD rats.The results suggest that hypoxic stress at the translation level can promote the translation of NRF2 protein in the brain and liver of E cansus,and inhibit the translation of KEAP1 and ?-TRCP in the liver.In conclusion,Antioxidant enzyme HO-1 exerts the same antioxidant effect in the brain and liver of E cansus and SD rats.NQO-1 is species-specific and tissue-specific.It is highly active under chronic hypoxia at 10.5%in E cansus liver to facilitate metabolism and detoxification.Under hypoxic stress,the NRF2-ARE pathway is tissue-specific in the antioxidant regulation of E cansus.KEAP1 is mainly in the brain and ?-TRCP is mainly in the liver,which is inhibited by hypoxia.Thus up-regulating free NRF2 can promote the expression of antioxidant enzymes in the brain and liver of E cansus and prevent oxidative damage.Compared with ground rodents SD rats,the antioxidant enzymes HO-1,NQO-1 and NRF2-ARE in the E cansus showed stronger hypoxia tolerance.HO-1 and NQO-1 up-regulate activity against oxidative stress in E cansus at 10.5%chronic hypoxia,and down-regulate activity at 6.5%acute hypoxia to reduce energy consumption and oxygen consumption,meet basic metabolism of the body,and achieve rapid low oxygen adaptation.The NRF2-ARE antioxidant pathway has the strongest regulation of downstream antioxidant enzymes in E cansus at 10.5%chronic hypoxia.This shows that the NRF2-ARE pathway is a key pathway for antioxidative stress in E cansus,and it has evolved a unique hypoxic regulatory mechanism that is different from SD rats in ground rodents.