Effects Of Cadmium And Lead On Keap1-Nrf2-ARE Pathway In Zebrafish

Danio rerio is a tropical ornamental fish widely distributed all over the world.Due to its advantages such as small size,transparent embryos,rapid development,short sexual maturity,and sequencing of the genome,zebrafish is attracted and become the world-recognized model of vertebrates,from which the remarkable results had been achieved.in many research fields such as toxicology,pathology,and developmental biology.Heavy metals are highly toxic,persistent,extensive,and difficult to degrade.Their entry into the water through natural pathways and human activities will result in water pollution and threat the survival of fish,ultimately leading to the destruction of the ecological environment and affecting humans Health.Cadmium and lead are the typical pollutants of water-borne metal pollutants.The Keapl-Nrf2-ARE pathway is an important anti-oxidative stress-related cellular pathway,and Nrf2(Nuclear factor erythroid 2/p45-related factor,Nrf2)is an important transcription factor for cell-mediated anti-oxidative stress response.The studies in our group have confirmed that the expression of nrf2a and nrf2b are down-regulated under lead treatment,indicating that Nrf2 may be involved in the metal lead poisoning stress response.Keapl(Kelch-like ECH associated protein 1,Keap1)is an important negative regulator of Nrf2 in vivo.Under normal physiological conditions,Keapl chelates with Nrf2 in the cytoplasm,making Nrf2 impossible to enter the nucleus and exert its activity,so the cell reaches a stable state;when under the action of stress,the body produces a large number of reactive oxygen species(ROS),then ubiquitination of Nrf2 protease is blocked,resulting in transient nuclear shift of Nrf2.Nrf2 enters the nucleus and binds to the small Maf protein on the anti-oxidant response element(ARE)located in the upstream promoter region of the cytoprotective gene,thereby initiating the gene expressions of a series of downstream detoxification and antioxidant factors(eg:Gstp1,Nqo1,Hsp70,etc.).Antioxidant system in the body,such as superoxide dismutase(SOD),glutathione(GSH)and other synergies can inhibit ROS damage to cells,and ultimately enhance the cell's defense capabilities.Zebrafish is selected in this thesis as experimental animals to investigate the effects of different types of heavy metals(such as cadmium and lead)and the roles of the Keapl and the Keapl-Nrf2-ARE pathway in the toxicity of heavy metals at the gene and protein levels.BLAST in the NCBI revealed that the zebrafish had two keapl homologue genes,such as keapla and keapl b.In this study,the open reading frames of zebrafish keapla and keap1b are 1806 and 1782 bp,encoding 601 and 593 amino acids,respectively.An alignment and a phylogenetic tree of Keapl protein sequences in different species were analyzed.The results showed that Keapl proteins of different species contain BACK,BTB,and multiple kelch domains and are conservative in different species.Fish Keapla is first gathered with those in other vertebrates,and then with fish Keap1b,suggesting that Keap1a is more conservative than Keap1b in fishes.The spatio-temporal expressions of zebrafish keapla and keaplb were analyzed by in situ hybridization during the ten periods(1-cell,2-cell,3.5 hpf,6 hpf,12 hpf,24 hpf,36 hpf,48 hpf,72 hpf,96 hpf).They expressed at various stages,which were regarded as maternal genes.In the early stage(before 24 hpf),They widely expressed throughout the body.After 24 hpf,they mainly expressed on the head.RT-PCR analysis showed that zebrafish keap1a and keap1b expressed differently in different tissues.Keap1a was mainly expressed in eyes,brain,heart,liver,muscle and skin,while keap1b was expressed in all tissues.Although keap1a and keap1b are paralogs,their expression patterns are different.After treated with different concentrations of cadmium and lead,keapla was more sensitive to cadmium before 48 hpf and more sensitive to lead after 48 hpf Keap1b is more sensitive to cadmium after 48 hpf,but does not respond to lead at 48 hpf,suggesting that the response of keap1a and keap1b to heavy metals has been different.In order to confirm that oxidative stress did occur in the body after treated with heavy metals,the SOD,MDA,and GSH levels related to antioxidant activity were measured,showed that the measured values of MDA and GSH were significantly changed except for SOD,of which GSH was increased highestly under lead stress.At the same time,the expressions of downstream antioxidant-related genes were analyzed after treatment with cadmium and lead by real-time fluorescent quantitative PCR and in situ hybridization,suggested that they all have an upward trend,which the gstp1 gene was the most typical and dose-dependent.Therefore,it is inferred that the treatment of heavy metals does indeed lead to the production of ROS in cells and up-regulates the expression of genes related to the Keap1-Nrf2-ARE pathway.The recombinant proteins Keap1a,Keap1b,Nrf2a,and Bach1b were finally purified to try to explore the role of heavy metals in the Keap1-Nrf2-ARE pathway at the protein level.These studies will strengthen the practicability of gene expression analysis in assessing chemical effects,helpful for the research of molecular toxicology of heavy metals,and for the more effective and rapid assessment of the early environmental risk of heavy metal.