| Objective:To study the effects of Nrf2 pathway on oxidative stress in New Zealand rabbits exposed to low,medium and high-doses of mixed arsenic,SFP and 5-Aza-DC.Methods:Eighty clean male and female New Zealand big ears rabbits were randomly divided into 10 groups according to their body weight:blank control group,low-dose arsenic group(0.075mg·kg-1·w-1),medium-dose arsenic group(0.15mg·kg-1·w-1),high-dose arsenic(0.75mg·kg-1·w-1),low-dose arsenic+SFP group,medium-dose arsenic+SFP group,high-dose arsenic+SFP group,low-dose arsenic+5-Aza-DC group,medium-dose arsenic+5-Aza-DC group and high-dose arsenic+5-Aza-DC group.The rabbits freely drank water and continued to infect for 14 weeks.The blood of the mid 9th week and the skin of the 14th weekend were tested.1.After 14 weeks of exposing arsenic,to use elect-ron microscopy to observe the morphological changes of the skin tissue,taking from the same back skin samples of the rabbits.2.ELISA was used to detect GSH,HO-1,COX-2,SAM,and HCY in rabbit serum and N6AMT1 in rabbit serum and skin.3.Quantitative real-time PCR was used to detect the mRNA expression levels of Nrf2,Keap1,Bach1,N6AMT1,As3MT and MRP2 in blood and skin of rabbits in each group.4.Western blot was used to detect the protein expression of Nrf2,Keap1,Bach1,As3MT and MRP2.Results:1.The results of electron microscopy of rabbits showed that arsenic can cause keratinization and basal cell abnormalities in skin.SFP slightly improves skin tissue dam-age,and 5-Aza-DC thickens the cuticle of the skin.2.ELISA Results:the expression of GSH in the low,middle and high-dose arsenic groups was significantly lower than that in the control group.Compared with the control group,the expression of N6AMT1 was increased?P<0.05?,the expression of HCY,COX-2 and HO-1 was increased in the middle and high-dose arsenic group?all P<0.05?.the expression of SAM expression was in-creased in high dose arsenic group?P=0.006?.In skin tissue,the expression of N6AMT1 in the medium-dose arsenic+5-Aza-DC group was lower than that in the middle dose ars-enic group.3.Gene level:1)In the rabbit blood,compared with control group,the mRNA expression levels of Nrf2 in the low-dose of arsenic group,Bach1 in the middle-dose group and high-dose of arsenic group,As3MT,N6AMT1 and MRP2 in the high-dose of arsenic group was increased?all P<0.05?,the mRNA expression level of Keap1 in the low-dose arsenic group was decreased?P=0.036?.2)in the skin of rabbits,the mRNA expression levels of Nrf2 in the low-dose arsenic group,Bach1 in the medium-dose arsenic group,As3MT and N6AMT1 in the medium-dose and high-dose arsenic group were significantly increased compared with the control group?all P<0.05?.Compared with the low-dose arsenic group,the expression of Nrf2 mRNA in the low-dose arsenic+5-Aza-DC group was decreased?P=0.005?and Bach1 was increased?P=0.017?.Com-pared with the high-dose arsenic group,the mRNA expression levels of As3MT,N6AMT1 and MRP2 increased in the high-dose arsenic+SFP group?all P<0.05?and the mRNA expression levels of As3MT decreased in the high-dose arsenic+5-Aza-DC group?P=0.023?.4.Protein levels:1)In the blood of rabbits,compared with the control group,the protein expression of Nrf2 in low-dose arsenic group and MRP2 in high-dose arsenic group was increased?all P<0.05?,in addition,the protein expression of Bach1 and Keap1in the low-dose arsenic group was decreased?all P<0.05?.2).In the Rabbits skin,compared with the control group,the protein expression of Nrf2 increased in the low,medium and high-dose arsenic group,the protein expression of Keap1 decreased?all P<0.05?,and with the increase of arsenic dose,the level of Nrf2 protein expression increased gradually,by contrast,the level of Keap1 protein expression decreased,the level of Bach1protein expression decreased in the medium-dose arsenic group?P<0.05?,MRP2 protein expression levels increased in the low and high-dose arsenic group?all P<0.05?.Arsenic can activate the Nrf2/Keap1 signaling pathways,and promote the Nrf2 downstream re-lated the expression of antioxidant index and arsenic metabolism index,SFP and 5-Aza-DC may also be involved in the Nrf2 pathway,SFP as activator of Nrf2,can reduce oxidative damage,and 5-Aza-DC mainly to inhibit enzymes activity of arsenic methy-lation metabolic and increase the body's oxidative damage,but the detailed mechanism still need further study.Conclusions:Arsenic can activate the Nrf2/Keap1 signaling pathways,and improve the Nrf2 downstream related antioxidant index and the activity of arsenic metabolism index.SFP and 5-Aza-DC can regulate the Nrf2 pathway,SFP as activator of Nrf2,reduces oxidative damage of the body clearly,and 5-Aza-DC also plays a role in inhibiting the activity of arsenic methylation metabolism enzyme,increasing the damage of arsenic on oxidative stress in the body,but its detailed mechanism of action needs further study. |
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