The Role Of Nrf2 Pathway In The Abnormal Keratinization Of The Skin Caused By Arsenic In Rabbits

Objective: By infecting the New Zealand big ear rabbits with low,medium and high doses of mixed arsenic,and intervened by sulforaphane(SFP)and 5-heterozygous nitrogen-2'-deoxycytidine(5-Aza-DC),to study on the regularity of continuous activation of Nrf2 and its role in derangement of skin.Methods: 1.80 male and female cleaned New Zealand big ears rabbits,randomly divided into 10 groups according to body mass.They were blank control group,low dose arsenic group(0.075mg·kg-1·w-1),middle dose arsenic group(0.15mg·kg-1·w-1),high dose arsenic group(3.75mg·kg-1·w-1),low dose arsenic+SFP group,middle dose arsenic+SFP group,high-dose arsenic+SFP group,low dose arsenic+5-Aza-DC group,middle dose arsenic+5-Aza-DC group and high dose arsenic+5-Aza-DC group.Provided free drinking water for the rabbits,made them be poisoned continuously for 14 weeks.Since the ninth week,intravenous injected the5-Aza-DC intervention group and SFP intervention group in the ear vein with0.125mg·kg-1 5-Aza-DC and 1.25mg·kg-1 SFP two times per week,lasts 6 weeks.2.After14 weeks of arsenic contamination,took back skin samples from the same position of all the rabbits,observed the morphological changes of skin tissue by electron microscopy.3.Enzyme linked immunosorbent assay(ELISA)was used to detect glutathione(GSH),heme oxygenase(HO-1),cyclooxygenase(COX-2),Sadenosylm-ethionine(SAM)and homocysteine(HCY)activity in rabbit's skin.4.Using Quantitative Real-time PCR detection of rabbit blood and skin of nuclear factor E2 related factor 2(Nrf2),Kelch like ECH associated protein 1(Keap1),BTB-CNC homology 1(Bach1),keratin 1,keratin 10,keratin protein(INV),involucre loricate protein(LOR),extracellular regulated protein kinases(ERK),nuclear factor-kappa B(NF-?B),c AMP response element binding protein(CBP)expression of m RNA.5.The level of expression of Nrf2,Keap1 and Bach1 protein in blood and skin of rabbits was detected by Western blot.Results: 1.The skin of rabbit is visible under electron microscope,compared with the control group,the skin tissue of each arsenic group changed obviously.As the dose of arsenic increases,the morphologicalchanges of the skin cells of the rabbit and the degree of the derangement of the cuticle in the skin of the rabbit deepened.2.In rabbits' skin,the expression of GSH protein in each group was downtrend compared with the control group,and the difference was statistically significant(P < 0.05).Compared with high dose arsenic group,high dose arsenic+SFP group and high dose arsenic+5-Aza-DC group showed a decreasing trend(P=0.003,P<0.001).In the expression of HCY protein,the medium dose arsenic group compared with the control group was on the rise(P=0.002).The medium dose arsenic group compared with the medium dose arsenic+SFP group and the middle dose arsenic+5-Aza-DC group was on the rise(P < 0.001,P=0.017).The expression of SAM protein in each group compared with the control group was up-regulated(P<0.05).The medium dose arsenic group compared with the medium dose arsenic+5-Aza-DC group was increased(P=0.048).The high dose arsenic group compared with the high dose+SFP group were decrease(P=0.022).The expression of HO-1 protein compared with the medium and high arsenic groups was up-regulated(P=0.016,P=0.002),and the medium and high arsenic groups compared with the medium and high dose of arsenic+5-Aza-DC were up-regulated(P=0.016,P=0.014).In the expression of COX-2 protein,there was an upward trend in each group compared with the control group(P<0.05).3.The expression of Nrf2 m RNA in the skin and blood of rabbits showed low dose of arsenic promotion.Keap1 m RNA rose in the blood of rabbit with the increase of arsenic dose,and showed low dose of arsenic inhibition in rabbit skin,while high dose of arsenic was decreased in the rabbit skin.Bach1 m RNA showed a middle dose of arsenic to promote expression and a high dose of arsenic to down regulate in rabbit skin,arsenic expression was up-regulated in the middle and high dose groups in the blood.The effect of 5-Aza-DC intervention on rabbit skin was inhibited the expression of Nrf2 m RNA in low dose group.4.The expression of K1 and K10 m RNA in the skin of rabbit increased with the increase of arsenic dose.The medium dose arsenic+5-Aza-DC group compared with middle dose arsenic group,INV m RNA showed up regulation(P=0.007).Lor m RNA showed down-regulation in low dose arsenic group(P=0.003).Low dose arsenic group was down regulated compared with low dose arsenic+SFP group(P=0.007).The expression of MAPK/ERK m RNA in the medium dose arsenic+5-Aza-DC group was up-regulated(P=0.002),and the expression in the high dose arsenic+5-Aza-DC group was down-regulated(P=0.001).In the expression of NF-kappa B m RNA,the high dose arsenic+5-Aza-DC group showed up-regulation(P=0.017).In the expression of CBP m RNA,the middle dose arsenic group showed down-regulation(P=0.036).5.The expression of Nrf2 protein in low dose and high dose of arsenic groupin rabbit blood was increased(P=0.002,P=0.036),and the expression of Nrf2 protein was increased in rabbit skin(P < 0.001).The expression level of Keap1 protein was down-regulated in rabbit blood and skin(P < 0.05,P < 0.05).Low and middle dose arsenic+5-Aza-DC was down-regulated compared with low and medium dose arsenic group(P<0.001,P<0.001)).In rabbit blood,the expression level of Bach1 protein was decreased in low dose and medium dose arsenic group(P < 0.001,P=0.033),and the expression of Bach1 protein in rabbit skin was down regulated(P < 0.05).The intervention of SFP in rabbit skin showed low dose promotion of Nrf2,and low dose inhibition of Keap1 and Bach1(P<0.05).The effects of 5-Aza-DC intervention on the expression of Nrf2,Keap1 and Bach1 in rabbit skin were all inhibited,and it also promoted the expression of Bach1 protein in high-dose group(P < 0.05).Conclusion:Arsenic can continuously activate Nrf2,and affect the related pathways and downstream antioxidant indicators,induced skin keratinization indicators,arsenic-induced increase in keratinizing genes(K1,K10,INV,Lor),plays an important role in arsenic induced keratoses,the combination of 5-Aza-DC,SFP intervention and arsenic induces the keratinization process.Arsenic can activate Nrf2 pathway,the Nrf2 pathway inhibits oxidative stress and exerts an anti-keratinization process,the mechanism of action of keratinization disorders in the skin needs further study.