Expression of NF-E2-related factor 2 and Kelch-like ECH-associated protein-1 in the brain of rats with chronic fluorosis and correlation with oxidative stress / 中华地方病学杂志

ABSTRACT

Objective To observe the expression of NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein-1 (Keap1) at mRNA and protein levels in the brain of rats with chronic fluorosis and to reveal the mechanism of brain damage induced by the factors.Methods Ninety SD rats were divided into three groups (30 rats in each group,half male half female) by the random number table method according to body weight.The control group was fed with normal tap-water,high-fluoride group with 50 mg/L fluoride (NaF) added in drinking water;and the high-fluoride plus vitamin E (Vit E) group with the same dose of NaF as the high-fluoride group,but giving 5 mg/kg Vit E by intragastric administration.The experiment period was 10 months.The fluoride contents in urine and bone were detected by fluoride-ion selective electrode.The protein and mRNA levels of Nrf2 and Keap1 in brain of rats were detected by Western blotting and quantitative real time PCR,respectively.The activity of superoxid dismutas (SOD) and the content of lipid peroxidation (MDA) were measured by biochemistry methods.Results Dental fluorosis was detected in high-fluoride group.The differences of fluoride contents in urine and bone were statistically significant between groups (F =6.87,182.87,all P ＜ 0.05).The urine fluoride [(2.16 ± 0.39),(2.07 ± 0.15)mg/L] and bone fluoride [(211.07 ± 40.52),(82.09 ± 28.60)mg/kg] in the high-fluoride and high-fluoride plus Vit E groups were higher than those of the control group [(1.70 ± 0.24)mg/L,(34.67 ± 11.15)mg/kg,all P ＜ 0.05].The differences of mRNA and protein levels of Nrf2 and Keap1 in brains of rats,SOD activity,MDA content were statistically significant between groups (F =654.33,432.87,447.45,398.88,68.34,68.34,all P ＜ 0.05).The mRNA levels of Nrf2 and Keap1 [(320.18 ± 6.83)％,(267.37 t 7.22)％] were increased compared to those of control group [(100.00 ±3.00)％,(100.00 ± 2.75)％,all P ＜ 0.05];the protein levels of Nrf2 and Keap1 [(283.28 ± 6.89)％,(196.32 ± 5.57)％]were also raised compared with those of control group [(100.00 ± 8.71)％,(100.00 ± 9.23)％,all P ＜ 0.05];the activity of SOD [(22.10 ± 2.10)μ,mol/kg] in brain of rats in fluoride group was significantly lower and the content of MDA [(8.63 ± 0.77) μmol/kg] was higher than those of control group [(35.05 ± 2.98),(1.25 ± 0.64) μmol/kg,all P ＜ 0.05].In high-fluoride plus Vit E group,the mRNA levels of Nrf2 and Keap1 [(243.23 ± 5.34)％,(180.54 ± 4.48)％] and the protein levels of Nrf2 and Keap1 [(210.88 ± 4.79)％,(150.68 ± 6.49)％] were lower than those of high-fluoride group (all P ＜ 0.05);the activity of SOD [(26.33 ± 1.84)μmol/kg] was significantly higher and the content of MDA [(4.88 ± 0.84)μmol/kg] was lower than that of high-fluoride group (all P ＜ 0.05).Correlation analysis showed that increased levels of Nrf2 and Keap1 were positively correlated with the level of MDA in rat brain (r =0.69,0.33,all P ＜ 0.05),but negatively correlated with the activity of SOD (r =-0.78,-0.80,all P ＜0.05).Conclusion The expression of Nrf2 and Keap1 in brain of rats with fluorosis is significantly increased and positively correlated with the content of fluoride in bone and the level of oxidative stress,whereas vit E can attenuate these abnormal changes induced by fluoride,which might be one of the mechanisms of brain damage of the disease.