Effect Of Coal-burning Fluorosis On Learning And Memory Ability Of Offspring And Its Mechanism

Objective:To study the influence of coal-burning fluorosis of female rats in puberty,pregnancy and lactation period on oxidative stress,protein and m RNA of neuronal nicotine receptors(n ACh Rs)of offspring rats,and to observe the changes of learning and memory ability of offspring rats and explore its possible mechanism.Methods:112 clean grade SD rats were weaned for two weeks(96 females and 16 males).According to body weight,female rats were randomly divided into control,low-fluorine,medium-fluorine and high-fluorine group with 24 rats in each group.The control group and male rats were fed with normal diet,while the fluoride group was fed with different fluoride contents(24.4,47.8,106.0 mg / kg)for 150 days,Before mating,the occurrence of dental fluorosis was observed and urine samples were collected for 24 hours.Eight female rats in each group were mated with four male rats(2:1)on the 90 th,120th and 150 th day after fluoride exposure.The female rats were exposed to fluoride after pregnancy until until offspring wean,and the offspring were given normal diet after weaning.After 30 days of age,offspring rats’ learning and memory ability were tested by Morris water maze,and 24-hour urine of the offspring was collected,then the offspring were killed by heart blood collection,the femur and brain tissue of the offspring were taken out.Fluorine in urinary bone and brain of neonatal rats,and female rats’ urinary fluorine detected by fluorine ion selective electrode method;the morphological changes of cerebral cortex and hippocampus were observed by hematoxylin eosin(HE)and Nissl staining;the content of malondialdehyde(MDA),the activities of superoxide dismutase(SOD)and catalase(CAT),the activity of total antioxidant capacity(T-AOC)and glutathione catalase(GSH-Px)in serum were detected by biochemical methods;the expression of neurotype nicotine receptor protein in the brain tissue of offspring rats was detected by Western blotting;the m RNA level of neural nicotine receptor in brain tissue of offspring was detected by real-time fluorescent quantitative PCR;Pearson correlation analysis was used for correlation analysis.Results:1.The model of coal-burning fluorosis puberty,pregnancy and lactation period of female rats was successfully reproduced;the contents of urinary fluoride,bone fluoride and brain fluoride in the fluoride exposed groups were higher than those in the control group,the contents of urinary fluoride,bone fluoride and brain fluoride increased with the prolongation of fluoride exposure time(P<0.05).2.HE staining showed that there were more neuron shrinkage,deeper staining and unclear boundary between nucleus and cytoplasm in the cerebral cortex and hippocampus in the fluoride group,and a small amount of vacuolation in the cortex in the high fluoride group.Nissl staining results showed that compared with the control group,the number of nissl bodies of a small number of neurons in the cortex decreased and the arrangement of cells in the hippocampus was disordered in the medium and high fluoride groups.The number of nissl bodies of a small number of neurons in the hippocampus decreased in the high fluoride group.3.Compared with the control group,the escape latency and the first time to reach the platform were prolonged and the times of crossing the platform were decreased in the fluoride exposed groups,The escape latency of the high fluoride group was significantly prolonged on the 3rd and 4th day of training(P<0.05),indicating that the ability of learning and memory was decreased.4.Compared with the control group,the content of MDA in serum increased in a dose-dependent manner(P<0.05);compared with 90 days,the content of MDA in serum increased in 120 days and 150 days,the difference was statistically significant(P<0.05).There was no significant difference in serum SOD activity among the groups exposed to fluoride for 90 days(P>0.05);compared with the control group,except for 150 days in the low fluoride group,the SOD activity in the groups exposed to fluoride for 120 and 150 days decreased,the difference was statistically significant(P<0.05).Compared with the control group,except for the low fluoride group for 90 days,the serum CAT activity of the other fluoride groups was decreased,the difference was statistically significant(P<0.05).Compared with the control group,the serum T-AOC activity of offspring rats exposed to middle and high doses of fluoride for 150 days decreased,and the difference was statistically significant(P<0.05);compared with the low dose of fluoride group,the serum T-AOC activity of offspring rats exposed to high doses of fluoride for 120 and 150 days decreased(P<0.05).At 90 days of fluoride exposure,the activity of serum GSH-Px in the high fluoride group was lower than that in the low fluoride group(P<0.05);at 120 and 150 days of fluoride exposure,compared with the control group,the activity of serum GSH-Px in the high fluoride group decreased significantly(P<0.05);compared with 90 days of fluoride exposure,the activity of serum GSH-Px in the 120 and 150 days of fluoride exposure groups decreased,the difference was statistically significant(P<0.05).5.Compared with the control group,the protein and m RNA expressions of a7,a4 and β2 subtypes of n ACh R in the fluoride exposed groups were decreased in varying degrees,especially in the high fluoride group(P<0.05).6.The results of correlation analysis showed that n ACh R a7,a4,β2 protein in brain tissue of fluorosis offspring rats was negatively correlated with escape latency and first reaching platform time(P<0.05),and positively correlated with crossing times(P<0.05);CAT was negatively correlated with first reaching platform time(P<0.05);MDA was positively correlated with escape latency and first reaching platform time(P<0.05),and positively correlated with n ACh R there was a negative correlation among a7,a4,and β2 subtypes(P<0.05),and a positive correlation between CAT,T-AOC and n ACh R a7,a4,and β2 subtypes(P<0.05).Conclusions:1.The spatial learning and memory ability of offspring was decreased by coal-burning fluorosis in female rats;2.The content of MDA in serum increased and the activities of SOD,GSH-Px,CAT and T-AOC decreased in the offspring of female rats exposed to coal-burning fluorosis;3.The expression of protein and m RNA of neuronicotine receptor in brain tissue of offspring rats was down regulated by coal-burning fluorosis in female rats;4.The decrease of learning and memory ability in the offspring of coal-burning fluorosis may be related to the increase of oxidative stress and the decrease of protein level of neuronicotine receptor.