| Fluoride is a nonmetallic chemical element,distributed widely in nature,is one of essential trace elements in human body,it can absorbed by the body through water,air and soil,resulting in long-term excessive intake of the human body,causing the accumulation of body injury,known as endemic fluorosis,mainly in bones and teeth,clinically known as skeletal fluorosis and dental fluorosis.Excessive fluoride in human body can enter the body of the offspring through the placenta barrier and accumulate in it,and effect the brain development.Low calcium diet may be one of the main inducing and promoting factors of fluorosis,it caused excessive stress of endoplasmic reticulum,increase the apoptosis rate of brain cells,enhance the neurotoxicity of fluoride.However,high calcium diet can significantly reduce body injury caused by fluoride poisoning.The related research of brain development and function injury induced by fluoride poisoning is thoroughly,the effects of dietary calcium on bone organ damage caused by fluoride poisoning have also been reported,but the effects of dietary calcium on nonbone organ damage caused by fluoride poisoning,especially the influence on brain injury is rarely reported.This experiment is based on previous studies of our laboratory,to investigate the role of endoplasmic reticulum stress in brain cells apoptosis induced by fluoride exposure in neonatal rats under different calcium nutrition conditions,to explore the molecular mechanism of dietary calcium effect on apoptosis of brain cells of offsrings induced by female rats exposed to fluoride,evaluate the intervention effect of fluorosis,provide the basis data for prevention and treatment of endemic fluorosis.Selected 50 clean healthy early weaning SD female rats,25 male rats(for reproduction),randomly divided into 5 groups after one week.The control group(Control),rats drinking tap water(fluoride content<0.2 mg/L),fed with a standard pellet diet(calcium content 0.79%);high fluoride group(F),rats drinking NaF solution(100 mg/L),fed with a standard pellet diet;low calcium group(LCa),rats drinking tap water and low calcium diet(0.063%calcium content);high fluoride with low calcium group(F+LCa),rats drinking NaF solution(100 mg/L)and low calcium diet;high fluoride and high calcium diet group(F+HCa),rats drinking NaF solution(100 mg/L)and high calcium diet(7%calcium content).The male rats were fed with tap water,and fed with the standard diet(fluoride content<0.2 mg/L,calcium content was from 7 to 10 g/kg).Solvents are tap water.After exposed to fluoride for 3 months,with maternal blood/dental fluorosis and urinary fluoride content as the replication,of successful animal model of subchronic fluorosis,male:female was 1:1 after mating,took fetal,14 day and 28 day old offsprings for experiment.The experimental groups detected the female/offsprings weight,blood/urine fluoride and blood/urine calcium content and apoptosis of hippocampal neural cells,endoplasmic reticulum chaperone BIP,CHOP,CRT protein/gene and hippocampus nerve cell apoptosis related signaling molecules Bcl-2,Caspasel2,JNK protein/gene expression level of offsprings as the observation index.The experimental results are as follows:(1)The detection results of the animal model of fluorosis:3 months after the female mice exposed to fluoride tooth dental fluorosis symptoms,compared with the control group,F group and F+LCa group were cutting the surface of yellow and white chalk color,clear stripes,showed obvious symptoms of dental fluorosis,and some white spots appear even tooth defect;F+HCa group were part of cutting surface the emergence of yellow and white stripes,showing mild symptoms of dental fluorosis.Compared with the control group,the fluoride content in blood group F+LCa increased significantly(p<0.05),urinary fluoride content in F group and F+LCa group were significantly increased(p<0.01),urinary fluoride content of F+HCa group were significantly decreased(p<0.05).The results showed that after 3 months of fluoride exposure,the animal model of chronic fluorosis was successfully reproduced.(2)Weighing results:compared with control group,LCa group and F+LCa group rats were weight were significantly reduced(p<0.01),there were no significant differences between F group and F+HCa group of maternal weight gain(p>0.05);14 days old rats in F group,LCa group and F+LCa group rat weight were significantly reduced(p<0.01),there was no significant difference in body weight of F+HCa group rats(p>0.05 weight);28 days old rats in LCa group and F+LCa group were significantly reduced(p<0.01),the body weight of F group and F+HCa group were significantly reduced(p<0.05).(3)Blood fluorine/calcium and urinary calcium/fluoride content determination results:Compared with control group,There was a significant increase of F+LCa(p<0.01)in the serum of rats in each group,The fluoride content in blood group F 14 day old male rats increased significantly(p<0.05),F group blood fluoride content of 28 day old male rats was significantly increased(p<0.01);the 14 day old male rats and 28 day old mice of LCa group and F+LCa group significantly reduced serum calcium(p<0.01),14 day old female rats F+LCa group of calcium content was significantly reduced(p<0.05),28 day old female rats in F+HCa group significantly increased serum calcium(p<0.05),28 day old male rats of F+HCa group of calcium content increased significantly(p<0.01);rats in each group F group and F+LCa group,the fluoride content in the urine were significantly increased(p<0.01),urinary fluoride the content of F+HCa group increased significantly(p<0.05);rats in each group LCa group and F+LCa group significantly reduced urinary calcium content(p<0.01),14 day old male rats and F+HCa rats of postnatal day 28 group urinary calcium content increased significantly(p<0.01),14 day old female rats F+HCa group urinary calcium content significantly increase(p<0.05).Compared with the F group,14 days old rats F+LCa group blood fluoride content increased significantly(p<0.01),the fluorine content of 28 days old rats and 14 day old male rats in LCa group were significantly decreased(p<0.05);the 14 and 28 day old male rats in F+LCa group significantly reduced serum calcium(p<0.01),14 and 28 day old female rats in F+LCa group were significantly decreased serum calcium(p<0.05),28 days old rats F+HCa group of calcium content increased significantly(p<0.01),28 days old rats LCa group of calcium content were significantly decreased(p<0.05);group LCa rats urine fluoride content were significantly reduced(p<0.01).The fluoride content in urine of group F+HCa were significantly decreased(p<0.05);rats in each group LCa group and F+LCa group significantly reduced urinary calcium content(p<0.01),28 days old rats and 14 day old male rats F+HCa group urinary calcium content increased significantly(p<0.01),14 day old male rats F+HCa group urinary calcium content significantly increase(p<0.05).(4)The determination of blood indicators related to oxidative stress results:Compared with the control group,the content of MDA in serum of F group and F+LCa group of 14 day old female rats was significantly higher than that of F+HCa group(p<0.01);The SOD,GSH-Px and T-AOC activity in serum of F group and F+LCa group were significantly decreased(p<0.01),the T-AOC activity in serum of LCa group and F+HCa group were significantly decreased(p<0.05).Compared with the F group,14 days old rats and 28 day old female rats in F+LCa group serum MDA content was significantly increased(p<0.01),28 days old rats and 14 day old male rats of LCa group and F+HCa group in serum MDA decreased significantly(p<0.05);group F+HCa rats serum SOD GSH-Px,and the activity of T-AOC increased significantly(p<0.05);F+LCa group,28 days old rats SOD,T-AOC and GSH-Px in 28 day old female rats and 14 day old female SOD rats and 14 day old male rats GSH-Px and T-AOC activity were significantly decreased(p<0.05),14 day old female rats and 28 days at the age of male rats,GSH-Px activity decreased significantly(p<0.01);LCa group in serum of female rats,T-AOC rats were GSH-Px and 28 days old rats and 14 day old and 14 day old rats SOD activity increased significantly(p<0.05),14 day old male GSH-Px rats and 28 day old male rats,the activity of SOD pole increased significantly(p<0.01).(5)The apoptosis of brain cells in hippocampus.Results:apoptotic cells in fetal rats and rats of Control group and LCa group hippocampus is relatively rare,F group and F+LCa group of apoptotic cells was significantly increased,the apoptotic cells in group F+HCa than in group F decreased.(6)PCR test results:compared with control group,F group and F+LCa group,fetal rats and neonatal rats BIP,CHOP,CRT,Caspase 12 and JNK gene expression levels were significantly increased(p<0.01),the expression level of Bcl-2 was significantly decreased(p<0.01);F+HCa group,fetal rats and neonatal rats BIP,CHOP,CRT,Caspasel2 and JNK gene expression levels were significantly increased(p<0.05),the expression level of Bcl-2 gene decreased significantly(p<0.05);group LCa,Bcl-2 gene in fetal rats and 14 day old female rats the expression levels were significantly decreased(p<0.05)and Caspase12 and JNK gene expression levels were significantly.Rose(p<0.05),CRT gene of 28 day old male rats the expression levels were significantly increased(p<0.05).Compared with F group,F+LCa group,BIP and CHOP genes in fetal rats and 14 day old female rats and 28 day old rats and 28 day old mice Caspasel2 gene and 28 day old female rats CRT gene expression levels were significantly increased(p<0.01),Bcl-2 gene in fetal rats and neonatal rats the expression levels were significantly decreased(p<0.05),Caspase12 and JNK genes in fetal rats and 14 day old female rats and 14 day old male rats Caspase12 gene and 28 days old rats JNK gene expression levels were significantly increased(p<0.05);group LCa,rat fetal rats and 28 days of age,BIP,CHOP,and CRT gene the 14 day old rat Caspase12 and JNK gene expression levels were significantly decreased(p<0.05),male Bcl-2 rats and 28 day old gene expression levels were significantly increased(p<0.05),Bcl-2 gene of 14 days old rats and 28 day old female rats were significantly increased the expression level(p<0.01).(7)Western blot analysis results:compared with control group,F group and F+LCa group,the expression level of fetal rats and 14 day old female rats of BIP,CHOP and CRT protein,14 day old male rats and 28 day old female rats of BIP and CHOP protein and CHOP protein of 28 day old male rats,fetal rats and 14/28 the age of male rats,Caspase 12 and JNK protein,14 day old female rats and 28 day old JNK protein Caspase12 protein were significantly increased(p<0.01),the expression level of fetal rats and neonatal rats Bcl-2 protein decreased significantly(p<0.01);group F+HCa,the expression level of BIP,CHOP and fetal rats,CRT protein 14 day old female rats of BIP and CRT protein,14 day old male rats,BIP,CHOP and Caspase 12 protein,28 day old female rats of BIP and Caspase 12 protein and 28 day old male rats,CHOP protein increased significantly(p<0.05),the expression level of 14 day old male rats and 28 day old female rats Bcl-2 protein decreased significantly(p<0.01).Compared with F group,F+LCa group,fetal rats and 28 day old male rats,BIP,CHOP and CRT protein,14 day old female rats of CHOP and CRT protein and BIP protein of 14 day old male rats and 28 day old female rats of CRT,Caspase 12 and JNK protein expression levels were significantly increased in LCa group(p<0.05);,the expression level of fetal rats,14 days old rats and 28 day old female rats of BIP and CHOP protein were significantly decreased(p<0.05),the expression level of fetal rats and 14 day old female rats and 28 day old male rats,Caspasel2 protein significantly decreased(p<0.05),the expression level of fetal rats and neonatal rats Bcl-2 protein significantly increased(p<0.01)and JNK protein expression decreased significantly(p<0.01).In summary,drinking water fluorosis of female rats will change the blood/urine fluoride concentration of offspring,reduce the activity of oxidation stress kinase in blood,increased MDA content,increased the content of chaperone BIP,CHOP,CRT gene/protein related to hippocampus nerve cell endoplasmic reticulum stress,increased the expression of apoptotic signaling molecule Caspasel2,JNK gene/protein related to hippocampal neuron cells,and decreased the expression level of Bcl-2 gene/protein,thus enhanced the apoptosis of hippocampal neuron cells,ultimately damage offspring brain cells.The results suggested that endoplasmic reticulum stress plays an important role in the apoptosis of offspring brain damage after female exposed to fluoride under different calcium nutrition conditions,the molecular mechanism of intervention effects of high dietary calcium(calcium content 7%)on the damage might be through up-regulated the expression of Bcl-2,down-regulated the expression of BIP,CHOP,CRT,Caspase12 and JNK,thus inhibiting the excessive apoptosis of brain cells in rats exposed to fluoride,and maternal consumption of low calcium diet(0.063%calcium content),and drinking high fluorine water,increased the toxic effects on brain cells in offsprings. |
PDF Full Text Request