Effect Of Chronic Fluorosis On Ca²⁺ Channel Signal In Rat Hippocampus

Moderate fluoride is the foundation of normal physiological activities. But the fluoride is a long-term accumulated poison, and excess intake of fluorine can cause skeletal fluorosis, dental fluorosis so far as to blood and nervous system damage. Nerve tissues are sensitive to fluorine particularly. Hippocampus as a target site of fluorosis in central nervous systems, which synaptic plasticity of nerve cells is vulnerable by affecting fluorine. In recent years, through the study on neurotoxic mechanism of fluorine, people put forward two theories that the theory of G protein signal transduction and the theory of oxidative stress.Generally thought, there is a close relation between the nervous system damage caused by nerve poison and Ca2+ steady-state disorder in G protein signal transduction pathways. The mechanism may relate to Nuclear factor-κB (NF-κB) which is sensitive in oxidative stress and regulated by Ca2+. Thus, we chose rat model of chronic fluorosis to explore the effects of different doses of fluoride on dental fluorosis, blood F and nerve cell structure firstly. Then combining calcium signaling pathways theory with oxidative stress theory for the first time, we detected calcium ion concentration ([Ca2+]) and the expression level of CaMKⅡ, c-fos, NF-κB, Bcl-2 and Bax.152, just weaning male Sprague-Dawley rats were randomly divided into four groups and given 15,30,60 mg/L NaF solution and distilled water respectively for 9 and 18 months. Rats were fed standard diet throughout the experiment and water was given ad libitum. The degree of dental fluorosis and blood F were tested when three months; animals were killed by cervical when exposure was stopped, then evaluated pathological change in hippocampus with HE staining, [Ca2+] in synaptosomes was measured by double wavelength fluorescence spectrophotometer, and expression level of CaMKⅡα,c-fos,NF-κBρ65,Bcl-2,Bax in hippocampus CA3 region with immunohistochemistry (IHC) method.1. As the concentration of fluoride increased, blood F increased in experimental groups and had differences or significant differences (P<0.05, P<0.01) compared with the control group, and the level and symptom of dental fluorosis in rats had increased. The teeth of high fluoride group rats became deformed and defected to some degree. Both indices also show that the fluorosis model worked in the research.2. Chronic fluorosis can impair rat's brain nervous cells. HE staining showed that, the distribution of rat's hippocampus pyramidal cells were sparse, cell contour disappeared and vacuole in cells.3. The test of [Ca2+] in synaptosomes showed that, synaptosomes [Ca2+] increased in the experimental groups with the fluoride concentration increased. Compared with the control group, moderate group had significant difference (P<0.01) and high-fluoride group had difference (P<0.05) in middle-term; moderate group and high-fluoride group had significant difference (P<0.01) in the end of experiment.4. IHC staning showed that, the expression level of protein CaMKⅡα,c-fos and NF-κBρ65 increased with the growth of fluoride concentration. Compared with control group, CaMKⅡαmoderate group and high-fluoride group had difference (P<0.05), c-fos moderate group and high-fluoride group had difference or statistical difference (P<0.05 or P<0.01), NF-κBρ65 had no significant difference in middle-term; CaMKⅡαand c-fos moderate group and high-fluoride group had statistical difference (P<0.01), NF-κBρ65 moderate group had difference (P<0.05) in the end of experiment.5. IHC staning also showed that, the expression level of protein Bcl-2 and Bax decreased with the growth of fluoride concentration. Compared with control group, Bcl-2 moderate group and high-fluoride group had difference or significant difference (P<0.05 or P<0.01), Bax moderate group and high-fluoride group had significant difference (P<0.01) in middle-term; Bcl-2 moderate group had difference (P<0.05), high-fluoride group had significant difference (P<0.01), Bax low-fluoride group had difference (P<0.05), moderate group and high-fluoride group had significant difference (P<0.01) in the end of experiment. And Bax/Bcl-2 moderate group and high-fluoride group had increased (P<0.05) in the end of experiment, compared with control group.Above all, chronic fluoride exposure had toxic effect on the central nervous system. Chronic fluorosis could injure the nerve cells in the hippocampus CA3 region to some degree. Our results show that the hippocampus synaptosomes [Ca+] were overloaded and the expression level of CaMKⅡα,c-fos,NF-κBρ65,Bcl-2 and Bax were abnormally in experimental groups. So chronic fluorosis could induction nerve cells tend to apoptosis eventually. Degenerative change also can cause nerve cell apoptosis, and fluorine aggravated this change. The study on Ca2+ channel signal may be the molecular basis of central nervous system damage by fluorosis. And NF-κB may be one of the related molecular target factors in central nervous system damage by fluorosis. So we can make some deeper research in early-animal-development and it can offer us a theoretical basis in research of NF-κB as an environmental neurotoxic substance biomarker.