Role And Mechanism Of L-type Calcium Channel In The Process Of Drinking Water-induced Cerebral Hippocampus Injury

Fluoride is an essential trace element for the human body,but excessive intake can cause toxic effects of multiple systems and multiple organs,which is called endemic fluorosis,referred to as fluorosis.Fluorosis can damage not only bone organs such as teeth and bones,but also non-bone organs such as brain.Fluorosis can cause intracellular calcium overload,induce abnormal expression of genes or proteins related to learning and memory function in the calcium signal transduction system,and then cause damage to the nervous system.Ca²⁺is an important information molecule in cells.L-type calcium channel plays an important role in regulating the level of intracellular Ca²⁺,However,the causes of intracellular calcium overload induced by fluorine,its effects on intracellular l-type calcium channels and downstream apoptosis-related molecules have rarely been reported.140 pre-weaned healthy ICR male mice were selected for both subchronic and chronic fluorosis experiments.After one week of adaptation,they were randomly divided into 7 groups.Control group?C?,high fluoride group?HF?,low fluoride group?LF?,high fluoride agonist group?HF+FPL?,high fluoride antagonist group?HF+NIF?,low fluoride agonist group?LF+FPL?,low fluoride antagonist group ?LF+NIF?.The control group was given tap water,and the high fluoride group and the low fluoride group were respectively given30 mg/L and 5 mg/L sodium fluoride solution.They were fed for 90 days and 180 days respectively,and one week before the end of fluoride exposure,the saline,calcium channel agonist FPL64176 and calcium ion channel antagonist Nifedipine were intraperitoneally injected at a dose of5 mg/?kg·d?.After the end of combined fluorosis,the mice were observed for their ability of self-exploration and learning and memory in space,the incidence of dental fluorosis,and the content of fluorine in blood/urine of each group,so as to verify whether the fluorosis model was successfully replicated in mice.Then to detect SOD,gsh-px activity and MDA content,morphology and hippocampal CA₁ area of nerve cells apoptosis,calcium ion channel Cav1.2,calmodulin CaM,calmodulin kinase CaMK?,Bcl-2 and Bax gene and protein expression level.Through above experiments,the molecular mechanism of brain function damage caused by drinking water fluoride exposure was explored,the role of L-type calcium channel in brain damage caused by drinking water fluorosis was preliminarily,and the theory of fluorine-induced"calcium contradiction"was further improved,New ways and methods to treat fluorosis were explored.The research results are as follows:?1?The blood/urine fluoride content measurement showed that compared with the control?C?group,the urinary fluoride content of the fluoride group at 3 months and 6 months was significantly increased?P<0.05?.In addition,the LF group has a lighter dental fluorosis.The symptoms of dental fluorosis in the HF group were significant.According to above indicators,It is considered that the fluorosis model in this study was successfully replicated.?2?The results of behavior test showed that,compared with group C,the number of running grid and standing times of mice exposed to different concentrations of fluorine at 3 months and 6 months showed a decreasing trend,the number of escape latency significantly decreased?P<0.05?,the number of crossing the platform significantly decreased,indicating that the space exploration and learning and memory ability of fluorine-poisoned mice decreased.?3?The results of brain tissue-related enzyme test showed that compared with group C,GSH-Px in brain tissue of mice exposed to fluoride at 3 months and 6months was lower than that of the control group.SOD activity and MDA content were significantly increased?P<0.05?.The MDA content in the HF+NIF group at 6 months was significantly lower than that in the 3 months old group?P<0.05?.?4?The morphology and apoptosis of nerve cells in the hippocampal CA₁ area showed that compared with the C group,the morphology of the cells in the hippocampus of the hippocampus of the mice exposed to fluoride for 3 months and 6months was changed.The apoptotic rate of hippocampal CA₁ region was significantly increased in the fluoride-treated group at different concentrations of 3 months and 6months?P<0.05?.The apoptosis rate of hippocampal CA₁ region in the antagonist group was significantly lower than that in the agonist group?P<0.05?,but higher than the control group.The apoptotic rate of hippocampal CA₁ area in the same concentration of fluoride treatment group was significantly increased at 6 months after fluoride exposure?P<0.01?.?5?PCR and Western blot analysis showed that compared with group C,the expression of Cav1.2 gene in hippocampus of HF and LF groups was significantly increased at 3 months and 6 months of age?P<0.05?,but the protein expression level was significant.decline.The expression levels of CaM gene at 3 months and 6 months were significantly decreased?P<0.05?,but the expression level of protein was increased,and the reason for the inconsistent expression level needs further clarification.At 3 months of age,CaMKII gene and protein expression levels decreased,while 6 months of age significantly increased.The expression of Bax gene and protein and the ratio of Bax/Bcl-2 gene and protein expression at 3 months and 6months were significantly increased?P<0.05?,while the expression of Bcl-2 gene was significantly decreased?P<0.05?.Compared with the same concentration of fluorine,the expression level of Cav1.2 gene and protein in the agonist group at 3 months was significantly decreased?P<0.05?,and the antagonist group had a decreasing trend.The expression levels of CaM genes and proteins increased at 3 months and 6 months,and the antagonist group showed a downward trend.The expression levels of CaMKII gene and protein in the agonist group and the antagonist group at 3 months of age decreased,but there was no statistical significance at 6 months.There was no significant difference in Bax gene and protein expression between the agonist group and the antagonist group at 3months?P>0.05?,but there was an upward trend in the 6-month-old agonist group and a downward trend in the antagonist group.The expression levels of Bcl-2 gene and protein in the agonist group at 3 months and 6 months were all decreased,and the antagonist group had an increasing trend.The ratio of Bax/Bcl-2 gene and protein expression levels in the agonist group at 3 months and 6 months was significantly increased?P<0.05?,and the antagonist group showed a downward trend.Based on the successful replication of drinking water fluorosis model,the results obtained in this study showed that the learning and memory,autonomous behavior and inquiry ability of mice with subchronic and chronic fluorosis were decreased,the antioxidant capacity of brain tissue was decreased,and the number of apoptotic cells in the hippocampus CA₁ region was increased.It may be related to the abnormal expression of Cav1.2 protein and apoptosis-related molecules bcl-2 and Bax protein,as well as the increased expression ratio of Bax/bcl-2,in the intracranial manna calcium channel,and the positive correlation between the expression level of the above molecules and the dose and time of fluorine exposure in the hippocampal nerve cells.It is suggested that the Bcl-2 and Bax apoptosis pathways in the hippocampus may be one of the molecular mechanisms of fluoride-induced behavioral changes and neuronal apoptosis in mice.In addition,the Ca²⁺channel agonist FPL64176 can further aggravate the damage of learning and memory in mice induced by fluoride,and abnormal expression of calcium channel Cav1.2 and downstream CaMKII,CaM and Bcl-2,Bax genes and proteins in hippocampus.The degree of deepening showed synergistic toxicity,while the calcium antagonist Nifedipine reversed the above behavior and molecular abnormality of fluorosis in mice,suggesting that the calcium antagonist Nifedipine may be a new effective anti-fluoride drug.