Molecular Mechanism Of Hippocampal Calcium Homeostasis In Fluorosis Mouse

Fluorine?F?is an essential trace element in the human body.Excessive fluorine accumulation causes systemic physiological and pathological changes in the body.It is called endemic fluorosis,referred to as fluorosis.Dental fluorosis and skeletal fluorosis are the main symptoms of bone-phase organ damage caused by fluorosis.Fluorosis can also cause damage to non-osseous organs such as the brain and kidney.Studies have shown that excessive fluoride intake can accumulate in the brain tissue through the blood-brain barrier and affect the physiological functions of the central nervous system.In recent years,the effects of fluorosis on the nervous system have become a hotspot in the research of fluorosis.Calcium is an important"second messenger"in the cells.Calcium homeostasis is the key to the cell's Ca²⁺signal transduction and completes a series of physiological functions.However,the molecular mechanism of calcium homeostasis in the hippocampus caused by fluoride has not been reported.This experiment is divided into two parts:subchronic fluoride exposure and chronic fluoride exposure.There are 120 newly weaned ICR male mice for each part.After 7 days of adaptation,they were randomly divided into 6 groups:Control group?C?:drinking tap water?fluorine content<0.2 mg/L?,eating common feed?calcium content 0.8%?;Fluoride group?F?:drinking 100 mg/L NaF water,eating common feed;High calcium group?HCa?:drinking tap water,eating high calcium feed?calcium content 2%?;Low calcium group?LCa?:drinking tap water,eating low calcium feed?calcium content 0.063%?;fluoride+high calcium group?F+HCa?:drinking 100 mg/L NaF water,eating high calcium feed;Fluoride+low calcium group?F+LCa?:drinking 100 mg/L NaF water,eating low calcium feed.After the fluoride exposure,the mice were measured for dental fluorosis,and their blood/urine fluoride and blood/urine calcium contents.Based on the successful replication of the fluorosis model,observe the morphological structure and apoptosis rate of CA₁ region of hippocampus;Fluo-4 fluorescent probe load was used to detect the intracellular Ca2+level in hippocampal;the hippocampus cell membrane was detected according to the kit method Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities;Western Blot,RT-PCR and other methods were used to detect the mouse hippocampus cell membrane L-type calcium channel Cav1.2,plasma membrane Ca²⁺pump PMCA,Na⁺-Ca²⁺exchanger NCS-1,Endoplasmic reticulum inositol triphosphate receptor IP3R and calcium pump ATP2A2/SERCA2 gene/protein expression levels.It is planned to systematically investigate the effects of different dietary calcium levels on fluoride-induced changes in hippocampus calcium homeostasis and molecular mechanisms for the first time,and provide basic data for exploring economically effective intervention pathways for fluoride-induced neurotoxicity and improving the theory of"fluorine-induced calcium paradoxical disease."The findings are as follows:?1?Replication of fluorosis modelCompared with group C,the symptoms of fluorosis were obvious and the fluoride content in urine increased significantly in each of the fluoride-treated groups of subchronic fluoride exposure and chronic fluorine exposure?P<0.05?,indicating that the mice of this study successfully replicated the model of chronic chronic fluoride exposure and chronic fluorine exposure..?2?Observation of morphology and structure of mouse hippocampusThe cell morphology of hippocampal CA1 region of mouse in subchronic/chronic fluoride exposure C group was regular,the cells were normal,the boundaries were clear,the structure was tight,and the layers were obvious.The mouse of F group was changed,and the intercellular space was wide and loosely arranged.Compared with the F group,the F+HCa group has a tighter cell arrangement and clear cell stratification,while the F+LCa group has lighter staining,a significantly reduced number of cells and a wider gap.?3?Results of apoptosis detection in mouse hippocampus CA1 regionCompared with C group,the number of apoptotic cells in subchronic/chronic fluoride exposure treatment groups in CA₁ region was significantly increased?P<0.01?.Compared with F group,the number of apoptotic cells in F+HCa group in the CA₁ region was significantly reduced?P<0.01?,while F+LCa group was significantly increased?P<0.01?.Compared with subchronic fluoride exposure,the number of apoptotic cells in CA₁ region in chronic fluoride exposure F group and F+LCa group significantly increased?P<0.05?.?4?Measurement results of intracellular Ca2+levels in mouse hippocampusCompared with C group,the levels of Ca²⁺in hippocampal in subchronic/chronic fluorine exposure were significantly increased?P<0.05 or P<0.01?.Compared with F group,the levels of Ca2+in the F+HCa group were significantly reduced?P<0.05 or P<0.01?,and the F+LCa group were significantly increased?P<0.05?.Compared with subchronic fluoride exposure,the F+HCa group,F+LCa group and F group in chronic fluoride exposure were significantly increased?P<0.05 or P<0.01?.?5?Detection results of Ca2+transport related molecules in mouse hippocampal cellsa.Detection results of related molecules of transmembrane intake of Ca2+Compared with C group,the expression level of Cav1.2 gene/protein was significantly increased?P<0.05 or P<0.01?,the ATP2A2/SERCA2 gene/protein was significantly reduced?P<0.05 or P<0.01?in hippocampal cell membrane of each fluoride-treated group.Compared with the F group,in the F+HCa group Cav1.2gene/protein expression level was significantly reduced?P<0.05 or P<0.01?,the expression level of ATP2A2/SERCA2 gene/protein was significantly increased?P<0.05 or P<0.01?;in the F+LCa group the expression level of Cav1.2 gene/protein was significantly increased?P<0.05 or P<0.01?and ATP2A2/SERCA2 gene/protein expression levels was significantly reduced?P<0.05 or P<0.01?.Compared with subchronic fluoride exposure,the expression level of Cav1.2 gene/protein in the chronic fluorine exposure F group,F+HCa group and F+LCa group was significantly increased?P<0.05 or P<0.01?,and the ATP2A2/SERCA2 gene/protein expression levels were significantly reduced?P<0.05 or P<0.01?.b.Results of Ca²⁺transmembrane release-related indicatorsThe activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase in the hippocampus tissue membrane of mice showed that compared with C group,the activities of Na⁺-K⁺-ATPase and Ca²⁺-ATPase in each treatment group were or significantly reduced?P<0.05 or P<0.01?;compared with the F group,the Na⁺-K⁺-ATPase and Ca²⁺-ATPase activities of the F+HCa group were significantly increased?P<0.05?,and while the F+LCa group were significantly reduced?P<0.05?;compared with the subchronic fluorine exposure,the Na⁺-K⁺-ATPase activity in the chronic fluorine exposure F group and F+LCa group was significantly reduced?P<0.05?.Compared with C group,the expression level of IP3R gene/protein in the endoplasmic reticulum membrane of hippocampus cells was significantly increased?P<0.05 or P<0.01?,the expression level of NCS-1,PMCA gene/protein was significantly reduced?P<0.05 or P<0.01?in the fluoride-treated group.Compared with the F group,the IP3R gene/protein expression level was significantly reduced?P<0.05 or P<0.01?,while the expression level of NCS-1,PMCA gene/protein was significantly increased in the F+HCa group?P<0.05 or P<0.01?;the expression level of IP3R gene/protein was significantly increased?P<0.05 or P<0.01?,NCS-1.PMCA gene/protein expression level was significantly reduced in the F+LCa group?P<0.05 or P<0.01?.Compared with subchronic fluoride exposure,the expression level of IP3R gene/protein in chronic fluorine exposure F group,F+HCa group and F+LCa group was significantly increased?P<0.05 or P<0.01?,NCS-1,PMCA gene/protein expression level was significantly reduced?P<0.05 or P<0.01?.To conclusion,the level of Cav1.2 gene/protein expression of L-type calcium ion channel in brain hippocampus cells caused by fluorosis,and the influx of extracellular Ca²⁺increased;meanwhile,the activity of Na⁺-K⁺-ATPase and Ca2+-ATPase was increased.The expression level of NCS-1 and the plasma membrane Ca²⁺pump PMCA gene/protein are decreased,so that the reverse Ca²⁺concentration in the cell is prevented from moving outside the plasma membrane;in the hippocampus cells,the IP3R gene/protein expression level was increased,and ATP2A2/SERCA2 gene/protein expression levels was decreased,increased Ca2+release from the endoplasmic reticulum of the main calcium pool in the cell,and hindered the reverse endoplasmic reticulum concentration from uptake of Ca2+from the cytosol.The above-mentioned multiple factors cause Ca²⁺overload in the cytoplasm,cause imbalance of Ca²⁺homeostasis in hippocampus cells,trigger abnormal expression of calcium signaling pathways and downstream apoptosis regulation-related molecules,induce abnormal apoptosis of hippocampus cells,and eventually damage brain hippocampus cells.The cerebral apoptotic rate was positively correlated with the fluoride exposure time.And dietary high calcium?2%?can reverse the abnormal expression of Ca2+transport molecules in the hippocampus cell membrane or endoplasmic reticulum membrane caused by the fluorine exposure to a certain extent,inhibit Ca²⁺overload in the cell,maintain calcium homeostasis,and reduce the rate of apoptosis.Dietary high calcium may be a cost-effective means of resisting fluoride.