The Role Of L-Type Calcium Channel In Fluoride In Duced PC12 Cell Injury And Its Mechanism

Fluorine(F)is a typical negative electricity,the most active non-metallic element,which has a strong oxidizing power,that can be directly or indirectly with almost all other elements to form the corresponding fluoride,mainly inorganic fluoride form exists in nature.Fluorine is present in almost all aspects of human living environment,but the main way the body's intake of fluoride is drinking water,food and air.Long-term intake of fluoride,will lead to excessive accumulation of fluorine in the body,causing damage to the body,which known as endemic fluorosis,referred to as fluoride,the body of bone and non-organoid organs have obvious damaged,but the specific mechanism remains unclear.In recent years,the study of calcium contradiction disease has gradually become a hot spot,which has aroused widespread concern in the study of the pathogenesis of fluoride disease.As a group of protein macromolecules that cross the cell membrane,calcium channels strictly control the process of calcium ions entering the cells.Changes in calcium channel activity may be one of the initiating mechanisms of neuronal apoptosis,so studying the steady state changes of calcium in nerve cells under the action of fluorine is very necessary in the study of the pathogenesis of fluorosis.L-type calcium channel is the main factor that causes calcium influx.It regulates calcium level in the cytoplasm and participates in nerve cells calcium homeostasis,synaptic plasticity,learning and memory and other aspects.PC 12 cells derived from rat adrenal medullary pheochromocytoma proliferate and differentiate into sympathetic neurons under the induction of nerve growthfactor(NGF),and are widely used in vitro studies of nervous system diseases.In this study,PC 12 cells exposed to high fluoride(5.00mM)/low fluoride(0.5mM)were treated with high fluoride/low fluoride combined L-type calcium channel agonist FPL64176(0.5umol/L)/antagonist Nifedipine(0.2umol/(Control group,high fluoride group,low fluoride group,high fluoride + agonist group,high fluoride + antagonist group,low fluoride + agonist group,low fluoride + antagonist group).After exposure,cell viability was detected by cck-8 staining at different time points(2,4,6,8,10,12 and 24 h);After exposed to fluoride for 2h,the level of reactive oxygen species(ROS)in cells was detected by DCF;the apoptosis status was detected by Hoechst-33342;the morphologicalchanges were observedby invertedmicroscope 24h after exposure to fluorine;Expression of mRNA/proteins Cav1.2 and CAMK?,c-fos,Bax and bcl-2.This study has great significance to explore the role and mechanism of L-type calcium channels in the damage of PC 12 cells induced by fluoride,which is great scientific value and clinical significance for further exploring the pathogenesis and prevention of low-fluorine disease.After 2/4/6/8/10/24h of fluoride staining,the survival rate of cells in the HF group was significantly(P<0.05)or extremely significantly lower(P<0.01)than that of the control group;the fluorine staining was 2/4/6/After 8/10 hours,the survival rate of HF+N cells increased significantly(P<0.05).PC12 cells showed a long fusiform shape.After 24 hours of exposure to fluorine,the cell density in the H F group decreased,some cells began to shrink and round,and the number of dead cells increased.Some could even see cell debris;HF+N group The number of dead cells was significantly smaller than that of the HF group.In the fluorosis group,intracellular calcium ion/intracellular reactive oxygen species/apoptosis level was significantly(P<0.05)or highly significant(P<0.0 1)increased,and intracellular calcium/intracellular activity in the HF+P/LF+P group Oxygen increased significantly(P<0.05)or significantly increased(P<0.0 1),and the level of intracellular calcium/intracellular reactive oxygen species in the H F+N/L F+N group decreased significantly(P<0.05)or significantly decreased.(P<0.0 1).L-type calcium ion channels and downstream related gene/protein expression test results showed that the expression levels of the Cavl.2 gene/CamKII gene in the HF group were significantly lower than those in the control group(P<0.01).The expression level of gene and Bax/bcl-2 gene/protein ratio increased significantly(P<0.01),and the expression level of Cavl.2 gene in H F+P group was significantly lower than that in H F group(P<0.01).The expression levels of Cav1.2,C AMKII,bcl-2 and Bax protein in F+N/LF+ group N increased significantly(P<0.0 1),and the cf os and Bax/b c1-2 ratio protein expression levels in water The level dropped significantly(P<0.0 1).In summary,fluoride can reduce the cell survival rate,within a certain period of exposure,the level of intracellular reactive oxygen species,intracellular calcium levels,apoptosis increased,which are in a dose-dependent manner,suggesting that fluoride exposure can cause increased cellular oxidative stress;FPL64176,an L-type calcium ion channel agonist,can exacerbate fluorosis to a certain extent.Nifedipine,an antagonist of L-type calcium channel,can improve fluorosis to a certain extent and the expression of Cavl.2 protein shows regular changes,suggesting Cavl.2 molecules may be an important molecular target in the treatment of fluorosis.The L-type cal cium channel inhibitor nifedipine may be one of the effective anti-fluorosis drugs.