| Fluorine is one of the trace elements related to human health.A proper intake of fluorine is beneficial to the development of body and to maintain the normal structure and physiological functions of bones and teeth.Once ingested in excess,it will enter the whole body through blood circulation,causing acute or chronic fluorosis effects,known as Endemic Fluorosis(or Fluorosis).The pathogenic mechanism of endemic fluorosis is complex,involving oxidative stress,mitochondrial changes,endoplasmic reticulum stress,signal pathways changes and so on.Studies have shown that the abnormal apoptosis caused by fluoride exposure may also be one of the mechanisms of fluoride-induced cells damage.Apoptosis is a kind of programmed death regulated by genes.Eukaryotic cells mainly mediate apoptosis through internal mitochondrial pathway,endoplasmic reticulum pathway and external death receptor pathway.Our previous studies suggested that endoplasmic reticulum and mitochondrial pathways are involved in the abnormal apoptosis induced by fluoride exposure,but the death receptor pathway of apoptosis induced by fluoride exposure is rarely reported.Fas /Fas L signaling pathway is one of the main death receptor pathways and plays an important role in the process of apoptosis.In many apoptotic processes,concentration of Ca2+ is often increased abnormally,especially in the early stage of apoptosis,which suggests that Ca2+ may be one of the early signals to initiate apoptosis.The phenomenon of intracellular calcium overload was detected in the animal model of fluorosis and in vitro cytotoxicity test,and it was confirmed that it was closely related to the increase of ROS level and apoptosis.In vivo electrophysiological studies have shown that intracellular calcium overload may be caused by the change of L-typecalcium channel activity on the cell membrane after fluoride exposure.L-type calcium channel blocker is the blocker of Ca2+ entering cells,which can effectively reduce the concentration of Ca2+ in cells and inhibit the signal of apoptosis initiation.However,whether L-type calcium channel antagonists can interfere with fluoride induced Fas /Fas L signaling pathway dependent apoptosis and its protective mechanism are rarely reported.The purpose of this study was to investigate the role of Fas/FasL signaling pathway in fluoride induced apoptosis of PC12 cells and its molecular mechanism,and to explore the intervention effect of Nifedipine in the above process.1.The effects of Fas/FasL apoptosis pathway in fluoride induced PC12 cell injuryIn order to investigate the role and molecular mechanism of Fas/FasL signaling pathway in PC12 cell apoptosis induced by fluorine,the PC12 cells were treated with20,40,80,160 mg / L Na F medium for 12,24,36,and 48 hours.Detect cell viability;after treating PC12 cells for 24 hours with Na F culture medium of different doses described above,intracellular reactive oxygen species level,apoptosis rate,intracellular Fas/FasL signal transduction pathway Fas,Fas L,FADD,Caspase 8,Caspase 3 and Bid gene / protein expression levels.Results:(1)Result of cell viability: After 12,24,and 36 hours of Na F exposure,the cell viability of each group showed a downward trend and was dose-dependent in fluoride exposure.The cell viability of PC12 cells in 160 F group were significantly decreased(P < 0.01),and it in 80 F group decreased significantly(P < 0.01)after fluoride exposure for 24 hours.The dose-survival curve of the cells in each group experienced a first significantly increasde(P < 0.01),and then significantly declined(P < 0.01)after fluoride exposure for 48 hours.(2)Result of ROS level: After 24 h of different doses of fluoride exposure,compared with the control group,the level of ROS in 40 F group increased significantly(P < 0.05),and it in 80 F and 160 F groups increased significantly(P <0.01).The level of ROS in PC12 cells in each group was fluoride dose-dependent.(3)Result of apoptosis rate: The rate of early and late apoptotic cells in each group were significantly increased when compared with the control group(P < 0.01).The higher the sodium fluoride dose,the higher the apoptosis rate(the sum of theearly and late apoptosis rates).(4)Expression of Fas/FasL signal pathway related molecular genes / proteins:Compared with the control group,the expression of Fas,Fas L,FADD,Caspase 8,Caspase 3 genes and proteins in PC12 cells were increasing(P < 0.05)after fluoride exposure for 24 hours,which was dependent on the fluoride exposure dose.While the expression of bid genes and proteins in each fluoride exposure group decreased gradually,especially in 80 F and 160 F groups(P < 0.05).In conclusion,fluoride exposure increased the level of ROS in PC12 cells,increased the expression level of Fas/FasL signaling pathway(P < 0.05),decreased the expression level of bid(P < 0.05),induced abnormal apoptosis of PC12 cells,and finally affected the cell viability,and the above indicators were dose-dependent.The results suggest that Fas/FasL signaling pathway plays an important role in the apoptosis of PC12 cells induced by fluoride exposure,and FADD may be an important target molecule in Fas/FasL signaling pathway.2.The effect of L-type calcium channel antagonists on the apoptosis of PC12 cells induced by fluorideTo investigate the effect of L-type calcium channel antagonist Nifedipine on the apoptosis of PC12 cells induced by fluorine through the Fas/FasL signaling pathway,an experimental dose-effective 80 mg/L Na F and 0.5,1.0,2.0,4.0 ?M Nifedipine were used to treat PC12 cells for 24 hours.The levels of reactive oxygen species and calcium ions,apoptosis rate,cell membrane L-type calcium channel Cav1.2 and Fas /Fas L signaling pathway related genes / proteins expression,and cell cycle distribution were detected in PC12 cells.Results:(1)Result of ROS level: Compared with the control group,ROS level in F and F+4.0N groups increased significantly(P < 0.01).Compared with the F group,the level of ROS in each combined treatment group decreased significantly(P < 0.01).ROS in different concentrations of Nifedipine treatment group showed a trend of first decreased and then increased.(2)Concentration of Ca2+ in cells: The intracellular Ca2+ level in each treatment group was significantly higher than that in the control group(P < 0.05).Comparedwith the F group,the intracellular Ca2+ concentration in F+1.0N group and F+2.0N group was significantly decreased(P < 0.01).Although there was no significant difference at F+0.5N group and F+4.0N group,there was a downward trend.(3)Result of apoptosis rate: Compared with the control group,the rate of early and late apoptotic cells in each group increased significantly(P < 0.01).Compared with the F group,the rate of apoptotic cells in F+0.5N group,F+1.0N group and F+2.0N group was drug-dependent.The rate of apoptotic cells in the F+4.0n group showed an upward trend.The rate of apoptosis decreased first and then increased.(4)Gene / protein expression results: Compared with the control group,the expression levels of Cav1.2 and Fas/FasL signaling pathway-related genes / proteins in F group and F+0.5N group were significantly increased(P < 0.05)or showed an upward trend.The expression levels of Bid genes and proteins in each treatment group were significantly reduced(P < 0.05).Compared with F group,the expression levels of Cav1.2 and Fas/FasL signaling pathway-related genes / proteins in F+1.0N group and F+2.0N group were significant decreased(P < 0.05).The expression levels of Bid genes and proteins in F+2.0N group increased significantly(P <0.01).(5)Result of cell cycle: Compared with the control group,the number of G1 phase cells in F group and F+4.0N group increased significantly(P < 0.01),and S phase cells decreased significantly(P < 0.01).Compared with F group,G1 phase cells in F+0.5N/ 1.0N/ 2.0N groups decreased significantly(P < 0.01),S phase cells increased significantly(P < 0.01),G2 phase cells increased significantly(P < 0.05)or showed an increasing trend.While G1 phase cells increased significantly(P < 0.01)and S phase cells decreased significantly(P < 0.01)in F + 4.0N group.In conclusion,Nifedipine,an L-type calcium channel antagonist,can effectively regulate the abnormal apoptosis of PC12 cells by inhibiting excessive ROS production,intracellular calcium overload,Fas/FasL signal pathway activation and cell cycle.It's suggested that Nifedipine may be a new and effective drug. |
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