| Objective:Moderate amount of fluoride promotes calcium and phosphorus metabolism and bone development.Long-term ingestion of excessive fluoride leads to fluorosis,which damages the normal function of various tissues and organs.Studies have found that fluorosis patients have abnormal lipid metabolism,and some scholars have found that sodium fluoride induces apoptosis of H9c2 cardiomyocytes through AMPK signaling pathway and mitochondrial pathway,but whether fluoride regulates energy metabolism of myocardial tissue by affecting AMPK/ACC/CPT1 signaling pathway has not been reported in the literature.In conclusion,in this study,the effects of fluoride on fatty acidβoxidation,glucose oxidation and mitochondrial oxidative phosphorylation marker were detected by establishing a fluorosis rat model and using H9c2 myocardial cell line.The role of AMPK/ACC/CPT1 signaling pathway was also discussed.This study provides new clues to elucidate the pathogenesis of myocardial damage caused by fluorosis.Methods:Thirty 6-week-old SD male rats were selected to establish a fluorosis model.The heart weight and body weight of rats were measured,and the organ index of each group was calculated.The contents of blood glucose,triglyceride,cholesterol,high density lipoprotein and low density lipoprotein in each group were detected.HE staining was used to observe the changes of myocardial tissue morphology and structure induced by fluoride in each group.The protein expression levels of fatty acidβoxidation,glucose oxidatio,mitochondrial oxidative phosphorylation marker and apoptotic protein were detected by Western Blot.The effect of fluoride on ATP content of H9c2 cardiomyocytes was tested in vitro.Western Blot and RT-PCR were used to detect the protein expression and m RNA levels of the above proteins.Results:1.Blood fluorine and urine fluorine contents of rats in each group:compared with control group,blood fluorine and urine fluorine contents of rats in 50 ppm and100 ppm fluoride groups were significantly increased(P<0.05).2.Effects of fluoride on myocardial tissue of rats:compared with the control group,there was no statistical difference in heart weight,body weight and organ index of rats in 50 ppm and 100ppm fluoride groups.3.Effects of fluoride on blood glucose and lipid related biochemical indexes in rats:compared with the control group,levels of blood glucose,triglyceride,cholesterol,high density lipoprotein and low density lipoprotein in rats in50 ppm and 100 ppm fluoride groups were significantly increased(P<0.05).4.The effect of fluoride on the morphology and structure of rat myocardial tissue:the myocardial fibers in the control group were neatly arranged and the intertissue space was small.In 50 ppm F~-and 100 ppm F~-groups,the arrangement of myocardial fibers was gradually disordered,the nuclei scattered in the interstitial space,the width of myocardial space gradually increased.5.Effects of fluoride on fatty acidβ-oxidation related proteins in rat myocardium:compared with control group,CD36 protein expression in myocardium of rats exposed to fluoride was significantly increased(P<0.05).Compared with control group,the expression levels of AMPK and ACC phosphorylated protein in 50 ppm F~-and 100 ppm F~-groups were significantly increased(P<0.05).The protein expression level of MCD in 100 ppm F~-group was significantly increased compared with control group(P<0.05).Compared with the control group,the protein expression levels of CPT1 and LCAD in 50 ppm F~-and 100ppm F~-groups were significantly increased(P<0.05).6,The effect of fluoride on the expression of Glut4,PDH and PDK4 protein in rat myocardial tissue:compared with the control group,100 ppm F~-group Glut4,PDH and PDK4 protein expression levels were significantly increased(P<0.05).7.The effect of fluoride on the protein of mitochondrial respiratory chain complexⅠNDUFAF1 in rat myocardial tissue:compared with the control group,the expression of NDUFAF1 protein in 100 ppm F~-group was significantly decreased(P<0.05).8.The effect of fluoride on the protein expression of apoptosis-related proteins in rat myocardial tissue:compared with the control group,the expression of anti-apoptosis-related protein Bcl-2 in 50 ppm F~-and100 ppm F~-groups had no significant change.The protein expression of proapoptotic factor Bax in 100 ppm F~-group was significantly increased compared with that in control group(P<0.05).9.The effect of fluoride on the survival rate of H9c2cardiomyocytes:H9c2 cells were treated with 0,6,12,24 and 48 mg/L Na F for 48 h.Compared with the control group,the cell survival rate of 6 and 12 mg/L Na F groups had no significant change,while the cell survival rate of 24 and 48 mg/L Na F groups was significantly decreased(P<0.05).10.The effect of fluoride on ATP content in H9c2 cardiomyocytes:Compared with the control group,ATP content in 9c2cardiomyocytes in 12 and 24 mg/L Na F groups was significantly decreased(P<0.05).11.The effect of fluoride on the expression of fatty acidβoxidation related proteins in H9c2 cardiomyocytes:Compared with the control group,the m RNA and protein expression levels of CD36 in 24 mg/L Na F group were significantly increased(P<0.05).Compared with the control group,m RNA and protein expression levels of AMPK in 6,12 and 24 mg/L Na F groups were significantly increased(P<0.05).Compared with the control group,ACC m RNA expression level in the 6 and 12mg/L Na F groups was increased,but significantly decreased in the 24 mg/L Na F group(P<0.05).Compared with control group,ACC protein expression level in 24 mg/L Na F group was significantly increased(P<0.05).Compared with the control group,m RNA and protein expression levels of MCD,CPT1 and LCAD in 24 mg/L Na F group were significantly increased(P<0.05).12.The effect of fluoride on the protein expression of PDH and PDK4 in H9c2 cardiomyocytes:Compared with the control group,the m RNA and protein expression levels of PDH in 24 mg/L Na F group were significantly increased(P<0.05).The protein expression of PDK4 in 12 and 24 mg/L Na F groups was significantly increased compared with control group(P<0.05).13.The effect of fluoride on the protein of H9c2 myocardial mitochondrial respiratory chain complexⅠNDUFAF1:Compared with the control group,the expression of NDUFAF1 protein in 24 mg/L Na F group was significantly decreased(P<0.05).14.The effect of fluoride on the apoptosis rate of H9c2 cardiomyocytes:compared with the control group,the apoptosis rate of H9c2 cells treated with Na F for 48h was significantly decreased in the 6 mg/L Na F group(P<0.05).Compared with the control group,the apoptosis rate in 12 and 24 mg/L Na F groups was significantly increased(P<0.05).15.Effects of fluoride on H9c2 cardiomyocyte apoptosis-related proteins:Compared with the control group,Bax m RNA and protein expression levels in 12 and 24 mg/L Na F groups were significantly increased(P<0.05).The m RNA and protein expression levels of anti-apoptotic protein Bcl-2 in 24 mg/L Na F group were significantly decreased compared with the control group(P<0.05).Compared with control group,m RNA and protein expression levels of Caspase-3 in 24 mg/L Na F group were significantly increased(P<0.05).Conclusion:1.Fluorine increases the intake of free fatty acids in cardiac tissue,activates AMPK/ACC/CPT1 signal pathway,and improves fatty acidsβOxidation.2.Fluorine leads to the disorder of energy metabolism in myocardial tissue,the damage of oxidative phosphorylation of mitochondria,and the reduction of ATP production. |
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