Fluoride Inhibits Glycolysis Via The PHD2/HIF1α Signaling Pathway In SW1353 Cells

Objective:Fluoride is an essential trace element for human growth and development.Fluoride has dual effects on the body.Appropriate intake of fluoride can enhance the ability of anti caries of teeth and promote the absorption of calcium and phosphorus in bones.Long term excessive intake of fluoride will lead to fluorosis.Because fluoride has a strong affinity for bone,the most representative symptom of fluorosis is skeletal fluorosis,and cartilage damage is the basic manifestation of the disease.Chondrocyte is an important component of growth plate cartilage.Maintaining the balance of chondrocyte anabolism and catabolism is the basic condition of cartilage growth and development.Glucose is an important metabolic material for all living cells.In chondrocytes,glucose not only provides energy source for chondrocytes,but also is an important structural premise for chondrocytes to synthesize proteoglycans,which is very important for the synthesis of extracellular matrix.Because growth plate cartilage is a kind of non vascular connective tissue,its oxygen content is very low,chondrocytes mainly provide energy by glycolysis.Hypoxia inducible factor HIF1αcan sense and adapt to hypoxic environment,and is a key factor in regulating glycolysis.However,it has not been reported whether fluoride regulates the glycolysis of chondrocytes through the PHD2/HIF1αsignaling pathway,thereby changing the homeostasis of anabolism and catabolism.Therefore,we detected the effect of fluoride on HIF1αexpression by tibial culture in vitro,and took SW1353 cells as the research object to observe whether fluoride can change the glycolysis of SW1353 cells by regulating the expression of related factors of PHD2/HIF1αsignaling pathway,and then affect the balance of anabolism and catabolism.Method:In this study,the tibia of fetal rats on the 20th day of embryo were selected and divided into control group and 10^-4M Na F group.The tibias were cultured inα-MEM medium containing 0.2%bovine serum albumin,0.05 mg/ml VC and 1 m Mβ-Sodium glycerophosphate.After 7 days,the tibia was fixed,paraffin embedded and sectioned,and the expression of HIF1αprotein was detected by immunohistochemistry.SW1353 cells were cultured in L-15 medium containing 10%fetal bovine serum,100μ/ml penicillin and 100μg/ml streptomycin.The activity of SW1353 cells was detected by CCK8 to determine the time and dose of fluoride staining;the synthesis of proteoglycan was detected by alcian blue staining;the production of ATP and lactic acid was detected by ATP and lactic acid detection kit;glucose consumption was detected by GOD-POD method;HIF1αexpression and cell localization were detected by cell immunofluorescence method;real-time fluorescence quantitative PCR and Western blot were used The m RNA and protein expression of Aggrecan,Col2a1,Col10a1,MMP13,HIF1αsignaling pathway（PHD2,HIF1α）and glycolysis related factors（Glut1,HK2,PKM,ENO1,LDHA,MCT4）were detected by blot;the expression of HIF1αwas up-regulated by Co Cl₂,then the related factors were detected.Results:1.Fluoride could reduce the expression of HIF1αin tibial chondrocytes in vitro:the expression of HIF1αin tibial chondrocytes of fluoride group was significantly lower than that of control group（P<0.05）.2.Effect of fluoride on SW1353 cell viability:SW1353 cells treated with 5×10^-4M Na F showed a significant decrease in cell viability,especially at 72 hours（P<0.001）.3.Effect of fluoride on the anabolism of SW1353cells:Alcian blue staining was used to detect the effect of fluoride on the synthesis of proteoglycan in SW1353 cells.The results showed that the intensity of Alcian blue staining in the fluoride group was weaker than that in the control group.The quantitative analysis of Alcian blue dissolved with guanidine hydrochloride showed that the absorbance value of the fluoride group was significantly lower than that in the control group（P<0.05）;the expression of anabolism related factors was detected,Fluoride treatment significantly inhibited the expression of Aggrecan,Col2a1 and Col10a1,（P<0.05）.4.Effect of fluoride on the catabolism of SW1353 cells:compared with the control group,fluoride promoted the expression of MMP13,the difference was statistically significant（P<0.05）.5.Effect of fluoride on glycolysis:the production of ATP,lactic acid and glucose consumption in the fluoride group were significantly lower than those in the control group（P<0.05）.6.Effects of fluoride on PHD2/HIF1αsignaling pathway and glycolysis related factors:compared with the control group,the signal of immunofluorescence staining was weaker in the fluoride group;the expression of PHD2 was up-regulated and the expression of HIF1αwas down regulated by fluoride at both m RNA and protein levels（P<0.05）;the expression of glycolysis related factors Glut1,HK2,PKM,ENO1,LDHA and MCT4 were decreased in the fluoride group（P<0.05）.7.Co Cl₂upregulated the expression of HIF1αand its downstream factor Glut1 by inhibiting the expression of PHD2,The expression of HIF1αand Glut1 was lower than that of the control group;after Co Cl₂ was added to the control group,the expression of PHD2 was inhibited,and the expression of HIF1αand its downstream target gene Glut1 was significantly up-regulated;after Co Cl₂ was added to the fluoride group,the expression of PHD2 was significantly lower than that of the fluoride group,and the expression of HIF1αand Glut1 was significantly increased（P<0.05）.8.Fluoride can reduce the glycolysis of SW1353 cells by inhibiting HIF1αin the fluoride group,the production of ATP,lactate and glucose consumption were lower than those in the control group,and the expression of glycolysis related factors were decreased;in the control group,the production of ATP,lactic acid and glucose consumption were increased,and the expression of glycolysis related factors was up-regulated after adding Co Cl₂;in the fluoride group,the production of ATP,lactic acid and glucose consumption were higher than those in the fluoride group ATP production,lactate production,glucose consumption and expression of glycolysis related factors were increased.9.Fluoride can reduce the anabolism and accelerate the catabolism of SW1353 cells by inhibiting HIF1αcompared with the control group,the expression of Col2a1 and Col10a1 in the fluoride group decreased,and the expression of MMP13increased.Compared with the control group,Co Cl₂ can up regulate the expression of Col2a1 and Col10a1,and down regulate the expression of MMP13.Compared with the fluoride group,the expression of Col2a1 and Col10a1 in the fluoride group was increased,and the expression of MMP13 was decreased.Conclusions:1.Fluoride inhibit the anabolism of SW1353 cells,accelerate its catabolism,and destroy the balance of anabolism and catabolism.2.Fluoride down regulates HIF1αand down regulates its downstream target gene by increasing the expression of PHD2.3.Fluoride can reduce the glycolysis of SW1353 cells through PHD2/HIF1αsignaling pathway,thus affecting its anabolism and catabolism.