| Objective: To lay the foundation for further clarify the role of Gs-AC-c AMP-PKA signaling pathway in skeletal fluorosis,rat osteosarcoma cells UMR-106 and Wistar rats were used to analyze the expression of each factor in this signal transduction pathway in vitro and in vivo. Methods: 1. Impact of Na F on UMR-106 cells phenotype and expression of Gs/Gi-AC-c AMP-PKA signal pathway. UMR-106 cells were treated with 0,50,100,500,1000,5000,10000,20000,40000 and 80000 mmol/L Na F. CCK-8 assay were used to detect the cell viability.(2)UMR-106 cells were treated with 0,1000,2000 and 4000mmol/L Na F. Flow cytometry was used to determine the cell cycle distribution and the apoptosis, disodium phenyl phosphate was used to detect ALP,In the same time,BGP,Gs,Gi,AC,PKA m RNA expression were detected by RT-PCR. 2. Impact of Na F on Wistar rats and expression of Gs/Gi-AC-c AMP-PKA signal pathway of bone tissue. 48 Wistar rats weaned 4 weeks were randomly divided into 4 groups according to body weight,control group(treated with tap water)and 3 Na F exposure groups(treated with 50,150,250 mg/L Na F),6 female rats and 6 male rats in each group. Na F was given through drinking water. The rats were executed after 6 months of treatment. The fluoride content of bone,tooth,serum and urinary and incidence rate of dental fluorine were detected. Colorimetry were used to test rat bone tissue inhibition of OH radical capacity and the activity of CAT,GSH-Px,SOD, the concentration of bone tissue 8-OHd G,PCO,MDA,Gs,Gi,AC,c AMP and PKA were assayed by ELISA. Results: 1. Impact of Na F on UMR-106 cells phenotype and expression of Gs/Gi-AC-c AMP-PKA signal pathway.(1)CCK-8 tested: treated 24h: UMR-106 cells survival rate in 1000~80000 μmol/L Na F groups were lower than control(P<0.05);treated 48h: UMR-106 cells survival rate in 50~80000 μmol/L Na F groups were lower than control(P<0.05).(2)ALP and BGP results: ALP activity inall Na F treated groups were obviously higher than control(P<0.05). BGP m RNA expression in 2000 and 4000mmol/L Na F groups were distinctly higher than control(P<0.05).(3)Cell cycle distribution and the apoptosis results: the cell number of G0/G1 phase proportion in1000,4000 mol/L Na F groups were lower than the control(P<0.05)and 2000 mmol/L Na F groups was higher than other groups(P<0.05);the cell number of S phase proportion in 2000 mmol/L Na F group was lower than other groups(P<0.05);the cell number of S phase proportion in 4000 mmol/L Na F group was lower than other groups(P<0.05).(4)Gs,Gi,AC,PKA m RNA expression results: the PKA m RNA expression in 2000 and 4000 mmol/L Na F groups and Gs in 4000 mmol/L Na F group were higher than control(P<0.05);Gi and AC m RNA expression in all groups were not observed difference(P>0.05). 2. Impact of Na F on Wistar rats and expression of Gs/Gi-AC-c AMP-PKA signal pathway of bone tissue.(1)The index of fluorosis model results: The incidence of dental fluorosis of each Na F exposure group was 100% and dental fluorosis was not found in control. The fluoride content of bone,tooth,serum and urinary in each Na F exposure group were higher than control(P<0.05).(2)The rat bone tissue oxidative damage index test results: To compare of control,the SOD activity in middle and the CAT activity and inhibition of OH radical capacity in middle and high group was lower, the concentration of PCO in each Na F exposure group were higher.(3)The content of bone tissue Gs,Gi,AC,c AMP,PKA test results: The content of bone tissue Gs,Gs,AC, c AMP and PKA were not found significantly differences among the groups(P>0.05). Conclusion: 1.Na F may prompt ALP activity and increase BGP m RNA expression of UMR-106 cells. 2.High content Na F can affect UMR-106 cell cycle, extending G0/G1 or S phase, inhibiting cell proliferation and induce apoptosis. 3. High content Na F can increase Gs, PKA m RNA expression of UMR-106 cells. 4. This study successfully established rat model of chronic fluoride poisoning. 5.150,250 mg/L Na F can cause bone tissue oxidative damage in rats. 6. The expressionchange of each factor in Gi/Gs-AC-c AMP-PKA signal transduction pathway in vivo were not observed by ELISA assay. |
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