Regulation Of P53 And FOXOs By SIRT1 Modulators On Apoptosis And Autophagy In Mouse MC3T3-E1 Cells Exposed To Fluoride

Fluoride is very crucial for the development of teeth and bones.Excessive fluoride,however,can cause damage to teeth,bones and other tissues resulting in serious public health problem.Related literatures showed that fluoride can induce cell apoptosis and autophagy,but the interaction between the two phenomenons needs to be clarified.SIRT1,as a deacetylation enzyme,regulates many physiological and pathological processes such as apoptosis,proliferation,autophagy,metabolism,cell cycle and so on.Therefore,this study was designed to investigate the involvement of autophagy and SIRT1 in fluoride-induced apoptosis in MC3T3-E1 cells and to study the regulatory mechanism of SIRT1 to apoptosis and autophagy.First of all,fluoride-induced apoptosis and autophagy was modelled after the cells were exposed to NaF at different concentrations.Morphological changes were observed under light microscope,cell apoptosis rate was determined using Annexin V-FITC/PI dual staining,cell cycle was detected with PI staining,intracellular ROS levels were measured with DCFH-DA probe,intracellular autophagosomes were evaluated with MDC staining,and apoptosis-and autophagy-related protein expressions were also determined using Western bloting technique.Results showed that there was an increase in apoptosis rate and Caspase-3 mRNA expression with increase in NaF concentrations.It was observed that there was a promotion in round and detached cells,intracellular ROS levels,the ratio of Bax/Bcl-2 and the apoptosis-related protein expression(Cyt c,Caspase-3 and p53)after the cells were exposed to NaF at high concentrations(1,2,3,4 and 5)× 10-3 mol/L;cell cycle analysis indicated blockage of S phase proportion as well as inhibition of mitosis.On the other hand,the mRNA and protein expression of autophagy marker LC3 was increased and the mRNA expression of autophagy-related protein Beclin-1 was also enhanced in the MC3T3-E1 cells after exposure with NaF at low concentrations(10-6,10-5,10-4 and 10-3 mol/L);it's also observed that various levels of NaF increased intracellular autophagosomes under fluorescence microscopy.Afterwards,the influence of SIRT1 on fluoride-induced apoptosis was further explored after MC3T3-E1 cells were pre-treated with SRT1720(SIRT1 activator)or Ex-527(SIRT1 inhibitor).Observation from fluorescence microscope,Annexin V-FITC/PI dual staining and Western bloting indicated that SRT1720 pretreatment significantly decreased the intracellular ROS levels,the protein expression levels of Caspase-3,cleaved-caspase-3,Ac-p53 and p21 and alleviated apoptosis and cell cycle arrest in S phase when compared with NaF treatment,while it was reversed using Ex-527.Furthermore,the effect of autophagy and SIRT1 on fluoride-induced cytotoxicity in MC3T3-E1 cells was also investigated.The cells were pre-treated with rapamycin(autophagy activator)or SRT1720(SIRT1 activator)or combined with 3-MA(autophagy inhibitor).Results of MTT assay showed that rapamycin and SRT1720 pretreatment alleviated the NaF-induced reduction of cell viability,whereas 3-MA abolished the rescue effect of two drugs.At last,the mechanism of SIRT1 involved with fluorine-induced autophagy in MC3T3-E1 cells was determined.In this part of experimentation,cells were pre-treated with SRT1720(SIRT1 activator)or Ex-527(SIRT1 inhibitor).The results revealed that SRT1720 pretreatment significantly enhanced the expression of LC3 mRNA and protein levels as well as the green fluorescence puncta(LC3)under fluorescence microscope and confocal laser scanning microscope,while Ex-527 had the contrary effect.Moreover,the protein expression levels of Ac-FoxO1 were inhibited by SRT1720,while Rab7 and Bnip3 protein expression was enhanced,and Ex-527 opposed the effect.In conclusion,NaF not only induced endogenous apoptosis of MC3T3-E1 cells by increasing the intracellular ROS levels and altering the cell cycle distribution,but also induced Beclin-1 mediated autophagy in MC3T3-E1 cells.SIRT1 activation reduced the intracellular ROS levels and relieved NaF-induced apoptosis;and autophagy and SIRT1 activation attenuated the cytotoxicity of fluoride.In addition,on one hand,SIRT1 activation inhibited apoptosis through SIRT1-p53-p21;on the other hand,it induced cell autophagy with the underlying pathways of SIRT1-Fox01-Rab7 and SIRT1-FoxO3-Bnip3.A theoretical basis for using SIRT1 as a key target for prevention and treatment of fluorosis was established according to the regulatory role of SIRT1 in cell apoptosis and autophagy.