Study On The Effect Of Fluoride On Different Stage Of Osteoblast And Metformin Against Fluoride-induced Bone Turnover

Fluorine is an inevitable of human growth.Fluorine is an osteophilic element,and more than90% of fluoride accumulates into bone tissues of body.Excessive intake of fluorides causes dental fluorosis and skeletal fluorosis.The deposition of fluoride in cancellous bone is more than that in compact bone.Meanwhile,bone turnover occurs in cancellous bone.The site where bone turnover occurred in fluorotic animal suggests that fluoride-induced bone turnover is related to the distribution of fluoride in bone tissue.The osteoclastic bone resorption and osteoblastic bone formation are contiguous during bone turnover,and active bone turnover is the essential pathological mechanism underlying fluoride-induced diversity of skeletal fluorosis.Transforming growth factor β1(TGFβ1)is a member of the TGFβ superfamily.It regulates the process of bone turnover by influencing osteoblasts and osteoclasts.A rather large number of studies prove that TGFβ1 modulates bone formation by affecting the proliferation and differentiation of osteoblasts.Recent studies have confirmed that metformin not only controls blood glucose,but also plays an important role in regulating bone turnover.It has been reported that metformin inhibits osteoclasts viability and bone resorption,but its efficacy on osteoblastic bone formation is inconsitent.Therefore,in this study,BMSCs and MC3T3-E1 cells were mineralized as early and late osteoblast differentiation models to observe the effects of different fluoride concentrations on osteoblasts at different period,and to analyze the role of TGFβ1 in the process of fluoride-induced osteoblast differentiation.In view of osteoblasts and osteoclasts being the target cells for the regulation of bone turnover,and the crucial role of bone turnover in the pathogenesis of skeletal fluorosis,we also studied the influence of metformin on fluoride-exposed osteoblasts and osteoclasts.1.Action of TGFβ1 on different stage of osteoblast exposed to fluorideThe BMSCs and MC3T3-E1 cells were induced into early differentiated and later differentiated osteoblasts as in vitro model through culturing with mineralization induction agents(β-glycerophosphate,dexamethasone and vitamin C).The effect of different fluoride concentrations with or without TGFβ1 on early differentiated and later differentiated osteoblasts was studied.The following experiments were performed:(1)Bone mesenchymal stem cells(BMSCs)were isolated from the femur of young ICR mice.When BMSCs were cultured with 0.5 mg/L and 4 mg/L of fluorine ion for 7 days,cell scratch assay was used to measure cell migration under different condition.(2)BMSCs were treated with 0.5 and 4 mg/L of fluorine ion with or without 10 ng/ml of TGFβ1for 4 days,respectively.The parameters including proliferation,apoptosis and DNA damage of early differentiated osteoblasts were detected by Flow Cytometry.(3)MC3T3-E1 cells were treated with different concentrations of fluorine ion from 0.001 to32 mg/L for 1,2,4,and 7 days,respectively.The cell viability of later stages of osteoblasts were detected by MTT assay.(4)MC3T3-E1 cells were treated with 0.5 and 4 mg/L fluorine ion with or without 10 ng/ml of TGFβ1 for 7 days.The expressions of osteogenesis and osteoclastogenesis related proteins in osteoblasts including ALP,Runx2,RANKL,Smad3/p-Smad3 and Wnt10 b were detected by Western Blot under different treatment conditions.The experimental results are as follows:(1)The migration assay showed that early differentiated osteoblasts treated with 0.5 and 4 mg/L of fluorine ion was significantly faster migration by comparison with the control.(2)Results of Flow Cytometry test indicated that TGFβ1enhanced cell proliferation and inhibited cell apoptosis in early differentiated osteoblasts when they co-exposed to a low dose of fluoride.However,high-dose fluoride combined with TGFβ1prompted the apoptosis of early differentiated osteoblasts.(3)The cell viability assay implied that the sensitivity of later differentiated osteoblasts to fluoride was increased with the extension of fluoride treatment time,which showed that the stimulation range of fluoride concentration was narrowed,while the inhibition range was gradually widened.The low-dose fluoride stimulated osteoblast viability,while high-dose fluoride inhibited osteoblast viability.(4)The protein expression in later differentiated osteoblast treated with various conditions wad investigated.It found that TGFβ1 and fluoride cotreatment increased the expression of ALP compared to the control,which affected the mineralization and maturation of later differentiated osteoblasts.TGFβ1 treatment increased RANKL expression in later differentiated osteoblasts,and then induced osteoclasts differentiation.Further analysis showed that fluoride stimulated intracellular phosphorylation of Smad3,a downstream factor of TGFβ1 signaling,and fluoride combined with TGFβ1 cotreatments enhanced the activation of TGFβ1 signaling pathway.However,TGFβ1 treatment blocked Wnt10 b expression in osteoblast,and affected the differentiation and maturation of osteoblasts.Conclusions: The above results indicated that the low-dose fluoride promoted the migration of early differentiated osteoblasts.The tolerance of fluoride dose was decreased with the advanced differentiation of osteoblasts.Effect of TGFβ1 signaling pathway had discrepancy on osteoblasts at different stage of fluoride exposure.It showed that TGFβ1 promoted the proliferation of the early differentiated osteoblasts;however,TGFβ1 inhibited the maturation of later differentiated osteoblasts.Further analysis found that TGFβ1 treatment promoted the differentiation and function of osteoblasts exposed to fluoride via activation of Smad3 signaling,and yet prevented differentiation to maturation of osteoblasts by blocking Runx2 and Wnt10 b expression in osteoblast.TGFβ1 treatment increased RANKL expression in later differentiated osteoblasts,thereby enhanced the effect of fluorine-exposed osteoblasts to induce osteoclast differentiation.2.Study on the effect of metformin against fluoride-induced bone turnoverIn view of osteoblasts and osteoclasts being the target cells for the regulation of bone turnover,we selected RAW264.7 cells and MC3T3-E1 cells as in vitro model of osteoclast and osteoblast by different condition of induction culture.MC3T3-E1 cells were induced into differentiated osteoblast by mineral induction medium culture.RAW264.7 cells were induced into osteoclast-like cells with culture medium containing 25 ng/ml of RANKL.The cell viability and differentiation of osteoclasts and osteoblasts were observed under various conditions with fluoride or combined with metformin treatment.The following experiments were performed:(1)RAW264.7 cells and MC3T3-E1 cells were exposed to different concentrations of metformin from 0.1m M to 40 m M for 1,2,and 4 days,respectively.Cell viability of osteoclasts and osteoblasts were detected by MTT assay.(2)The above experimental result showed that 0.5 m M of metformin significantly inhibited cell viability of osteoclast,but affected osteoblast little.The 0.5 m M of metformin combined with respective fluorine ion from 0.1 mg/L to 32 mg/L was administered to RAW264.7 cells and MC3T3-E1 cells for 7 days.The cell viability of osteoclasts and osteoblasts were detected by MTT assay.(3)RAW264.7 cells and MC3T3-E1 cells were treated with 0.5,4,and 16 mg/L of fluorine ion with or without 0.5 m M of metformin for 2 days,respectively.Western Blot technique was used to detected protein expression of RANK,TRAP,Wnt10 b,ALP,Runx2,Smad3,p-Smad3,p-AMPK and p-GSK3βand observed the effect of metformin on expression of these factors in osteoclasts and osteoblasts exposed to fluoride.The experimental results are as follows:(1)Metformin from low to high concentration significantly inhibited the viability of osteoclasts compared to the control.With the extension of low-dose metformin exposure time,the viability of osteoclasts was significantly inhibited by comparison with the control,while the viability of osteoblasts was inhibited only under the highdose metformin.(2)The viability of osteoclasts and osteoblasts cotreated with fluoride and metformin was detected.It showed that metformin had an inhibitory effect on osteoclast viability stimulated by fluoride.Under the same conditions,metformin treatment showed little effect on osteoblast viability.The specificity of inhibitory effect of metformin on osteoclasts exposed to fluoride was clarified in this study.(3)Protein expression of bone turnover related factors showed that metformin had an inhibitory effect on osteoclast differentiation induced by fluoride treatment,but had no obvious inhibitory effect on osteoblast differentiation induced by fluoride treatment.Conclusions: Metformin specifically inhibited cell viability of osteoclast,but had little effect on osteoblast.Metformin inhibited cell viability of fluoride-stimulated osteoclasts,but had little effect on cell viability of fluoride-treated osteoblasts.In addition,metformin activated the AMPK/GSK3β signaling pathway,and inhibited the expression of osteoclast differentiation related factors,such as RANK and TRAP,and then suppressed the differentiation of fluoride-treated osteoclasts.These results preliminarily revealed that metformin probably acted as an antagonist of bone turnover.Metformin mainly inhibited osteoclast viability and differentiation in the initial stage of bone turnover,and then blocked fluoride-induced bone turnover.