The Effect Of Zoledronate On The Proliferation And Differentiation Of Osteoclasts Under High Glucose Condition And The Mechanism Of Signaling Molecules

Objectives Bisphosphonates(BPs)is the first choice for the treatment of osteoporosis(OP).Diabetic osteoporosis(DOP)as a secondary bone metabolic disease can effect the stability of bone system.The purpose of this study was to explored the effects of zoledronate(ZOL),the third generation of BPs,on osteoclast differentiation and bone resorption under high glucose condition,and the regulation mechanism of p38 mitogen activated kinase(p38 MAPK)pathway,so as to provide experimental basis for the treatment of diabetic osteoporosis with ZOL.Methods The precursor osteoclast RAW264.7 cells were induced to differentiate into mature osteoclasts with 50ng/m L RANKL.The cells were divided into 4 groups: low glucose group(glucose concentration: 5.5 mmol/L),low glucose +ZOL group(glucose concentration: 5.5 mmol/L,ZOL concentration :0.1 mol/L),high glucose group(glucose concentration: 16.5 mmol/ L),high glucose +ZOL group(glucose concentration: 16.5mmol/L,ZOL concentration: 0.1 mol/L).The cell proliferation activity of each group was determined by MTT assay.Transwell culture system was used to investigate the migration ability of RAW264.7 cells in each group and the formation of cell sealing zones in each group was detected by immunofluorescence cytochemical staining.After adding group 5:high +ZOL+ SB203580(16.5mmol/L glucose +0.1μmol ZOL+10μmol p38 MAPK inhibitor SB203580),and the number of positive osteoclasts in the five groups was identified by TRAP staining.The number and area of bone resorption lacunae in 5 groups of osteoclasts were measured by scanning electron microscopy(SEM)after RAW264.7cells were co-cultured with dentin slices.The m RNA and protein expression of osteoclast related factors: p38 MAPK,p-p38 mitogen activated protein kinase(p-p38MAPK),nuclear factor activated T cell cytoplasmic 1(NFATc1),Cathepsin K(CTSK)were detected by realtime PCR and Western blot.Results(1)High glucose condition had no effect on the proliferation activity of RAW264.7 cells,promoted the migration ability of RAW264.7 cells(P < 0.05),inhibit the formation of cell sealing zones.The number of TRAP positive osteoclasts was inhibited,and the number and area of bone resorption lacunae were reduced(P < 0.05).Downregulation of p38 MAPK,p-p38 MAPK,NFATC1,CTSK m RNA and protein expression(P < 0.05),which can inhibit the differentiation and function of osteoclasts.(2)After the addition of ZOL,the proliferation of RAW264.7 cells was not affected,the migration ability of RAW264.7 cells was inhibited(P < 0.05),the formation of osteoclast sealing zone was further destroyed.The number of TRAP positive osteoclasts and the number and area of bone resorption lacunae were further reduced(P < 0.05).The m RNA and protein expressions of p38 MAPK,p-p38 MAPK,NFATc1 and CTSK were decreased(P < 0.05),which further inhibited the differentiation and function of osteoclasts.After addition of SB203580,osteoclast differentiation and bone resorption function were inhibited,the expression of p38 MAPK,p-p38 MAPK was inhibited,and the expression of osteoclast related factors: NFATC1,CTSK was decreased(P < 0.05).Conclusions(1)High glucose had no effect on the proliferation activity of RAW264.7cells,promote osteoclast migration ability,inhibit osteoclast differentiation and bone resorption,and down regulate the m RNA and protein expression levels of p38 MAPK,pp38 MAPK,NFATc1 and CTSK.(2)ZOL had no effect on the proliferation activity,inhibited the migration ability of RAW264.7 cells,further inhibited the differentiation and bone resorption of osteoclasts on the basis of high glucose,and reduced the m RNA and protein expression levels of p38 MAPK,p-p38 MAPK,NFATC1 and CTSK of osteoclasts.By regulating the p38 MAPK pathway,ZOL inhibited osteoclast differentiation and bone resorption under the condition of high glucose.Figure12;Table10;Reference 174...