| Objective: In this study,the molecular mechanism of fluoride and arsenic-induced bone injury was preliminaries investigated by the expression of RANK/TRAF-6/NF-κB1 signaling pathway-related genes and proteins in osteoblasts and osteoclast co-culture systems.Methods: 1.The co-culture system of mouse pre-cranial osteoblast MC3T3-E1 induced by osteogenic inducer and mononuclear macrophage RAW264.7 cells was established.After 7 days of culture,it was treated with tartrate-resistant acid phosphatase(TRAP)staining identifies osteoclasts.2.The detection of cell viability: sodium fluoride(Na F)or sodium arsenite(Na As O2)medium exchange medium exposure,fluoride dose of 0.1,0.5,1.0,1.5,2.0,2.5,3.0mmol F-/L,arsenic doses were 0.1,0.5,1.0,5.0,10.0,15.0,20.0 μmol As3+/L.After exposure for 24 h,the relative survival rate of cells was measured by CCK-8 method,and the toxicity of osteoclasts.3.the design of the dose group: according to the above cell viability test results,select the dose of fluoride and arsenic: sodium fluoride(0,0.1,0.4,1.6 mmol /L Na F),sodium arsenite(0,0.5,2.5,12.5μmol /L Na As O2)and different doses of arsenic and arsenic combined medium were cultured for 24 h,according to 2 × 4 factorial experimental design,divided into 16 groups,each group of 3 duplicate wells.The infected cells were divided into control group(F0As0),low fluoride group(F0.1),medium fluorine group(F0.4),high fluorine group(F1.6),low arsenic group(As0.5),and medium arsenic.Group(As2.5),high arsenic group(As12.5),low fluorine low arsenic group(F0.1As0.5),low fluorine medium arsenic group(F0.1As2.5),low fluorine high arsenic group(F0.1As12.5),medium fluorine low arsenic group(F0.4As0.5),medium fluorine intermediate arsenic group(F0.4As2.5),medium fluorine high arsenic group(F0.4As12.5),high fluorine low arsenic group(F1.6As0.5),high fluorine arsenic group(F1.6As2.5),high fluorine high arsenic group(F1.6As12.5).4.Morphological characteristics of cells were observed under a microscope.The expressions of pathway-related factors OPG,RANKL,RANK,TRAF-6,NF-κB1,NFATc1,TRAP and MMP-9 were detected by real-time quantitative PCR(RT-q PCR).The levels of RANK,TRAF-6,NF-κB1,C-FOS,NFATc1 and TRAP protein were detected by Western blot.The changes of OPG and RANKL protein in cell culture medium were detected by enzyme-linked immunosorbent assay(ELISA).Results: 1.After co-culture of osteoblasts with RAW264.7 cells for 7 days,the cells were fused by TRAP staining,and the volume became larger and multinucleated,indicating that RAW264.7 cells could differentiate into multinucleated osteoclasts.2.Expression of RANK/TRAF-6/NF-κB1 signaling pathway-related m RNA in fluoride and arsenic in co-culture system: Fluoride-only group: OPG and RANKL m RNA expression in osteoblasts with increasing dose of fluoride both increased first and then decreased.The difference between the groups was statistically significant(F=177.269,747.167,P <0.05).With the increase of fluoride exposure,RANK,TRAF-6,NF-κB1 in osteoclasts the expression of m RNA increased gradually.The difference was statistically significant(F=529.402,373.569,77.832,P<0.05).The expression of NFATc1,TRAP,MMP-9 m RNA increased gradually in osteoclasts increased with the dose of fluoride,and the difference was statistically significant(F=147.324,17.187,160.212,P<0.05).In the arsenic exposure group alone,with the increase of arsenic exposure dose,the expression of OPG in osteoblasts decreased first and then decreased,while the expression of RANKL m RNA increased first and then decreased,and the difference between the groups was statistically significant(F=339.115,66.774,P<0.05);with the increase of arsenic dose,the expression of RANK,TRAF6 and NF-κB1 m RNA in osteoclasts increased first and then decreased,and the difference was statistically significant(F=100.696,302.554,24.183,P<0.05);With the increase of arsenic dose,the expression of NFATc1,TRAP and MMP-9 m RNA in osteoclasts increased first and then decreased,the difference was statistically significant(F=88.497,17.187,306.870,P<0.05).3.Expression of RANK/TRAF-6/NF-κB1 signaling pathway-related protein in co-culture system:Fluoride-infected group: the expression of OPG protein in osteoblasts decreased withthe increase of fluoride concentration.The differences between the groups were statistically significant(F=9.78,P<0.05).The expression of RANKL protein in osteoblasts increased with the concentration of fluoride,the difference was statistically significant(F=9.996,P<0.05);The expression of RANK protein increased first and then decreased in osteoclasts,and the expression of TRAF-6 and NF-κB1 protein increased gradually with the dose of fluoride,the differences between the groups were statistically significant(F=111.430,40.732,59.950,P<0.05).In the osteoclasts,the expression of C-FOS and NFATc1 increased first and then decreased,while the expression of TRAP protein decreased first and then increased,the difference between the two groups was statistically significant(F=309.918,156.172,156.106,P< 0.05).In the arsenic exposure group alone,the expression of OPG protein in osteoblasts decreased with the increase of arsenic concentration,the difference between the two groups was statistically significant(F=21.889,P<0.05).The expression of RANKL protein in osteoblasts was observed.The concentration of arsenic increased,and the difference was statistically significant(F=4.539,P<0.05).The expression of RANK,TRAF6 and NF-κB1 protein in osteoclasts increased with the dose of arsenic.The increase was followed by the decrease,and the difference between the groups was statistically significant(F=591.273,263.791,38.509,P<0.05).The expression of C-FOS protein in osteoclasts is increased with the dose of arsenic.The expression of NFATc1 and TRAP protein increased first and then decreased with the increase of arsenic dose,the difference between the groups was statistically significant(F=251.103,141.830,295.826,P<0.05).Fluorine-arsenic combined exposure group: In the osteoblasts,the interaction analysis of OPG and RANKL m RNA and protein expression: when fluorine alone acts,it has a main effect on the expression of OPG and RANKL m RNA and protein(F=126.14,170.70,7.57,7.38,P<0.05);Arsenic alone had a main effect on each index(F=302.90,30.47,19.86,10.22,P<0.05);The combination of fluoride and arsenic there were interactions(F=117.49,109.08,4.93,11.61,P<0.05);Analysis of RANK/TRAF6/NF-κB1 signal-related gene interactions in osteoclasts: fluorination alone,RANK,TRAF6 The expressions of NF-κB1,NFATc1,TRAP and MMP-9m RNA had the main effects(F=219.588,367.415,82.143,199.921,50.062,319.830,P < 0.05).There are main effects(F=81.615,410.033,131.927,314.042,48.102,94.602,P<0.05);The combination of fluoride and arsenic has an interaction effect on each gene index(F=125.110,239.495,63.853,97.947,21.508,317.906,P<0.05).In osteoclasts,interaction analysis of RANK/TRAF6/NF-κB1 signal-related proteins:when fluorescein alone,it has a major effect on the expression of RANK,TRAF6,NF-κB1,C-FOS,NFATc1,and TRAP(F=63.330,92.208,85.433,17.454,21.571,29.952,P <0.05);When arsenic acts alone,it has a main effect on each index(F=335.063,136.335,181.131,222.703,230.121,231.321,P<0.05);The combination of fluoride and arsenic had an interaction effect on each protein index(F=113.899,50.363,50.894,115.165,61.149,219.012,P<0.05).Conclusion: 1.Co-culture of MC3T3-E1 osteoblasts with RAW264.7 monocyte macrophages,RAW264.7monocyte macrophages can be induced to differentiate into osteoclasts.2.Fluoride and arsenic can regulate OPG protein and up-regulate the expression of RANKL factor to regulate the ratio of OPG/RANKL,participate in the differentiation and maturation of OC,and the interaction of arsenic and arsenic on the expression of OPG and RANKL mainly manifested as antagonism.3.Fluoride alone can activate the expression of RANK/TRAF-6/NF-κB1 signaling pathway-related factors and promote osteoclast proliferation and differentiation;Medium dose arsenic can also promote their expression and up-regulate bone resorption function factors TRAP and MMP-9,but high arsenic mainly inhibits its expression;The interaction of arsenic and arsenic combined with RANK/TRAF-6/NF-κB1 signaling pathway-related factors is mainly antagonistic. |
