The Mechanism Of Bone Differentiation Induced By Orthosilicic Acid Through Non-Classical BMP Pathway In Vitro

BackgroundOsteoporosis(OP)is a systemic bone disease characterized by low bone mass,destruction of bone microstructure,and increased bone fragility.In the process of osteoporosis,the dynamic balance between bone formation and absorption is disturbed,and a vicious cycle is gradually formed,resulting in a significant increase in fracture susceptibility.Meanwhile,osteoporosis,as a degenerative disease,is closely related to aging.With the prolongation of human life,osteoporosis has become an important public health problem.Therefore,exploration of new treatments for osteoporosis and its complications has become a hot topicSilicon is an important trace element in the human body,which is closely related to the occurrence and development of osteoporosis.Silicon,liking vitamin D,may significantly increase the rate of bone mineralization.Previous studies have found that lack of silicon dietary harms bone growth and decreases bone density.Orthosilicic Acid,with low molecular weight and good water solubility,is the most suitable form of silicon to be absorbed by human and animal.Recent studies have confirmed the important role of bone morphogenetic protein(BMP)in bone differentiation mediated by orthosilicic acid,but whether there are other non-classical BMP pathways involved in the bone differentiation mediated by orthosilicic acid has not been fully clarifiedStudies have confirmed that PI3K-Akt-mTOR pathway plays a positive role in the regulation of osteogenesis.However,there is no date on the relationship between bone differentiation mediated by orthosilicic acid and PI3K-Akt-mTOR pathway.Therefore,our present study focuses on the effect of orthosilicic acid on bone differentiation through the PI3K-Akt-mTOR pathway in adipose-derived mesenchymal stem cells(ADMSCs)and human osteoblast-like cells(MG-63 and U2-OS).Considering the potential of multiple-differentiation of ADMSCs,this study further examined the mechanism of orthosilicic acid in osteoclast differentiation through RANKL/OPG pathway in ADMSCs.The novelties of this study are as follows:1.The relationship between bone differentiation and PI3K-Akt-mTOR pathway after the intervention of orthosilicic acid on ADMSCs and human osteoblast-like cells was investigated firstly.2 Orthosilicic acid was applied to ADMSCs to explore the mechanism in both osteogenic differentiation and osteoclastic differentiation for the first time.The results of this study will provide clues for the combined application of orthosilicic acid and ADMSCs in the treatment of osteoporosis and complications,and provide a new theoretical basis for the application of orthosilicic acid in the treatment of osteoporosis.Part IThe Mechanism of Orthosilicic Acid Influencing Bone Differentiation of Adipose-Derived Mesenchymal Stem Cells Through the PI3K-Akt-mTOR and RANKL/OPG Pathways in VitroObjective(1)To detect the effects of orthosilicic acid on cell viability of ADMSCs.(2)To detect the effects of orthosilicic acid on bone differentiation of ADMSCs.(3)To explore the potential mechanism of orthosilicic acid on the bone differentiation of ADMSCs.Methods(1)In order to verify the stability of purchased ADMSCs,the 6th generation ADMSCs in good growth status were stained by alizarin red S staining and oil red O staining to identify their osteogenic and adipogenic differentiation abilities.Meanwhile,the surface specific markers were identified by flow cytometry.(2)The cell viabilities of ADMSCs were detected by CCK-8 assay at 1,3,5 and 7 days after intervened by variational concentrations of orthosilicic acid.(3)Wstern blot analysis was used to detect the expressions of osteogenic differentiation indicators(COL1 and RUNX2)in ADMSCs at 7 days after intervened by variational concentrations of orthosilicic acid.The relationship between the expressions of COL1 and RUNX2 and the concentrations of orthosilicic acid were analyzed to determine its optimal concentration to induce osteogenic differentiation of ADMSCs.(4)Alizarin red S staining was used to detect the formation of calcium nodules in ADMSCs after 28 days intervened by orthosilicic acid.(5)The changes of PI3K-Akt-mTOR pathway in ADMSCs at 7 days intervened by variational concentrations of orthosilicic acid were detected by Wstern blot analysis,and the relationship between the changes and the concentrations of orthosilicic acid was analyzed.(6)Cellular immunofluorescence technique was used to detect the expressions of P-Akt and P-mTOR in ADMSCs at 7 days intervened by orthosilicic acid to further verify the effects of orthosilicic acid on the PI3K-Akt-mTOR pathway.(7)According to the presence or absence of orthosilicic acid and LY294002,inhibitor of PI3K,the cells were grouped as follows:control group,control+LY294002 group,orthosilicic acid group,and orthosilicic acid+LY294002 group.The expressions of PI3K-Akt-mTOR pathway and osteogenic differentiation indicators(COL1 and RUNX2)were detected by Wstern blot to analyze the relationship between orthosilicic acid-mediated osteogenic differentiation of ADMSCs and PI3K-Akt-mTOR pathway.(8)The BCIP/NBT ALP staining was used to detect the effects of orthosilicic acid and LY294002 on the expressions of ALP of the four groups to further explore the relationship between orthosilicic acid-mediated osteogenic differentiation of ADMSCs and PI3K-Akt-mTOR pathway.(9)Wstern blot analysis was used to detect the expressions of RANKL and OPG in ADMSCs at 7 days intervened by orthosilicic acid.The relationship between the expressions of RANKL and OPG and the concentrations of orthosilicic acid was analyzed to determine the optimal concentration of orthosilicic acid to inhibit the osteoclastic differentiation of ADMSCs according to the ratio of RANKL/OPG.(10)According to the presence or absence of the active protein sRANKL and ortho silicic acid,the cells were grouped as follows:control group,sRANKL group,and sRANKL+orthosilicic acid group.The expressions of osteoclast differentiation indicators(NFATcl,CALCR,CTSK)were detected by Wstern blot to analyze the role of orthosilicic acid in the sRANKL-mediated osteoclast differentiation of ADMSCs.(11)The TRACP staining was used to detect the expressions of osteoclast differentiation indicators TRACP of the three groups to further analyze the role of orthosilicic acid in the sRANKL-mediated osteoclast differentiation of ADMSCs.Results(1)After the 6th generation ADMSCs were induced by osteogenic and adipogenic,both staining results were positive.Meanwhile,the specific protein on the cell surface identified by flow cytometry showed that the CD90 positive rate was more than 90%,and the CD45 and CD11 positive rates were less than 5%.The results indicated that ADMSCs owned the ability of osteogenesis and adipogenic differentiation,as well as the surface characteristics of mesenchymal stem cells.(2)The cell viability of ADMSCs showed no change with the variational concentration and time of orthosilicic acid.It was indicated that orthosilicic acid(≤40μM)had no significant effect on the cellular viability of ADMSCs.(3)Orthosilicic acid can up-regulate the expressions of COL1 and RUNX2 and the most obvious effect was observed at the concentration of 20μM,which represented the optimal concentration of orthosilicic acid for inducing osteogenic differentiation of ADMSCs.(4)After 28 days of ADMSCs intervened by orthosilicic acid(20 μM),the formation of calcium nodules were significantly promoted.(5)Orthosilicic acid could up-regulate the expressions of PI3K,P-Akt,and P-mTOR.P-Akt was most significantly upregulated when the orthosilicic acid concentration was 20 μM.The expressions of total Akt and total mTOR showed no significantly change with the variations of orthosilicic acid concentration.(6)Orthosilicic acid could significantly increase the number of fluorescent positive cells and the fluorescence intensity of P-Akt and P-mTOR.The results were consistent with the trends of Wstern blot analysis above.(7)Orthosilicic acid could up-regulate the expressions of PI3K,P-Akt,P-mTOR,COL1 and RUNX2.The expressions of these proteins were down-regulated when the PI3K inhibitor LY294002 was added.The results indicated that the orthosilicic acid-mediated osteogenic differentiation of ADMSCs was also inhibited after the inhibition of PI3K-Akt-mTOR pathway.(8)Orthosilicic acid could increase the expression of ALP,and the expression of ALP was inhibited when LY294002 was added.These results were consistent with the trend of other osteogenic differentiation indicators(COL1 and RUNX2).It was reconfirmed that the PI3K-Akt-mTOR pathway played a positive role in orthosilicic acid-mediated osteogenic differentiation of ADMSCs.(9)Orthosilicic acid could down-regulate RANKL and up-regulate OPG.These results indicated that orthosilicic acid inhibited the osteoclast differentiation of ADMSCs by reducing the ratio of RANKL/OPG.The ratio of RANKL/OPG was the lowest when the concentration of orthosilicic acid was 20μM,which could be used as the optimal concentration to inhibit the osteoclastic differentiation of ADMSCs.(10)The active protein sRANKL could up-regulate the expressions of osteoclast differentiation indicators(NFATcl,CALCR and CTSK),and the expressions of these indicators were all down-regulated when orthosilicic acid was added.These results indicated that orthosilicic acid could inhibit sRANKL-mediated osteoclast differentiation of ADMSCs.(11)The active protein sRANKL could enhance TRACP staining positive reaction and increase the number of positive cells.When combined with orthosilicic acid,the TRACP staining positive reaction was weakened and the number of positive cells were decreased.These results reconfirmed that orthosilicic acid could inhibit sRANKL-mediated osteoclast differentiation of ADMSCs.Conclusion(1)Orthosilicic acid(≤40μM)has no significant effect on the cell viability of ADMSCs.(2)Orthosilicic acid can up-regulate the expressions of osteogenic differentiation indicators(RUNX2,COL1 and ALP)and promote the osteogenic differentiation of ADMSCs.(3)Orthosilicic acid can activate the PI3K-Akt-mTOR pathway during the orthosilicic acid-mediated osteogenic differentiation of ADMSCs.When this pathway is inhibited,the osteogenic differentiation is also inhibited,indicating that PI3K-Akt-mTOR pathway plays a positive regulatory role in the orthosilicic acid-mediated osteogenic differentiation of ADMSCs.(4)Orthosilicic acid can down-regulate RANKL and up-regulate OPG expression.It can inhibit the osteoclast differentiation of ADMSCs by reducing the ratio of RANKL/OPG.(5)Orthosilicic acid can inhibit the sRANKL-mediated osteoclast differentiation of ADMSCs and down-regulate the expression of osteoclast differentiation indicators(NFATcl,CALCR,CTSK and TRACP).This result further indicates that orthosilicic acid can not only reduce the expression of RANKL,but also inhibit the RANKL-mediated osteoclast differentiation.Part ⅡThe Mechanism of Orthosilicic Acid Accelerating Bone Formation in Human Osteoblast-Like Cells Through the PI3K-Akt-mTOR Pathway in VitroObjective(1)To detect the effects of orthosilicic acid on cell viabilities of human osteoblast-like cells(MG-63 and U2-OS).(2)To detect the osteogenesis mediated by orthosilicic acid on human osteoblast-like cells.(3)To explore the relationship between the orthosilicic acid-mediated osteogenesis and PI3K-Akt-mTOR pathway.Methods(1)The cell viabilities of MG-63 and U2-OS was detected by CCK-8 assay at 72 hours intervened by variational concentrations of orthosilicic acid.(2)Wstern blot analysis was used to detect the expressions of osteogenic indicators(COL1 and RUNX2)in MG-63 and U2-OS at 72 hours intervened by variational concentrations of orthosilicic acid.The relationship between the expressions of COL1 and RUNX2 and the concentrations of orthosilicic acid was analyzed to detennine the optimal concentration of orthosilicic acid to induce osteogenesis of human osteoblast-like cells.(3)Cellular immunofluorescence technique was used to detect the expressions of P-Akt and P-mTOR in MG-63 and U2-OS at 72 hours intervened by orthosilicic acid to explore the effect of orthosilicic acid on the PI3K-Akt-mTOR pathway.(4)According to the presence or absence of orthosilicic acid and LY294002,the cells were divided into the following four groups:control group,control+LY294002 group,orthosilicic acid group,and orthosilicic acid+LY294002 group.The expressions of PI3K-Akt-mTOR pathway and osteogenic indicators(COL1 and RUNX2)in the 4 groups were detected by Wstern blot to analyze the relationship between orthosilicic acid-mediated osteogenesis and PI3K-Akt-mTOR pathway.(5)The BCIP/NBT ALP staining was used to detect the effect of orthosilicic acid and LY294002 on the expression of ALP of the 4 groups and to analyze the relationship between the expressions of ALP and PI3K-Akt-mTOR pathway.(6)ALP microenzyme assay method was used to further detect the effect of orthosilicic acid and LY294002 on ALP activity of the 4 groups and to explore the relationship between the activities of ALP and PI3K-Akt-mTOR pathway after long-term intervention of orthosilicic acid for 7 days.(7)ELISA was used to detect the effects of orthosilicic acid and LY294002 on the expressions of OCN and P1NP in cell supernatant of the 4 groups and further to detect the relationship between the expressions of OCN and P1NP in orthosilicic acid-mediated osteogenesis and PI3K-Akt-mTOR pathway.Results(1)The cell viability of MG-63 and U2-OS showed no change with the concentration of orthosilicic acid.This indicated that orthosilicic acid(≤40 μM)had no significant effect on the cell viabilities of MG-63 and U2-OS within 72 hours.(2)Orthosilicic acid can up-regulate the expressions of COL1 and RUNX2 and the most obvious effects was observed at the concentration of 10μM,which could be used as the optimal concentration of orthosilicic acid for promoting osteogenesis.(3)Orthosilicic acid could significantly increase the number of fluorescent positive cells and the fluorescence intensity of P-Akt and P-mTOR in MG-63 and U2-OS.(4)The expressions of PI3K,P-Akt,P-mTOR,COL1 and RUNX2 were up-regulated by orthosilicic acid,and down-regulated by LY294002.The results indicated that the PI3K-Akt-mTOR pathway plays a positive role in the orthosilicic acid-mediated osteogenesis.(5)The expression of ALP was increased at 72 hours after intervention,and can be inhibited by LY294002.These results were consistent with the trends of COL1 and RUNX2 detected by Western-blot,which further verified the active role of PI3K-Akt-mTOR pathway in the orthosilicic acid-mediated osteogenesis.(6)The activity of ALP was enhanced by orthosilicic acid after prolonged intervention for 7 days,and decreased by LY294002.The similar expression trend of ALP at 72 hours and 7 days intervened by orthosilicic acid indicated that ALP was an important indicator of early and middle stage of osteogenesis and the PI3K-Akt-mTOR pathway played a positive role in the early and middle stage of orthosilicic acid-mediated osteogenesis.(7)The expressions of OCN and P1NP were increased by orthosilicic acid,and decreased by LY294002.As the decomposition product of COL1 in the early stage of synthesis,the expression trend of P1NP was consistent with that of COL1.These results further indicated that the PI3K-Akt-mTOR pathway played an important role in the orthosilicic acid-mediated osteogenesis.Conclusion(1)Orthosilicic acid(≤40 μM)has no significant effects on the cell viabilities of human osteoblast-like cells(MG-63 and U2-OS within 72 hours.(2)Orthosilicic acid can promote the expressions of osteogenic indicators(RUNX2,COL1,ALP,OCN and P1NP)and play a positive role in the osteogenic process.(3)Orthosilicic acid can activate the PI3K-Akt-mTOR pathway during the orthosilicic acid-mediated osteogenesis.(4)The PI3K-Akt-mTOR pathway is involved in the orthosilicic acid-mediated osteogenesis and plays a positive regulatory role.