| Purpose:Osteoporosis is a common and complex disease.There are studies suggest that its pathological mechanism is related to the interexchange of osteoblasts and osteoclasts,as well as the imbalance between the transformation of stem cells into lipid cells and stem cells into osteogenic cells,and so on.Some reports reveal that Icariin,the extract from a Chinese traditional medicine(Epimedium)can affect osteoblasts,osteoclasts and stem cells,by stimulating the formation of osteoblasts while inhibiting osteoclasts formation,but the mechanism on the coupling of of osteoblasts and osteoclasts to the stem cell adipogenic division is still not clear.The present study is aimed to investigate 1)the effect of Icariin to the activity in the formation of osteoblasts and osteoclasts,2)the effect of Icariin intervening the co-culturing of osteoblasts-osteoclasts-stem cells to the Apidogenic division of stem cells,by the means of establishing an in vitro mouse osteoblasts MC3T3-E1 and mouse osteoclasts(RAW264.7 induced by RANKL)two cell co-culture system,as well as the mouse osteoblast MC3T3-E1,mouse osteoclasts and mouse BMSCs three cell co-culture system.Another aim of this study is to investigate the inter-relationship between genetic expression of stem cell lapidating division and osteoporosis,by examing human bone sample.Methodologies:1.Acquire and cultivate in vitro mouse osteoblast MC3T3-E1 and mouse monocytes RAW264.7,RANKL-induced mouse RAW264.7 monocytes division into mature osteoclasts,in a Transwell chamber to establish osteoblast-osteoclast cell co-culture system.By CCK-8 experimenting,Alizarin Red staining,TRAP staining to detect the cell activity of osteoblasts andosteoclasts.By using the PCR and Western blot method,detectthe influence of the co-culture system to the genetic expression of mouse osteoblastic MC3T3-E1 of OPG,TGF-b1 and the protein expression of osteoclasts RANKL,and also to exam the influence of the co-culture system to genetic and protein expression of RANK and NF-κb in osteoclasts.2.Apply different concentrations of icariin to intervenethe mouse osteoblast-osteoclast co-culture system;Use CCK-8 method,Alizarin Red staining andTRAP staining to exam cell activity.PCR and Western blot methodologies are used to detect the influence of icariin on the gene expression of mouse osteoblastic MC3T3-E1 of OPG.TGF-bl.as well as on the protein expression of osteoclasts RANKL;and to exam the influence of co-culture system to gene expression and protein expression of mouse osteoclasts RANK,and NF-κb.3.In a Transwell room,establish the co-culture system of mouse osteoblast cells MC3T3-E1-osteoclast cells-BMSCs,then intervene by different concentration of Icriin-By using the Oil Red staining,detect the BMSCs lipid cell division;By HE staining to detect the mouse osteoblast MC3T3-E1 and osteoclasts.By PCR,exam the gene expression change in mouse osteoblasts MC3T3-E1 and osteoclasts OPG,ALP,RANKL,TGF-bl,RANK,NF-κb,as well as BMSCs’ PPARγ,C/EBPb,RUNX2 in the co-culture system.,.Then apply the Western blot method to detect protein expression change of OPG,NF-κb.and PPARy.4.By using PCR and Western bolt method,exam the PPARy gene and protein expression change in clinical patients’ bone samples,and study the correlation for patients’ bone mineral density BMD.Result:Mouse MC3T3-E1 cells and RAW264.7 cell co-culture system may be established,within the co-culture system of osteoblasts and osteoclasts differentiation activity was significantly enhanced in the Transwell chamber.Icariin acts on mouse osteoblast-osteoclast co-culture system,improve MC3T3-E1 cell proliferation and osteogenic differentiation activity,increase and improve OPG gene-protein expression,reduced RANKL gene expression and NF-Kb,and ALP decreased level of gene expression and gene expression of TGF-bl and RANK;decreased osteoclast activity in RAW264.7 cells,and reduce the RANK gene expression and NF-Kb reduced NF-Kb expression.Mouse MC3T3-E1 cells-RAW264.7 cells co-cultured with BMSCs,weakened osteogenic activity(OPG,NF-Kb,RUNX2 and other transcription levels decline)and enhance osteoclast activity(RANK and RANKL transcription level rise),enhanced BMSCs adipogenic differentiation(PPARγ,C/EBPβ,C/EBPα gene transcription level rise).Icariin may act at the non-contact BMSCs cultured MC3T3-E1 cells were co-cultured cells-RAW264.7 body,promote osteogenic differentiation levels(promoting gene expression of OPG,RUNX2,etc.),inhibit osteoclast differentiation level(RANK and RANKL inhibition of gene expression,etc.),reduced adipogenic differentiation level(inhibit the formation of lipid area,suppressing C/EBPα and C/EBPβ gene transcription,etc.).Similarly,PPARy gene and protein levels in patients with clinical BMD was negatively correlated.Results:1.The co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell may be established in a Transwell chamber.Within the co-culture system the cell division activities of osteoblasts and osteoclasts are significantly enhanced Under the microscope one can observe the proliferation of the osteoblasts and slight de-acceleration of the osteoclast division,which are demonstrated as the decrease of both protein OPG expression and ALP gene expression,and the increase of gene expression in TGF-b1 and RANKL of the osteoblasts;whereas in the meantime for the related osteoclasts proteins,the gene expression of RANK and NF-Kb are decreased,and the protein expression of OPG and NF-Kb are increased,while the RANKL protein expression is decreased,which is in agreement of the trend of gene expression change.2.Icariin acts on mouse osteoblast-osteoclast co-culture system,which in return improves MC3T3-E1 cell proliferation and osteogenic division activity,enhances OPG gene-protein expression;whereas it also reduces gene expression of TGF-bl and RANKL.This further reduces osteoclasts activity and RANK and NF-Kb gene expression.The trend of protein expression in OPG,RANKL and NF-Kb is similar to their gene expression,it is also decelerated.3.It’s possible to establish the mouse osteobalsts MC3T3-El-Osteoclasts-BMSCs co-culture system in a Transwell chamber.In this system,the activity of osteoblasts MC3T3-E1 is reduced,along with level of gene expression in OPG,ALP and TGF-bl.Only RANKL gene expression level is increased;Osteoclasts activity increases,and NF-Kb gene expression level and the average protein expression level are reduced,but the RANK gene expression is significantly improved.The co-culture system enhances BMSCs adipogenic division,inhibits the division of osteoblast cells.The gene expression related to PPARy,C/EBPa and C/EBPb all increase,as well as the gene and protein expression of PPAPy,whereas the RUNX2 gene transcription level related to osteoblast adipogenic division is decreased.4.Icariin may act on the co-culture system of mouse osteoblast MC3T3-El-osteoclast-BMSCs,increase the bone formation activity of osteoblast cell MC3T3-El.This effect is appeared to be the gene transcription level improvement of OPG,ALP and TGF-bl as well as the enhancement of OPG protein expression,whereas only the level of RANKL gene transcription is decreased.The inhibition of osteoclast cell activity results in the decrease of gene transcription level of RANK and NF-Kb,as well as the protein expression of NF-Kb;weakening of BMSCs lipid adipogenic division level inhibits the lipid formation area,decreases the gene transcription of PPARy,C/EBPa and C/EBPb,and reduces the protein expression of PPARy.5.PPARγ gene and protein expression levels in clinical patients are negatively correlated with BMD.Conclusions:Mouse osteoblast MC3T3-E1 cells and RANKL-induced RAW264.7 cells can be co-cultivated in vitro,and they can be further co-cultivated with mouse BMSCs.Icariin maygenerally promote the regeneration of bone tissue in mouse co-culture system of osteoblast cell MC3T3-El-osteoclast cells,and decrease osteoclast activity.Icariin acts on the mouse osteoblast cell MC3T3-E1-osteoclast cells-BMSCs co-culture system to promote the osteoblast cell division,inhibit the osteoclast cell division as well as lipid adipogenic activity.Clinically patient’s PPARy gene and protein expression levels are negatively correlated with BMD. |
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