| Icariin（ICA）,the most important flavonoid active component of Epimedium,can increase bone mineralization density,biomechanical strength and thickness of bone trabecular in osteoporosis rats,and its osteogenic effect is comparable to Estrogen（E2）.With the development of medical research,the application of Traditional Chinese medicine has been gradually introduced into the field of orthodontics.In our previous study,ICA was applied to the rat rapid expansion animal model,and it was found that ICA could promote bone remodeling in the expansion area and accelerate the formation of new bone,but its intrinsic osteogenesis mechanism needs to be further clarified.CXC Motif chemokine receptor Type 4（CXCR4）is a g-protein-coupled chemokine receptor that interacts with Stromal cell-derived factor-1（SDF-1）.The SDF-1/CXCR4 signaling axis formed by SDF-1 binding not only plays a role in stem cell mobilization and homing,but also plays an important role in the osteogenic differentiation of Mesenchymal stem cells（MSCs）.CXCR4 deficiency hinders osteoblast maturation and inhibits bone growth and development,which is manifested by decreased bone mass in bone trabeculae and cortex and decreased bone mineral density.Guang et al.found that the expression of CXCR4 gene and protein in BMSCs of C57BL/6J wild-type mice aged from 3 to 23 months decreased with age.Compared with young mice,CXCR4 deficiency decreased Alkaline phosphatase（ALP）activity,Osteocalcin（OCN）synthesis and calcium deposition in elderly mice,which hindered the osteogenic differentiation potential of elderly BMSCs.Overexpression of CXCR4 can reverse the impaired degree of BMSCs osteogenic differentiation in aged mice to a large extent.These results suggest that CXCR4 is critical to the osteogenic potential of MSCs.Silencing CXCR4 or using CXCR4-specific receptor inhibitors to inhibit BMP downstream signaling pathway Smad signaling pathway.Inhibit transcription of transcription factors such as JunB,Plzf and runt-related transcription factor 2（Runx2）,and down-regulate the expression of OCN,a marker of late osteogenic differentiation.It also blocked the transcription of Msx2,Dlx5 and its downstream Osterix and the formation of ALP in MPAKp38 signaling pathway and Erk signaling pathway.Therefore,CXCR4 signaling pathway participates in BMP-2 induced osteogenic differentiation and plays an important role in its osteogenic differentiation.Our previous study showed that ICA can promote the migration of rBMSCs to SDF-1α by up-regulating the expression of CXCR4.Therefore,we speculated whether CXCR4 signaling pathway is synergistically involved in ICA-induced osteogenic differentiation.STAT3,a key Signal transduction protein,is a member of JAK-STAT signaling pathway.It is involved in signal transduction of various cytokines,growth factors and tumor proteins,and is the most important transcription factor in the STAT family that mediates bone formation and osteoclast absorption.It is expressed in osteoblasts,osteoblasts and osteoclasts.STAT3 inactivation resulted in decreased bone density and bone strength in embryonic mice.Inhibition of JAK-STAT3 phosphorylation inhibits the formation of BMP-2 induced ALP and inhibits osteogenic differentiation.The cDNA microarray showed that the expression of CXCR4 was continuously upregulated in cells with continuous STAT3 activation.It was found that phosphorylated STAT3 was transferred into the nucleus and bound to the nuclear SIE sequence to participate in gene transcription.SIE sequence happened to be located in the CXCR4 promoter,so the activation of SIE sequence CXCR4 expression will be continuously upregulated.Lim et al.found that ICT,another active component of Icariin,can up-regulate CXCR4 expression in BMSCs by enhancing the binding of p-STAT3 to SIE sequence,thereby promoting osteogenic differentiation of BMSCs.Therefore,we speculated that ICA might enhance the expression of CXCR4 protein by up-regulating STAT3 expression,thus promoting icA-induced cell proliferation and osteogenic differentiation.Therefore,the present study explored the mechanism of ICA promoting osteogenic differentiation by establishing in vivo and in vitro experimental models,mainly including the following parts:（1）based on the rat rapid expansion animal model,we detected whether ICA promoted osteogenesis in the expansion region while up-regulating CXCR4 protein expression.（2）An in vitro cell culture model of rBMSCs was established to detect the effect of ICA on osteogenic differentiation ability of rBMSCs,and the optimal concentration of ICA was screened;（3）Si-CXCR4 was constructed and transferred into rBMSCs to explore the effect of interfering CXCR4 on ICA-induced cell proliferation and osteogenic differentiation.（4）The expression of STAT3 and P-STAT3 protein by ICA was detected by WB,and the regulation of CXCR4 and STAT3 protein expression by ICA was detected by ER inhibitors ICI 182,780.（5）By interfering CXCR4 and STAT3 respectively,explore the mechanism of CXCR4 and STAT3 synergistically participating in ICA to promote cell proliferation and osteogenic differentiation,providing experimental basis for ICA clinical application.Methods1.ICA promoted the expression of CXCR4 in the enlarged region of RME in ratsRME animal model was established,and 24 SD rats were randomly divided into Control group（Control group）,OE group（OE group）and ICA group（IE group）,with 8 rats in each group.The Control group received only local injection of α-MEM solution containing 0.01%DMSO without arch expansion or local injection of ICA.The rats in OE group and IE group were installed with two eye ring reed extender device in the upper jaw.In OE group,αMEM solution containing 0.01%DMSO was locally injected into the soft tissue of the palatal suture in the extender area.In IE group,ICA solution of 1μM was locally injected into soft tissue of palatal suture in the expansion area,and samples were collected 7 days after expansion.Three SD rats were randomly selected from each group to perform microCT scanning and reconstruct 3D images of the maxillary.Software was used to measure the width of the maxilla palatal suture to determine whether the pedicle extender could achieve the purpose of expanding the maxilla palatal suture of rats.Bone volume（BV）in the target region of the palatal suture was measured to evaluate the osteogenesis of ICA on the palatal suture region 7 days after arch expansion.HE staining and Masson staining were used to observe the morphology of the palatal suture in the expanded area.The expression of CXCR4 protein in RME expansion region was detected by CXCR4 immunohistochemical staining and immunofluorescence staining.It was proposed that ICA could promote the expression of CXCR4 protein in rat expansion region,which laid a foundation for cell experiments in vitro.2.The effect of Icariin on osteogenic differentiation of rBMSCsThis part of the experiment mainly describes obtaining cell sources and exploring the optimal drug concentration of ICA on cells.rBMSCs were isolated and cultured in vitro by whole bone marrow adherent method.CD44,CD45,CD90,CD34 were detected by flow cytometry to identify cell origin.Identification of multidirectional differentiation potential of stem cells by osteogenic and adipogenic induction.CCK-8 assay was used to detect the effects of ICA(0.1 μM,1μM,10μM on the proliferation activity of rBMSCs.Flow cytometry was used to detect the effects of ICA on cell apoptosis at different concentrations（0.1μM,1μM,10μM）,and CCK-8 assay and apoptosis detection were used to screen the optimal drug concentration of ICA on rBMSCs.ALP staining and quantitative detection of the effects of ICA at different concentrations（0.1 μM,1 μM,10μM）on the ALP activity of rBMSCs were performed.Alizarin red staining was used to detect the effects of ICA at different concentrations（0.1μM,1μM,10μM）on the ability to form mineralized nodules in vitro.The effect of ICA at different concentrations on osteogenic differentiation ability of rBMSCs was detected to provide a cell model basis for later in vitro cell research.3.ICA regulates the osteogenesis of rBMSCs through CXCR4rBMSCs were isolated and cultured in vitro by whole bone marrow adherent method.CD44,CD45,CD90 and CD34 were detected by flow cytometry to identify the cell origin.Identification of multidirectional differentiation potential of stem cells by osteogenic and adipogenic induction.CCK-8 assay was used to detect the effects of ICA（0.1μM,1μM,10μM）on the proliferation activity of rBMSCs.Flow cytometry was used to detect the effects of ICA at different concentrations（0.1μM,1μM,10μM）on cell apoptosis.ALP staining and quantitative detection of the effects of ICA at different concentrations（0.1μM,1μM,10μM）on the ALP activity of rBMSCs were performed.Alizarin red staining was used to detect the effects of ICA at different concentrations（0.1 μM,1μM,10μM）on the ability to form mineralized nodules in vitro.The effects of ICA at different concentrations on osteogenic differentiation ability of rBMSCs were detected to provide a cell model basis for later in vitro cell studies,and to screen the optimal ICA drug concentration.4.ICA regulates the osteogenic differentiation of rBMSCs by STAT3/CXCR4qRT PCR and WB were used to detect p-STAT3 and STAT3 protein expression at different time points of ICA.Estrogen receptor inhibitors ICI 182,780 were used to detect whether ICA regulated CXCR4 and p-STAT3 protein expression through ER.The last part of the experiment explored the synergistic participation mechanism of CXCR4 and STAT3 in ICA to promote osteogenic differentiation.In this part of the experiment,CXCR4 and STAT3 were interfered respectively to explore their relationship.First,si-RNA interference with CXCR4 was used to detect the regulation of ICAinduced p-STAT3 and STAT3 protein expression by CXCR4 interference.Subsequently,the regulation of CXCR4 protein expression by STAT3 was detected by using si-RNA interference of STAT3 and STAT3 inhibitor WP1066 to inhibit STAT3 phosphorylation,and the mechanism of CXCR4 on ICA promoting osteogenic differentiation of rBMSCs was discussed.Results1.ICA promoted CXCR4 expression in the enlarged region of RME in ratsIn animal experiments,the rat RME animal model was successfully established in accordance with the previous model established by the research group.Micro CT,HE staining,Masson staining,immunohistochemical and immunofluorescence detection of CXCR4 were performed on the maxillary palatal suture area of rats after 7 days of arch expansion.Micro CT results showed that the width of maxillary palatal suture in OE group and IE group was significantly wider than that in Control group,indicating that the rapid expansion animal model was successfully established.The BV value in the target region of palatal suture was higher in the group of patients than in the group of patients with OE,and the bone volume in the group of IE was stronger than that in the group of OE.HE staining showed that the palatal suture area of the Control group was composed of long and narrow fibrous tissue bands in the central part and chondrocyte bands on both sides,and the outermost part was the edge of the palatal bony plate,where scattered osteoblasts could be seen.In OE group and IE group,the fibrous tissue in the dilated area was stretched in the direction consistent with the direction of the pedicle force,and chondrocytes were extended to the fibrous tissue.Osteoblasts were increased at the edge of the bony plate in the dilated area,suggesting active bone remodeling in the palatine raphe area,in which the number of osteoblasts in IE group was more than that in OE group.Masson staining results showed that the blue staining of collagen fibers in IE group was significantly more than that in OE group.In conclusion,ICA promoted osteogenesis in the expansion area.Immunofluorescence and immunohistochemical staining results showed that the positive expression of CXCR4 in the expansion area of IE group was significantly higher than that in the OE group,suggesting that ICA could promote osteogenesis in the expansion area and at the same time up-regulated the expression of CXCR4 protein.2.The effect of Icariin on osteogenic differentiation of rBMSCsThe results of cell immunophenotype identification showed that CD90 and CD44 were positive,and CD34 and CD45 were negative,which was in line with the identification of cell immunophenotype.The cells could induce mineralized nodules and lipid droplets respectively after the continuous culture of osteogenic and lipid induction solution,indicating that the rBMSCs cultured in vitro in this study had the potential of multidirectional differentiation such as osteogenic and lipid formation,and the in vitro culture model of rBMSCs was established.CCK-8 showed that ICA promoted the proliferation of rBMSCs at 0.1 μM,1 μM and 10μM,and 1μM ICA had the most significant effect on cell proliferation compared with control group.Cell apoptosis detection results showed that 0.1μM,1μM and 10μM ICA had no significant effect on cell apoptosis compared with the Control group.The effect of ICA on osteogenic differentiation of rBMSCs was further detected.The results of ALP staining showed that ICA with different concentrations was deeply stained compared with the Control group,and the ICA group with 1μM had the deepest staining.The quantitative detection of ALP activity showed a consistent trend with ALP staining results.Alizarin red staining showed that ICA staining at different concentrations was darker than that in the Control group,in which ICA staining at 1μM was the darkest.The results of quantitative analysis of mineralized nodules were consistent with alizarin red staining,indicating that ICA at low concentrations（0.1μM,1μM,10μM）could promote cell proliferation and osteogenic differentiation of rBMSCs cultured in vitro.Moreover,ICA at 1μM had the strongest effect on promoting osteogenesis.3.ICA regulates the osteogenesis of rBMSCs through CXCR4Firstly,the expression of CXCR4 protein at different time points of ICA was detected by qRT PCR and Western blot.qRT PCR results showed that CXCR4 mRNA expression was upregulated at 6h,12h and 24h after ICA treatment,and the mRNA expression level was the highest in the 12h group.Western blot results showed that the expression of CXCR4 protein increased gradually at 6h,12h and 24h after ICA treatment,indicating that ICA promoted the expression of CXCR4 protein in rBMSCs,which was consistent with the phenotype of animal model in vivo.Si-CXCR4 was then established and transferred into rBMSCs to explore the effect of interfering CXCR4 on ICA-induced cell proliferation and osteogenic differentiation.CCK-8 results showed that the promoting effect of ICA on cell proliferation was significantly downregulated after CXCR4 interference compared with ICA group,suggesting that interfering CXCR4 inhibited the promoting effect of ICA on cell proliferation.In terms of osteogenic differentiation,interference with CXCR4 inhibited the mRNA and protein expressions of Col I,Runx2 and OCN,ALP staining and ALP activity of rBMSCs were down-regulated,and the deposition of mineralized nodules in vitro was reduced,indicating the potential of interference with CXCR4 to block ICA in promoting osteogenic differentiation.4.ICA regulates the osteogenic differentiation of rBMSCs by STAT3/CXCR4First,WB was used to detect ICA’s expression of STAT3 protein at different time points,and estrogen receptor inhibitors were used to explore ICA’s regulation of CXCR4 and STAT3 protein expression.The results showed that the expression of P-STAT3 was gradually enhanced at 6h,12h and 24h after ICA treatment,while the expression of STAT3 basic protein was basically unchanged.The ICA-induced expression of P-STAT3 and CXCR4 was inhibited by the addition of estrogen receptor inhibitor ICI,suggesting that ICA can regulate the expression of p-STAT3 and CXCR4 simultaneously through ER.At the end of the experiment,we focused on the collaborative participation mechanism of CXCR4 and STAT3 in ICA osteogenic differentiation.First,a si-CXCR4 interference cell model was established to detect ICA-induced STAT3 and p-STAT3 protein expression,so as to explore whether ICA regulates STAT3 protein expression through CXCR4.The results showed that after CXCR4 interference,ICA-induced CXCR4 expression was decreased correspondingly,but ICA-induced STAT3 and p-STAT3 protein expression were not down-regulated correspondingly,suggesting that CXCR4 may not be involved in ICA regulation of P-STAT3 and STAT3 protein expression.Secondly,the si-STAT3 interfering cell model was established to detect the regulation of ICA-induced CXCR4 protein expression by interfering STAT3.The results showed that after interfering STAT3,the ICA-induced p-STAT3 and STAT3 protein expression were significantly decreased,and the corresponding CXCR4 protein expression was also significantly down-regulated.These results suggest that STAT3 is involved in the regulation of CXCR4 protein expression during ICA-induced osteogenic differentiation of rBMSCs.At the same time,WP1066,a phosphorylation inhibitor of STAT3,was used,and it was found that WP1066 inhibited the expression of p-STAT3 protein while blocking the ICA-induced expression of CXCR4 protein.Therefore,it was concluded that ICA regulated the expression of CXCR4 protein in rBMSCs through p-STAT3.In order to verify our experimental results,we used WP1066 to detect the ICA-promoted cell proliferation and osteogenic differentiation potential of p-STAT3.The results showed that WP1066 inhibited the ICA-promoted cell proliferation of rBMSCs and blocked the up-regulated expression of osteogenic related genes Runx2,Col I and OCN proteins.Decreased ALP activity and reduced the deposition of mineralized nodules,further demonstrating that ICA promotes the osteogenic differentiation potential of rBMSCs through STAT3/CXCR4.Conclusion1.ICA promoted osteogenesis in the RME region of rats and up-regulated CXCR4 expression.2.ICA at low concentration（0.1 μM,1 pM,10μM）promoted osteogenic differentiation of rBMSCs.3.ICA regulates osteogenic differentiation of rBMSCs through CXCR4.4.During ICA-induced osteogenic differentiation of rBMSCs,STAT3 was involved in the regulation of CXCR4 protein expression. |
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