Effects Of Icariin On The Proliferation And Differentiation Of Osteoblast And Its Possible Mechanism Of Action In Vitro

Mandibular defect reconstruction is a cutting-edge research focus inmodern maxillofacial surgery. The use of traditional Chinese medicinehasbecome a routine method in treatment of acquired mandibular defects.According to the “Shen Bugu” theory, the osteoblastic mechanism of“kidney-tonifying”herbshas aroused great interest to researchers in worldwide.Epimediiherba (Yinyanghuo) is one of the most frequently usedtraditional Chinese medicine that is prescribed for invigorating kidney tostrengthen yang and strengthening sinews and bone. Icariin (ICA) is believedto be the major active ingredient of Epimediiherba. Although there are amplestudies concerning the potential effects of ICA on osteoblast proliferation anddifferentiation, the definite conclusive resultshave yet to be elucidated.Especially, the understanding of its mechanism of action, target, and signaltransduction of ICA in osteogenesis remains incomplete. Bone morphogeneticproteins (BMPs) have widely recognized a major inducer during bone repair inthe body. Mechanistic analysis revealed that the BMPs can bind to theirreceptors BMPR, promoting its association with the coactivator Smads, p38-MAPK, which can activate downstream transcription factor, such as RunX2and Osterix, to directly target the promoter region of alkaline phosphatase(ALP) and osteocalcin to regulate osteoblastgenetic programs anddifferentiation. Therefore, in the present study, we investigate the effects ofICA on osteoblast differentiation from at least four perspectives, so as toobtain an understading of its mechanism of action and elucidate the molecularmechanisms of this drug, which might provide an insight into the rationalityand in-depth theoretical basis for the clinical application of ICA in promotingosteogenesis. Part Ⅰ Effects of ICA on proliferation ofosteoblastObjective: to observe the proliferative and growth conditions ofMC3T3-E1cells after different concentrations of ICA treatment at differenttime points.Methods: MC3T3-E1cells were cultured and passaged according to themethod from ATCC. After incubating MC3T3-E1cells by differentconcentrations (0,0.01,0.1and1μM), MTT assay was employed to detectproliferative activity at different time points (24h,48h, and72h). Theproliferation markergene PCNA and Ki67expression was measured byfluorescence quantitative PCR and Western blot method, respectively.Results: We first investigated whether cell proliferation could be regulatedby ICA in MC3T3-E1cells. Different concentrations of ICA have no obviouseffects on the morphology of MC3T3-E1cells. MC3T3-E1cells were treatedwith ICA for different times and each time point with different doses. MTTassay showed that ICA had no effect on MC3T3-E1cell proliferation. Next,we preformed a real-time PCR assay and a Western blot assay to detect theproliferation-relatedgenes PCNA and Ki67mRNA and protein expression. Asa result, neither their mRNA expression nor their protein expression wasaffected by ICA treatment.Conclusion: It turned out that ICA had no direct influence on MC3T3-E1morphology and proliferative index. We speculate that ICA might exert itsosteogenic effects through induction of cell apoptosis and differentiationsignaling.Part ⅡEffects of ICA on the osteogenic differentiation of osteoblastObjective: to observe the effects of different concentrations of ICAon the morphology of MC3T3-E1cells by transmission electronmicroscope and to detect the activity of acid phosphatase at different timepoints and osteoblast cell differentiation and associated gene expression,such as typeⅠcollagen (COL1), BGP, OPN, and BMP2at48h.Methods: MC3T3-E1cells were cultured and passaged according to themethod from ATCC. After MC3T3-E1cells treated with1μM ICA for14d,transmission electron microscope was employed to observe the changes of cell ultrastucture. The activity of acid phosphatase (ALP) was determinedby methods of pyrocatechol phosphate of MC3T3-E1cells after treating withdifferent concentrations of ICA at different time points (24h,48h, and72h).Fluorescence quantitative PCR and Western blot method was employed tomeasure major gene expression alteration, including COL1, BGP, OPN andBMP2.Results: After observation by scanning electron microscopy, MC3T3-E1cells present the following characteristics, such as, lack of cell surfaceprotrusions, cytoplasm Golgi apparatus, endoplasmic reticulum andmitochondria underdeveloped, which seems like pre-osteoblast phenotype.When treated with ICA for14d, there were more cell surface protrusions onMC3T3-E1cells. The nuclear was located in the side of the cell and thenucleolus is scattered in the medullary structure of glycogen and lysosomes.There were more Golgi apparatus, expanded endoplasmic reticulum andmitochondria in MC3T3-E1cells after ICA treatment, which seems likeosteoblast phenotype. MC3T3-E1cells were treated with ICA for differenttimes and each time point with different doses. ALP activity was significantlyincreased in MC3T3-E1cells treated with0.1μM ICA for24h. In addition,when MC3T3-E1cells were stimulated with0.01μM ICA for48h, ALPactivity was also significantly increased. Besides, the optimal concentration iswithin1μM.Next, we preformed a real-time PCR assay and a Western blotassay to detect the mRNA and protein expression ofdifferentiation-relatedgenes BGP, OPN, COL1, and BMP2. As a result, ICAcould significantly induce these genes expression in a dose-dependent manner.Conclusion: After MC3T3-E1cells treated with ICA, the ultrastructurewas osteoblast-like phenotype. These data suggest that ICA could increaseALP activity of MC3T3-E1cells in time and dose-dependent manners, andthat its optimal concentration is within0-1μM. In addition, ICA couldupregulate the expression of osteogenic differentiation relatedgenes, includingBGP, OPN, COL1and BMP2, which had a dominant-positive effect onosteogenic microenvironment and thus promoting bone differentiation and formation.Part Ⅲ Effects of ICA on BMP2-Smads-RunX2signaling pathway inosteroblastObjective: to explore the potential effects of ICA on promotingosteogenic differentiation by detecting BMP2, Smad1, Smad5, Smad8, RunX2and Osterix gene expression alterations after ICA treatment.Methods: To determine whether ICA induced MC3T3-E1celldifferentiation was mediated by BMP2-Smads-RunX2signaling pathway.After preincubation for48h in ahumidified incubator, fresh Media containing1μM of icariin was added to MC3T3-E1cells. Fluorescence quantitative PCRand Western blotting method was employed to measure major gene expressionalteration, including BMP2, Smad1, Smad5, Smad8, RunX2and Osterix.Delivery of siRNA targeting RunX2, downstream transcription factors wereevaluated.Results: MC3T3-E1cells were treated with0,0.01,0.1, and1μM ICA for48h and the expression of BMP2, Smad1, Smad5, Smad8, RunX2, and Osterixwere examined. Real-time PCR and Western blot assay showed that ICA couldsignificantly upregualte these genes expression. The mRNA and protein levelsof these genes pretreated with ICA were much higher than control group(p<0.05). Besides, the optimal concentration is within1μM.To further investigate whether BMP2-Smads-RunX2signaling pathwaywas involved in ICA-induced MC3T3-E1cell differentiation, we knockeddown the expression of RunX2by transfection of MC3T3-E1cells withRunX2-specific siRNA (siRNA-RunX2). After delivery of siRNA targetingRunX2, the mRNA and protein level of RunX2was reduced significantly by40nM RunX2-siRNA compared with control group (p<0.05). This level ofgene silencing was sufficient to inhibit ALP activity in MC3T3-E1cells.Besides, BMP2and osteogenic differentiation related genes including Smad1,Smad5, Smad8, RunX2and Osterix, were also downregulated accordingly.Conclusion: BMP2is a determinant factor in MC3T3-EI cell osteogenicdifferentiation. ICA’s role in the promotion of MC3T3-EI cell differentiation is closely related to BMP2-Smads-RunX2-Osterix signaling pathway. Besides,we found that RunX2played a pivotal role in MC3T3-EI cell differentiation,which directly regulated the downstream transcription factors, such as BGP,COL1and OPN.Part Ⅳ Effects of ICA onBMP2-p38MAPK signaling pathway inosteroblastObjective: to evaluate p38MAPK, ERK and JNK expression andcorresponding phosphorylation levels after treating with ICA. Afterp38MAPK signaling pathway was blocked by SB203580, BMP2-p38MAPKsignaling was analyzed to further elucidate the pro-osteogenesis mechanismcaused by ICA.Methods: To determine whether BMP2-p38MAPK signaling pathwaywas also involved in ICA induced MC3T3-E1cell differentiation. Afterpreincubation for48h in ahumidified incubator, fresh Media containing1μMof icariin was added to MC3T3-E1cells. Western blotting was used to detectp38MAPK, ERK, and JNK expression and phosphorylation levels inMC3T3-E1cells after treating with1μM ICA at48h. After p38MAPK wasblocked by SB203580specifically, MC3T3-E1cell differentiation wasobserved after treating with ICA.Results:1μM ICA significantly increased phospho-p38MAPK levelwithout affecting the levels of phospho-ERK, phospho-JNK, total p38MAPK,total ERK, and total JNK. Since ICA treatment leads to p38MAPKphosphorylation, we decided to test whether activated p38MAPK is involvedin ICA-induced MC3T3-E1cell differentiation. The effect of p38MAPKinhibitor SB203580on ICA-induced p38MAPK phosphorylation andMC3T3-E1cell differentiation were detected. SB203580largely blockedICA-induced p38MAPK phosphorylation without affecting total p38MAPKlevel. In addition, SB203580significantly inhibited ICA-increased ALPactivity. Furthermore, the mRNA and protein expression ofdifferentiation-related genes BGP, COL1，OPN induced by ICA were alsocompletely blocked by SB203580treatment. Conclusion: In the differentiation process, ICA could upregulatep38MAPK phosphorylation, while exert no effect on ERK and JNK. ICAmight promote osteoblast differentiation by BMP2-p38MAPK signalingpathway.