| Objective:Cynomorium songaricum Rupr is a traditional Chinese medicine for tonifying kidney-yang,which has the effects of tonifying the kidney and filling essence,nourishing body fluid and moistening dryness,etc.It can enhance sexual function and immunity,and has a certain anti-osteoporosis effect.It is widely used in the treatment of male,gynecological and senile osteoporosis and other diseases Osteoblasts are mainly responsible for bone formation and are one of the important cell components to maintain bone balance in vivo.Among them,the positive regulation of osteoblast differentiation is of great significance to bone homeostasis Osteogenic differentiation is regulated by many signaling pathways and factors Under the guidance of the theory of "kidney dominates bone and generates marrow"of traditional Chinese medicine,we choose the commonly used kidney-tonifying traditional Chinese medicine Cynomorium songaricum to prepare different concentrations of drug-containing serum of Cynomorium songaricum,to preliminarily discuss the effect of drug-containing serum of Cynomorium songaricum on osteogenic differentiation and its possible molecular mechanism in the process of osteogenesis based on Wnt/?-catenin signaling pathway,and to enrich the scientific connotation of the theory of "kidney dominate bone and generate marrow" in traditional Chinese medicine,to promote the the development and application of Cynomorium songaricum resources and guide the clinical medicationMethod:In this experiment,SPF kunming mice were divided into control group and high,middle and low dose groups.After gastric administration with different concentrations of Cynomorium songaricum Decoction(control group was replaced by saline),the corresponding serum containing Cynomorium songaricum was prepared to interfere with MC3T3-E1 cells cultured in vitro.Cell proliferation was detected by CCK-8 method and alkaline phosphatase secretion was detected by Ca-Co staining The secretion level of stromal cell-derived factor-1(SDF-1)was detected by enzyme-linked immunosorbent assay(ELISA),and the calcification of osteoblasts was detected by alizarin red staining.The expression of osteoblast-related genes,such as ?-catenin,Runx2 and Osterix(OSX)in Wnt/?-catenin signaling pathway was detected by real-time fluorescence quantitative PCR.Results:The results of this study showed that:1.At 24h,48 h and 72h,compared with the control group,each dose group of Cynomorium songaricum drug serum could promote the proliferation of MC3T3-E1 cells in mice,and there was a certain dose correlation.At the same time point,the higher the concentration of drug-containing serum,the stronger the cell proliferation ability.At 24h,compared with the control group,the ability of promoting cell proliferation in each dose group was significantly higher than that in the control group(p<0.01),but at 48 h and 72 h,there was no significant difference between the low dose group and the control group(p>0.05).All the cells proliferated significantly in the middle dose group and the high dose group compared with the control group(p<0.01).At 48h and 72h,compared with the low dose group,the middle dose group had no statistical significance(p>0.05),but the high dose group had significant difference(p<0.01).2.Compared with the control group,each dose group could significantly promote the secretion of alkaline phosphatase by mouse MC3T3-E1 cells(p<0.05),and with the increase concentration of drug-containing serum of Cynomorium songaricum,the secretion of alkaline phosphatase increased in a dose-dependent manner.The effects of medium dose group and high dose group were more obvious.3.Compared with the control group,the calcification of MC3T3-E1 cells was significantly promoted in each dose group(p<0.05),and the area of formed calcified nodules was dose-dependent with the concentration of the drug-containing serum.The higher the drug-containing serum concentration was,the more calcified nodules were,and the larger the calcified area was.4.Compared with the control group,the middle and high dose groups of Cynomorium songaricum drug-containing serum could significantly promote the secretion of SDF-1 by MC3T3-E1 cells in mice(p<0.01).Compared with the low dose group,the level of SDF-1 secretion in the high dose group was significantly different from that in the low dose group(p<0.05).5.Compared with the control group,each dose group of the drug-containing serum of Cynomorium songaricum could promote the mRNA expression of ?-catenin,Runx2 and OSX in MC3T3-E1 cells in varying degrees.Among them,the high dose group could promote the mRNA expression of ?-catenin and the difference was statistically significant(p<0.05),but there was no significant difference in the low dose group and the middle dose group(p>0.05).All dose groups of the drug-containing serum of Cynomorium songaricum could promote the mRNA expression of Runx2 and OSX in MC3T3-E1 cells to a certain extent,and the high dose group could significantly promote the expression of Runx2 and OSX mRNA(p<0.01).There was no significant difference in low and middle dose groups(p>0.05).And there is a certain dose dependence,in a limiting concentration range,the higher the concentration,the more obvious the promoting effect.Conclusion:1.the drug-containing serum of Cynomorium songaricum could promote the proliferation of mouse MC3T3-E1 cells in a time-dose dependent manner.Within 72 hours,the longer the intervention time was,the more obvious the cell proliferation was.2.Alkaline phosphatase(ALP)and mineralized nodules are significant markers of osteogenic differentiation.After 7 days of drug-containing serum of Cynomorium songaricum intervention,the expression level of ALP increased in a dose-dependent manner,and after 14 days of intervention,the mineralized nodules of the cells in each group of drug-containing serum of Cynomorium songaricum also increased in a dose-dependent manner,which means that the drug-containing serum of Cynomorium songaricum could significantly promote the osteogenic differentiation of mouse MC3T3-E1 cells.3.Wnt/?-catenin signaling pathway is one of the main pathways of osteogenic differentiation.The drug-containing serum of Cynomorium songaricum can up-regulate the expression of ?-catenin,Runx2 and OSX mRNA,which are the key genes of osteogenic differentiation in a concentration-dependent manner in Wnt/?-catenin signaling pathway,which suggested that the mechanism of osteogenic differentiation induced by drug-containing serum of Cynomorium songaricum is related to Wnt/?-catenin signaling pathway.4.Stromal cell derived factor 1(SDF-1)can regulate the expression of Runx2,which is the key gene of osteogenic differentiation through BMP2,and interact with Wnt/?-catenin signaling pathway to regulate osteogenic differentiation.The drug-containing serum of Cynomorium songaricum can significantly promote the secretion of stromal cell-derived factor 1(SDF-1)by mouse MC3T3-E1 cells in a concentration-dependent manner,indicating that its role in promoting osteogenic differentiation may also be related to the secretion of SDF-1.5.The results of this study showed that drug-containing serum of Cynomorium songaricum could significantly promote the osteogenic differentiation of mouse MC3T3-E1 cells,which may be related to the following two ways.It may be related to the following two ways,firstly,the expression of key genes in osteogenic differentiation is up-regulated by Wnt/?-catenin signaling pathway to promote osteogenic differentiation.Secondly,it promotes the secretion of SDF-1 by mouse MC3T3-E1 cells and participates in osteogenic differentiation.Furthermore,Wnt/?-catenin signaling pathway and SDF-1 may play a synergistic role through BMP2,but its mechanism needs to be further studied. |
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