| Osteoporosis（OP）has become one of the three major diseases affecting the elderly after diabetes and senile dementia.At present,the first-line treatment drugs of osteoporosis generally have problems such as long treatment cycle,large side effects,high price,etc.It is still urgent to find new,safe,effective and cheap anti-osteoporosis drugs.Bone marrow mesenchymal stem cells（BMSCs）are adult stem cells in the bone marrow cavity and are the main source of osteoblasts.Try to stimulate BMSCs to differentiate more into osteoblasts is an important strategy to prevent and control OP.Eucommia ulmoides is a kind of traditional Chinese medicine（TCM）,which has a variety of pharmacological effects in treating low back pain,nourishing qi,strengthening bones and muscles.Recent studies have shown that Eucommia ulmoides,as a phytoestrogen,can promote the activity of osteoclasts to slow down the rate of bone resorption,and can stimulate the osteogenic differentiation of BMSCs to promote the formation of new bone,so as to achieve the prevention and treatment of OP.It is known that pininol diglucoside（PDG）is its main active component,but whether PDG can promote the osteogenic differentiation of BMSCs has not been reported before.The following series of studies were conducted in this paper.1.Rat BMSCs were extracted by whole bone marrow adherence method and expanded to establish an in vitro culture system;2.The effect of different concentrations of PDG on the proliferation of BMSCs was detected by CCK-8 method;3.The optimal action concentration of PDG was determined by the influence of different concentrations of PDG on ALP activity;4.The effect of PDG on BMSCs osteogenic differentiation was determined by alkaline phosphatase positive clone staining,calcification nodule alzarin red staining,Western Blotting detection of osteogenesis-related protein expression,and detection of osteogenesis-related gene expression by RT-PCR;5.The effect of PDG on BMSCs adipogenic differentiation was determined by oil red O staining,Western Blotting for adipogenic-related protein expression,and adipogenic-related gene expression by RT-PCR;6.Add Noggin,a key protein inhibitor of BMP2/Smad1/5/8 signaling pathway,and explore the effect of PDG on BMP2/Smad1/5/8 signaling pathway by measuring ALP activity,western blotting,RT-PCR,calcification nodule alzarin red staining and immunofluorescence staining.The main results of this experiment are as follows:1.PDG has no effect on BMSCs’proliferation;2.PDG can significantly improve the ALP activity of BMSCs with an optimal concentration of 1×10-6 mol/L;3.PDG can significantly increase the number of clones with positive staining and calcified nodules;4.After PDG treatment,the expression of bone formation related proteins Collagen I,Runx2,OPG,OSX,and BMP2 were significantly upregulated ang the expression of bone resorption key protein RANKL was significantly downregulated,while the m RNA expression of bone formation related genes BMP2,Runx2,OSX,collagen I,and OPG were still significantly increased;5.PDG can significantly reduce the formation of oil droplets;6.After PDG treatment,there was a significant downregulation of adipogenic related proteins and genes;7.Adding Noggin,the ability of PDG to promote ALP activity in BMSCs,upregulate the expression of osteogenic related proteins,downregulate the expression of lipogenic related proteins,and increase the number of calcified nodules disappeared;8.After PDG treatment,the expression of BMP2/Smads signaling pathway related proteins BMPR II,BMPRⅠA,BMPRⅠB and p-Smad1/5/8 were significantly upregulated.The m RNA expression levels of related genes BMPR II,BMPRⅠA,BMPRⅠB and BMP2 were also significantly upregulated.Smad1/5/8 was phosphorylated and aggregated in the nucleus.While Adding Noggin reverses the above results.Based on the above results,the following conclusions are obtained:1.PDG has no effect on BMSCs’proliferation;2.PDG can significantly promote the osteogenic differentiation of BMSCs,and inhibit its adipogenic differentiation,and its optimal concentration is 1×10-6mol/L;3.PDG promotes osteogenic differentiation of BMSCs by activating the BMP2/Smads signaling pathway.This study lays the foundation for developing new anti osteoporosis drugs derived from Eucommia ulmoides Oliv. |
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