| Osteoporosis（OP）is a kind of metabolic bone disease which seriously perplexes the health of middle-aged and elderly people.At present,there is no ideal treatment drug.Although bisphosphonates are very popular,their oral absorption is not good,and there are obvious side effects.Therefore,it is still an urgent task to find new safe and effective anti-osteoporosis drugs.In recent years,Chinese herbal medicine has aroused great interest in the prevention and treatment of osteoporosis due to its low cost,easy access,small side effects and other characteristics.In this paper,according to the literature reports and previous research results,four Chinese herbal medicine monomers,icariin（ICA）,pinoresinol diglucoside（PDG）,costunolide（COS）and dehydrocostunolide（DEH）,were selected to compare their effects on the osteogenic differentiation of rat calvarial osteoblasts（ROBs）cultured in vitro,In order to lay a foundation for the development of new anti-osteoporosis drugs,the signal pathway of osteogenic activity was studied after determining the best one.The main experimental methods and steps of this paper are as follows:1.The osteoblasts from calvaria of neonatal rats within 48 hours of birth were extracted,isolated and cultured by enzymatic hydrolysis;2.Six concentrations of ICA,PDG,cos and DEH(0,1×10-5,1×10-6,1×10-7,1×10-8,1×10-9mol/l)were used for comparative study,and the activity of alkaline phosphatase（ALP）and the expression of osteogenic related genes and proteins were used as indicators to screen and determine the best monomer;3.Histochemical staining was used to explore the effect of the best monomer compound on the formation of ALP positive clones and calcified nodules of osteoblasts;4.Western blot and real time PCR were used to study the effects of the best monomers on the gene and protein expression of BMP-2,Runx-2,collagen I and OSX;5.Western blot,real time PCR and immunofluorescence staining were used to explore the best monomer pair for Wnt and BMP-2 signaling pathway on the activity of BMSCs were studied.The main results are as follows:1.According to the effect of PDG on ALP activity,the increase of ALP activity by PDG is the largest,followed by ICA.2.ICA,PDG and COS can increase the expression of osteogenic marker protein of ROBs,but DEH has no obvious effect.The result of gene expression was similar to that of protein.In general,the improvement effect of PDG is the highest and the most stable.3.PDG significantly increased the number of ALP positive clones and calcified nodules（P<0.01）.4.The gene expression of OSX,OCN and collagen I in PDG treated ROBs for 24 hours was significantly higher than that in the control group,and the expression of OSX and collagen I began to decrease after 48 hours.5.After PDG treatment of robs for 24 hours,The protein expression of Wnt 10b andβ-catenin increased,The nuclear translocation ofβ-catenin was obvious;The expression levels of BMP-2 and Runx-2 in BMP-2signaling pathway increased 24h after PDG treatment,and further increased 48h after PDG treatment,but Runx-2 decreased slightly.The above results showed that PDG was the best way to promote the formation of osteoblasts in vitro among the four Chinese medicine monomers,ICA,PDG,COS and DEH.The optimal concentration(1×10-6mol/l)of PDG significantly increased the ALP activity of ROBs.ALP staining was used to identify positive clones and calcified nodules,and promote the expression of osteogenic genes and proteins.PDG not only activated Wnt signal pathway,but also increased the activity of BMP-2 signal pathway,indicating that the promotion of osteogenic activity might be completed by the cooperation of the two pathways. |
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