Effects On Proliferation And Differentiation Of Osteoblast And Influence On Its OPG/RANKL Signaling Pathway Of Icariin

Obeject:To determine the effects on proliferation and differentiation of h FOB1.19 osteoblast and study the influence on its OPG/RANKL signaling pathway of icariin. It provides a experimental basis for the clinical application of icariin and medicine therapy of bone metabolic diseases.Methods:The h FOB1.19 osteoblast was treated with different concentrations(0, 0.001, 0.1, 10, 50, 100, 500, 1000ng/m L) to cultivating 24 hours. Then we assessed the proliferation of these cells by MTT method and determined its alkaline phosphatase activiby by alkaline phosphatase kit. The Alizarin Red staining was used to evaluate the mineralization of h FOB1.19 osteoblast by different concentrations of icariin. The m RNA expression of osteoblast’s Col I and BGP as the marker gene of differentiation were detected by q RT-PCR. The levels of TGF-β1 factor and NF-ΚB of h FOB1.19 osteoblast after treating icariin we measured by ELISA method. The q RT-PCR was used to estimate m RNA levels of osteoblast’s OPG and RANKL and the western blot was used to detect its protein levels. Afer treating ICI as estrogen receptor antagonist or estradiol as positive program or icariin, the osteoblast’s OPG and RANKL also were detected by q RT-PCR to its m RNA levels and western blot to its protein levels.Results:1. We found that the concentrations of icariin in the range of 0.001 to 1000 ng/m L have effect on the promoting proliferation of h FOB1.19 osteoblast(p<0.05 vs control).From the range of 0.001-50 ng/m L,the effect on proliferation increased along with the increasing concentrations of icariin. While the larger dose of icariin like 100ng/m L,500 ng/m L, 1000 ng/m L could decreased the proliferation effects. The osteoblast group treating 50ng/m L icariin was the strongest proliferation to compare with control group(p<0.01) and The increment effect is 1.45 time than control group. This results explain that icariin can promote effetct on the osteoblast proliferation in the certain concentration range. 2. Afer dealing with 0.1,10,50,100,and 1000 ng/m L icariin to h FOB1.19 osteoblast,the ALP activity of osteoblast was enhanced,the formating calcium nodules were increased significantly and the m RNA expression of Co1 I and BGP were increased in 0.1,10,50 ng/m L icariin groups(p<0.05 vs control).The ALP value and the Co1 I,BGP m RNA expression of 50 ng/m L icariin group were 2.14, 2.75 and 2.15 times to control group(p<0.01 vs control),respectively. The formating calcium nodules increased most significantly in 50 ng/m L icariin group. The results indicate that the concentrations of 0.1-50ng/m L icariin have the role of promoting differentiation and the 50ng/m L icariin was the best effect on differentiation of osteoblast. 3. Icariin can promote the growth factor TGF-β1 secretion and inhibit the nuclear factor NF-ΚB expression of h FOB1.19 osteoblast. 4. The concentrations of 0.1-50ng/m L icariin can enhance the OPG m RNA expression,to some extent concentration can increase the OPG protein expression, while the RANKL have not clearly expression in protein.From the results, 50ng/m L group can express hightest in m RNA and protein. the 50 ng/m L icariin group was the maximum OPG/RANKL ratio of osteoblast m RNA and protein levels being 2.03 and 1.24 times compared with control group,respectively(p<0.01 vs control). 5. Estrogen receptor blockers(ICI) have significantly inhibitory effect on icariin increasing the expressions of OPG m RNA and protein and the OPG / RANKL ratio(p<0.01 vs control). The results showed that the founction of icariin on regulation of OPG and RANKL is related to the estrogen receptor.Conclusion:1. Icariin has effect on promoting proliferation, differentiation and calcium nodules formation of h FOB1.19 osteoblast.It can increase the amount of osteoblast’s alkaline phosphatase(ALP), osteocalcin(BGP), type I collagen and transforming growth factor TGF-β1. However, it reduced expression of NF-κB. The roles of icariin promoting proliferation,differation and mineralization of osteoblast are in a concentration-dependent manner. 2. Icariin can up-regulate the expression of OPG m RNA and protein, and obviouly upregulate the OPG/RANKL ratio. Icariin has related to estrogen receptor when it comes to the regulation of OPG and RANKL. The founction of icariin on up-regulation of OPG expression and OPG/RANKL ratio is related to the estrogen receptor.