The Effect Of Osthole On The Activity And Expression Of IL-6 MRNA,Cbfa1mRNA Of Osteoblast In The Osteoblasts-osteoclasts Co-culture System

Objectives:To investigate the effects of Osthole on proliferation of osteoblast,ALP activity,expression of interleukin-6mRNA and Cbfa1mRNA in the osteoblasts-osteoclasts co-culture system.Based on the experimental results,we have discussed the mechanism of action of Osthole in bone metabolism.Methods:1.Osteoblasts and osteoclasts were isolated respectively from SD rats,1 and 5 days old. The osteoblasts-osteoclasts co-culture system was built to prevent the two kinds of cells from contact and allow the media to exchange.2.The proliferation of osteoblast,ALP activity and mineralization in co-culture and mono-culture groups were detected respectively by the MTT and PNPP methods,and alizarin staining.3.The MTT method was used to detect effect on the proliferation of osteoblasts in order to determine the best concentration of Osthole in the co-culture.4.The proliferation of osteoblasts,ALP activity,and osteoclasts TRAPase activity in the co-culture system were detected respectively by the MTT,PNPP,and TRAPase detection reagent, so that we know the effects of Osthole in the co-culture.5.The expressions of interleukin-6 and Cbfa1 were detected respectively by reverse transcriptase PCR.Results:1.After detection by Coomassie Brilliant blue staining,we demonstrated that the protein in OB culture room dispersed to OC culture room through membrane within 8 hours,which suggested that the establishment of plane osteoblast and osteoclast co-culture system was successful.2.The Osthole's concentration from 10^-5mol/L to 10^-8mol/L all can enhance the proliferation of osteoblasts,but the concentration of 10^-7 mol/L is the most obviouse.3.In the concentration of 10^-7mol/L Osthole co-culture system group,osteoblasts proliferation,ALP activity,were higher than those of control group(P＜0.05).However,the osteoclasts TRAPase activity was lower than that of the control group(P＜0.05).4.In the concentration of 10^-7mol/L Osthole co-culture system group,the expression of interleukin-6 were lower than those of control group(P＜0.05),and Cbfa1 were higher than those of control group(P＜0.05).Conclusion:1.Comparing to the mono-culture system,proliferation,differentiationand,mineraliziation of osteoblasts were enhanced in the co-culture system.2.Osthole can obviously enhance the proliferation of osteoblasts,ALP activity and up-regulate Cbfa1 mRNA and down-regulate IL-6mRNA.Meanwhile,it can also inhibit osteoclasts TRAP activity.