The Effects Of Genistein Derivatives On HFOB1.19 Activity And OPG/RANKL Expression

Object:Study effect of different concentrations and time of genistein derivatives on proliferation?differentiation and apoptosis of hFOB1.19 cells and investigate high viable genistein derivatives affect viability of hFOB1.19 cells whether regulate level of OPG/RANKL protein via ER? and ER? signaling pathway.It provides a novel direction of new drug and base of theory and research for treatment of postmenopausal osteoporosis by estrogen replacement therapy.Methods: After treated with various concentrations(0?10-5?10-3?10-1?10?M) of four genistein derivatives(WH-3-39?WH-4-20?WH-4-40 and WH-4-41) and genistein for respectively 24,48,72 h,hFOB1.19 cells were detected by MTT to estimate cell activity and choose effective compounds.The hFOB1.19 cells were collectively cultivated with different doses of WH-3-39 and WH-4-20(0?10-5?10-3?10-1?10?M)for 24,48 h,then action of cell apoptosis was determined by AO/EB and DAPI staining and its alkaline phosphatase(ALP) activity was detected by kit.Effect of mineralized nodule was measured and quantified by 0.1% Alizarin Red S.The protein level of OPG/RANKL of hFOB1.19 cells originated from treatment with different concentrations of WH-3-39 and WH-4-20 for separately 24,48 h,was adopted by western blot,then ICI and estradiol respectively were regarded as estrogen receptor blocker and positive drug and they were put into hFOB1.19 cells that were disposed of more suitable concentrations of 10-3?M WH-4-20 for 24 h and 10?M WH-3-39 for 48 h,which affected expression of OPG and RANKL.What's more,kinds of selective antagonist ER? and ER? incubation with hFOB1.19 cells for 24 h was filtrated the best inhibition concentration and impacted on OPG and RANKL expression.Furthermore,after treatment with selective antagonist ER? and ER?,the expression of OPG and RANKL on hFOB1.19 cells was influence on genistein derivatives of WH-4-20 and WH-3-39.Results:1.We observed higher concentration group of WH-4-40 and WH-4-41 only had effect on cell proliferation compared with control group,but all doses of genistein?WH-4-20 and WH-3-39 stimulated cell viability and were greater effective proliferation than WH-4-40 and WH-4-41,in addition,the better concentration was 10-3?M.The cell growth curve that hFOB1.19 cells were disposed by 10-1?M of WH-4-40 and WH-4-41 and 10-3?M of genistein ? WH-4-20 and WH-3-39 for respectively 24,48,72 h indicated the function of cell viability of WH-4-20 and WH-3-39 was significantly stronger than other compounds and control group.2.Compared with control group,after treatment with different doses of WH-4-20 and WH-3-39(0?10-5?10-3?10-1?10?M)to hFOB1.19 osteoblast for separately 24,48 h,all groups of the ALP activity was clearly increased and the highest ALP density was 10-3?M group and its ALP density was enhanced as time increased,but dose of other groups' ALP density was descended in different degree as time increased.In comparison with control group,mineralization of nodule formation was promoted by each group of doses of WH-4-20 and WH-3-39 combination with osteoblast.3.In condition of serum starve-induced osteoblast apoptosis,each concentration of WH-4-20 and WH-3-39 could reduce cell apoptosis compared to control group.The best ability of inhibition concentration was 10-3?M.4.We got the following results by means of western blot.(1)in 24 h,compared to 0?M group, the expression of OPG that belonged to 10-5 and 10-3?M group of WH-4-20 and WH-3-39 was significantly increased and its RANKL expression was obviously declined and the density of maximum expression of OPG was 10-3?M,while the expression of OPG didn't been changed by 10-5 and 10-3?M group and its RANKL expression was further reduced.in 48 h,each group of OPG expression was apparent difference in comparison with control group and there was no noticeable change among drug groups,but quantity of RANKL expression was gradually brought down.Compared to control group,The osteoblastic expression of OPG was improved by various concentrations of WH-3-39 as a dose-response way and the maximum group of relative OPG expression was 10-3?M,then RANKL expression was no regular changes of the whole groups,but 10?M of RANKL protein was slowly reduced with time lasting and was lower to 0?M,while it compared with other groups,there was no difference.(2)The ER antagonist could clearly decrease OPG expression and elevate RANKL expression,but also invert effect of OPG and RANKL of WH-4-20 and WH-3-39.(3)After hFOB1.19 cells were treated with different concentrations of selective receptor blocker of ER? and ER?,We found expression of ER? protein with the increase of concentration was added,but OPG expression was reduced.The protein of RANKL expression was not regularity,but compared to 0?M,other groups was clearly decreased.The groups of ER? and OPG protein compared with 0?M was shown as a dose—dependent way to apparently decline.RANKL protein was not regularly expressed.(4)We detected inhibiting ER? and ER? signaling pathway could downregulate effect of OPG produce and OPG/RANKL ratio of WH-4-20 on hFOB1.19 cells.In context of blocking ER? signaling pathway,OPG expression was enhanced and content of RANKL was reduced by WH-3-39,but suppressing the ER? pathway simultaneously declined protein levels of OPG and RANKL.Conclusion:1.Genistein derivatives of WH-4-20 and WH-3-39 could enhance cell activity and differentiation and inhibit cell apoptosis on hFOB1.19 osteoblast.2.A certain concentration of WH-4-20 and WH-3-39 obviously facilitated OPG expression and increased portion of OPG/RANKL.This function was interfered by ER antagonist,thereby, WH-4-20 and WH-3-39 may made impact on OPG and RANKL expression of hFOB1.19 via ER? and ER? pathway.