The Effect And Molecular Mechanism Of Bone-targeted Estrogen On Human Osteoblasts

Objectives: With the aging of human society, postmenopausal osteoporosis (PMOP) has an impact on the life quality of older women as a frequently-occurring disease, its primary cause is post-menopausal estrogen levels decrease. With bone targeting estrogen drugs can avoid the toxicity of estrogen on breast, ovarian and other non-target organ, while using them to treat the PMOP. Therefore, these drugs are a hot research currently. Our room use bone-targeted molecule rhein as the carrier, estrone as a parent, synthesis of bone-targeted estrogen (rhein-piperizinyl-estrone, named LC). Previous studies have confirmed that LC has significant anti-osteoporosis activities on ovariectomized rats. In this experiment, further study the effect of LC on human osteoblast-like cell line MG-63, include proliferative activity, differentiation function, the expression of osteoprotegerin (OPG)/ the receptor activator of NF-κB ligand (RANKL)/ receptor activator of NF-κB, (RANK) system, the expression of interleukin-6 (IL-6) and its receptor, also explore possible molecular mechanism of LC.Methods: 1.The proliferation activity assay with MTT method. 2.Cell cycle assay with flow cytometry. 3. Alkaline phosphatase (ALP) activity assay with colorimetric method. 4.The expression levels of OPG, RANKL, IL-6, IL-6 receptor (IL-6Rα, gp130) mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR). 5.The expression level of IL-6 protein was detected by enzyme-linked immnosorbent assay (ELISA). 6.The expression levels of OPG, RANKL, IL-6Rα, gp130 protein were detected by Western Blot. 7.Using RNA interference (RNAi) technology for estrogen receptor-α(ERα), estrogen receptor-β(ERβ) of MG-63 to conduct gene silencing, G418 screened for positive monoclonal expansion of cultured, then using Western Blot identification to choose ERα/ERβhigh and medium inhibit expression clones for exploring the affinity of LC to ERαand ERβ. 8. Using Luciferase reporter gene assay and mitogen activated protein kinase (MAPK) specific antagonist to explore the signaling pathways related the effect of LC.Results:1.The effect of LC on Biology nature of human osteoblast-like cell line MG-63LC could markedly promote MG-63 cell proliferation in dose-dependent manner, also advanced cell growth by altering cell cycle distribution, it made MG-63 cells a significant decrease in the G1 phase and increase in the G2+S phase. LC stimulated cell differentiation through promoting the activities of ALP of MG-63 cell. LC could markedly up-regulated mRNA and protein levels of OPG in dose-dependent manner of MG-63 cells, markedly down-regulated RANKL mRNA and protein expression levels,the most effective dose was 100 nM. Besides,LC could inhibit the expression of IL-6 mRNA and protein of MG-63 cells; it had depressant effect on IL-6RαmRNA expression,but had no effect on IL-6Rαprotein expression; LC had no effect on both transcriptional and protein expression levels of gp130.2.The molecular mechanism of LC on human osteoblastEstrogen receptor (ER) antagonist ICI182780 (Brand name: Fulvestrant) could completely abolish the effect of LC on human osteoblast, it indicated that LC affect on osteoblast through estrogen receptors. The effect of LC on the clones of super-inhibit ERαexpression and super-inhibit ERβexpression had no significant difference, so its affinity to ERαand ERβwere similar. LC could markedly enhance ER promoter activation, accounting that LC might work through the classical Genomic mechanism (ERE means); while MAPK specific antagonist PD98059 (ERK inhibitors),SP600125(JNK inhibitors),SB202190(p38MAPK inhibitors) could partly block the effect of LC, suggesting that the effect of LC on human osteoblast also depended on non-Genomic mechanism (MAPK signaling pathway).Conclusions: 1.LC could stimulate human osteoblast proliferation and differentiation, also advance cell growth by altering cell cycle distribution. 2.LC could promote the expression of OPG and decrease the expression of RANKL and IL-6 of osteoblast. 3.The Effect of LC on osteoblast through estrogen receptors, and it had no selectivity between ERαand ERβ. 4. LC play its role in osteoblast through activating ERE means and MAPK pathway.