Study On The Pathogenesis Of PMOP And The Intervention Of Icari In Based On ER?/AMPK/Sirt1 Signaling Pathway

BackgroundWith the tendency of aging society,the incidence of Postmenopausal Osteoporosis(P MOP)is increasing year by year,as a result,its related complications and costs.have becom e a heavy burden of the health care system.In nowadays,pharmacological treatment is still the main treatment of the disease,despite the obvious clinical efficacy of these drugs,we sh ouldn't neglect the side effects,hence,it is necessary to carry out more basic research in ord er to offers a firm foundation for the development of anti-osteoporosis medicine.The studie s of signaling pathways have become a hotspot among the basic researches associated with PMOP,however,the pathogenesis of PMOP is a complex pathological process,the known molecular signaling pathways can not fully elucidate its pathogenesis,there may be some u nproven signaling pathways which involved in the pathogenesis of PMOP.Previous studies indicated that compared with Liuwei Dihuang Pills,Jinkui Shenqi Pills can significantly i mprove the bone mass and serum estradiol level of ovariectomized rats as well as promote t he proliferation and differentiation of osteoblasts,the underlying mechanism may be related to the up regulation of ER? and down regulation of p-AMPK by Jinkui Shenqi Pills.More over,si-ERa could increase the expression of p-AMPK which indicatad ERa may act as an upstream protein to regulate the expression and phosphorylation level of AMPK in osteobla sts.In addition,according to research reports,in the regulatory network of cells,AMPK is t he main upstream kinase of Sirtl and Sirtl is a positive protein of bone mass regulation.Th ese suggest that ER?,AMPK and Sirtl may regulate bone metabolism through a certain cas cade manner and participate in the pathogenesis of PMOP.Therefore,the present study pro poses the concept of ER?/AMPK/Sirtl signaling pathway for the first time.Ovariectomized mice,osteoblast lines was established in the study to verify this hypothesis from the level o f histomorphology and molecular biology and to explore the relationship between ER?/AM PK/Sirtl signaling pathway and PMOP,at last,ICA was used to elucidate the pathogenesis of PMOP and provide new ideas for clinical prevention and treatment of PMOP.ObjectiveTo confirm ER?/AMPK/Sirtl signaling pathway and explore its possible relationship with PMOP,in addition.ICA was used to investigate whether it can prevent PMOP through ER?/AMPK/Sirtl signaling pathway.Methods1.To obtain the optimal concentration of ICA.Mouse osteoblasts(MC3T3-E1 cells)w ere treated with different concentrations of ICA(ranged from10-6mM?10-3mM).Cell viabi lity,ALP activity,the number of mineralized nodules,the formation of autophagosomes,ap optosis rate,the expression of osteogenic differentiation markers such as Runx2,osterix,O CN,OPG and the expression of ERa were detected by CCK8,ALP staining or ALP activat e Kit,Alizarin red staining,MDC stain ing,ANNEXIN V-FITC/PI Kit,RT-qPCR respectiv ely,then,the optimal concentration of ICA was obtained by these experiments;2.To confirm ER?/AMPK/Sirtl signaling pathway,MC3T3-E1 cells were divided int o four groups,blank control group,ERa agonist group,ER? agonist group,ER?+ER? agon ist group.The specific agonists of ERa and ER?,PPT and DPN were used to overexpress E R? and ER? in osteoblast,cells viability,the number of autophagosomes,the level of osteo genic differentiation,the expression of Runx2,osterix,OPG,OCN,and the key markers of AMPK signaling pathway such as LKB1,CaMKK?,AMPK?1,Sirtl were measured by M TT,MDC staining,ALP staining and Alizarin red staining,RT-qPCR respectively.Morever,Western blotting was used to detect the expression of LKB1,CaMKK?,AMPK?1,p-AMP K?1,Sirtl in protein levels.At last,Co-Immunoprecipitation was performed to detect the in teraction between ERa and LKB1,CaMKK?,AMPK?1,and between ER? and LKB1,Ca MKK?,AMPK?1;3.To understand if ICA promotes osteogenic differentiation through ER?/AMPK/Sirt 1 signaling pathway,cells were divided into four groups:blank control group,ICA group,I CA+IC1 182780 group and ICA+Compound C group.Cells were pretreatmented by ERa sp ecific antagonist ICI 182780 or AMPK specific inhibitor Compound C in order to inhibit th e expression of ERa or AMPK,then RT-qPCR was used to detect the expression of collage nI,OPN,OCN,Runx2 and ERa as well as LKB1,CaMKK?,AMPK?1,Sirt1,the key mar kers of AMPK signaling pathway.Futhermore,Western blotting was also used todetect the expression of LKB1,CaMKK?,AMPK?1,p-AMPK?1,Sirt1 in protein level.4.To investigate if ICA prevents PMOP through ER?/AMPK/Sirtl signaling pathway,ovariectomised rat model was established and divided into four groups:Sham group,OVX group,ICA group,E2 group.After 12 weeks of treatment,the bone parameters of the distal femur were detected by Micro-CT and the expression of ER?,LKB1,CaMKK?.AMPK?1 and Sirtl were detected by RT-qPCR and Western blotting.Results1.Compared with the control group and other groups,ICA at the concentration of 10-5 mM can significantly increase the cell viability of osteoblasts as well as promote the ALP a ctivity and the formation of mineralized nodules,futhermore,ICA at the concentration of 10-5mM can increase the number of autophagosomes and inhibit the apoptosis of osteoblasts,at last,itcan also significantly increase the expression of ERa and the osteogenic differentia tion markers such as Runx2,osterix,OCN and OPG.2.Compared with the blank control group,ALP activity,mineralized nodules and auto phagosomes were increased in ERa agonist group and ER?+ER? agonist group,as well as t he expression level of osteogenic differentiation markers such as Runx2,Osterix,OPG,OC N and LKB1,CaMKK?,AMPKal,Sirt1,the key factors of AMPK signaling pathway.At t he same time,the result of CO-IP indicated that there was a direct interaction between ERa and LKB1 or AMPK?1,but no interaction was found between ER? and CaMKK?,in additi on,ER? interacted with AMPK?1 but not with CaMKK? and LKB1.3.Compared with the blank control group,ICA can significantly promote osteogenic d ifferentiation and upregulate the expression of ER?,LKB1,CaMKK?,AMPKal and Sirtl,while after pretreated by ICI 182780 or compound C,the expression of these markers was s ignificantly decreased.4.Compared with sham group,the bone micro parameters of OVX group were worse,accompany with the downregulation of ER?,LKB1,CaMKK?,AMPK?1 and Sirt1,while compared with OVX group,ICA group and E2 group could improve the expression of thes e markers.Conclusion1.In this study,the optimal concentration of ICA was 10-5mM.2.The present study had confirmed ER?/AMPK/Sirtl signaling pathway in osteoblasts,the activation of this signaling pathway can promote the proliferation and differentiation of osteoblasts.3.ICA can promote osteogenic differentiation through ER?/AMPK/Sirtl signaling pat hway.4.ER?/AMPK/Sirtl signaling pathway is involved in the pathogenesis of PMOP in rat s model and ICA can prevent PMOP in rat through ER?/AMPK/Sirtl signaling pathway.