Chlorogenic Acid Prevents Osteoporosis By Shp2/PI3K/Akt Pathway In Ovariectomized Rats

Objective:The aim of this study was to determine whether chlorogenic acid (CGA) can prevent estrogen deficiency-induced osteoporosis and to analysize CGA's mechanism of bioactivity.Methods:(1) Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups:saline vehicle,17a-ethinylestradiol (E2), or CGA at 9,27, or 45 mg/kg/d.(2) Two-dimensional total bone mineral content (t-BMC) and total bone mineral density(t-BMD) of the right femur were measured by Lunar Prodigy Advance system(3) The rats'femoral metaphyses were evaluated by micro-computed tomography (?CT).(4) Serum calcium (S-Ca), phosphorus (S-P), ALP Serum osteocalcin (OC) and Urine calcium (U-Ca), phosphorus (U-P), creatinine (Cr), Urinary deoxypyridinoline (DPD) were be analyzed.(5) Using the MTT assay CGA proliferation of BMSCs, CGA at concentrations effective the protein expression of Akt, p-Akt, cyclinD 1 were be detected(6) BMSCs induced osteogenic differentiation for 7 days to detect the ALP activity levels, analysis CGA osteogenic differentiation of BMSCs.(7) Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002, Detect BMSCs cell activity and cyclinD 1 protein expression.(8) CGA at 1 or 10 ? M increased BMSC differentiation to osteoblasts, Shp2 RNAi suppressed CGA-induced osteoblast differentiation by decreasing Shp2, p-Akt, and cyclin D1.Results:(1) CGA at 27 and 45 mg/kg/day inhibited the OVX-induced decrease in femur bone mineral density (BMD) and significantly decreased levels of bone turnover markers, prevented decreases in the bone volume/tissue volume (BV/TV), connect density (Conn.D), trabecula number (Tb.N) and trabecula thickness (Tb.Th), and prevented increases in the trabecular separation (Tb.Sp) and structure model index (SMI).(2) MTT showed CGA (concentration between 0.1-10 ?mol/L) significantly promoted BMSCs proliferation and ALP activity, and promote p-Akt cyclin D1 protein expression, and the effect of different concentrations were significantly different (P<0.05), in a dose-dependent manner.(3) In the presence of a PI3K inhibitor LY294002, CGA (10?mol/L) on BMSCs proliferation had no significant effect (P> 0.05), but the CGA alone under BMSCs proliferation activity was significantly increased (P<0.05) and the expression of cyclin D1 was significantly increased.(4) In Sph2 RNAi presence, CGA ((10 ? mol/L)) on BMSCs proliferation had no significant effect (P> 0.05), but under the CGA alone condition BMSCs proliferation activity was significantly increased (P<0.05) and Sph2, p-Akt and cyclin D1 protein expression was significantly increased.Conclusion:(1) CGA improved bone quality by modifying the BMD and trabecular micro-architecture.(2) CGA promoted proliferation of osteoblast precursors and osteoblastic differentiation of BMSCs via the Shp2/PI3K/Akt/cyclin D1 pathway.(3) CGA might be an effective alternative treatment for postmenopausal osteoporosis.