Discovery Of Bioactive Constituents From Absorbed Constituents Of XLGB With Promoting Differentiation And Mineralization Activity On MC3T3-E1 Cells And Preliminary Study On Action Mechanism Of Sweroside

Objective:Xianlinggubao Capsule(XLGB)consists of six herbs: Fructus Psoraleae,Radix dipsaci,Herba epimedii,Radix salvia miltiorrhizae,Rhizoma anemarrahenae,and Rehmannia glutinosa.XLGB is mainly used for the prevention and treatment of osteoporosis.Our research group previous reported that XLGB-treated plasma sample promoted osteoblastic differentiation and mineralization on MC3T3-E1 cells.To further explore bioactive constituents from absorbed constituents of XLGB with promoting cell differentiation and mineralization,and to reveal its possible action mechanism,our research group carried out the following study.Six representative absorbed constituents(psoralen,neobavaisoflavone,icariin,sweroside,magnoflorine,asperosaponin VI)were selected,and then employed to prepare a combination of these six constituents according to their concentrations in rat plasma.We found that the combination significantly promoted cell mineralization on MC3T3-E1 cells,while each constituent at this concentration had no obvious osteoblastic mineralization,which suggested that six constituents might synergistically promote cell mineralization.However,the bioactive constituents of the combination and their underlying mechanisms were still unclear,so our study aimed to determine the active constituents of the combination as well as to explore the action mechanisms.Methods:1.Discovery of bioactive constituents from absorbed constituents of XLGB.Six representative constituents(psoralen,neobavaisoflavone,icariin,magnoflorine,sweroside,asperosaponin VI)were selected due to chemical structural diversity and their concentrations in rat plasma,and then employed to prepare a combination of these six constituents according to their concentrations in rat plasma.The combination was evaluated by osteoblastic proliferation,differentiation and mineralization assay on MC3T3-E1 cells.The effects of each component on differentiation and mineralization of MC3T3-E1 cells were explored by the systemic evaluation of each component and the combination with knocking out components,and finally determined the bioactive constituents from absorbed constituents of XLGB.2.The effects of sweroside on MC3T3-E1 cells.Based on previous exploration of the concentrations of sweroside in proliferation,differentiation and mineralization on MC3T3-E1 cells,osteoblastic activity of sweroside was further evaluated at the concentrations of 0.5 ?M to 10 ?M by cell proliferation,differentiation and mineralization assay.3.The effects of sweroside on estrogen receptors(ERs).The interaction between sweroside and estrogen receptors was predicted by the means of molecular docking,and further validated on sweroside induced ALP activity by treating with ER-? antagonist MPP and ER-? antagonist PHTPP on MC3T3-E1 cells.The effect of sweroside on ER-? expression was evaluated by ER antagonist ICI 182780 treatment.4.The study on the distribution of sweroside in the intracellular and extracellular fluids of MC3T3-E1 cells.To reflect the actual absorption degree of sweroside on MC3T3-E1 cells,the concentrations of sweroside in intracellular and extracellular fluids at different time points were measured by previous constructed UPLC/TQ-XS-MS method.5.The study on the effect of sweroside on estrogen membrane receptor GPR30.The effect of sweroside on GPR30 expression was evaluated.Cell differentiation and GPR30 expression were investigated by treating with a selective GPR30 agonist G1 and GPR30 antagonist G15.6.The effect of sweroside in MAPK signaling pathway.Western blot was performed at different time points after treated with sweroside at 1 ?M on MC3T3-E1 cells,and further validated via co-treatment with the antagonists.Results:1.The combination(6Mix 20×)of six constituents(sweroside 0.0298 ?M,psoralen 0.0269 ?M,magnoflorine 6.2 n M,asperosaponin VI 0.201 n M,icariin 3.730 p M,neobavaisoflavone 0.039 n M)could maximally promote osteoblastic differentiation and mineralization on MC3T3-E1 cells,while each constituent had not osteogenic activity.The differentiation and mineralization of the combination of six constituents were differently decreased after knocking out sweroside(0.0298 ?M),or psoralen(0.0269 ?M),or magnoflorine(6.2 n M),respectively.However,the differentiation and mineralization of the combination of psoralen,sweroside and magnoflorine(PMS)was comparable to the combination of six constituents,and osteoblastic activity was decreased after PMS knocked out any constituents.2.Sweroside promoted the osteoblastic differentiation and mineralization on MC3T3-E1 cells,especially sweroside at 1 ?M induced a maximal enhancement of osteoblastic differentiation and mineralization,which was equivalent to the effects of 17?-estradiol at 10 n M.3.The results of molecular docking indicated that sweroside has high binding affinities to estrogen receptors,and sweroside induced ALP activity were significantly inhibited by ER-? antagonist MPP and ER-? antagonist PHTPP.Sweroside also promoted the expression of ER-?.4.The results of intracellular and extracellular fluids showed the intracellular concentration of sweroside was only 2.5 n M and extremely lower than the extracellular concentration of 1 ?M.5.Sweroside promoted the expression of estrogen membrane receptor GPR30,and a GPR30 antagonist G15 significantly decreased sweroside-induced ALP activity.6.Sweroside activated the phosphorylation of p38(p-p38),while did not obvious affect the phosphorylations of extracellular signal-regulated kinase(ERK)and c-Jun N terminal kinase(JNK).Moreover,the improved ALP activity,mineralization and the expression of p-p38 increased by sweroside were markedly reduced by a p38 antagonist SB203580 treatment,and the expression of p-p38 was obvious inhibited by G15 treatment.Conclusion:1.Psoralen,sweroside and magnoflorine are bioactive constituents from absorbed constituents of XLGB with promoting differentiation and mineralization on MC3T3-E1 cells.The combination of them has better osteoblastic activity than each component,which indicates they synergistically promote osteoblastic activity.2.Sweroside promotes osteoblastic differentiation and mineralization via estrogen membrane receptor GPR30 mediates p38 signaling pathway on MC3T3-E1 cells.Moreover,GPR30 interacts with ER-? to promote osteoblastic activity,and the mechanism needs further study.