| Existing research shows that new natural products and bioactive substances directly stimulating bone formation may be an effective way to treat osteoporosis.This paper selects twenty glycopolymers extracted from Humulus lupulus(HL),Achyranthes bidentata(AB),Polygonatum sibiricum(PS),Alhagi pseudlhagi(AP),Morinda officinalis(MO)and Curculigo orchioides(CO)in the early stage of the research team to evaluate the osteogenic effects in vitro.The intrinsic mechanism of Achyranthes bidentata glycopolymer ABW90-1 and Morinda officinalis glycopolymer MOPB-2 with better activity was studied.Twenty refined glycopolymers were separated from the early research in our group,including Humulus lupulus refined glycopolymers(HLP50-1,HLP70-2-1,HLP70-2-2,HLP70-3,HLBP-2 and HLBP-4),Achyranthes bidentata refined glycopolymers(ABPB-4 and ABW90-1),Polygonatum sibiricum refined glycopolymers(PSP90-1,PSP90-2 and PSP50-2-2),Alhagi pseudlhagi refined glycopolymers(APP90-1-1,APP90-1-2,APP90-2,APP70-3-1,APBP-2-2 and APBP-2-3),Morinda officinalis refined glycopolymers(MOW50-1 and MOPB-2)and Curculigo orchioides refined glycopolymers(COP50-4).Twenty refined glycopolymers were applied to MC3T3-E1 cells and then to detect the effects on the proliferation,differentiation and mineralization,including MTT experiment,ALP activity experiment and Alizarin red staining experiment.The experimental results showed that twelve glycopolymers had the effect on promoting the proliferation of MC3T3-E1 cells including HLP50-1,HLP70-2-1,HLP70-2-2,HLP70-3,HLBP-2,HLBP-4,ABPB-4,PSP90-1,PSP90-2,APP90-2,APBP-2-2 and MOPB-2;twenty refined glycopolymers could increase the ALP activity and mineralization ability of MC3T3-E1 cells,and a certain concentration of HLP50-1,HLP70-2-1,ABPB-4 and MOPB-2 promoted ALP activity and mineralization as much as E2,and a certain concentration of HLP70-2-2,ABW90-1 and COP50-4’s promotion effect is stronger than that of positive drug E2.The above results showed that these twenty refined glycopolymers had good bone-promoting activity in vitro.In this project,combining the activity and the actual accumulation of refined glycopolymers,ABW90-1 and MOPB-2 were selected for subsequent mechanism research.In order to clarify the mechanism of the glycopolymers ABW90-1and MOPB-2 in promoting bone formation,qRT-PCR,Western blot,molecular docking and immunofluorescence methods were used to explore the effects of the two glycopolymers on osteogenesis-related pathways.Molecular docking experiments showed that ABW90-1 had a strong interaction with WNT pathway complex(-5.9394 kcal/mol)or BMP2(-5.9622 kcal/mol).In addition,ABW90-1(25 μM)activated WNT/β-catenin and BMP2/SMAD1 signaling pathway by promoting the expression of active-β-catenin,p-GSK-3β,LEF-1,BMP2,p-SMAD1 and the nuclear transfer of β-catenin.Studies revealed that the bone-promoting effect of ABW90-1 could be partially inhibited by DKK-1(specific inhibitor of WNT pathway)and Noggin(specific inhibitor of BMP pathway).Collectively,these findings indicated that ABW90-1 promoted bone formation through WNT/β-catenin and BMP2/SMAD1 signaling pathways.Moreover,studies indicated that MOPB-2 activated the BMP2/SMAD1 pathway by significantly up-regulating the expression of BMP2 and p-SMAD1 in the BMP pathway.LDN-193189(BMP pathway inhibitor)could significantly inhibit MOPB-2’s promotion effects of proliferation,differentiation,mineralization,and osteogenic-related gene protein expression levels,further confirming that MOPB-2 promoted osteogenesis through the BMP2/SMAD1 pathway.In summary,this thesis preliminarily clarified the active parts and ingredients of Humulus lupulus,Achyranthes bidentata,Polygonatum sibiricum,Alhagi pseudlhagi,Morinda officinalis and Curculigo orchioides.It systematically explained that ABW90-1 promoted bone formation through WNT/β-catenin and BMP2/SMAD1 signaling pathway and MOPB-2 had effects on promoting bone formation through BMP2/SMAD1 pathway.All of the above have laid the foundation for the research of glycopolymers on anti-osteoporosis. |
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