The Effect Of β-sitosterol On The Inhabition Of Osteoclast Differentiation And Prevention Of Bone Loss In Ovariectomized Mice

Objective:(1)To analyze the effective components and possible molecular mechanism of Angelica sinensis in the treatment of osteoporosis by the network pharmacology.(2)To explore the effect of β-sitosterol on the growth and differentiation of osteoclasts.(3)To explore whether β-sitosterol can improve the bone quality in ovariectomized mice.Method:(1)Screening the active ingredients and targets of Angelica by Traditional Chinese Medicine System Pharmacology Database(TCMSP).By using GeneCards,OMIM,PHARMGKB and other databases to find the relevant targets of osteoporosis.Take the intersection of the points and the target points of the active ingredients of Angelica sinensis to obtain the potential targets of the active ingredients of Angelica sinensis for the treatment of osteoporosis.Then,using the Clusterprofiler package in the R language to perform KEGG pathway enrichment analysis about the potential therapeutic targets and pathways of the active ingredients of Angelica.Finally,the active ingredients of Angelica sinensis were molecularly docked with the therapeutic targets to predict the binding ability of the two.(2)First,the cytotoxicity of different concentrations of p-sitosterol on bone marrow mesenchymal stem cells and RAW264 were tested by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoIiumromide(MTT).Subsequently,the effect of β-sitosterol on the osteogenic differentiation of mesenchymal stem cells was evaluated by alizarin red staining.Through tartrate-resistant acid phosphatase assay(TRAP),cell migration assay,immunofluorescence,bone resorption Lace assay,etc.,to evaluate the effect of(3-sitosterol on osteoclast differentiation,migration,actin ring formation and bone resorpion in RAW264.7 cells.Finally,the expression levels of related genes and proteins in osteoclasts were compared by real-time fluorescent quantitative PCR and Western blotting(WB)to explore the molecular mechanism of β-sitosterol inhibiting osteoclasts.(3)Twenty-four C57 BL/6J mice were randomly divided into sham operation group,ovariectomy group and β-sitosterol intervention group(20 mg/kg).After 6 weeks of intervention,the bilateral femurs of the mice were harvested,and the bone microstructure parameters,including femoral bone mineral density,bone volume fraction,trabecular bone number and bone trabecular thickness,were compared by Micro-CT scanning.The numbers of trabecular bone and osteoclasts in bone tissue sections were compared by hematoxylin-eosin staining(HE)and TRAP staining.Result:(1)The screening results of TCMSP database show β-sitosterol has many common targets with osteoporosis.The result means that β-sitosterol is a potential anti-osteoporosis drug.The biological processes involved in β-sitosterol mainly include aging,reactive oxygen species metabolism and angiogenesis.Important genes involved in the regulation of osteoporosis by β-sitosterol include HSP90AA1,PIK3CG,ADRB2,BCL2 and c-Jun.The KEGG pathway enrichment analysis of 13 common targets showed that the cAMP pathway was associated with osteoporosis,and c-Jun was a downstream expressed gene of the cAMP pathway.The binding ability of β-sitosterol to the above important genes is c-Jun,ADRB2,PIK3CG,BCL2 and HSP90AA1 from strong to weak.Therefore,β-sitosterol may play a role in the treatment of osteoporosis by regulating the cAMP pathway and its downstream gene c-Jun.(2)MTT results showed that β-sitosterol at concentrations of 0.1 μM-20 μM had no obvious toxicity to bone marrow mesenchymal stem cells and RAW264.7 cells.1 μM,10μM and 20 μM of β-sitosterol did not significantly promote or inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells.With the increase of β-sitosterol concentration,the number and volume of osteoclast-like cells induced by RANKL gradually decreased,indicating that its inhibitory effect on osteoclasts was concentration-dependent.In terms of the effect on cell migration ability,β-sitosterol can effectively inhibit the migration and fusion of osteoclast precursor RAW264.7 cells.In terms of bone resorption function,compared with the RANKL-induced group,the number and volume of the osteoclast bone resorption function ring-F-actin ring in the β-sitosterol group decreased,indicating that it can inhibit the bone resorption function of osteoclasts.generation of the ring.Further,the bone resorption lacuna experiment showed that the number of bone resorption lacuna decreased after β-sitosterol intervention,indicating thatβ-sitosterol can not only reduce the production of F-actin ring,but also inhibit the bone resorption function of osteoclasts.Real-time fluorescence quantitative PCR showed thatβ-sitosterol dose-dependently inhibited the expression of c-Fos,NF-κB and NFATc1,the related landmark genes in the cAMP pathway and NF-κB pathway,and promoted the NF-κB pathway inhibitor IκB-α expression level.WB results showed that the protein expression levels of PKA,NFATc1,NF-κB and c-Jun in the cAMP pathway and NF-κB pathway were significantly down-regulated on the 3rd and 5th day after β-sitosterol intervention compared with the RANKL group;In addition,β-sitosterol also attenuated the phosphorylation of CREB protein in osteoclasts.The above results indicate that β-sitosterol can inhibit the generation and function of osteoclasts by regulating the cAMP pathway and the NF-κB pathway.(3)Micro-CT scan results showed that the femoral trabecular bones in the drug intervention group were denser than those in the ovariectomized group,with some thicker trabecular bones and smaller trabecular spacing.The software quantitative analysis showed that compared with the ovariectomized group,the femur bone mineral density(BMD),bone volume per tissue volume(BV/TV),and trabecular bone density(BV/TV)of the mice in the drug intervention group were compared with those in the ovariectomized group.Number,Tb.N),trabecular thickness(Tb.Th),trabecular spacing(Tb.Sp)and cortical thickness(Ct.Th)have been improved to varying degrees trend.The results of HE staining and TRAP staining further confirmed that β-sitosterol can improve the bone loss state of ovariectomized mice by increasing the number of bone trabeculae and reducing the number of osteoclasts.Conclusion:(1)Network pharmacology analysis indicated that β-sitosterol may be a potential anti-osteoporotic supplement.The results of target gene prediction,pathway enrichment analysis,and molecular docking showed that β-sitosterol may affect osteoporosis by regulating the cAMP pathway and c-Jun gene.(2)Cell experiments showed that β-sitosterol can inhibit the migration of osteoclast precursor cells,reduce the generation of RANKL induced osteoclast-like cell,suppress the formation of F-actin rings,and finally attenuate the bone absorption of osteoclast.The molecular mechanism is related to the regulation of cAMP pathway and NF-κB pathway.(3)Animal experiments showed that β-sitosterol can prevent the bone loss of ovariectomized mice by reducing the number of osteoclasts and inhibiting the resorption of trabecular bone.Therefore,β-sitosterol is a potential therapeutic drug for the prevention of osteoporosis.