| Objective Osteoporosis is one of the main causes of pathological fractures in postmenopausal women,which is mainly caused by the excessive activation of osteoclasts in the body causing the disorder of bone metabolism.Inhibition of osteoclast differentiation and maturation has become an important direction in the study of inhibiting osteoporosis.There are many kinds of drugs currently used for the treatment of osteoporosis,but the long-term use of them has relatively large side effects and is easy to cause damage to the body.Therefore,it has become a hot research topic in recent years to find effective targets targets for drugs that act on osteoporosis to treat osteoporosis-related diseases.With the development of traditional Chinese medicine,traditional Chinese medicine and natural products have gradually entered people’s field of vision and become the focus of anti-osteoporosis treatment research.Iseliensinine(Iso)is a bisbenzylisoquinoline alkaloid with antioxidant,anti-inflammatory,and anticancer activities.However,whether it can be used as a potential drug for the treatment of osteoporosis remains undiscovered.Here,we investigated whether Iso might suppress the differentiation of osteoclasts in vitro and in vivo to play an anti-osteoporosis role.Methods ⑴ The potential role of Iso in osteoporosis-related diseases was analyzed based on the relevant data of network pharmacology.⑵ In vitro experiments,the bone marrow macrophages(BMMs)of mice were cultured and treated with different concentration gradients of Iso.CCK-8test was used to determine the safe concentration dose of the drug.BMMs were induced by nuclear factor kappa B receptor activator ligand(RANKL),and cultured with different concentrations(0,0.625,1.25,2.5,5 μM)of Iso.The time dependence of osteoclast formation was detected by 5 μM Iso at different time periods.Osteoclasts were stained with rhodamine-phalloidin and DAPI to observe the effect of isoliensinine(2.5 and 5 μM)on F-actin ring formation.BMMs were inoculated on hydroxyapatite bone plates,and the intervention effect of Iso(2.5 and 5 μM)was observed through bone resorption experiments.Bio Tek was used to take photos and analyze and calculate the bone resorption area.Real-time quantitative PCR(q RT-PCR)was used to detect the expression of Iso on osteoclast formation-related genes,including c-fos,Ctsk,Trap,Mmp9,Dc-stamp and Nfactc1.at the same time,molecular docking technology was used to dock the osteoclast classical MAPK and NF-κB related proteins,and the immunofluorescence staining test was used to explore the inhibition of p65 nuclear translocation of osteoclasts by Iso.Finally,Western Blot technique was used to detect the effect of Iso on RANKL-induced cell signaling pathways MAPK and NF-κB,and to explore the effect of Iso on the molecular mechanism of osteoclasts.⑶ In vivo experiments,10-week-old female C57BL/6 mice were randomly divided into 3 groups: blank control group,OVX group,and OVX+Iso group.Ovarian ovariectomy was performed on both sides of the mice to construct an animal model of ovarian castration.Then,the mice in the OVX group were intraperitoneally injected with Iso 5 mg/kg,and the C57BL/6J mice in the sham-operated group and the OVX group were intraperitoneally injected with the same amount of normal saline dissolved in the same amount of DMSO as the control group.The whole injection course lasted for 6 weeks.Micro-CT scanning and pathological analysis(HE and TRAP staining)were used to analyze the changes of bone mass in the distal femur in each group,and to explore the relationship between Iso’s intervention in osteoclastogenesis and bone loss in animals.Results ⑴ Through database search,it was found that the potential targets of Iso in vivo and the targets of osteoporosis-related diseases have 15 targets that overlap.KEGG signaling pathway molecules can be enriched in the osteoclast differentiation pathway.⑵ In vitro experiments,CCK-8 results showed that Iso had no toxic inhibitory effect on BMMs cells in the concentration range of 0.625 μM-5 μM.Osteoclast differentiation experiments showed that different concentrations of Iso(0,0.625,1.25,2.5 and 5 μM)inhibited osteoclastogenesis in a dose-dependent manner,and and inhibited the formation of osteoclast in the early stage(1-3 days);The results of F-actin staining showed that Iso(2.5 and 5μM)could effectively inhibit the formation of F-actin rings in osteoclasts in a concentration-dependent manner.The results of bone resorption showed that the concentrations of Iso(2.5 and 5 μM)could inhibit the bone resorption area of hydroxyapatite by osteoclasts.The results of q RT-PCR showed that compared with the control group,Iso could inhibit the expression of osteoclast-related genes c-fos,Ctsk,Trap,Mmp9,Dc-stamp and Nfactc1.Molecular docking results showed that compared with the MAPK pathway,Iso can form a more stable conformation with the NF-κB pathway,with higher hydrogen bonding.Immunofluorescence assay confirmed that Iso inhibited the nuclear translocation of p65 in osteoclasts.Western Blot results showed that Iso could inhibit the phosphorylation of p65 and IκBα,and at the same time inhibited the expression of long-acting proteins NFATc1,c-Fos and CTSK in the downstream pathway of osteoclasts,thereby inhibiting the generation and activation of osteoclasts.⑶ In vivo experiments In the OVX animal model of ovariectomized mice,Micro-CT results showed that Iso reduced bone loss in the distal femur of OVX mice,and significantly promoted BV/TV,Tb.N,Tb.Th,Conn.Dn and BS indicators.Conclusion This study identified drug targets associated with osteoporosis through network pharmacology.network pharmacology,in vivo and in vitro experiments confirmed that Iso can inhibit RANKL-induced osteoclast formation,differentiation and bone resorption through NF-κB signaling pathway,thereby protecting bone loss in ovariectomized mice effect.In conclusion,Iso is an alkaloid with good safety,which can be used as an inhibitor of osteoclast differentiation in the treatment of osteoclast-related diseases,and has great potential in the treatment of osteoporosis. |
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