| Objective1 To investigate of the anti-osteoporotic pharmacological effects from a system of osteoblast/osteoclast-maintained bone homeostasis and to verify the possible anti-osteoporotic mechanisms of myricitrin in vitro.2 To investigate the effects of myricitrin on the expression of genes and proteins related to bone metabolism in the bone tissue of ovariectomize mice and to verify the possible antiosteoporotic mechanisms of myricitrin in vivo.Methods1 Cell research(1)Immortalized mouse bone marrow mesenchymal stem cells(imBMSCs)extracted from mouse long bones and immortalized were used to examine the effect of different concentrations of myricitrin on cell viability,as determined by CCK-8 assay.An osteogenic induction medium group with myricitrin was established by assessing alkaline phosphatase(ALP)activity and staining.Alizarin red staining was performed to assess mineralization.The expression of genes and proteins related to osteoblast differentiation was analyzed through qPCR and Western blot.(2)In order to investigate the mechanism underlying osteogenic differentiation of imBMSCs,we extracted RNA from treated cells and performed high-throughput transcriptome sequencing.The sequencing results were analyzed using KEGG and GO enrichment to identify key pathways and processes involved in osteoblast differentiation.Based on these results,we found that the PI3K/AKT signaling pathway was implicated in the regulation of osteoblast differentiation.This finding was further validated through molecular docking,protein protein interaction(PPI)network analysis and Western blot analysis.(3)To further confirm the involvement of the PI3K/AKT signaling pathway in promoting osteoblast differentiation of imBMSCs via myricitrin,we conducted a rescue experiment using the PI3K inhibitor,copanlisib.We examined the effect of different concentrations of copanlisib on cell viability using the CCK-8 assay.After treatment with copanlisib and myricitrin,we performed ALP and alizarin red staining to detect changes in osteogenic differentiation.We also analyzed the expression of genes and proteins related to osteoblast differentiation using qPCR and Western blot.We used immunofluorescence to detect the expression levels of Runx2 and Collal in the presence of copanlisib.We performed a dual luciferase reporter gene assay to evaluate the promoter region levels of Myc.(4)To investigate the impact of myricitrin on the adipogenic differentiation of imBMSCs,an adipogenic induction group was established with the addition of myricitrin.Oil Red O staining was performed to assess the inhibitory effect of myricitrin on adipogenic differentiation.Furthermore,qPCR and Western blot analyses were conducted to evaluate the expression levels of genes and proteins associated with adipogenic differentiation.(5)The effects of myricitrin on mouse monocyte macrophage leukemia cells(RAW264.7)were investigated.The effect of different concentrations of myricitrin on cell viability was detected using a CCK-8 assay.An osteoclastic induction group was established by inducing RANKL osteoclastic differentiation in RAW264.7 cells with myricitrin.The osteoclastic differentiation was examined by quantification of Tartrate Resistant Acid Phosphatase(TRAP)activity and staining to analyze the ability of myricitrin to inhibit osteoclastic differentiation.The expression of filamentous actin(F-actin)and cytoskeletal protein Vinculin at different concentrations was examined using immunofluorescence.qPCR and Western blot analyses were used to investigate the effects of myricitrin on the expression of genes and proteins related to osteoclastic differentiation.(6)To investigate the underlying mechanism of myricitrin in inhibiting osteoclast differentiation of RAW264.7 cells,we extracted RNA and performed high-throughput transcriptome sequencing.The results were analyzed using KEGG and GO enrichment.To further verify the potential targets of myricitrin,we used molecular docking,protein protein interaction(PPI)network analysis and Western blotting.2 Animal experimentAn ovariectomy model was established in 8-week-old C57BL/6J mice,with all groups except the sham-operated group undergoing ovariectomy.The sham and model groups received an appropriate volume of PBS solution,while the remaining two groups were given intraperitoneal injections of myricitrin for intervention.After 8 weeks,femoral and lumbar tissues were collected from each group for analysis.Femoral tissues were fixed with paraformaldehyde and scanned with Micro-CT to observe the bone microstructure of the distal femur and analyze relevant parameters.Hematoxylin-eosin(HE),Masson,and Trap staining were performed to assess relevant changes.Immunohistochemistry and Western blot were used to determine the expression levels of osteoblastic and osteoclastic proteins and the pathways involved in mice.Results1 Cell research(1)Effects of myricitrin on proliferation and osteoblast differentiation of imBMSCsThe CCK-8 assay revealed that treatment of imBMSCs with myricitrin in concentrations ranging from 1-50 μM for 24 h,48 h,72 h and 96 h had promoted cell growth and no cytotoxic effects.ALP staining and activity assessment showed no formation of blue deep dye in the control group,whereas ALP-positive blue dye and activity level were observed in both OIM and myricitrin groups,with the most profound color and activity level at a concentration of 25 μM.Alizarin red staining showed no apparent calcium deposition in the control group,while red calcium deposition was observed in some cells in both OIM and myricitrin groups,with the most notable bone mineralization and the highest number of red calcium deposition at a concentration of 25 μM.Moreover,qPCR results indicated that myricitrin significantly up-regulated the expression of ALP,OPN,OCN,Collal,Runx2,and Osterix in a dose-dependent manner at concentrations below 25 μM,and Western blot results revealed that myricitrin up-regulated the expression of OPN,OPG,Collal,Runx2,and Osterix in a dose-dependent manner within a concentration range of 1-25 μM.(2)High-throughput transcriptome sequencing was conducted to investigate the effect of myricitrin on the osteogenic mechanism.A volcano map revealed that 77 genes were upregulated,and 169 genes were down-regulated.The top 20 up-or-down-regulated differential genes were visualized using the cluster heat map.Results indicated that the expression levels of genes,including ler2,Slc23a3,Nyx,Sox11,Kcnj4,and Slc39a12,were significantly higher in the MYR group compared to the OIM group,while the expression levels of genes such as Cd93,Gbp5,Gm9780,Trim34a2,and Spire2 were significantly lower in the MYR group compared to the OIM group.Analysis of Gene Ontology(GO)Biological Process showed that angiogenesis was involved in the osteogenic differentiation process.KEGG pathway analysis revealed that the PI3K/AKT,HIF-1,and Rapl signaling pathways were also involved in the osteogenic differentiation of myricitrin.Western blot results demonstrated that the expression of p-PI3K and p-AKT increased after myricitrin intervention.Additionally,molecular docking analysis showed a high degree of affinity between myricitrin and its corresponding key target mouse phosphatidylinositol 3-kinase p110δ(PIK3CG)(PDB ID:5ngb),with an affinity score of-8.5 kcal/mol.The network diagram of PPI protein interactions suggested that Ace,Ednl,Cd163,Ccn1,Adm2,and Ramp1 may be the target proteins for the action of myricitrin.(3)The inhibitory effect of Copanlisib on Myricitrin-induced osteogenic differentiationIn order to investigate the involvement of the PI3K/AKT signaling pathway in the process of myricitrin-induced osteogenic differentiation,the inhibitory effect of this pathway on osteogenic induction in the myricitrin group was evaluated.The results of the CCK-8 assay demonstrated that at 72 h and 96 h,7.8125 nM and 15.625 nM Copanlisib groups began to inhibit cell proliferation when compared with the control group.ALP staining and activity analysis demonstrated that the expression of ALP blue dye and activity level in the 25μM myricitrin group was higher than that in the 25μM myricitrin+10 nM Copanlisib group.Similarly,alizarin red staining showed that the red calcium deposition in the 25 μM myricitrin group was higher than that in the 25 μM myricitrin+10 nM Copanlisib group.The qPCR results showed that the mRNA expression levels of ALP,OPN,OCN,Collal,Runx2,and Osterix in the 25 μM myricitrin+10 nM Copanlisib group were significantly lower than those in the 25 μM myricitrin group.Western blot analysis showed that the protein expression levels of OPN,OPG,Collal,Runx2,and Osterix in the 25 μM myricitrin+10 nM Copanlisib group were significantly lower than those in the 25 μM myricitrin group.Moreover,the fluorescence intensity of Runx2 and Collal in the 25 μM myricitrin+10 nM Copanlisib group was significantly lower than that in the 25 μM myricitrin group.Lastly,the results showed that the fluorescence intensity of Myc in the 25 μM myricitrin+10 nM Copanlisib group was significantly lower than that in the 25 μM myricitrin group.(4)Effects of myricitrin on adipogenic differentiation of imBMSCs:Through Oil Red O staining,no obvious lipid droplets were observed in the blank group after 18 days of adipogenic induction,while red lipid droplets were observed in some cell colonies of the Adipo group and Myricitrin group.Myricitrin was found to significantly downregulate the number of lipid droplets in a dose-dependent manner,as observed through Oil Red O staining.Additionally,the mRNA expression levels of PPARγ,CEBPα and FABP4 were significantly down-regulated in a dose-dependent manner by Myricitrin,as shown by the results of qPCR.Similarly,the protein expression levels of PPARγ,CEBPα and FABP4 were significantly down-regulated in a dose-dependent manner by Myricitrin,as observed in the Western blot results.(5)Effects of myricitrin on osteoclast differentiation of RAW264.7:The CCK-8 assay revealed that treatment of RAW264.7 cells with myricitrin in concentrations ranging from 1-50 μM for 24 h,48 h,72 h and 96 h had promoted cell growth and no cytotoxic effects.Results from trap staining showed that there were no pink dark-stained substrates in the blank group,but pink dark-stained substrates of varying shades were observed in the RANKL group and myricitrin group.Notably,the size,activity level and maturity of osteoclasts induced by RAW264.7 were significantly reduced when treated with myricitrin at a concentration of 25 μM.Additionally,Vinculin immunofluorescence and phalloidin fluorescence staining showed that the fluorescence expression levels of cytoskeletal protein and filamentous actin in myricitrin group were lower than those in the RANKL group.Furthermore,the qPCR results showed that myricitrin significantly down-regulated the mRNA expression levels of Acp5,NFAT2.c-fos,atp6v0d2,CTSK and MMP9 in a dose-dependent manner.Similarly,Western blot results showed that myricitrin significantly down-regulated the protein expression levels of NFAT2,c-fos,CTSK and Acp5 in a dose-dependent manner.Additionally,myricitrin significantly down-regulated the expression levels of p-p38 MAPK,p-JNK,p-ERK in the MAPK signaling pathway in a dose-dependent manner.(6)High-throughput transcriptome sequencing was conducted to investigate the effect of myricitrin on the mechanism of osteoclasts.A total of 93 genes were up-regulated and 159 genes were down-regulated,as demonstrated by the volcano plot.The top 20 up-or down-regulated differential genes were illustrated by the cluster heat map,revealing a significant increase in the expression levels of Hsh2d,Itgax,Ccl22,Tsku,Pdcdl,Ccl4,Tmem51,and Egr3 in the MYR group compared with the PC group.Meanwhile,the expression levels of Extll,Avil,Itgb7,Celsr1,Arhgap22,Ypel3,and Lims2 were significantly decreased in the MYR group compared with the PC group.KEGG pathway analysis suggested that myricitrin may inhibit osteoclast differentiation in RAW264.7 cells through cytokine-cytokine receptor interaction and chemokine signaling pathway.GO enrichment analysis revealed that these differentially expressed genes were highly correlated with intracellular processes,biological regulation,cellular structural bodies,and protein binding.The results further suggested that the inhibition of osteoclast differentiation by myricitrin was primarily involved through the chemokine pathway,and the MAPK pathway was one of its downstreams.Myricitrin was found to target three sub-pathw ays of the MAPK signaling pathway,including extracellular signal-regulated kinase ERK1/2,c-Jun N-terminal kinase(JNK1/2),and mitogen-activated protein kinase(p38MAPK)protein binding.The corresponding key targets,including ERK1/2(PDB ID:5LCJ),JNK1/2(PDB ID:3OY1),and p38MAPK(PDB ID:1 CM8),exhibited a higher degree of linkage,with affinities of proteins with 51cj,3oy1,and lcm8 being-7.9 kcal/mol,-8.3 kcal/mol,and-6.8 kcal/mol,respectively.The network diagram of PPI protein interactions showed that Snap25,Syt1,Gria2,Grial,Ccl2,Cxc110,Ntrk2,Rtn1,and Itgax had higher scores,suggesting that these proteins could be the target proteins for the action of myricitrin.2 Animal experimentThe study utilized micro-CT three-dimensional reconstruction analysis to evaluate the impact of myricitrin on ovariectomized mice.Results showed that compared to the sham group.the OVX group exhibited decreased bone volume fraction(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th),and bone mineral density(BMD)values,while the values of trabecular separation(Th.Sp)and structure model index(SMI)were significantly increased.The distal femur exhibited notable bone loss and trabecular bone fracture.However,both low-dose and high-dose myricitrin interventions significantly reduced bone loss in the mice.Additionally,Western blot results showed significantly lower expressions of osteopontin(OPN),osteoprotegerin(OPG),osteocalcin(OCN),p-PI3K,and AKT in the model group compared to the sham group.In contrast,the expression levels of NFATc1,Acp5,CTSK,c-fos,MAPK signaling pathway p-p38 MAPK,p-JNK,p-ERK were significantly increased.Intervention with low-dose and high-dose myricitrin significantly increased the expression of OPN,OPG,OCN,p-PI3K,and AKT and decreased the expression levels of NFATc1,Acp5,and MAPK signaling pathway p-p38 MAPK,p-JNK,p-ERK in comparison to the model group.HE and Masson staining results indicated that myricitrin partially alleviated bone tissue disorder and loss and promoted the regeneration of fibrous tissue in OVX mice.Trap staining results showed that myricitrin inhibited osteoclast numbers in low-dose and high-dose myricitrin groups to reduce bone mass loss.Immunohistochemistry results demonstrated that the contents of OPG,OPN,OCN,and p-PI3K in the femoral tissue of the model group were significantly lower than those in the sham group.In contrast,the contents of OPG,OPN,OCN,and p-PI3K were significantly higher in low-dose and high-dose myricitrin groups compared to the sham group.Similarly,the contents of osteoclast-related genes NFAT2,c-fos,Acp5,and CTSK in the femur of the model group were significantly higher than those in the sham group.The contents of NFAT2,CTSK,and Acp5 in low-dose and high-dose myricitrin groups were significantly lower than those in the sham group.Conclusion1.Myricitrin was found to promote osteoblast differentiation of imBMSCs by partially depending on the PI3K/AKT signaling pathway,while inhibiting adipogenic differentiation of imBMSCs.2.Myricitrin was found to inhibit osteoclast differentiation of RAW264.7 cells mediated by the MAPK signaling pathway.3.In vivo,myricitrin was found to alleviate bone mass loss caused by estrogen deficiency and to reduce the numbers and activity of osteoclasts,demonstrating an excellent antiosteoporotic effect. |
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