| ObjectivesThe effects of Tarasaponin IV extract from the root bark of Aralia echinocaulis Hand.-Mazz.on the formation and bone resorption of primary osteoclasts and differentiation and mineralization of primary osteoblasts were studied.The Tarasaponin IV was used to detect differentially expressed genes in osteogenic differentiation by Gene Chip technology.The cellular and molecular mechanisms of its regulation of osteogenic differentiation was analyzed.Tujia minority medicine Aralia echinocaulis Hand.-Mazz.in clinical treatment of osteoporosis was to provide pharmacodynamic and pharmacological experimental support.MethodsThe primary BMMs from SD male rats at 4 weeks old were induced by M-CSF and RANKL in vitro.The primary osteoclasts were obtained and identified.The suitable range of concentration of Tarasaponin IV intervention BMMs was tested by CCK-8 assay.The effects of osteoclasts formation and bone resorption of Tarasaponin IV intervention were tested by TRAP staining and toluidine blue staining.The calvarial bone of SD rats at 24 hours old were digested.The primary osteoblasts were collected.They were cultured and identified in vitro by beta-Glycerophosphate disodium and L-Ascorbic acid.The effects of Tarasaponin IV on the proliferation of primary osteoblasts were tested by MTT assay.The effects of osteoblasts differentiation and Mineralization function of Tarasaponin IV intervention were tested by alkaline phosphatase activity and alizarin red S staining.The differentially expressed genes of osteogenic differentiation in Tarasaponin IV intervention were tested by the Gene Chip and screened the key genes by bioinformatics analysis.Western Blot was used to detect the expression of key gene-coding proteins to study the molecular mechanism of Tarasaponin IV involvement in regulating signal pathways.ResultsCompared with the control group,after 4 and 11 days,the concentration groups of Tarasaponin IV were intervented osteoclasts.The results showed that the number of osteoclasts and the area of bone resorption pits were decreased significantly(P<0.01)in 0.5?g/mL concentration.After 2,4,6 and 14 days,the concentration groups of Tarasaponin IV were intervented osteoblasts.The results showed that ALP activity of osteoblasts and the area and number of mineralized nodules were increased significantly(P<0.05 or P<0.01),Gene Chip Operating Software screened 1541 differentially expressed genes(P<0.05).They in top 10 of Fold Change(|FC|)was JAK3 genes associated with osteogenic differentiation.With P<0.01 as the further screening condition,the differentially expressed genes were 301(up 142 genes and down 159 genes).Associated with osteogenic differentiation include TRPC6,TCL1A and BLNK significantly(P<0.001).Gene Ontology database function enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that differential genes were mainly enriched functions in the nucleus,facies lateralis of cytoplasmic membrane and metabolic pathways,osteoclast differentiation,rheumatoid arthritis,NF-?B signaling pathway and calcium signaling pathway.Compared with the control group,after 2 days,Western Blot assay showed that TRPC6 and TCL1A protein expression were decreased significantly(P<0.05)and JAK3 protein expression was increased.The Western Blot results were consistent with the genes expression results.The mechanism that Tarasaponin IV could regulate TGF?/BMP,PI3K/AKT and JAK-STAT signaling pathway by regulating the proteins expression of TRPC6,TCL1A and JAK3.ConclusionTarasaponin ? can inhibit the formation and bone resorption of primary osteoclasts,and promote the differentiation and mineralization of osteoblasts in a certain concentration range.Tarasaponin IV can regulate the differentiation of osteoblasts by down-regulating TRPC6,TCL1A and up-regulating JAK3 proteins expression,involved in the regulation of TGF-(3/BMP,PI3K/AKT and JAK-STAT signaling pathway respectively,and in the regulation of osteoblast differentiation process. |
PDF Full Text Request