Effect Of Er-xian Decoction Drug-containing Serum On Osteoblast Bone Formation By Regulating BK Channel And Osteoclast-derived Extracellular Vesicles

Objective:Network pharmacology predicts the mechanism of EXD in the prevention and treatment of osteoporosis（OP）.To explore the effect of EXD-containing serum on the bone formation of osteoblasts or H₂O₂-stimulated osteoblasts through BK channel and the downstream PTEN/AKT/FOXO1 pathway.To investigate the effect of osteoclast derived vesicles treated with EXD-containing serum on the bone formation of osteoblasts.Methods:1.The active ingredients of EXD,OP disease targets and intersection targets were obtained through TCMSP database,Gene Cards database and Venn diagram,and the"traditional Chinese medication-active ingredient-target"network was constructed by Cytoscape3.9.1 software.Protein-protein interaction（PPI）network was generated on the STRING website,and GO and KEGG pathway enrichment analysis was performed.2.Under the treatment of EXD containing serum and 3 mmol·L-1 tetraethylamine chloride（TEA）,the proliferation activity of MC3T3-E1 cells was detected by CCK-8 method.alkaline phosphatase（ALP）activity and ALP staining were detected by ALP kit.Western blot was used to detect protein expression（BMP2,OPG,and COL1）,Real-time PCR was used to detectm RNA expression（Runx2,OPG,and BMP2）,and alizarin red staining was used to detect the mineralized area of osteoblasts.In addition,the protein expressions of BK channel BKαand downstream PTEN/AKT/FOXO1 pathway were detected by Western blot.3.The oxidative damage model of MC3T3-E1 cells stimulated by H₂O₂was constructed,and BK channels of osteoblasts were blocked by TEA,and the effect of EXD on osteogenic differentiation of osteoblasts stimulated by H₂O₂was detected.4.TRAP staining and bone resorption assay were used to observe the effect of EXD on osteoclast proliferation and differentiation.EVs secreted by osteoclasts were extracted by ultrafiltration method.Transmission electron microscopy,nanoparticle size analyzer and Western Blot were used to observe the morphology,size and markers CD9,CD63 and CD81 of EVs.At the same time,the effect of osteoclast derived EVs intervened by EXD drug-containing serum on the bone formation of osteoblasts was explored.Results:1.32 active components,184 component targets and 1252 OP-related targets of EXD were identified,and 70 intersection targets were screened;Network topology analysis showed that the key active ingredients of EXD included quercetin,luteolin,apigenin and rutin.PPI network screening showed that interleukin-6,tumor necrosis factor,protein kinase B1,vascular endothelial growth factor A were the key targets of EXD in the treatment of OP.GO function analysis showed that it was mainly related to protease activity,regulating cellular inflammatory response,oxidative damage,aging,proliferation,apoptosis and angiogenesis.KEGG pathway analysis showed that it was mainly related to cancer pathway,HIF-1,VEGF,FOXO and reactive oxygen species signaling pathways.2.EXD-containing serum can improve the cell viability of MC3T3-E1 cells by reducing the expression of BKαprotein.EXD drug containing serum can increase ALP activity and calcium nodule formation in osteoblasts,promote the protein expression of BKα,COL1,BMP2 and OPG,and promote the m RNA expression of RUNX2,OPG and BMP2.In addition,Ex D-containing serum upregulated the phosphorylation of AKT and FOXO1 and decreased the expression of PTEN in osteoblasts,but these effects of EXD were reversed by TEA.3.Under the stimulation of H₂O₂,the proliferation and ALP activity of osteoblasts decreased,the expression of BKα,COL1,BMP2,OPG and phosphorylated AKT decreased,the m RNA expression of RUNX2,BMP2 and OPG decreased,and the area of calcium nodules decreased significantly.EXD drug containing serum could increase H₂O₂-stimulated osteoblast proliferation and ALP activity,up-regulate the protein expression of BKα,COL1,BMP2,OPG and phosphorylated AKT and FOXO1,promote the m RNA expression of RUNX2,BMP2 and OPG,and increase the area of calcium nodules.However,TEA could reverse these effects of EXD.4.EXD-containing serum can inhibit osteoclast proliferation,TRAP activity and bone resorption ability of thin bone slices.The EVs derived from osteoclasts were oval in shape with a particle size of about 45nm,and the marker proteins CD9,CD63 and CD81 were highly expressed.EXD-containing serum can improve the inhibitory effect of osteoclast derived EVs on the proliferation of osteoblasts and the production of ALP,OPG,COL1 and calcium nodules.Conclusion:1.EXD may exert its anti-OP effect by acting on AKT,FOXO,VGEF and reactive oxygen species signaling pathways through active components such as quercetin,luteolin,apigenin,rutin and nohodin A.2.EXD-containing serum promoted osteoblast bone formation by promoting BK channel protein expression and activating the downstream PTEN/AKT/FOXO1 signaling pathway,while TEA attenuated these effects of EXD.3.EXD-containing serum protected osteoblasts against H₂O₂-stimulated oxidative damage by increasing BK channel protein expression and affecting the downstream AKT/FOXO1 signaling pathway,while TEA attenuated these effects of EXD.4.EXD-containing serum can inhibit the proliferation,differentiation and bone resorption of osteoclasts,and promote the bone formation of osteoblasts through osteoclast derived vesicles.