| Objective: The target and related biological signal pathway of Mongolian medicine Eechinops latifolius in the treatment of osteoporosis were screened by network pharmacology-molecular docking method.Based on the results of network pharmacology and molecular docking studies,the drug-containing serum of Mongolian medicine Eechinops latifolius was used to intervene rat bone marrow mesenchymal stem cells to observe the effect of the serum on the expression of key regulatory factors of Wnt/β-catenin pathway,and to elucidate the mechanism of action of Mongolian medicine Eechinops latifolius in the treatment of postmenopausal osteoporosis from the perspective of molecular biology.Method:1.Screening the effective components of Mongolian medicine Eechinops latifolius based on Pharm Mapper platform,Search and analysis of "postmenopausal osteoporosis" or "PMOP" in Drugbank,TTD,Pharm GKB,Genecards,Dis Ge NET,OMIM database;The targets of drug components and PMOP related therapeutic pathways were intersected into STRING database for protein interaction network(PPI)analysis;Molecular docking was carried out according to the enrichment analysis results of GO and KEGG pathways,and the combination of active components and biological targets was comprehensively evaluated,which provided the basis for the selection of key mediating factors in the regulation of Wnt/β-catenin pathway by drugcontaining serum of Mongolian medicine Eechinops latifolius.2.Bone marrow mesenchymal stem cells(BMSCs)were extracted from 4-week-old SD rats for culture,and the rats were divided into groups to receive serum containing the concentration of Mongolian medicine Lanacitum.CCK8 and ALP were used to analyze the effects of the medicated serum of Mongolian medicine Eechinops latifolius on the proliferation and osteogenic differentiation of BMSCs.3.RT-PCR and Western blot were used to detect the effects of different concentrations of drug-containing serum on the protein and m RNA expression of Wnt3 a,β-catenin,Runx2,the key regulatory factors of Wnt/β-catenin pathway.To further elucidate the mechanism of promoting the proliferation and osteogenic differentiation of BMSCs by the medicated serum of Mongolian medicine Eechinops latifolius.Results:1.According to the network pharmacological analysis of the main components in the Mongolian medicine cyanophora,they may pass the target of albumin(ALB),serine/threonine kinase 1(AKT1),β-catenin(CTNNB1)and epidermal growth factor receptor(EGFR).PMOP can be improved by regulating the expression of PI3K-Akt,MAPK,Wnt and EGFR pathways,and it can be intuitively seen in the molecular docking diagram that it has very good binding activity with CTNNB1,RUNX2,CASP3 and WNT3.In conclusion,it can be concluded that Mongolian medicine Eechinops latifolius can regulate the expression of CTNNB1,RUNX2 and WNT3 proteins in the signaling pathway through the Wnt pathway to treat PMOP,providing scientific theoretical basis for the following cell experiments and exploring the possible mechanism of action of Mongolian medicine Eechinops latifolius on PMOP from the molecular level.2.Effects of different concentrations of Mongolian medicine Eechinops latifolius on proliferation and osteogenic differentiation of BMSCsCompared with blank control group,the drug intervention of low,medium and high doses of Mongolian medicine Eechinops latifolius medicated serum groups significantly promoted the proliferation of BMSCs,and the high dose group had the best effect on cell proliferation.Compared with the low,medium and high doses of Mongolian medicine Eechinops latifolius medicated serum groups,the proliferation of BMSCs was inhibited in the DKK-1 medicated serum group,but the proliferation ability of BMSCS was increased with the increase of concentration.Compared with the control group,all dosages of Mongolian medicine Eechinops latifolius serum had higher ALP activity in the intervention of BMSCs,and could promote osteogenic differentiation,with statistical significance(P<0.05).The highest activity and the strongest osteogenic differentiation ability were found in the high dose serum of Mongolian medicine Eechinops latifolius.Compared with the same dose of Mongolian medicine Eechinops latifolius serum group,the activity of ALP in DKK-1 medicated serum group was significantly decreased(P<0.05).3.Western blot and RT-PCR resultsCompared with the control group,the protein and m RNA expression levels of Wnt3 a,β-catenin and Runx2 were significantly increased in low,medium and high dose serum groups(P<0.05);Compared with the same dose of Mongolian medicine Eechinops latifolius serum group,the protein and m RNA expression levels of Wnt3 a,β-catenin and Runx2 in the DKK-1inhibitor group were significantly down-regulated(P<0.05).Conclusion:1.Network pharmacokinetics and molecular docking analysis showed: that the main components of Mongolian medicine Eechinops latifolius had good binding activity with β-catenin(CTNNB1)and RUNX2,suggesting that Mongolian medicine Eechinops latifolius may improve PMOP by regulating the expression of Wnt/β-catenin pathway.2.The medicated serum of Mongolian medicine Eechinops latifolius can significantly promote the proliferation and osteogenic differentiation of BMSCs.3.BMSCs were interfered with different concentrations of medicated serum of Mongolian medicine Eechinops latifolius.The results showed that BMSCs were observed and detected by modern scientific research methods such as Western blot and RT-PCR,and the results showed that the genes and proteins of upstream wnt3 a,β-catenin and downstream Runx2 were upregulated,which promoted osteogenic differentiation,inhibited osteoclast formation,and increased bone mass and bone formation.4.At the cellular and molecular level,Mongolian medicine Eechinops latifolius may regulate BMSCs differentiation into osteoblasts through Wnt/β-catenin pathway,thereby improving postmenopausal osteoporosis. |
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