Network Pharmacological Analysis Of Bushen Jianpi Huoxue Recipe And Its Effect On JMJD2B Promoting Osteogenic Differentiation Of Rats BMSC

ObjectiveBushen Jianpi Huoxue recipe is an empirical prescription with good clinical effect in the treatment of osteoporosis.In order to further study the possible molecular mechanism of Bushen Jianpi Huoxue recipe in the treatment of osteoporosis,the method of network pharmacology was used to analyze it.Then,bone marrow mesenchymal stem cell(BMSC)from rat was used to study the effect of Bushen Jianpi Huoxue recipe on histone demethylase JMJD2B and bone formation,so as to provide new experimental basis and research ideas for the mechanism of Bushen Jianpi Huoxue recipe in the prevention and treatment of osteoporosis.Methods1.Network Pharmacological Analysis of Bushen Jianpi Huoxue recipe for Prevention and treatment of OsteoporosisThe active components and potential targets of Bushen Jianpi Huoxue recipe were screened based on TCMSP database.The known genes related to osteoporosis were screened by GeneCards,OMIM,PharmGkb,TTD and DrugBank databases.To determine the drug active components of Bushen Jianpi Huoxue recipe and the potential common target of osteoporosis.The drug composition-target relationship network was constructed by Cytoscape software,and the protein-protein interaction diagram was constructed by String online software,and the core targets were screened by network topology.The biological function of GO and the enrichment of KEGG pathway of the common target were analyzed.2.Effect of Bushen Jianpi Huoxue recipe on osteogenic differentiation of rat BMSC induced by JMJD2BThe drug-containing serum was obtained by intragastric administration of Bushen Jianpi Huoxue recipe in SD rats.Different concentrations of serum were used to interfere with rat bone marrow mesenchymal stem cells.The cell activity,proliferation and alkaline phosphatase activity were detected,and the protein expression of histone demethylase JMJD2B and osteoblast proliferation and differentiation related protein RUNX2 were detected by WesternBlot.Results1.Network Pharmacological Analysis of Bushen Jianpi Huoxue recipe for Prevention and treatment of Osteoporosis(1)Through the screening of Bushen Jianpi Huoxue recipe by TCMSP,167active components and 250potential targets were obtained.A total of 4161 osteoporosis-related targets were screened by GeneCards,OMIM,PharmGkb,TTD and DrugBank databases.165 common drug-disease targets and 118 corresponding drug active components were obtained.(2)the protein-protein interaction diagram was drawn by String online software,and 16 network core targets such as IL6,TP53,FOS,JUN,STAT3,RELA,CCND1,MYC,MAPK1,MAPK8,MAPK14,VEGFA,AKT1,ESR1,TNF and NR3C1 were screened by(3)GO biological function enrichment analysis showed that the biological functions such as response to drugs,regulation of steroids,oxidative stress,response to metal ions and nutrition level,regulation of DNA transcription and enzyme binding were closely related to the common target.(4)KEGG pathway enrichment analysis showed that AGE-RAGE signal pathway,atherosclerosis,virus infection,IL-17 signal pathway,cancer-related pathway,TNF signal pathway,hepatitis,HIF-1 signal pathway,cell differentiation,apoptosis,thyroid hormone signal pathway were closely related to the signal pathway of Bushen Jianpi Huoxue recipe involved in osteoporosis.(5)In the biological function analysis of GO,it was found that the core targets of PPI,such as MAPK8,VEGFA and TP53,were related to histone modification function.In KEGG pathway analysis,it was found that multiple PPI core targets such as TP53,FOS,JUN,RELA,CCND1,MYC,MAPK1,MAPK8,MAPK14,VEGFA,AKT1,ESR1 and TNF were associated with MAPK,Estrogen,Wnt and other bone formation pathways.2.Effect of Bushen Jianpi Huoxue recipe on osteogenic differentiation of rat BMSC induced by JMJD2B(1)The proliferation of BMSC:compared with the serum containing Bushen Jianpi Huoxue recipe,the serum containing 5%,10%,15%and 20%of the concentration of Bushen Jianpi Huoxue recipe could promote the proliferation of BMSC for 24 hours(P<0.05),and the proliferation effect of 15%and 20%concentration groups was stronger than that of 10%concentration group and 5%concentration group(P<0.05).There was no significant difference between 15%concentration group and 20%concentration group(P>0.05).(2)The changes of ALP activity of BMSC:the ALP activity in the low,middle and high drug-containing serum groups was higher than that in the blank serum group,and the ALP activity in the high drug-containing serum group was higher than that in the middle-drug-containing serum group and the low-drug-containing serum group(P<0.05),and the effect of Bushen Jianpi Huoxue recipe drug-containing serum on ALP was concentration-dependent after intervention of BMSC7 in rats.(3)Changes of protein expression of JMJD2B and RUNX2 in BMSC:after BMSC of each group was interfered with different serum for 7 days,the relative expression of JMJD2B and RUNX2 protein in low drug-containing serum group,middle-drug-containing serum group and high-drug-containing serum group was higher than that in blank serum group(P<0.05).In the intervention of the drug-containing serum of the three groups,the relative expression of JMJD2B protein in the high drug-containing serum group was higher than that in the middle-drug-containing serum group and the low-drug-containing serum group,and the relative expression of RUNX2 protein in the high-drug-containing serum group was higher than that in the middle-drug-containing serum group and low drug-containing serum group(P<0.05.There was no significant difference between the middle drug-containing serum group and the low drug-containing serum group(P>0.05).ConclusionBushen Jianpi Huoxue recipe may have a multi-target effect on osteoporosis,which may promote the osteogenic differentiation of bone marrow mesenchymal stem cells by affecting cell proliferation and JMJD2B protein expression,so as to prevent and treat osteoporosis.