Study On The Therapeutic Effect And Mechanism Of Mongolian Medicine Sugemule-7 On Retinoic Acid-induced Osteoporosis In Mice

Osteoporosis(OP)is the most common systemic skeletal disease and has become a major global medical and socioeconomic burden.It also seriously restricts the healthy development of modern breeding industry and has been paid more and more attention by pet raising families.Therefore,the prevention and treatment of OP is an urgent problem all over the world.The current strategy for treating OP mainly focuses on suppressing osteoclast activity or inducing bone formation.Traditional Mongolian medicine has a long clinical practice and a lot of clinical experience in preventing and treating OP,and it has great potential to be explored.Mongolian medicine Sugemule-7(SGML-7)has good therapeutic effects on heart and kidney Heyi disease,lumbago due to kidney-asthenia,lumbago and scelalgia;however,the mechanism of its anti-OP action is still unclear.Firstly,this study was aimed to explore the potential mechanism of SGML-7 in the treatment of OP by using a network pharmacology approach.The therapeutic effect of SGML-7 on bone loss in retinoic acid(RA)-induced OP model mice was then evaluated.Next,the effects of SGML-7 medicated serum on osteogenic activity and function of mouse MC3T3-E1 cells were investigated.Meanwhile,the molecular mechanism of SGML-7against OP was further studied in animal and cell experiments.The contents and results of the research are as follows:(1)According to the screening criteria,a total of 55 active compounds and 203 corresponding targets of SGML-7 were obtained from the TCMSP database.In addition,1329 OP-related targets were retrieved from the Gene Cards,OMIM,TTD,Pharm GKB and Drug Bank databases.Further,by taking the intersection of the OP-related targets and SGML-7 targets,77 SGML-7–OP common targets were collected.Then,a PPI network of the common targets was constructed with the STRING database and Cytoscape software,and 71 key common targets and 8 hub targets were acquired.Finally,the GO and KEGG enrichment analysis of the key common targets were performed using the Bioconductor package and cluster Profiler package in R language.The GO analysis enriched 1810 biological processes,15 cellular components and 99 molecular function items.The KEGG pathway analysis revealed that the targets were involved in 157 signaling pathways.The results showed that SGML-7 contains a variety of active compounds,which may prevent and treat OP mainly by acting on multiple hub targets such as MAPK1,JUN,ESR1,TP53,AKT1,NCOA1,and FOS,affecting multiple biological processes such as response to drug,multi-multicellular organism process,and response to steroid hormone,and regulating multiple signaling pathways such as MAPK,estrogen,and TNF.Among them,MAPK signaling pathway has the highest enrichment degree.(2)A high-conversion OP mouse model was induced by RA.SPF-grade female KM mice were randomly assigned into control group and model group,respectively.The model group was given gastric gavage of 75 mg/Kg/d RA,1% carboxymethylcellulose(CMC)as solvent,while the control group was administrated with equal volume of 1% CMC.After 2weeks of administration of RA,serum samples were collected to measure the levels of Ca,P,bone-specific alkaline phosphatase(BALP),tartrate-resistant acid phosphatase 5b(TRACP-5b),osteoprotegerin(OPG)and soluble receptor activator of nuclear factor-κB ligand(s RANKL).The left and right femurs of the mice in each group were weighed to calculate the organ indexes,and their lengths and diameters were also measured.Bone mineral density(BMD)and bone mineral content(BMC)of the right femurs were measured by dual energy X-ray absorptiometry.The left femurs were prepared into paraffin-embedded sections for HE staining and bone histomorphometric analysis.The results showed that compared to the control group,the mice in the model group had slower weight gain,significantly increased bone turnover rate,significantly shortened femur length and diameter,significantly decreased femur BMD and BMC,and significantly degraded microstructure of femoral trabecular bone.The mouse OP model was successfully established by RA induction,which provided foundation for studying the role of SGML-7 on the treatment of bone loss.(3)SPF-grade female KM mice were randomly assigned into control group and model group.After successful modeling,the mice in the model group were randomly divided into five groups: model group,alendronate sodium group(Positive,10 mg/Kg/week),SGML-7low-dose group(SGML-L,5 g/Kg/d),SGML-7 medium-dose group(SGML-M,10 g/Kg/d)and SGML-7 high-dose group(SGML-H,20 g/Kg/d).The mice in each group were given the corresponding drug by gavage for 8 weeks.Serum samples were collected to measure the levels of bone mineral metabolism and turnover markers.The heart,lung,liver,spleen,left and right kidney of the mice in each group as well as the left and right femur were weighed to calculate the organ indexes,and the length and diameter of the left and right femurs were also measured.Liver and kidney tissue homogenate were prepared for detection of liver and renal function.The right femurs were used to measure BMD and BMC.The left femurs were made into paraffin-embedded sections for HE staining,bone histomorphometric and immunohistochemical(IHC)analysis.The results showed that as compared with the model group,SGML-7 reduced bone turnover,increased femur BMD and BMC,and effectively improved the microstructure of femoral trabecular bone,and the optimal concentration for therapeutic effect is 10 g/Kg/d.Meanwhile,it had no toxic effect on organs,and liver and renal function of mice.IHC analysis suggested that SGML-7 may promote bone formation by regulating ERK1/2 and p38 MAPK pathways,OPG/RANKL/RANK pathway and Runx2,thereby treating OP.(4)To further understand the molecular mechanism of SGML-7 against OP,SGML-7medicated serum was prepared in this study.5% blank serum was for use as a control,and MC3T3-E1 cells were cultured with different concentration of(0.3125%,0.625%,1.25%,2.5% and 5%)SGML-7 medicated serum,the cell viability,differentiation and formation of mineralized nodules were then detected.Next,5% blank serum was still used as the control,and MC3T3-E1 cells were cultured with 5% SGML-7 medicated serum(in the presence or absence of PD98059 or SB 202190).Expressions of osteogenic differentiation related genes were detected by q RT-PCR,and expressions of ERK1/2 and p38 MAPK pathways,OPG/RANKL/RANK pathway related proteins and Runx2 were detected by Western blotting.The results showed that compared to 5% blank serum,SGML-7medicated serum promoted the proliferation and differentiation of MC3T3-E1 cells in a concentration-and time-dependent manner;5% SGML-7 medicated serum clearly promoted the formation of mineralized nodules,increased the phosphorylation levels of Erk1/2 and p38,and increased the expression of OPG,RANKL,Runx2 proteins and OPG/RANKL ratio,and the use of PD98059 or SB 202190 can inhibit the above results.SGML-7 medicated serum promoted osteogenic function and bone formation of MC3T3-E1 cells by activating ERK1/2 and p38 MAPK pathways,and then regulating OPG/RANKL/RANK pathway and Runx2.In summary,SGML-7 regulated bone formation by promoting osteoblast differentiation and maturation through multiple signaling pathways,which reflected its characteristics of ―multi-target and multi-pathway‖ in the treatment of OP.So it has the potential to be developed as an anti-OP drug.