Study On The Mechanism Of Erzhi Wan On Postmenopausal Osteoporosis Based On Biological Network

Er Zhi Wan(EZW)also named Nv Zhen Dan,firstly described in Fu Shou Jing Fang by Wu Min Ji in China during Ming Dynasty.It contains only two herbs,Fructus Ligustri Lucidi(FLL,Ligustrum lucidum Ait.)and Ecliptae Herba(EH,Eclipta prostrata L.),which have the functions of tonifying the kidney and strengthening the waist and knees.In Chinese clinical practice,EZW is mainly used for postmenopausal osteoporosis(PMOP)with definite therapeutic effect.Specnuezhenide(SPN)was quality ingredient of EZW in the book of Chinese Pharmacopoeia and its content is relatively high,however,there are few study related SPN treating PMOP.Salidroside(SA)is not only a characteristic component of Erzhi Wan,but also a primary active metabolite of SPN.There have been no reports that compare the pharmacodynamic action and the difference in efficacy of EZW,SPN and SA in treating PMOP.In addition,the specific therapeutic effects and mechanism of EZW with its main compounds in treating PMOP have not been fully understood or studied systematically.In this paper,the active ingredients in EZW for osteoporosis were screened based on molecular docking technology and verification.Moreover,the traditional pharmacological method,the powerful metabolomics and proteomics were combined to investigate the pathological mechanism of PMOP and the intervention effects of EZW with its main compounds based on the mice model induced by ovariectomy.The main content of this paper includes the following six parts:Part one:Literature SearchBased on the related study in recent years,the pathological mechanism of PMOP were investigated.In addition,the application of systems biology in osteoporosis were summarized,which provided a theoretical basis for the further research on systems biology.Part two:Screening of active ingredients in EZW for osteoporosis based on molecular docking technology and verificationIn this study,we used molecular docking technology and validation experiments in vitro to screen the active ingredients of EZW for treating osteoporosis.Firstly,the compound in EZW was docked with four osteoporosis-related targets by molecular docking technology,and the results were compared with the binding energy of the target with anti-osteoporosis drugs.As a consequence,the five active compounds,SA,SPN,tyrosol,quercetin,and wedelolactone,were initially selected.Then,MC3T3-E1 osteoblasts were cultured in α-Mem medium containing different concentrations of compounds,and the cell proliferation rate and mineralized nodules were tested.The verification results showed that the proliferation of MC3T3-E1 cells with SA,SPN and quercetin were more obvious,and SA has a better effect on the mineralization of MC3T3E1 cells than those of SPN and tyrosol.The molecular docking technology coupled with validation experiment in vitro can be used for the screening of anti-osteoporotic active ingredients of EZ W,and it also can provide a method for the study of effective ingredients of traditional Chinese medicine.Part three:Study on the intervention effects of EZW on PMOP miceOvariotomy were used to establish the PMOP mice model.As many as 6 substances in the serum were detected to evaluate the PMOP mice model and the therapeutic effect of EZW with its main compounds,including osteoprotegerin(OPG),Nuclear factor K receptor activator ligand(RANKL),estradiol(E2),osteocalcin(OC),Alkaline phosphatase(AKP)and Type Ⅰ collagen Cterminal peptide(CTX-I).In addition,the pathological morphological changes of left tibia and microstructure changes of epiphysis of distal femur were also observed.Comparing with shamoperated group,the ovariectomy group showed a significant increase in the level of OCN and RANKL(P<0.05)and a decrease in the level of OPG and OPG/RANKL(P<0.01 or 0.05),while the level of AKP,CTX-1 and E2 didn’t exhibited significant changes.Moreover,the tibia showed more broken trabecular bones and adipocytes in medullary space,and the femurs showed a decrease in the level of BMD,BV/TV and Conn.D(P<0.05).Above all,the PMOP mice model was successfully established.Based on the above mice model,the therapeutic effect of EZW with its main compounds were investigated.Contrast to model group,EZW-prevention group could reduce AKP,CTX-1 and RANKL(P<0.00001)or 0.05)and increase E2 and OPG/RANKL significantly(P<0.05),EZW-treatment group could significantly reduce CTX-1,OCN and RANKL(P<0.0001 or 0.01),SA group could significantly reduce AKP,OCN and RANKL(P<0.01 or 0.05)and increase OPG and OPG/RANKL(P<0.05),and SPN group could significantly reduce AKP and OCN(P<0.0001 or 0.001),positive group could significantly increase E2 and OPG(P<0.00001 or 0.01).EZW and its main compounds also showed protective effect on the injury tibia and femur tissue to some extent.Part four:Study on the femur metabolomics of PMOP mice and the intervention effects of EZW with its main compundsBased on the PMOP mice model induced by ovariectomy,changes of metabolic profile in mice femur and the therapeutic effect of EZW with its main compunds towards abnormal metabolism of PMOP mice were investigated by GC-MS.Principal Orthogonal partial least square-discriminate analysis(OPLS-DA)and Mann-Whitney test adjusted by FDR were used for pattern recognition and univariate statistical analysis,respectively.The VIP value,Fold change and the P value were taken into account for selecting the significant variables.As a result,a total of 15 potential biomarkers were identified in the femur,which were associated with PMOP.And these metabolites were all or part recovered in different treatment groups at different levels,among which 2 metabolites showed significant callback trend in EZW-prevention group,5 metabolites showed significant recovered in SA group,3 metabolites showed significant recovered in SPN group.Pathway analysis showed that these significantly different metabolites were involved in 13 metabolic pathways including synthesis and degradation of ketone bodies,inositol phosphate metabolism,and sphingolipid metabolism et al.Metabolic network analysis showed that cytidine and phosphate were selected as key metabolites because of the high degree-value and closeness centrality.EZW and its main compounds may achieve the protective effect on PMOP mice by acting on these pathways including galactose metabolism,sphingolipid metabolism and glycerophospholipid metabolism et al.Part five:Study on the femur proteomics of PMOP mice and the intervention effects of EZW with its main compundsBased on the PMOP mice model induced by ovariectomy,changes of protein in mice femur and the therapeutic effect of EZW with its main compunds towards abnormal protein expression of PMOP mice were investigated by quantitative proteomic analysis with tandem mass tag(TMT)technology.The softwares of Mascot2.2 and ProteomeDiscovererl.4 were used for identification and quantitative analysis of protein.The Fold change and the P value were taken into account for selecting the differential protein.As a result,a total of 30 proteins were screened in the femur,which were associated with PMOP.And these proteins were all or part recovered in EZW and SA groups at different levels,among which Q9D4H1 showed significant recovered in EZWprevention group,and Q9R1Q7,P15535,Q9D110,Q9D4H1 and P01821 showed significant recovered in SA group.Pathway analysis showed that these differential proteins among the shamoperated and model groups were involved in 43 metabolic pathways,and the significant enrichment metabolic pathways were 7,including nicotinate and nicotinamide metabolism,galactose metabolism,glycosphingolipid biosynthesis-ganglio series,Mannose type O-glycan biosynthesis,glycosaminoglycan biosynthesis-keratan sulfate,and glycosphingolipid biosynthesis-lacto and neolacto series.In addition,GO analysis showed that these proteins involved in 431 significant biological process,52 molecular function entry and 39 cellular component entry.EZW and its main compunds may achieve the protective effect on PMOP mice by callbacking abnormal protein,acting on pathways including nicotinate and nicotinamide metabolism,galactose metabolism,glycosphingolipid biosynthesis-ganglio series et al.Part six:Study on the mechanism of EZW with its main compunds on postmenopausal osteoporosis based on biological networkFirstly,based on the 30 of proteins and 15 of metabolites screened from proteomics and metabolomics,respectively,the metabolic pathways were analyzed by means of different proteins and metabolites.Pathway analysis showed that PMOP may related to glucose metabolism pathways such as galactose metabolism,glycolysis and glycosaminoglycan degradation,lipid metabolism such as sphingolipids metabolism,fatty acid metabolism and glycerophosphate metabolism,and energy metabolism such as purine metabolism,arachidonic acid metabolism and tyrosine metabolism.Then,the "protein-metabolite" network diagram was established by CytoScape,and the method revealed the action of PMOP may be the result of multi-targets,multipathways interaction and interconnection.Finally,partial proteins verified by Western blot and ELISA,and the results were consistent with the data of proteomics,which can be seen the veracity of proteomics results.The result indicated that EZW and its main components may have a certain regulatory effect on the "protein-metabolite" biological network,thus playing a crucial role in treating the disease of PMOP.