Study On The Material Basis Of Erxian Decoction In Regulating Iron Homeostasis Against Postmenoposal Osteoporosis

Postmenopausal osteoporosis(PMOP)is a chronic metabolic disease that causes loss of bone mass as ovarian function declines and estrogen levels in menopausal women decrease.It causes changes in bone microarchitecture and fracture susceptibility,resulting in a significant health and economic burden for patients.There is increasing clinical evidence that PMOP is often associated with iron overload,including elevated serum ferritin(SF)levels and decreased levels of hepcidin.Iron accumulation in the body can directly or indirectly inhibit bone formation and promote bone resorption,leading to further bone loss.Therefore,the regulation of iron metabolism is crucial to the treatment of PMOP.Erxian decoction(EXD),a scriptural formula documented in the clinical treatment guidelines for PMOP,consists of Epimedii Folium(Yinyanghuo,YYH),Curculiginis Rhizoma(Xianmao,XM),Morindae Officanalis Radix(Bajitian,BJT),Phellodendri Chinensis Cortex(Huangbo,HB),Anemarrhenae Rhizoma(Zhimu,ZM),and Angelicae Sinensis Radix(Danggui,DG).It has good efficacy without significant adverse effects in PMOP.However,there are no reports on the modulation of iron overload risk factors by EXD in PMOP treatment.Therefore,the present study was the first to investigate the material basis of EXD regulation of iron homeostasis in reversing PMOP and to explore its possible mechanisms.Chapter 1 Introduction This chapter provided a comprehensive review of PMOP,iron metabolism and EXD by reviewing the literature.The epidemiology of osteoporosis,the physiological processes and major regulators of bone metabolism were described;the pathogenesis of PMOP and the current major clinical therapeutic agents,including chemical and biological drugs and traditional Chinese medicine,were briefly described;the mechanisms of iron metabolism and regulation in the body and the association between iron overload and PMOP were outlined;finally,the sources,composition and research progress of EXD were summarized.Chapter 2 Prediction of active ingredients and mechanism of action based on network pharmacology In order to initially screen potential active ingredients in EXD,a network pharmacology study was conducted based on the chemical composition of EXD aqueous extracts.Methods: HPLC-Ion Trap-MS/MS was used to qualitatively identify the chemical components in EXD aqueous extract.The targets of components were obtained with the help of Pharm Mapper and TCMSP databases;the target genes of iron overload and osteoporosis were obtained by Drug Bank,Gene Cards,OMIM,Dis Ge Net and TTD databases.The target genes of EXD and disease were then intersected to obtain common targets.GO and KEGG enrichment analyses were performed with the help of Metascape database to acquire the physiological processes and signaling pathways of EXD down-regulating iron overload against osteoporosis.Based on the KEGG secondary classification annotation and literature retrieve in Pubmed,20 regulatory signaling pathways were screened with the highest association with iron metabolism and bone metabolism;the "component-target-signaling pathway network" of EXD downregulating iron overload against PMOP was constructed by Cytoscape software,thus initially screened the potential material basis components and key signaling pathways.Results: One hundred and thirty-five chemical components,mainly flavonoids,saponins and alkaloids,were identified from the aqueous extracts of EXD.A total of 166 signaling pathways were enriched by KEGG analysis,and 20 key pathways were obtained by pathway annotation and literature search,among which the BMP-SMADs pathway of TGF-β signaling pathway was identified as the main regulatory pathway of hepcidin,and was identified as the target pathway in the follow-up study.Finally,three key target genes(BMP2,BMP7,MAPK1)and 69 potential active components of BMP-SMADs signaling in TGF-β signaling pathway were obtained by "component-target-signaling pathway network".Chapter 3 Metabolomic study based on zebrafish model To elucidate the effect of EXD on organismal metabolism during the regulation of iron homeostasis against osteoporosis,an untargeted metabolomic study was conducted with the help of iron overloadinduced and osteoporotic zebrafish model.Methods: Osteoporosis was induced in zebrafish using 100 μg/m L of ferric ammonium citrate(FAC).Zebrafish at 3 dpf(days post fertilization)were divided into blank control(E3 culture),model(FAC,100 μg/m L),positive control Desferrioxamine(DFO,67 μg /m L)and different concentrations of EXD group(6.25-400μg/m L),and the optimal concentration of EXD administration was screened by bone mineralization area(BMA)and cumulative optical density(IOD)of zebrafish skull after EXD intervention.Then suitable concentrations were selected and metabolomic analysis of zebrafish was performed by GC-MS;significant differential metabolites were screened by PLS-DA and OPLS-DA analysis and identified with the help of NIST.14 and HMDB databases;pathway analysis of differential metabolites was performed by Metabo Analyst 5.0platform to explore the metabolic reversal of osteoporosis by EXD regression mechanism.Results: EXD achieved 100% lethality in zebrafish in the concentration range of 400-200μg/m L,43.33% lethality at a concentration of 100 μg/m L,and no significant toxicity was observed below 50 μg/m L;meanwhile,EXD at 50 μg/m L significantly improved BMA and IOD in zebrafish compared with the model group(P < 0.0001).Therefore,the optimal concentration for EXD zebrafish administration was determined to be 50 μg/m L.Twenty-six endogenous differential metabolites were obtained and identified by analysis of GC-MS data.Pathway analysis enriched 17 metabolic pathways,and three key metabolic pathways were finally identified based on P values,false discovery rates and pathway impact values,including Aminoacyl-t RNA biosynthesis,Valine,leucine and isoleucine biosynthesis and Glycine,serine and threonine metabolism.and threonine metabolism.Chapter 4 Optimization of potential active ingredients To further optimize the composition of active ingredients,a joint analysis of network pharmacology and metabolomics was performed.Methods: Twenty-six differential metabolites obtained from metabolomics studies were imported into the Metabo Analyst 5.0 database for network analysis,and gene-metabolite interaction network of differential metabolites were constructed to obtain metabolite-associated genes.These genes were merged with target genes from network pharmacology to obtain a collection of metabolic target genes and component target genes for EXD downregulating iron overload against osteoporosis,which were imported into the Metascape database for KEGG pathway analysis.The key pathways were screened by comparing with the network pharmacology results,and a joint analysis network was constructed to integrate multiple factors such as degree value and relative content to screen potential active ingredients.Finally,the potential affinity between the candidate active ingredients and the target genes was explored with the help of Autodock Vina-based molecular docking technology.Results: 19 key signaling pathways were obtained by combined network pharmacology and metabolomics analysis;8 differential metabolites were obtained as biomarkers;16 potential candidate components were obtained by screening the degree and relative content of the TGF-β pathway;the molecular docking results showed that8 of the 16 chemical components(Baohuoside I,Epimedin C,Icaritin,Berberine,Palmatine,Timosaponin BII,Anemarrhenasaponin I and Stigmasterol)had strong affinity to the target genes BMP2,MAPK1 and BMPR1 A compared with the positive control molecule BMP receptor agonist(SY-BL-35);and 5 other active components(Icariin,Epimedin A,Epimedin B,Curculigoside and Ferulic acid)were reported in the literature for iron overload and osteoporosis.Finally,the above 13 compounds were identified as the main candidates for anti-PMOP components in EXD.Chapter 5 Validation of potential active ingredients based on zebrafish model In order to validate the 13 candidate compounds screened above,the activity of individual compounds and the pharmacodynamic and mechanism of action of mixtures of active ingredients were investigated with the help of iron overload-induced osteoporosis zebrafish model.Methods: Zebrafish at 2 dpf were divided into a blank control group(E3),a model group(FAC,100 μg/m L),an EXD group(50 μg/m L),a DFO group(67 μg /m L)and a group administered with different concentrations of active ingredients(0.1-100 μM,singlecomponent administration of 13 active compounds),and screened by zebrafish mortality and skull BMA,IOD for components with anti-osteoporosis activity and determine the effective concentration range of the components.Then,a mixture solution of active ingredients was prepared according to the relative content of each ingredient in the aqueous extract of EXD,and a blank group(E3),a model group(FAC,100 μg/m L),an EXD group(50 μg/m L)and a mixture of active ingredients were administered(high,medium and low doses),and the equivalence of EXD and the mixture of active ingredients was compared by the results of zebrafish skull mineralization analysis.Finally,zebrafish were treated with BMP receptor inhibitor K02288 at 2-3 dpf to induce BMP inhibition model,and zebrafish were divided into blank group(E3),model group(K02288,20 μM),EXD group(50 μg/m L)and active ingredient mixture administration group(high,medium and low doses),and after 3 days of treatment EXD and the screened active compounds were elucidated by zebrafish bone mineralization analysis.The effects of EXD and the screened active compounds on BMP receptors were elucidated by zebrafish bone mineralization analysis after 3 days of treatment.Results: Pharmacodynamic studies of individual components showed that no significant reversal of the effect of Timoaponin BII was observed compared with the model group,while the remaining 12 components significantly increased BMA and IOD in zebrafish(P < 0.05),among which Icariin,Epimedin A,Stigmasterol,Berberine and Palmatine were the most potent,which significantly improved BMA and IOD in zebrafish at 0.1 μM(P < 0.05);followed by Baohuoside I,Icaritin and Anemarrhenasaponin I,all of which reversed osteoporosis at 1 μM(P < 0.05);the mixture of 12 active ingredients significantly improved BMA and IOD in zebrafish compared to the model group(P < 0.0001);the mixture of 12 active ingredients was equipotent with no significant efficacy difference compared to the EXD extract(P > 0.05).In addition,both EXD extract and the mixture of candidate active ingredients(high,medium and low)significantly reversed K02288-induced osteoporosis(P <0.0001),and the high-dose mixture of active ingredients and EXD extract were equipotent.Chapter 6: Validation of the mechanism of EXD down-regulating iron overload against osteoporosis A rat model was constructed to investigate the effects of EXD on bone metabolism and iron metabolism in osteoporotic rats with concomitant iron overload,and to investigate the effect of EXD on BMP-SMADs signaling.Methods: Fifty-six female SD rats(200 ± 20 g)were used to construct a rat osteoporosis model by ovariectomy combined with intraperitoneal injection of ferric ammonium citrate,and the rats were divided into blank group,sham-operated group,model group,positive control group(Alendronate 1.0mg/kg or Deferasirox 124.0 mg/kg)and EXD group(9.3 g(raw material)/kg for the highdose group and 4.7 g(raw material)/kg for the low-dose group).The treatment was started at the 6th week after surgery and continued for 2 months,and serum,liver and femur samples were taken from the rats after the last administration for testing.ELISA kits were used to detect serum biochemical indexes related to bone metabolism and iron metabolism,Micro-CT was used to detect the microstructure of bone,and the gene and protein expressions of the offbuild targets in the pathway obtained by network pharmacological enrichment were detected by Western blotting and RT-qPCR.Results: Compared with the model group,EXD treatment significantly increased bone mineral density and improved bone microarchitecture(P < 0.01);levels of osteogenic markers(OCN,BALP)and hepcidin(P < 0.05)were significantly increased,and levels of iron-level markers SF and osteoclast markers(TRAP)(P < 0.0001)were significantly decreased.Moreover,the expression of key genes of the BMP-SMADs pathway,including BMP2,BMP7,SMAD1/5/8,ID2 and HAMP,as well as osteogenesisrelated factors RUNX2 and OSX,were also significantly up-regulated by EXD(P < 0.05).Conclusion The active components in EXD,including Icariin,Baohuoside Ⅰ,Epimedin A,Epimedin B,Epimedin C,Icaritin,Stigmasterol,Ferulic acid,Curculigoside,Berberine,Palmatine and Anemarrhenasaponin Ⅰ,are the important material basis for its ability to improve PMOP by promoting bone formation,inhibiting bone resorption,increasing hepcidin expression,and regulating amino acid metabolism of the body.Promoting the signaling of BMP-SMADs to increase the expression of hepcidin is an important mechanism of EXD down-regulation of iron overload against PMOP.