Preliminary Investigation Of Potential Active Ingredients And Molecular Mechanisms Of Total Flavonoids Of Drynariae Rhizoma-mediated Treatment Of Glucocorticoid-Induced Osteoporosis

Glucocorticoids(GCs)with wide clinical application were mostly used to treat allergic disorders,autoimmune disease and respiratory illness.However,a prolonged and heavier GCs use often leads to secondary osteoporosis,which is called glucocorticoidinduced osteoporosis(GIOP).According to the large-scale epidemiological investigations in China,the rheumatic patients which received GCs,present a over 80%prevalence of osteoporosis.Due to the features of low side-effect and long-term administration,the traditional Chinese medicines(TCMs)are gaining widespread attention for the past few years.The traditional Chinese medicine preparations with the effect of tonifying kidney have increasingly been used in treating osteoporosis.As the represent of "Kidneytonifying" TCMs,Drynariae Rhizoma(Davallia marisii Moore ex bak)with bitter flavor and warm nature is attributed to the liver and kidney meridians,which has the effect of strengthening the kidney,reinforcing bone,treating disorders and relieving pain.Modern pharmacological studies show that the Drynariae Rhizoma has great anti-osteoporosis,anti-inflammatory,fracture healing,kidney protection,tooth growth and hypolipidemic effects,owing a good prospect for application.The predominant active ingredients of Drynariae Rhizoma are flavonoids called total flavonoids of Drynariae Rhizoma(TFDR).TFDR is widely applied to treat postmenopausal osteoporosis,however,its effect on treating GIOP is not well defined.Besides,the studies about TFDR often take it as a whole,while the exact chemical composition of TFDR is not clear leading to more difficulties for its intensive investigation.Therefore,this study firstly designed in-vivo experiment to confirm efficacy of TFDR against GIOP.Then the LC-MS/MS was employed to perform qualitative and quantitative analysis in order to identify the potential active ingredients of TFDR.The network pharmacology and quantitative real-time PCR(qRT-PCR)were used to primly predict and verify the molecular mechanisms.Furthermore,this study preliminarily investigated the pathogenesis of GIOP Based on label-free mass proteomics.The specific experiment details were displayed as follows:1.The pharmacodynamic study of TFDR-induced treatment against GIOP.Using female Sprague-Dawley rats as subjects,the GIOP rat model was established by alternatively injecting dexamethasonethe on the left and right leg.After confirming successful modeling,the rats were gavaged TFDR solutions at a clinical equivalent dose.After 9 weeks,the plasma,femur,and tibia of different groups were collected for the subsequent detection.The representative indicators of osteogenesis and osteoporosis in different groups were detected using enzyme-linked immunosorbent assays.The parameters on bone structural mechanics and changes in the bone micro-structure were detected by micro-CT scanning and HE staining.The results showed that TFDR demonstrated high efficacy against GIOP,which was able to significantly enhance BMD of rats and improve bone macroscopic structure.Therefore,it was valuable to further investigate the potential active ingredients and exact molecular mechanisms of TFDR against GIOP.2.The in-vivo and in-vitro qualitative analysis of chemical constituents in TFDR based on UHPLC-QE-Orbitrap-MS/MSBased on LC-MS/MS technology,this experiment performed qualitative analysis aiming to collecting in-vivo and in-vitro compounds of TFDR.After administrated with TFDR in clinical equivalent doses by gavage,the plasma samples at different time points were collected and fixed to identify in-vivo compounds of TFDR.TFDR extractions were dissolved by 50%methanol to identify in-vitro compounds of TFDR.The information of molecular weight and fragment ion was collected and was compared with related database.A total of 192 compounds were identified in-vivo and 45 were in-vitro.The compounds such as naringin,naringenin,neoeriocitrin,eriodictyol,eriocitrin,kaempferol,scutellarin,kaempferol-7-O-glucoside,epicatechin were detected in both in-vivo and invitro,which were with a semi-quantitative abundance and wer worth exploring in depth for further analysis.3.The in-vivo and in-vitro quantitative analysis of chemical constituents in TFDR based on UHPLC-Q-TRAP-MS/MSReferring to the above results of qualitative analysis,9 flavonoid compounds were selected to perform in-vitro quantitative analysis.Among all the compounds,naringin had the highest content,which was up to 134.08 mg/g,followed by neoeriocitrin,eriodictyol,and naringenin.4 flavonoid compounds with higher content in vitro and in blood were selected as the subjects.The pharmacokinetic studies were performed in rats of SHAM group and GIOP group with 2 g/kg administration,and the blood samples in different group were collected.The results showed that the metabolic process of 4 flavonoid compounds were similar between SHAM group and GIOP group,while the pharmacokinetic parameters such as Cmax,Tmax,and AUC showed significant differences.It meant that the absorption of flavonoid compounds were changed in pathological states,and the pharmacokinetic behavior of GIOP group significantly changed compared with it of SHAM group.4.The prediction and validation of molecular mechanisms on TFDR-induced treatment against GIOP based on network pharmacology and quantitative real-time PCR(qRT-PCR)Based on the above results of quantitative and qualitative analysis,integrating literature,and searching in different databases,a total of 13 compounds were selected as candidates for subsequent analysis.The related targets of TFDR and GIOP were selected according to the Swiss Target Prediction,OMIM,GeneCards and Drugbank databases.After integrating the information of different targets,the veen was displayed and 67 overlapped targets were screened as the focuses of subsequent studies.The core targets with high degree such as AKT1,SRC,TNF,VEGFA,ESR1,PPARγwere selected by constructing protein-protein interaction network.These targets might have the potential effects in TFDR-mediated treatment of GIOP.The results from the GO,KEGG bioinformatics analysis showed that the overlapped targets more enriched in cancer related signaling pathway and PI3K/AKT signaling pathway.These pathway might be closely related to TFDR-mediated treatment of GIOP,which can provide references for further investigation on pharmacological mechanisms.5.Pathogenesis of GIOP Based on label-free mass proteomicsIn order to lay the foundation for applying TCMs to treat GIOP,it was necessary to further understand the exact pathogenesis.The label-free mass proteomics was used to investigate the pathogenesis.To ensure the successful modeling,the bone samples of different groups were compared with each other by the histopathological analysis.After extraction,enzymatic hydrolysis,and desalination,the total femur proteins were detected using LC-MS/MS technology for performing qualitative and quantitative analysis.It was shown that a total of 20 up-regulated and 27 down-regulated differentially expressed proteins(DEPs)were finally identified.The expression of protein NPM1,APMAP,COX6A1 and ACP5 displayed a significant difference between two groups,deserving to be explored intensively.The expression of protein APMAP was shown to be most significantly up-regulated,while protein COX6A1 to be most significantly down-regulated.Our bioinformatics analysis results showed that the AMPK signaling pathway were expected to become potential core pathway to revealing GIOP pathogenesis.According to the protein-protein interaction analysis,the target protein ALB with highest degree showed a close relationship with other targets,which can be a potential candidate for GIOP treatment.Overall,this study confirmed the TFDR efficacy against GIOP expanding the clinical application of TFDR for the first time;The potential active ingredients of TFDR were analyzed through qualitative and quantitative analysis,which complemented the basic chemical research for the first time;The molecular mechanisms of TFDR against GIOP were preliminarily predicted and validated using network pharmacology integrating quantitative(real-time)RT-PCR for the first time,which preliminarily addressed the question of the molecular mechanisms was not poorly defined;The pathogenesis of GIOP was investigated using label-free mass proteomics for the first time,which provide references for TCMs’ application in treating GIOP.