Study On The Anti-senile Osteoporosis Pharmacodynamics Substances And Mechanism Of Shenrong Guben Pill

Objective: Ginseng and Antler Solidifying Pill is a classic formula recorded in Feiheting Jifang,which is one of the representative formulas of the tonic category in the collection,with the effects of benefiting essence and filling marrow,strengthening tendons and bones.However,there is no clinical study on Ginseng Antler Solid Pill,and its pharmacodynamic substances and mechanism of action in the treatment of osteoporosis are still unclear.Therefore,based on the active ingredient research idea of "entering the bloodstream and entering the target",this study applied quantitative effect-weighted network pharmacology and machine learning to screen the active ingredients and core targets of Ginseng Antler Solid Pill against osteoporosis in the elderly,and established the network of "active ingredient-core target-intercellular pathway" to determine the active ingredient and core target,and identified the active ingredient and core target.The network of "active ingredient-core target-intercellular pathway" was established to determine the composition of the active components,and the efficacy and mechanism of Ginseng Antler Solid Pill in intervening osteoporosis were investigated through in vitro co-culture cell experiments and animal experiments in osteoporotic rats,so as to ultimately explore the material basis and mechanism of action of the compound against osteoporosis in old age,and to lay the foundation for the further development and utilisation of Ginseng Antler Solid Pill.Methods: 1.Examination of the in vitro activity of Ginseng Antler Solidifying Pill on BMDM and BMSC: Ginseng Antler Solidifying Pill-containing serum was prepared using SD rats,and the effects of the serum on BMDM M2 polarisation and BMSC osteogenic differentiation were examined in co-culture and separate culture systems;2.Analysis of the targeting components of Ginseng Antler Solidifying Pill on BMDM M2 polarisation and BMSC osteogenic differentiation: Based on the research idea of "blood-into-targeting",firstly,the blood-into-targeting components of Ginseng Antler Solidifying Pill-containing serum were analysed by UPLC-QE-MS/MS,and then the intracellular targets that can be associated with BMDM and BMSC were obtained by cell-affinity ultrafiltration.Secondly,by cell-affinity ultrafiltration,we obtained the target components that can bind to the intracellular targets of BMDM and BMSC,and finally,we analysed the composition of the "target" components in the serum of Ginseng Antler Solidago Pills by UPLC-QE-MS/MS;3.Screening and validation of the active ingredients and core targets of Ginseng Anti-bone Pills against osteoporosis: Screening the active ingredients and core targets of Ginseng Anti-bone Pills against osteoporosis through quantitatively weighted network pharmacology and machine learning,validating the attribution of the active ingredients and core targets through molecular docking and in vitro activity experiments,and confirming the core genes of the inter-cellular communication of BMDM and BMSC by integrating the measured data and the PPI analysis,and finally constructing the "core target" gene of BMDM and BMSC.The core genes of BMDM and BMSC inter-cellular communication were identified through the combination of measured data and PPI analysis,and the network of "active ingredient-core target-inter-cellular pathway" was finally constructed;4.In vitro validation and mechanism analysis of the active components of Ginseng and Antler Pills: Based on the ratio of the peak areas of the active components in the ion flow diagram of the drug-containing serum,the active components of Ginseng and Antler Pills for the treatment of osteoporosis were obtained by medium-pressure liquid chromatography,and at the same time,the active components were combined in equal amounts to form an equiproportionate group for the purpose of comparing with alcoholic extracts and the active components.The in vitro activities of BMDM and BMSC in the co-culture system were investigated to clarify the effects of the active components on inflammatory factors and osteogenic markers,and the effects of the active components on the gene expression of inflammatory factors and osteogenic markers were investigated by RT-q PCR;Pharmacodynamic changes and mechanism study of Ginseng Antler Solidifying Pill intervention in aged rats with osteoporosis: Natural aging combined with hydrocortisone injection was used to create an aged rat model of osteoporosis,and then the alcoholic extract of Ginseng Antler Solidifying Pill and the active components were given to the model rats for intervention,and the BMD,bone microstructure,HE,Von Kossa,and Senkaku Pathology were investigated to evaluate the efficacy of the compound in the treatment of aged rats with osteoporosis.The efficacy of the compound in treating osteoporotic rats was evaluated by examining bone mineral density,bone microstructure,HE,Von Kossa,and reddish green pathological tests,and the effects of Ginseng and Guubenwan on serum metabolic profiles of osteoporotic rats were investigated by measuring the gene expression of inflammatory factors and related osteogenic markers in the femur of the rats.Results: 1.The results of in vitro activity investigation of Ginseng and Ginseng Pills:after administration of BMDMs and BMSCs in the co-culture system,the content of IL-6secreted in the supernatant of the cells was decreased,the content of IL-10 was elevated,and the content of BALP was increased as compared with that of the model group.Meanwhile,after the administration of BMDMs cultured alone,the content of IL-10 secreted in the cell supernatant was increased and the content of IL-6 was decreased compared with the model group;the content of BALP in BMSCs was increased,which had the same trend as the results of the activity of the co-culture system mentioned above but was slightly lower than that of the activity of the co-culture system.The results of RT-q PCR further demonstrated that the inflammatory indicators of BMDM,CD86,IL-6 expression decreased,and M2 polarisation indicators CD206 and IL-10 expression increased.2.Analysis of the entry components of Ginseng antler solidifying pill on BMDM M2 polarisation and BMSC osteogenic differentiation: UPLC-Q Extractive MS was applied to characterise the entry components of Ginseng antler solidifying pill,and the structures of 89 compounds were deduced by consulting the database and combining with the analysis of mass spectrometry cleavage patterns,which mainly included 21 ginsenosides such as ginsenoside and 16 cycloenetheretherpene glycosides such as catalpol.cyclic enol ether terpene glycosides,9 terpenoids such as genipin glycosides,10 steroidal saponins,6 amino acids and 5 phenolic acids,and others.Among the 89 compounds,14 compounds were from ginseng,15 compounds were from antler velvet,22 compounds were from asparagus,15 compounds were from maitake,and 23 compounds were from dioecious or ripe dioecious.Based on the analysis of the target components by live cell affinity ultrafiltration-mass spectrometry(UF-MS),17 chemical components were found in BMDM,including Diosgenin,Diosgenin D,and Ginsenoside Rd,and 10 chemical components were found in BMSC,including Ginsenoside Rd,Maitake Saponin D,and Ginsenoside Rh2,which initially clarified the target components of Ginseng and Antiherbic Pills in the prevention of osteoporosis in the elderly.3.Screening and validation of the active ingredients and core targets of Ginseng Antler Solid Pill against osteoporosis in the elderly: The results of the quantitatively weighted network pharmacology showed that the serum containing Ginseng Antler Solid Pill induced M2 polarisation in BMDM through 22 core targets such as AKT1,PRKCA and EGFR,and promoted the osteogenic differentiation of BMSC through 17 core targets,such as MAPK1,MMP9,and EGFR;the KEGG enrichment results showed that the core targets of BMDM were enriched to the HIF-1 signalling pathway,and the direct targets in BMSCs were enriched to the VEGF signalling pathway;through the actual data of supernatant VEGF in the co-culture-containing serum group and the comprehensive analysis of the PPIs,it was found that the BMDMs might promote the differentiation of BMSCs towards osteogenesis by releasing VEGF,which constitutes a core gene structure based on TLR4/HIF-I/VEGF/Runx2 as the core gene.Runx2 as the core genes of the intercellular pathway.Then,the anti-osteoporosis active ingredients of the formula were screened by machine learning through the core target back-propagation of the ingredients,which were sarsasaparilla saponin,kynurenine glycoside,ginsenoside Rf,digitonin D,hypoxanthine,maitake saponin D,catalpol,guanosine,adenosine,and ginsenoside Rd,respectively;the molecular docking verified the binding effect of the entry components and effector targets,and the results of the in vitro activity experiments proved the accuracy of the screening of the active ingredients.Finally,the network of "active ingredient-core target-intercellular pathway" was constructed.4.In vitro verification and mechanism analysis of the active components of Ginseng and Antler Pills: After the intervention of BMDM and BMSC in the co-culture system,the content of IL-6 secreted in the supernatant of the cells decreased,the content of IL-10 increased,and the content of BALP increased compared with that of the model group,and the order of the strengths of the pharmacological effects was as follows: active component > alcohol extract group > equal proportion preparation group.Gene expression results showed that the expression of pro-inflammatory factor IL-6 gene was down-regulated and the expression of IL-10 anti-inflammatory factor gene was up-regulated in BMDM,and the expression of BALP gene,a marker of early osteogenesis,was up-regulated in BMSC.5.Research on the mechanism of ginseng antler solid pill to promote bone formation in aged osteoporotic rats: the results of rat bone morphology showed that compared with the model group,after the administration of ginseng antler solid pill to rats,the femur bone density increased,the structural cavity of cancellous bone was improved in a concentration-dependent manner,the distribution of bone trabeculae was uniform and regular and the thickness of the trabeculae was increased,the ratio of the volume of bone tissue was elevated,the total porosity of the cancellous bone was reduced,and the width of the medullary gap between the bone trabeculae was narrowed;the HE,Von The results of HE,Von Kossa,and Senna Green showed that compared with the model group,lipogenesis was significantly improved in the femur,the thickness of trabeculae was increased,the inflammatory infiltration was reduced,the expression of calcium deposition was increased,and the expression of osteoblasts and chondroplasts was significant.The expression of relevant targets in the femur was analysed by RT-q PCR,and the expression of IL-6 and TLR4 was down-regulated,and the expression of IL-10,HIF-1,VEGF,Runx2 and COL1 were all up-regulated to a certain extent,and the gene expression level of the drug administration group tended to be higher than that of the blank group.The results of non-targeted metabolomics of rat serum showed that Ginseng and Antler Solid Pill could adjust the expression trend of serum metabolites such as amino acids and sterols,and ultimately normalise the metabolic profiles of aged osteoporotic rats.Conclusion: 1.Ginseng antler solid pill has the effect of regulating the homeostasis of inflammatory bone microenvironment,promoting the shift of BMDM to M2-type polarisation and the differentiation of BMSC to osteogenesis.2.The cell affinity ultrafiltration-mass spectrometry(UF-MS)technique can efficiently analyse and confirm the target components in the serum of Ginseng and Antler Solidifying Pills that directly bind to the target cell proteins,which provides actual data for the screening of the active ingredients of Ginseng and Antler Solidifying Pills for the treatment of osteoporosis in the elderly.3.The targeting components of Ginseng Antler Solidifying Pill not only directly act on BMSCs to promote osteogenic differentiation,but also synergistically promote the osteogenic differentiation of BMSCs by inducing the release of VEGF through M2 polarisation of BMDMs,thus constituting an intercellular pathway with TLR4,HIF-1,VEGF and Runx2 as the core genes.4.The active components,consisting of 11 active ingredients,can promote the osteogenic differentiation of BMSCs under the co-culture system,induce the polarisation of BMDM M2,and regulate the gene expression of the core targets in the inter-cellular pathway of TLR4/HIF-1/VEGF/RUNX2.Based on the results of in vitro experiments in which the active components were superior to the alcoholic extracts of the original formula and the components of the equivalent proportion of the ingredients,the scientific mechanism of the ginseng and antler pill has been proved to be the best.The scientific validity of the mechanism of Ginseng and Antler Pills.5.Ginseng and antler solid pill and its active components can regulate the homeostasis of bone microenvironment in rat femur inflammation and promote osteogenic differentiation,mainly by sarsaparilla saponin,jingnipinoside acid,ginsenoside Rf,digitonin D,hypoxanthine,maitake saponin D,catalpol,guanosine,adenosine,ginsenoside Rd,and ginsenoside Rh2,etc.,and modulate the components such as TLR4,MAPK1,EGFR,HIF-1,and KDR,VEGF and other targets.Finally,the regulation of osteogenic differentiation of rat BMSCs was achieved at the whole animal level through the TLR4/HIF-1/VEGF/RUNX2 intercellular pathway.The results of rat serum non-targeted metabolome showed that this formula could up-regulate the content of amino acid metabolites such as aspartic acid and glycine to promote the participation of amino acid metabolites in osteogenic differentiation,and down-regulate the content of sphingomyelin to inhibit the lipid differentiation of mesenchymal stem cells to improve osteoporosis.In conclusion,Ginseng and Antler Solid Pill and its active components can act on the target of inducing M2 polarisation of BMDMs and osteogenic differentiation of BMSCs,and play a synergistic effect through the intercellular pathway TLR4/HIF-1/VEGF/Runx2,which can improve the chronic inflammatory microenvironment of bone marrow and promote bone formation,and achieve the treatment of osteoporosis by "tonifying the kidney and filling in the bone marrow,and strengthening the tendons and bones".It can achieve the goal of "tonifying the kidney,filling the marrow,strengthening the tendons and bones" in the treatment of osteoporosis in the elderly.