| Objective:There are more than 200 million patients suffering from osteoporosis worldwide,which is a disease that seriously affects the quality of life of middle-aged and elderly people,but various therapeutic drugs have certain toxic and side effects.Ginseng is a kind of safe and reliable homologous medicine,and has the treatment of osteoporosis,the subject of the hot processing products Black ginseng,screening the activity of osteoporosis and analysis of its possible mechanism,through the construction of mesoporous calcium silicate drug delivery system,its osteoporosis activity and mechanism,for the development of natural drugs and clinical transformation to provide more possible.Methods:1.GEO gene chip technology was used to analyze the pathogenesis of osteoporosis,system biology and molecular docking techniques were used to study the mechanism of ginsenoside in the treatment of osteoporosis,and its active components were screened by virtual screening.2.HPLC-Q-TOF-MS/MS,pyrolysis pathway and multivariate statistical analysis were used to reveal the dynamic changes of ginsenosides in unprocessed ginseng and 9 different stages of processed black ginseng.The dynamic changes of anti-osteoporosis activity of black ginseng in different stages were investigated by zebrafish osteoporosis model combined with multivariate statistical analysis,and the ginsenosides with strong anti-osteoporosis activity were screened.3.The"content coefficient"and"efficacy coefficient"were introduced,and the higher content components and core action targets were located to the center of the network by the Content-Effect weighted network pharmacology.nuclear magnetic resonance metabonomics combined with principal component analysis,partial least square discriminant analysis and other multivariate statistical methods to explore the differential metabolites between black ginseng group and prednisolone model group.The expression of related genes in anti-osteoporosis core effect target of black ginseng was detected by real-time fluorescence quantitative PCR.4.The core target of Ginsenoside Rh1 against osteoporosis was selected by network pharmacology,and its molecular docking and molecular dynamics simulation were carried out.The interaction mode of trace protein and small molecule RMSD,RMSF and trajectory stability interval was extracted to investigate the mechanism of Ginsenoside Rh1anti-osteoporosis.5.Mesoporous calcium silicate nanomaterials were synthesized by in situ self-assembly of surfactant supramolecules by template method.HPLC difference method was used to investigate the loading and release process of Ginsenoside Rh1 mesoporous calcium silicate drug delivery system.X-ray diffraction,Fourier transform infrared spectroscopy,nitrogen adsorption and desorption,scanning electron microscope and transmission electron microscope were used to construct and characterize mesoporous calcium silicate nanomaterials and Ginsenoside Rh1-loaded mesoporous calcium silicate drug delivery system,and zebrafish osteoporosis model was used to investigate their biological safety and anti-osteoporosis activity.6.The concentration gradients of mesoporous calcium silicate nanomaterials,Ginsenoside Rh1 monomers and Ginsenoside Rh1 mesoporous calcium silicate drug delivery system were set,and their anti-osteoporotic activity was studied by alizarin red staining,ALP activity detection,ALP staining test,MTT test and surface marker detection of human bone marrow mesenchymal stem cells.The effects of Q-RT-PCR and Western Blot on the expression of m RNA and protein related to osteogenic differentiation of human bone marrow mesenchymal stem cells were investigated,and the possible mechanism of their anti-osteoporosis activity was revealed.Results:1.A total of 168 differential genes in osteoporosis GSE161361 dataset were obtained by GEO microarray technology,including 92 up-regulated genes and 76 down-regulated genes.There were 57 ginsenosides and 17 core targets virtually screened by system biology and molecular docking technique.The results showed that all 57 ginsenosides had certain anti-osteoporotic activity.among them,triol ginsenoside>diol ginsenoside,and the anti-osteoporosis activity of ginsenoside with high molecular weight is lower than that of ginsenoside with low molecular weight.Ginsenoside B,Ginsenoside Rh1 and other compounds may be potential anti-osteoporotic compounds.2.Through HPLC-Q-TOF-MS/MS,pyrolysis pathway and multivariate statistical analysis,the processing of black ginseng can be divided into three stages:the first stage is unprocessed,when the ginsenosides in garden ginseng are mainly macromolecular primary saponins;the second stage is processed for 5 times,during which the content of components changes greatly,and the above macromolecular primary saponins are decomposed into medium molecular weight ginsenosides by pyrolysis.The third stage is 6-9 times of processing,when the change of component content tends to be stable,rich in small molecular weight rare saponins.At the same time,25 ginsenosides were selected as differential components.Through the zebrafish osteoporosis model combined with principal component analysis,the activity intensity of black ginseng in the processing process can be divided into three stages:the first stage is the unprocessed panax ginseng,that is,it has anti-osteoporosis activity,but the degree is relatively weak;the second stage is processed for5 times,during which the activity decreases at first and then increases,and changes greatly.In the third stage,the activity was processed for 6 to 9 times,and the activity increased significantly and tended to be stable.Based on the principal component load analysis index factor load value and the results of virtual screening in the first chapter,the monomer activities of 13 kinds of ginsenosides were verified.The results showed that triol ginsenosides had good anti-osteoporosis activity,while diol ginsenosides showed toxicity to zebrafish,and Ginsenoside Rh1 showed better anti-osteoporosis activity.This is basically consistent with the results of virtual filtering.3.Based on the Content-Effect weighted network pharmacology,it is found that there are great differences in the core targets in the processing of black ginseng,which can be summarized into three stages:when the first stage is unprocessed,the targets are quite different from those after processing,and the unique core targets are CNR1 and FGFR1,mainly from primary ginsenosides.In the second stage,the core targets were processed for 5times,and the core targets were similar to those in the first stage,and the core targets in the third stage were processed for 6 times,and the core targets were basically the same and no longer changed.the anti-osteoporotic active targets in the above process were mainly enriched in the PI3K-Akt signal pathway,while cooperating with MAPK,Wnt and other signal pathways to play a role in the treatment of osteoporosis.A total of 17 differential metabolites were screened based on metabonomics and multivariate statistical analysis.the analysis of related metabolic pathways showed that they were closely related to lipid metabolism,glycolysis and tricarboxylic acid cycle.The expression of genes related to the anti-osteoporosis core effect target of black ginseng was investigated by q PCR.Finally,it was found that the anti-osteoporosis activity of black ginseng may be through up-regulating the expression of STAT3 and EGFR genes and down-regulating the expression of IL2 and PPARγgenes,which has an active effect on zebrafish osteoporosis model.4.The mechanism of anti-osteoporosis activity of Ginsenoside Rh1 was explored by means of network pharmacology,molecular docking and molecular dynamics simulation.It was found that Ginsenoside Rh1 may exert its anti-osteoporosis activity mainly by affecting the targets and pathways of ATK,PPARG and IGF1R.5.The mesoporous calcium silicate nanomaterials were successfully synthesized by X-ray diffraction and Fourier transform infrared spectroscopy.The specific surface area,pore volume and average pore diameter of the mesoporous calcium silicate synthesized by BET nitrogen adsorption were 575.2 m2 g-1,the pore volume is 1.76 m3 g-1,and the average pore diameter of 11.52 nm,indicating a large number of slit mesorous structure in the material.Scanning electron microscope（SEM）and transmission electron microscope（TEM）show that the central cavity of the synthesized mesoporous calcium silicate nanomaterials is very obvious,and the honeycomb pores are closely arranged,uniformly distributed and regular.The particle size is about 50 nm and the mesoporous pore diameter is about 7-8 nm,which is close to the results calculated by BET theory.After Ginsenoside Rh1 was successfully loaded with mesoporous calcium silicate,Ginsenoside Rh1 mesoporous calcium silicate drug delivery system was constructed,and the biological safety and anti-osteoporosis activity of Ginsenoside Rh1 mesoporous calcium silicate drug delivery system were investigated by zebrafish osteoporosis model.The results showed that the anti-osteoporosis activity of Ginsenoside Rh1 mesoporous calcium silicate drug delivery system was better than that of unloaded mesoporous calcium silicate nanomaterials.6.The cell viability of human bone marrow mesenchymal stem cells treated with different concentrations of mesoporous calcium silicate nanomaterials,Ginsenoside Rh1monomers and Ginsenoside Rh1 mesoporous calcium silicate drug delivery system was investigated by MTT method.It was found that the cell viability did not change,which confirmed its biosafety.Ginsenoside Rh1 and Ginsenoside Rh1 mesoporous calcium silicate drug delivery system can promote the expression of alkaline phosphatase activity during osteogenic differentiation of human bone marrow mesenchymal stem cells,and Ginsenoside Rh1 mesoporous calcium silicate drug delivery system is the most effective.Ginsenoside Rh1 and Ginsenoside Rh1 mesoporous calcium silicate drug delivery system can promote osteogenic differentiation of human bone marrow mesenchymal stem cells,and Ginsenoside Rh1 mesoporous calcium silicate drug delivery system has the strongest osteogenic differentiation ability.Mesoporous calcium silicate nanomaterials,Ginsenoside Rh1 and Ginsenoside Rh1 mesoporous calcium silicate drug delivery systems can all promote the m RNA and protein expression of osteogenic differentiation-related genes ALP,Runx2,OSX and OPN of human bone marrow mesenchymal stem cells.The results show that all of them are involved in the pre-,middle and late stages of bone formation,and all have the role of promoting expression,of which Ginsenoside Rh1 mesoporous calcium silicate drug delivery system has the strongest promoting effect.Conclusion:In this study,the anti-osteoporotic active substances of black ginseng were screened by dry-wet method.Combined with the techniques of many disciplines,such as genomics,the Content-Effect weighted network pharmacology,nuclear magnetic metabonomics and molecular dynamics simulation,the mechanism of anti-osteoporosis activity of black ginseng was explained from the perspective of whole network,metabolic pathway and molecular level.At the same time,a more efficient mesoporous calcium silicate drug delivery system was constructed to improve the utilization rate of active components,prolong the action time of drugs,and improve the therapeutic effect of osteoporosis,which provides a methodological reference for the development and utilization of traditional Chinese medicine. |
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