| Objective:Isopsoralen(Isopsoralen,IP)is a natural small molecular drug and one of the effective components of traditional Chinese medicine psoralen.It can promote bone formation,inhibit bone resorption and improve the state of chondrocytes and so on.It has the potential to become an effective drug for the treatment of osteoporosis.Our project predicts the target of isopsoralen in the treatment of osteoporosis on the basis of network pharmacology,and the prediction results are verified by in vitro and in vivo experiments.To further study the mechanism of isopsoralen in the treatment of osteoporosis.Method: Experiment 1: the network pharmacological study of isopsoralen in the treatment of osteoporosis.(1)using Pub Chem Compound database and CTD database to predict isopsoralen target genes,and then using STRING database and DAVID database to unify the gene names and IDs.(2)Searching osteoporosis related genes by using Gene database and Gene Cards database,in which the search results of Gene Cards database need ID conversion in DAVID database;(3)overlapping drug action targets with disease genes to obtain intersection targets,importing target information into STRING database and Cytoscape software to construct PPI network,performing visualization processing,and analyzing the interaction relationship between targets;(4)GO enrichment analysis related to cell composition,molecular function and biological process and KEGG analysis related to target enrichment pathway were carried out.Experiment 2: experimental study on the effect of isopsoralen on MC3T3-E1 cells.The mouse osteoblast cell line MC3T3-E1 was divided into control group and experimental group.the control group was only added with complete culture medium,while the experimental group was added with isopsoralen of 10μM,20μM and 50μM.after 48 h and 72 h,q RT-PCR was performed to detect the expression of PI3 K,AKT and mTOR,which were predicted by network pharmacology,respectively,to verify the results of network pharmacology.Experiment 3: experimental study on the effect of isopsoralen on chondrocytes in SD rats.SD rats were divided into experimental group and control group,with 6 rats in each group.The experimental group was given 40mg/kg isopsoralen for 7 days,while the control group was given the same amount of solvent for 7days.After 24 hours,the rats were killed by neck removal,and enough knee cartilage tissue was taken,which was quickly frozen in liquid nitrogen and ground into powder.After extracting RNA by Trizol method,q RT-PCR was performed to detect the expression of target genes PI3 K,AKT and mTOR predicted by network pharmacology,and verify the results of network pharmacology.Results: Experiment 1:87 targets of isopsoralen were predicted by Pub Chem Compound database and CTD database,and 252 targets related to diseases were obtained by Gene database and Gene Cards database.Finally,19 target proteins of isopsoralen for osteoporosis were obtained by intersection of disease targets and drug targets.After constructing PPI network,it was found that AKT1 gene had the highest Degree value and was at the core of the network.GO analysis showed that the biological processes related to osteoporosis mainly included osteoblast differentiation,protein kinase B signal transduction,cell proliferation,osteoclast differentiation and so on.KEGG analysis showed that PI3K/AKT/mTOR signaling pathway was highly correlated with osteoporosis,and AKT1,PIK3 CA and mTOR were all enriched in this pathway.Therefore,we chose PI3 K,AKT and mTOR genes for in vitro and in vivo experiments in subsequent experiments.Experiment 2:the results of cell experiment showed that compared with the control group,the expression level of PI3 K in 48 h group at 10,20 and 50μM could be significantly increased,but with the increase of dose,the effect of promoting PI3 K expression did not increase significantly,and even showed a downward trend(P＜0.05);In 72 h group,the expression of PI3 K showed a downward trend at 10,20 and 50μM concentrations,showing an inhibitory effect(P＜0.05).Compared with the control group,10,20 and 50μM concentrations in the 48-hour group significantly increased the expression level of AKT,which was positively correlated with the dose(P＜0.05).In 72 h group,10μM concentration can promote AKT expression,while 20μM and 50μM can inhibit AKT expression(P＜0.05).Compared with the control group,all the three concentrations in the 48-hour group promoted the expression of mTOR significantly,and there was a positive correlation with the concentration(P＜0.05)In 72 h group,10μM and50μM can promote the expression of mTOR,while 20μM can inhibit the expression of mTOR(P＜0.05).Experiment 3:the results of animal experiment showed that compared with the control group,the PI3 K gene expression level of SD rat chondrocytes was significantly higher than that of the control group after 40 mg/kg isopsoralen gavage for 7 days,showing a promoting effect(P＜0.05);compared with the control group,the AKT gene expression level of the SD rat chondrocytes was significantly higher than that of the control group after 40mg/kg isopsoralen was intragastrically administered for 7 days,showing a promoting effect(P＜0.05);compared with the control group,40 mg The mTOR gene expression level of SD rat chondrocytes was higher than that of the control group after 7 days of intragastric administration with isopsoralen/kg,which showed a promoting effect(P＜0.05).Conclusion:(1)Isopsoralen plays a role in the treatment of osteoporosis through AKT1,PIK3 CA,mTOR and other multi-targets,and its related pathway is PI3K/AKT/mTOR signal pathway;(2)Isopsoralen acts on 48 to promote PI3 K,AKT,and mTOR gene expression in MC3T3-E1 cells in a concentration-dependent manner.Its action pathway is PI3K/AKT/mTOR signaling pathway;(3)Isopsoralen at 10,20,and 50μM concentration for72 h has an effect on MC3T3-E1 cells have cytotoxic effects;(4)40mg/kg isopsoralen can promote the increase of PI3 K,AKT,mTOR gene expression in SD rat chondrocytes after 7days of intragastric administration,and its action pathway is PI3K/AKT/mTOR signal path. |
PDF Full Text Request