Investigate The Regulation Of PI3K/AKT/eNOS Signaling Pathway By Osteoking In Treating Osteoporosis Based On Network Pharmacology

Objective:In this study,the effective active components、key targets and signal pathways of Osteoking in treating osteoporosis were identified through network pharmacology analysis.Then,the binding stability between effective active components and core targets were determined by molecular docking technology.Finally,the therapeutic effects of Osteoking on the prevention and treatment of the ovariectomized rats and the regulation of PI3K/AKT/e NOS signaling pathway in it were investigated.Methods:1.Network pharmacology and molecular docking analysisThe active ingredients of Osteoking were retrieved by TCMSP,TCMID database and literature mining method,and the active ingredients were screened by ADME(absorption,distribution,metabolism,excretion)criteria,and the active ingredients were extracted by TCMSP,Pharm Mapper and Swiss Target Prediction database to query the targets corresponding to the active ingredients;use the Gene Cards database to obtain the targets of osteoporosis,and extract the common targets of drugs and diseases by Tbtool software;use Cytoscape software to construct PPI networks for the common targets,and use the David Bioinformatics Resources platform to obtain GO and KEGG enrichment analysis of common targets with P value less than 0.05,and then use Omic Share cloud platform for visualization and mapping.Meanwhile,the chemical structures of the core targets were retrieved in the PDB database and the 3D structures of the active ingredients were retrieved in the Pub Chem database for molecular docking by Auto Dock software.2.The ovariectomized osteoporosis rat modelThirty SPF-grade 6-month-old female SD rats were randomly divided into sham-operated group(Sham,n=10),ovariectomized group(OVX,n=10),and Osteoking group(Osteoking,n=10).After one week of acclimatization,each group of rats was anesthetized and bilateral ovariectomy was performed to establish a devitalized osteoporotic rat model,except for the sham-operated group where adipose tissue was removed.One week after the operation,the Osteoking group was gavaged every other day at a dose of 0.9 ml/time;the sham group and OVX group were given saline gavage,and the material was taken after 12 weeks.The left tibia was taken for HE staining to observe the trabecular structure of rats in each group;Micro-CT was performed on the fifth lumbar vertebra to observe the bone density and bone microstructure parameters(BMD,BV/TV,Tb.N,Tb.Sp,BS/BV,Tb.Th)of rats in each group;the right femoral was taken for TRAP staining to observe the differentiation and number of osteoclasts;RT-PCR was used to detect the left femoral tissue of the m RNA expression levels of CTSK 、 MMP9 and RANKL,proteins characteristic of osteoclast differentiation;the protein expression levels of PI3 K,p-PI3 K,AKT,p-AKT e NOS and p-e NOS were detected by western blotting in the right femoral tissue.Results:1.Network pharmacology and molecular docking analysisThere are a total of 85 active ingredients and 263 target proteins for Osteoking.The GO enrichment analysis included 624 entries for biological processes,68 entries for cell composition,and 123 entries for molecular functions.125 signaling pathways were analyzed by KEGG enrichment,including PI3K/AKT signaling pathway,MAPK signaling pathway,TNF signaling pathway,and osteoclastic differentiation.The PPI network showed AKT1 as the core target gene,and molecular docking results showed strong affinity of AKT1 with quercetin,luteolin and kaempferol.2.The ovariectomized osteoporosis rat modelHE staining results showed that,compared with the sham group,the number of bone trabeculae in the OVX group decreased,broke,widened gaps,and increased fat droplets in the medullary cavity,and other pathological changes of osteoporosis in the rats.After the intervention of Osteoking,the trabecular structure of bone in the Osteoking group was significantly improved compared with that in the OVX group,suggesting that Osteoking had a protective effect on bone loss in the ovariectomized osteoporosis rats.The results of Micro-CT showed that,compared with the sham group,the fifth lumbar vertebrae of the OVX group showed deterioration of bone junction,bone microarchitecture deterioration,BMD,BV/TV,Tb.N,Tb.Th decreased significantly and BS/BV,Tb.Sp increased significantly(p<0.01);after treatment with Osteoking,BMD,BV/TV,Tb.N,Tb.Th increased significantly and BS/BV,Tb.Sp decreased significantly in the Osteoking group(p<0.05 or p<0.01),suggesting that Osteoking can effectively improve bone mass and bone microarchitecture in the ovariectomized osteoporosis rats.TRAP staining results revealed that the number of osteoclasts increased in the OVX group compared with the sham group(p<0.001),while the number of osteoclasts was significantly reduced in the Osteoking treatment compared with the OVX group(p<0.01),suggesting that Osteoking was able to inhibit the formation of osteoclasts.RT-PCR showed that the expression of osteoclast differentiation characteristic genes CTSK and MMP9 were up-regulated in bone tissue of the OVX group(p<0.05),while the expression of the above m RNA was significantly down-regulated in the Osteoking group compared with the OVX group(p<0.05),suggesting that Osteoking could inhibit bone resorption in the ovariectomized osteoporosis rats.Western Blotting assay demonstrated that Osteoking could increase the phosphorylation levels of p-PI3 K,p-AKT,and p-e NOS.(p<0.05),suggesting that the activation of PI3K/AKT/e NOS signaling pathway is involved in the prevention and treatment of osteoporosis by Osteoking.Conclusion:1.Osteoking has multi-component,multi-target and multi-pathway characteristics for the treatment of osteoporosis,among which,AKT1 is one of the key targets of Osteoking for the treatment of osteoporosis.2.Osteoking has a significant protective effect against ovariectomy-induced bone loss,and by a mechanism involving activation of PI3K/AKT/e NOS signaling pathway.