The Mechanism Of Gu-tong Formula In The Inhibition Of The "Pain-bone Destruction" Of The Tumor Osteolytic Metastasis Based On Network Pharmacology

As a patent prescription of traditional Chinese medicine,Gu-tong formula has been used for many years in clinic for its stable curative effect,with the function of warming Yang,dispersing cold,dredging collaterals and relieving pain.In this study,we mainly used network pharmacology,molecular docking,molecular dynamics and animal experiments to study the biological mechanism and pharmacodynamic components in the treatment of cancer related pain and osteolytic bone metastasis.This study preliminarily explored the mechanism of "multi-target and multi-channel" of Gu-tong formula,and laid a foundation for further research.Research 1:Multitarget mechanism of Gu-tong formula in the treatment of cancer related pain based on network pharmacology and molecular dockingGu-tong formula has achieved good curative effect in the treatment of cancer related pain.However,its potential mechanisms have not been explored.We used network pharmacology and molecular docking to investigate the molecular mechanism and the effective compounds of the prescription.Through the analysis and research in this paper,we obtained 74 effective compounds and 125 drug-disease intersection targets to construct network,indicating that quercetin,kaempferol and β-sitosterol were possibly the most important compounds in Gu-tong formula.The key targets of Gu-tong formula for cancer related pain were Jun Proto-Oncogene(JUN),Mitogen-activated Protein Kinase 1(MAPK1)and RELA Proto-Oncogene(RELA),etc.2204 GO entries and 148 pathways were obtained by GO and KEGG enrichment respectively,which proved that Chemokine,MAPK and Transient receptor potential(TRP)channels can be regulated by Gu-tong formula.The results of molecular docking showed that stigmasterol had strong binding activity with Arginine Vasopressin Receptor 2(AVPR2),C-X3-C Motif Chemokine Ligand 1(CX3CL1),and cholesterol was more stable with p38 MAPK,ProstaglandinEndoperoxide Synthase 2(PTGS2)and Transient receptor potential vanilloid-1(TRPV1).In conclusion,the therapeutic effect of Gu-tong formula on cancer related pain is based on the comprehensive pharmacological effect of multi-component,multi-target and multichannel.This study provides a theoretical basis for further experimental research in the future.Research 2:Key compounds and targets of Gu-tong formula in treatment of osteolytic bone metastasis based on network pharmacology Bone metastasis is a common complication in patients with advanced cancer,leading to cancer pain and bone destruction,which seriously affects the quality of life of patients.For a long time,alternative medicine with unique theoretical guidance has played an important role in the treatment of tumor related complications.As a representative prescription of traditional Chinese medicine in the treatment of cancer pain,Gu-tong formula has a good effect in preventing bone destruction in clinical using.However,the molecular mechanism was still unclear.This study explored the mechanism of Gu-tong formula in the treatment of osteolytic bone metastasis through network pharmacology and chemical space analysis.The results showed that clove and Asarum in Gu-tong formula were the most important herbs in the treatment of osteolytic bone metastasis.Quercetin,kaempferol,sesamin and rhamnetin may play more important roles.Among the kernel targets,IL-6 and TGFβ1 are the core targets of Gu-tong formula in the treatment of osteolytic bone metastasis.Regulating the content of IL-6 and TGFβ1 may be the key of Gu-tong formula in the treatment of osteolytic bone metastasis.The study of pathway enrichment found that Gutong formula mainly played a role by affecting immune,proliferation and inflammation related pathways.Chemical space analysis showed that the compounds in Gu-tong formula had similar pharmacodynamic activities with those targeting IL-6 and TGFβ1 respectively,and there was no significant difference in IC50 values among them.This study provides clues for understanding the synergistic effect of Gu-tong formula in relieving osteolytic bone metastasis.It lays a foundation for further research in the future.Research 3:Exploration the mechanism of gutongtie in regulating osteolytic bone metastasis by IL-6/TGFβ1IL-6 and TGF-β,both of them can be produced by tumor cells,which can promote tumor bone colonization and activate osteoclasts.In the previous study,Gu-tong formula showed the potential of regulating IL-6 and TGFβ1.In this study,the content of IL-6 and TGFβ1 in serum and bone tissue were measured by ELISA and Western blot,and the bone mineral density of tumor bearing tibia was measured by micro CT.The results showed that contents of IL-6 and TGFβ1 in serum were significantly decreased and the bone mineral density was significantly increased after the application of Gu-tong formula.Gu-tong formula was involved in the treatment of osteolytic bone metastasis by regulating the interaction between IL-6 and TGFβ1.Through experiments,this study laid a foundation for further understanding the intrinsic biological mechanism of Gu-tong formula and its clinical promotion in the future.Research 4:Molecular docking and dynamics simulation of key compounds contained in Gu-tong formula binding with IL-6 and TGFβ1As the key proteins of bone remodeling,IL-6 and TGFβ1 can stimulate tumor cells to colonize on bone surface and directly stimulate RANKL to cause osteolytic bone destruction.Because of the key role of IL-6 and TGFβ1 in skeletal related events,targeted therapy was considered to be meaningful.As a multi-target and multi-mechanism traditional Chinese medicine preparation,Gu-tong formula has been proved to regulate IL6 and TGFβ1 in previous studies.In this study,molecular docking and molecular dynamics simulation were used to explore the mode of effective compounds targeting IL-6 and TGFβ1 in Gu-tong formula from the perspective of microscopic interaction between residues and key pharmacodynamic molecules.The results showed that Sesamin and Deltoin could bind IL-6 and TGFβ1 stably with binding energies of-7.28 and-7.35 kcal/mol,respectively.Molecular dynamics studies showed that Sesamin and Deltoin could change the fluctuation of protein residues,indicating stable binding.Binding interaction analysis showed that Arg179 residue of IL-6 and Arg25 residue of TGFβ1 bound to small molecule compounds respectively,which affected the further binding of protein to receptor and hinder the downward signal transmission.From the perspective of microscopic force,this study explained the molecular mechanism of Gu-tong formula in the treatment of osteolytic metastasis,and laid a foundation for drug screening of IL-6 and TGFβ1 targets and further study of Gu-tong formula.