Based On AMPK-mTOR Signaling Pathway Explore Mechanism Of Self-made Traditional Chinese Medicine Herbal Formula On Colorectal Cancer

Background:Colorectal cancer(CRC)is one of the most common malignant tumors of the digestive system.Only 40%of CRC can be detected in the early stage,and CRC has a high rate of recurrence after surgery.However,drug resistance often occurs in almost all CRC patients who undergo systemic chemotherapy,limiting the therapeutic effect of anticancer drugs.Traditional Chinese medicine(TCM)plays an important role in the treatment of CRC due to its multi-component,multi-target and multi-signal pathway.Mammalian target of rapamycin(mTOR)is an atypical serine/threonine protein kinase.Adenosine 5 ’-monophosphate(AMP)-activated protein kinase(AMPK)is a major regulator of cellular energy metabolism,and AMPK is a negative regulator of mTOR pathway.AMPK-mTOR pathway plays a central role in cancer and is associated with tumorigenesis,angiogenesis,tumor growth and metastasis.Network pharmacology can transform the model of "one target,one drug" into "multiple targets,multiple drugs".From the perspective of network pharmacology,the interaction of genes,proteins and proteins between diseases and drugs is elucidated,which provides the possibility to study the relationship between traditional Chinese medicine and diseases.Objective:The aim of this study is to investigate the effect of a self-made traditional Chinese medicine herbal formula on the fate of colorectal cancer cells and AMPK-mTOR signaling pathway,so as to further study the anti-colorectal cancer effect of the Chinese herbal compound and provide reliable theoretical data for its clinical application.Methods:In this study,the effect of the self-made TCM compound on the viability of CRC cells was investigated by traditional Chinese medicine serum pharmacology,network pharmacology,Western blot,cell scratch,Transwell and AV-PI experiments.Effects of traditional Chinese medicine compound on the migration and apoptosis of CRC cells;The network pharmacology method was used to predict the target and mechanism of action of the compound,and the results were verified at the cellular level.Results:(1)There were 189 components in the compound,mainly from triterpenoids,flavonoids,saponins and organic acids.Among them,there were 28 triterpenoids,36 flavonoids,13 alkaloids,9 saponins,organic acids and other compounds.In addition,a total of 71 blood components were identified,among which medicinal compounds such as evodia officinalis,osthole,Platycodon grandiflorum,and Rhizoma polyflorum were detected more in the serum of mice.(2)The migration ability of CRC cell lines RKO and DLD-1 was significantly decreased after treatment with the compound by wound healing and Transwell assay.Western blot showed that the expression of E-cadherin in HCT116 and DLD-1 cells gradually increased,and the expression of Ncadherin and Vimentin gradually decreased after the gradient treatment of traditional Chinese medicine compound.The results of AV-PI and Western blot showed that the apoptosis of RKO and DLD-1 cells was significantly increased after the gradient treatment of traditional Chinese medicine compound.(3)Through TCMSP,TCMID,ETCM and other databases,using Swiss TargetPrediction to predict its targets,180 active ingredients in the compound were screened,1340 related targets and 1117 CRCrelated genes were predicted and screened,and 313 common targets were obtained by comparison mapping.The "drug-disease-target" network was constructed and found.For example,PI3K,Akt,mTOR and other targets are involved in the treatment of CRC with TCM compound.(4)The expression of p-AMPK protein was increased,and the expression of mTOR protein and mTOR downstream protein p-p70S6K was decreased in HCT116 and RKO cells treated with the compound.However,after knocking down AMPK by siRNA and then treating RKO and DLD-1 cells with traditional Chinese medicine compound,the expression level of p-AMPK protein decreased,and the expression levels of mTOR and mTOR downstream protein p-p70S6K were compensated.Crystal violet assay confirmed that knocking down AMPK reversed the inhibitory effect of the traditional Chinese medicine compound on DLD-1 cell proliferation.Conclusion:The self-made TCM compound can significantly inhibit the migration and promote the apoptosis of CRC cells.It may affect the progression of CRC by up-regulating AMPK-mTOR signaling pathway.Among them,triterpenoids,flavonoids and saponins are the main compounds in the compound.Among them,Wuzhuyu(Evodia officinalis),osthole(Osthole),Platycodon grandiflorum(Platycodon grandiflorum),and Chonglou(Polyphylla verticollis)might be the main herbs with antiCRC effects.