Study On The Mechanism Of Yiqi Jiedu Prescription And Its Effective Ingredient Quercetin Against Nasopharyngeal Carcinoma

Objective:Based on the analysis of ancient and modern literature,the theoretical origin of traditional Chinese medicine treatment of nasopharyngeal carcinoma（NPC）was traced;The pharmacodynamic material basis and potential molecular mechanism of Yiqi Jiedu（YQ）prescription and its effective ingredient quercetin in the treatment of NPC were investigated through network pharmacology,molecular docking technology and in vitro and in vivo experiments.Methods:1.Through"Chinese Medical Dictionary","CNKI","Wanfang Medical Network","VIP Chinese Journal","Chaoxing Journal","Duxiu"database or literature library,with"lost honor"or"nasopharyngeal cancer"as the search term,to statistics and analysis the principles,methods and prescriptions,syndromes and treatment laws of ancient and modern traditional Chinese medicine for NPC.2.Combined use GEO,Gene Cards,OMIM,Pharm Gkb,TTD,TCMSP,BATMAN-TCM and other databases to retrieve chemical active ingredients of YQ prescription and their targets for NPC;Cytoscape software to construct"active ingredient-target"interaction network;STRING database to build a visual PPI network;GO,KEGG and DO enrichment analysis of core genes.3.CCK-8 assay,RTCA and Cytation TM5 cell imaging system were used to detect the proliferation inhibition of NPC cell lines CNE-1 and CNE-2 by YQ prescription and its active compound quercetin;Hoechst 33342 staining,JC-1mitochondrial membrane potential and Annexin V-FITC/PI double Fluorescence staining were used to detect the apoptosis-promoting.PI staining was used to detect the effect of drugs on cell cycle.Western blot was testd drugs on the expression levels of proliferation,apoptosis and cyclin-related proteins;The effects of drugs on the m RNA expression of core genes were detected by RT-q PCR;The expression levels of related proteins in the PI3K-AKT,MAPK,and NF-κB signaling pathways were detected by Western blot.The subcutaneous transplanted tumor model was constructed,the cell morphology was observed by HE staining,and the protein expression levels of Ki-67,PCNA and cleaved Caspase-3 were detected by IHC.4.Quercetin was selected as a small molecule ligand,and target genes enriched in PI3K-AKT signaling pathways such as TP53,MYC,MDM2,AKT1 were used as protein receptors for simulated molecular docking to verify the possibility of binding with each other;The mechanism of PI3K-AKT signaling pathway in the antiproliferative effect of quercetin was preliminarily studied by cell experiments.Results:1.NPC can belong to the category of"loss"and other categories,The etiology machine is summarized as"virtual","toxic","phlegm","stasis"four aspects,the syndrome types in the ancient and modern literature of NPC are mostly:Qi Deficiency of yin and yin,flaming heat poison,fire poison stagnation,accumulation of phlegm and turbidity,heat poison attacking the lung;in the treatment of NPC,there are 26 ancient prescriptions in total,including 127 traditional Chinese medicines.and according to the modern literature search,there are 61 prescriptions that meet the inclusion criteria,and a total of 83 traditional Chinese medicines.According to the frequency of drug use,There are 28 common medicines in ancient and modern documents.2.In this study,the active ingredients were screened according to conditions,including 14 in Coptis chinensis,25 in Astragalus,5 in Hedyotis diffusa,42 in Codonopsis pilosula,12 in Poria,14 in Trichosanthin,106 in Licorice,total 197active ingredients after removing duplicate compounds.Obtained 493 drug targets,Screened out 173 potential targets for drug treatment of NPC,top five genes were TP53,STAT3,AKT1,JUN,MAPK1,etc.;GO analysis mainly involved chemical stress,reactive oxygen species response,serine/threonine protein kinase complex,glutathione Transferase activity,DNA-binding transcription factor binding,etc.;and mainly enriched in PI3K-AKT,MAPK and other signaling pathways;DO analysis showed that YQ prescription could also have a synergistic therapeutic effect on breast cancer,thymic carcinoma,benign cellular tumors,and urinary system cancer.3.Based on the presumed results of network pharmacology,in vitro and in vivo experiments were used to evaluate the effect of YQ prescription on inhibiting the proliferation of NPC cells and promoting apoptosis.the study found that YQ prescription and quercetin inhibited the proliferation of CNE-1 and CNE-2 cells in a time-dependent and dose-dependent manner.YQ prescription could significantly block CNE-1 and CNE-2 in G₂/M phase after treatment for 36 h（P<0.05）;RT-q PCR results showed that different doses of YQ prescription could significantly down-regulate the m RNA expression levels of JUN,CCND1,VEGFA,and IL-1βin CNE-1 and CNE-2.（P<0.05 or P<0.01）;Western blot results showed that compared with the control group（solvent control group）,the cell proliferation-related proteins XIAP,PCNA,Survivin and cycle-related proteins CCND1 and CDK2 were downregulated to varying degrees,and the expression of apoptosis-related proteins like Bax,cleaved Caspase-3 and cycle-related proteins p21 were up-regulated in different degrees,and the differences were statistically significant,（P<0.05 or P<0.01）;.After different concentrations of YQ prescription acted on CNE-1 and CNE-2 for 36 h,The protein expression levels of PI3K,AKT1,p-AKT,m TOR,Ras,ERK1/2,p-MEK1/2,p-c-Raf,NF-KB p65 and IKK were down-regulated in different degrees,while the protein expression level of PTEN and IKB-αwere up-regulated to varying degrees.Compared with the Control group,the difference was statistically significant,（P<0.05 or P<0.01）;HE staining showed that,compared with Control group,YQ prescription group had loose tissue arrangement,disordered structure,and some cells tended to degeneration and necrosis.The results of IHC showed that after drug intervention in NPC,PCNA and Ki-67 decreased to varying degrees,while the expression of cleaved Caspase-3increased.4.The molecular docking results showed that quercetin had good binding sites with TP53,MYC,MDM2,and AKT1,different concentrations of quercetin could inhibit the proliferation of CNE-1 and CNE-2 cells to different degrees and induce cell apoptosis;Quercetin can significantly block CNE-1 and CNE-2 in G₂/M phase（P<0.05）;RT-q PCR results show that quercetin can significantly down-regulate the m RNA expression levels of TP53,MYC,MDM2,and JUN in CNE-1 and CNE-2,（P<0.05 or P<0.01）;Western blot results showed that compared with the Control group,quercetin could down-regulate the cell proliferation-related protein XIAP,PCNA,Survivin and cycle-related proteins CCND1,CDK2 expression,up-regulated apoptosis-related protein Bax and cycle-related protein p21 protein expression level,the differences were statistically significant（P<0.05 or P<0.01）;PI3K-AKT signal Studies on the regulatory role of quercetin in the inhibition of NPC cell proliferation by quercetin have shown that quercetin can significantly down-regulate the expression of key proteins PI3K,AKT1,p-AKT1 and m TOR in the PI3K-AKT signaling pathway,and up-regulate the expression of PTEN;while after activating PI3K-AKT signaling pathway（plus activator SC79）,the effect of quercetin on inhibiting proliferation and inducing apoptosis of NPC was weakened.Conclusions:1.Drawing on the valuable experience of the sages,inheriting the principles,methods and prescriptions of the doctors of the past dynasties in the treatment of loss of glory,Theoretical tracing of TCM treatment of NPC has good practical significance for the academic development of integrated traditional Chinese and Western medicine.2.Use network pharmacology to systematically excavate the pharmacodynamic material basis,potential molecular mechanism and action targets of the successfully obtained YQ prescription in the treatment of NPC,and combine in vitro and in vivo experiments to verify the molecular mechanism of YQ prescription in the treatment of NPC,It is verified that YQ prescription has the effect of multi-target,multi-signaling and multi-pathway inhibiting NPC.3.Molecular docking technology can laterally reflect the binding ability of small molecule receptors and protein receptors.In vitro experiments show that quercetin may exert its anti-NPC effect by targeting the PI3K-AKT signaling pathway to inhibit the proliferation and promote apoptosis,but more experiments are needed to verify.