Study On Anticancer Effect Of Astragali Radix Total Flavonoid Combined With Cisplatin On Laryngeal Squamous Cell Carcinoma

Objective:Laryngeal squamous cell carcinoma(LSCC)is the most common head and neck cancer.Astragali radix extracts play crucial roles in the regulation of cancer progression.However,the role of Astragali radix(AR)extracts in LSCC and the related mechanisms remains unclear.Flavonoids are the main types of bioactive substances in AR extracts.This study mainly:(1)investigates the effect of Astragali radix total flavonoid(TFA)combined with cisplatin(CDDP)to LSCC xenograft model in nude mice,laying a foundation for the chemosensitization and a high-efficiency and low-toxicity clinical therapeutic strategy of LSCC;(2)explores the possible regulatory mechanism of anticancer of TFA on LSCC.Methods:Firstly,the LSCC xenograft model was constructed,and the study evaluates the inhibitory effects of the combined use of TFA and cisplatin(CDDP)on an LSCC mouse model by pharmacodynamics.Then,Ultra-high-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)was employed to define the prototype of TFA in vivo.Finally,the potential drug targets were identified through the integrative analysis of LSCC microarrays,RNA sequencing data and the main bioactive component of TFA.Furthermore,a protein-protein interaction network(PPI),compound target network(C-T network)and target pathway network(T-P network)were constructedbased on the prototype and potential drug targets to identify the main targets and pathways.Results:Animal experiments showed that TFA has significant synergistic antitumor activity with cisplatin and attenuates the nephrotoxicity caused by CDDP chemotherapy,and improving the survival of LSCC-bearing mice.The tumor inhibition rate was higher with24 mg/kg TFA+1 mg/kg CDDP and 48 mg/kg TFA+1 mg/kg CDDP compared to that with CDDP alone(29.3% and 40.2% vs.26.8%,respectively).Using UPLC-MS/MS,the study identifies 8 constituents of TFA in experimental mice serum: formononetin,ononin,calycosin,calycosin-7-0-?-D-glucoside,7,2'-dihydroxy-3',4'-dimethoxyisoflavan,7,2'-dihydroxy-3',4'-dimethoxyisoflavaneglucoside,3-hydroxy-9,10-dimethoxypterocarpan and 9,10-dimethoxyptercarpan-3-0-?-D-glucoside.Next,a network pharmacology strategy was introduced to predict 184 protein targets(drug targets)for the 8 primary TFA constituents,and a total of 1096 LSCC-associated targets(disease targets)were collected from two datasets,of which 114 targets were from the network database DisGeNet and 996 targets were from the intersection of microarray data and RNA sequencing data.19 candidate targets were identified by Venn analysis,and they were EGFR,ERBB2,ERBB4,MMP1,MMP3,NRAS,NOX4,TOP2 A,DRD1,MAOB,ACHE,CA9,ALDH1A1,ALDH1A2,ALOX12 B,ALOX15,DCT,CYP1B1 and DAPK1.By constructing a PPI network of the 19 candidate targets of TFA,this study found that EGFR might be the core target of TFA,which interacted with ERBB2,ERBB4,MMP1,MMP3,NRAS,NOX4,TOP2 A,CA9,and ALDH1A1.By constructing a C-T network,this study found that 8 components of TFA could be docked with the candidate targets EGFR,ERBB2,ERBB4,MMP3,MMP1,ALDH1 A,ALDH1A2,TOP2 A,MAOB,ALOX15,NRAS,ACHE and DAPK1 with high docking scores(docking score > 4.52),especially the flavonoid glycoside components ononin,calycosin-7-0-?-D-glucoside,7,2'-dihydroxy-3',4'-dimethoxyisoflavaneglucoside and9,10-dimethoxyptercarpan-3-0-?-D-glucoside and 3-hydroxy-9,10-dimethoxypterocarpan could bind to targets tightly.To better understand the pharmacological mechanisms of the TFA synergistic effect with CDDP to inhibit LSCC,the 19 targets were annotated through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.GO analysis suggested that the target genes are involved in the process of oxidation-reduction,regulation of apoptosis,and cell proliferation.Furthermore,the KEGG pathway analysis results revealed that the 19 targets are enriched in PI3K-Akt,ErbB,and calcium signaling pathways.Conclusion:Astragali radix total flavonoid combined with cisplatin can significantly inhibit the proliferation and enhance the chemosensitivity of laryngeal squamous cell carcinoma.Integrative analysis predicted 19 target genes for TFA constituents,and the target genes were mainly involved in the EGFR-related cancer signaling,metabolism and oxidative stress.Collectively,these findings highlight the role of TFA in the regulation of LSCC and provide potential targets for a high-efficiency and low-toxicity therapeutic strategy of LSCC.