Anti-cancer Effect Of The Total Flavonoids Of Astragalus Combined With Cisplatin On Lewis Lung Cancer Mice

Rationales:Cancer is now the world's number one cause of death,with lung cancer being the top three among all cancers.At present,chemotherapeutic drugs also have large toxic side effects while exerting anti-tumor effects.Recent studies have shown that Chinese medicine has significant advantages in the synergistic treatment of tumors.Astragalus as a traditional Chinese medicine is commonly used in traditional Chinese medicine.The flavonoids are one of the main components of Astragalus.It has been shown that in addition to anti-inflammatory,anti-viral and myocardial protection,Astragalus flavonoids also have certain anti-cancer effects.Previous studies by our group showed that the total flavonoids of Astragalus membranaceus had a significant increase in leukopenia caused by the chemotherapy drug cyclophosphamide.Based on the previous research,this study is to study the in vivo pharmacodynamics and mechanism of total flavonoids of Astragalus membranaceus and cisplatin against lung cancer.Objective:To analyze the pharmacodynamic effect and mechanism of total flavonoids of Astragalus membranaceus on LLC-bearing mice with lung cancer,and to provide basis for further development of synergistic and attenuated drugs for cancer chemotherapy.Method:(1)The total flavonoids of Astragalus membranaceus were prepared by the method established by our research group,and the content of the main flavonoids was determined.(2)Animals were divided into blank group,model group,positive drug group,low-dose TFA combination chemotherapy regimen group,medium-dose TFA combination chemotherapy regimen group,high-dose TFA combination chemotherapy regimen group;Lewis tumor-bearing mice were established by subcutaneous injection of LLC lung cancer cells.In the lung cancer model,the pharmacodynamics of total flavonoids of Astragalus membranaceus against cisplatin were analyzed by tumor size,tumor volume curve,tumor weight and tumor inhibition rate.The attenuating effect of total flavonoids of Astragalus membranaceus on cisplatin was analyzed by organ index.(3)Anti-lung cancer activity of the total flavonoids of Astragalus membranaceuswas predicted by network pharmacology Targets,western blot was used to validate and the predicted target protein.(4)Serum metabonomics based on UHPLC-MS System was used to determine the biomarkers and metabolic pathways of normal and lung cancer mice,and the mechanism of Astragalus flavonoids was further analyzed through the regulation of Astragalus flavonoids on biomarkers of lung cancer models.Results:(1)The content of eight flavonoids in the total flavonoids of Astragalus membranaceus: the content of calycosin-7-O-?-D-glycoside is 9.82%,the content of ononin is 8.62%,the content of(6a R,11 a R)--9,10-dimethoxypterocar pan-3-O-?-Dglucoside is 11.24%,and the content of 7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-?-D-glucoside is 3.71%,the content of calycosin is 0.54%,the content of formononetin is 0.02%,the content of(6a R,11 a R)-9,10-dimethoxypterocarpan is 0.02%,the content of7,2'-dihydroxy-3',4'-dimethoxyisoflavan is 0.01%.The total content of 8 kinds of xanthine is 33.98%.(2)Through the results of the curve of tumor volume,the weight and size of the tumor,and the tumor inhibition rate,it was found that the anti-cancer effect of the combined drug group was stronger than that of the positive drug cisplatin,which indicated that the total flavonoids of Astragalus membranaceus could enhance the anti-cancer effect of cisplatin.According to organ index analysis,total flavonoids of Astragalus membranaceus can alleviate the immunosuppressive effect of cisplatin.In conclusion,total flavonoids of Astragalus can not only enhance the anti-cancer effect of cisplatin,but also reduce the toxic and side effects of cisplatin,that is to say,total flavonoids of Astragalus combined with cisplatin has significant synergistic and attenuating effects on Lewis lung cancer mice.(3)The 46 target targets of total flavonoids of Astragalus membranaceus were predicted by network pharmacology,and the component-target-path-disease network and the protein-protein phase-assisting network were further constructed.The results showed that the six components of calycosin,calycosin-7-glucoside,(6a R,11 a R)-9,10-dimethoxypterocarpan,(6a R,11 a R)-9,10-dimethoxypterocar pan-3-O-?-D-glucosideand 7,2'-dihydroxy-3',4'-dimethoxyisoflavan-7-O-?-D-glucoside,Astragaloside IV were active ingredients.From degree value and Betweenness value,the correlation between calycosin-7-glucoside and activity was the greatest.In addition,STAT3,ERBBA2,NOTCH1,TRP53,FGF2,ALB,VEGFA and other targets are associated with the inhibition of cisplatin by total flavonoids of Astragalus.The levels of IL-6 in the high-and medium-dose groups were significantly lower than those in the model group,which was close to the blank level.The level of STAT3 protein in the high,medium and low dose groups was lower than that in the positive group,indicating that the total flavonoids of Astragalus membranaceus could be down-regulated.STAT3 levels are dose dependent.It is concluded that the total flavonoids of Astragalus membranaceus may inhibit the expression of STAT3 protein by down-regulating IL-6 levels,that is to say,total flavonoids of Astragalus may inhibit tumor growth through the IL-6/STAT3 pathway.(4)Thirty different metabolites,including fatty acids and amino acids,were determined by multivariate statistical analysis between the blank group and the model group.Then,the contents of these differential metabolites were compared in all groups(blank group,model group,positive drug group,combined low-dose group,combined drug-administered group,combined high-dose group).After the combination of flavonoids,5 metabolites could be better corrected.Cisplatin alone could not be adjusted,but combined with total flavonoids of Astragalus membranaceus could call back 4metabolites.Meta PA metabolic pathway analysis showed that in the four metabolic pathways associated with the Lewis lung cancer model,total flavonoids of Astragalus membranaceus could achieve synergistic and attenuating effects by regulating the primary bile acid biosynthetic metabolic pathway.Conclusion: Total flavonoids of Astragalus membranaceus can significantly enhance the anti-tumor effect of cisplatin on Lewis lung cancer mice.Metabonomics and network pharmacological analysis show that its mechanism is related to IL-6/STAT3 pathway and primary bile acid biosynthesis metabolic pathway.This study provides a basis for further development of synergistic and attenuated drugs for cancer chemotherapy using Astragalus membranaceus as raw material.