Studies On The Effects And Mechanism Of Lvjiao Buxue Granules On Cyclophosphamide-induced Leucopenia Of 4T1 Mammary Carcinoma Mice

Rationales:Cancer has become the most serious public health problem in China,and one of the major diseases that seriously threaten human health.Statistics in 2019 show that there are more than 3.929 million people with cancer in China,and the cancer growth rate is 3.2%,which means that more than 10,000 people are diagnosed with cancer every day,and 7.5 people are diagnosed with cancer every minute.In recent years,the application of chemotherapy has greatly improved the treatment of cancer.However,the cytotoxicity of chemotherapy drugs often causes patients with leukopenia,which seriously affects the patient's tolerance,leading to the termination of chemotherapy and increased cancer mortality.Therefore,how to effectively improve leukopenia and enable cancer chemotherapy to proceed smoothly has become one of the major issues that need to be solved urgently.Existing chemotherapeutic drugs such as vitamin B4,sharkol,and serotonin,etc.,most patients have the phenomenon of leukocytosis recurrence after discontinuation of drugs.New drugs,such as recombinant human granulocyte colony stimulating factor,will have side effects on musculoskeletal and digestive system,which will seriously affect the continuity of chemotherapy.In addition,it is unclear whether these drugs affect the development of tumors while treating leukopenia caused by chemotherapy.Therefore,it is of great clinical significance and value to research and develop drugs with clear efficacy,little toxicity and side effects,and low price for the treatment of leukopenia after cancer chemotherapy.Lvjiao Buxue Granules(LBG),a good medicine for treating deficiency of vital energy and hemopenia,has the characteristics of nourishing yin and supplementing blood,strengthening spleen and Qi-nourishing,regulating menstruation and activating blood,and often used to treat diseases such as chronic illness,deficiency of vital energy and hemopenia.In traditional Chinese medicine,leukopenia is often classified as "deficiency of blood" and "deficiency of labor".These symptoms coincide with the effect of LBG.There is clinical evidence that LBG can increase the leukocyte level of cancer chemotherapy patients,improve the compliance of chemotherapy,and improve the quality of life of patients.However,the pharmacological mechanism of LBG inincreasing leukocyte is not clear,which limits the rational application of the drug in the treatment of leukopenia.Therefore,in this study,the experimental animal model of cyclophosphamide(CTX)-induced 4T1 tumor-bearing mouse leukopenia was established,and LBG were given to observe its effect on improving leukopenia in mice.The mechanism of LBG was elucidated by metabonomics,biological network analysis and molecular biology,which provided scientific basis for the rational clinical application of LBG in adjuvant cancer chemotherapy.Objective: The present study was aim to investigate the exact effect on the treatment of LGB to the CTX induced leukopenia in 4T1 tumor bearing mice,and to explore the internal mechanism using the integrated analysis of the metabolomics,biological targets network and molecular biology.Methods:(1)Leukopenia model of 4T1 tumor bearing mice induced by CTX.The effect of LBG on leukopenia was studied with blood routine test,organ index,tumor inhibition rate and the number of lymphocyte subsets in spleen as pharmacodynamic indexes.(2)~1H-NMR metabonomics was used to analyzed the metabolites collected from blood and spleen of mice.First,principal components analysis(PCA)was performed on the standardized data to identify the degree of dispersion among the control group,model group and administration group,and to eliminate the abnormal values.Next,partial least squares discrimination analysis(PLS-DA)was used to visually distinguish the metabolic spectrum differences among the control group,model group and drug group.PLS-DA was used to find the VIP value of metabolites between the control group and the model group.Finally,the metabolites with VIP>1 and P<0.05 were identified as differential metabolites by ANOVA analysis in SPSS 21.0.(3)The chemical components of LBG were analyzed by UHPLC-Q Exactive orbital hydrazine high resolution mass spectrometry.The chemical components were supplemented and screened by TCMSP and TCMID,and the chemical components database of LBG was established.The selected chemical components are imported into the pharmapper server to further identify the target of the active components,and the target obtained is associated with the target of the differential metabolites to form an intersection,so as to establish the "active components target network".The targetwas imported into string database to construct protein interaction network and elucidate the relationship between metabolic enzymes.KEGG and go analysis were carried out to find out the key metabolic pathway by using cluego plug-in.Finally,systems dock software was used to study the molecular docking between the target in the key metabolic pathway and the active components of donkey glue blood replenishing granules,in order to verify the accuracy of the results.(4)The results of KEGG pathway analysis show that branched chain amino acids(BCAAs)decomposition may be the most important way for LBG to improve leukopenia in 4T1 tumor-bearing mice induced by CTX.First,phenylbutyric acid,an inhibitor of BCKDK kinase,was administered to PBMC and RAW264.7 cells,and the cell activity was observed,and to verify the effect of key enzymes in BCAAs catabolic pathway on cells in vitro.Secondly,PBMC and RAW264.7 cells were exogenously supplemented with a certain concentration of BCAAs to observe whether they had the effect of increasing cell viability.Finally,the content of acyl-Co A dehydrogenase(ACADS)and branched-chain ketoate dehydrogenase(BCKDHA),the key rate-limiting enzymes in the BCAAs decomposition pathway,was determined by ELISA kit.Result:(1)The body weight,blood routine parameters and organ indexes levels of leucopenia mice were improved by LBG.After LBG intervention,the proportion of splenic lymphocyte subsets and immunity was improved in leukopenia mice.While leukopenia was improved,LBG increased the anti-tumor effect of CTX to a certain extent,with the characteristics of "attenuating and increasing efficacy".(2)Results: 31 endogenous metabolites including amino acids,sugars and organic acids were identified by serum metabonomics analysis.The content of lactate,lipid and glutamic acid in the serum of the model group increased significantly compared with the control group,while the content of ?-glucose,betaine,pyruvate,glutamine,o-acetylglycoprotein and creatinine decreased significantly.Abnormal serum metabolism can be reversed by LBG.The content of WBC was positively correlated with the levels of glutamine,o-acetylglycoprotein,pyruvic acid,creatinine,?-glucose and betaine,and negatively correlated with the levels of lactate andglutamic acid.32 endogenous metabolites were identified by spleen metabonomics analysis.The levels of TMAO,pyruvate,GPC,taurine and glutamic acid in the spleen of the model group were significantly higher,and the contents of choline,inositol,tyrosine,valine,isoleucine,leucine,PC,?-glucose and phenylalanine were lower compared with the control group.Abnormal spleen metabolism can be reversed by LBG.WBC,RBC,HGB,HCT,NE,ly,Mo and MCH were negatively correlated with glutamate,pyruvate,aspartic acid,GPC,taurine,glutamate and TMAO,and positively correlated with valine,isoleucine,leucine,choline,PC,inositol,?-glucose,tyrosine and phenylalanine.In addition,there is also a significant correlation between spleen index and differential metabolites,which indicates that LBG may play a role in increasing leukocytes by balancing the disorder of spleen metabolites.The results of metabolomics studies show that the differential metabolites that can be regulated by LBG are mainly related to amino acid metabolism,including the conversion of glutamic acid and glutamine,phenylalanine metabolism,BCAAs metabolism,etc.In addition,it also involves multiple biological pathways such as energy metabolism and choline metabolism,reflecting the mechanism of multi-component,multi-target and overall regulation of LBG.(3)After the LC-MS chemical composition analysis results were combined with TCMSP and TCMID to find the chemical components,a total of 35 active ingredients were screened based on literature reports.The chemical composition of LBG was imported into the Pharm Mapper server database to obtain targets,and 872 targets were obtained after deduplication.The target of the metabolite and the target of the active ingredient are intersected to obtain 32 active targets of the LBG active ingredient to regulate the metabolite.The active ingredients,target targets,and differential metabolites of LBG were imported into Cytoscape software to draw an "active ingredient-target-metabolite" network,involving 35 active ingredients,32 targets,and 14 differential metabolites of LBG.Through topological analysis,Rehmannioside A,Catalpol,Astragaloside ? and Astragaloside ? were found to play an important role in the target network.IL4I1,BCAT2,BCAT1,PLA2G4 A and LYPLA3 may be the key targets for LBG to increase leukocytes.The main targets of CTX induced leukopenia in 4T1 tumor bearing mice improved by LBG includenuclear oxidoreductase,transferase,hydrolase,isomerase and lyase.In addition,it also contains calcium binding proteins,enzyme regulators,receptors and signal molecules,among which oxidoreductase,transferase and hydrolase are more frequent,which are closely related to amino acid catabolism.The KEGG pathway analysis results show that the LBG's 32 target targets are mainly involved in the valine,leucine and isoleucine decomposition pathways(BCAAs decomposition),and also participate in cysteine and methionine metabolism,glycolysis metabolic pathways such as the solution/gluconeogenesis pathway and pyruvate metabolism fully reflect the multi-component,multi-target,and overall regulatory effects of LBG.The results of GO enrichment analysis showed that the catabolism of small molecules was found to be the most important biological function of these targets,involving a total of 19 targets.In addition,biological pathways such as carboxylic acid decomposition,organic acid decomposition,and cellular amino acid catabolism are also regulated by LBG.Based on the results of KEGG and GO analysis,it is known that the BCAAs decomposition may be the main pathway for LBG to increase leukocytes.(4)When the concentration of benzoic acid was 180-2880 ?M/L,the activity of RAW264.7 cells was significantly inhibited.When the concentration of benzoic acid was 720-2880 ?M/L,PBMC cells were significantly inhibited.The reason for the above results is that BCKDK kinase is inhibited by phenylbutyric acid,which results in BCKDH being activated,the consumption of BCAAs in the whole BCAAs catabolic pathway is increased,thus the cell viability is weakened,which is consistent with the result of metabonomics.The experimental results of exogenous BCAAs supplementation showed that when the concentration of BCAAs was 180-2880 ?m/L,the proliferation of RAW 264.7 cells was significantly promoted.When the concentration of BCAAs was 1440 ?m/L and 2880 ?m/L,the cell viability of PBMC was significantly promoted.The results showed that the improvement of CTX-induced leucopenia of 4T1 mammary carcinoma mice was caused by LBG regulating BCAAs content.The content of ACADS and BCKDHA in the model group increased significantly compared with the control group,which indicated that BCAAs metabolic pathway was activated and BCAAs was over consumed,which confirmed the resultsof biological target network and metabonomics analysis.,The content of ACADS and BCKDHA in mice were significantly decreased after the administration of LBG,which indicated that LBG could improve the BCAAs by interfering with BCAAs decomposition pathway.Conclusion:CTX-induced 4T1 tumor-bearing mice with reduced WBC,organ damage,and decreased immune function and other symptoms can be significantly improved by LBG,and it has a certain inhibitory effect on tumor development.Its mechanism of action may be to play a role by interfering with the expression of ACADS and BCKDHA enzymes,regulating the BCAAs degradation pathway,and recalling the level of BCAAs.