| Rationales:In recent years,our country present a tendency of increasing the incidence of cancer,and the treatment of cancer still depends mainly on radiotherapy and chemotherapy.While radiotherapy and chemotherapy will bring a series of side effects,of which leukopenia is the most obvious.The continuous decline of white blood cells will not only affect the patient's treatment,but even more life-threatening.When the number of white blood cells in peripheral blood is lower than 4×10~9 for a long period of time,it is called leukopenia,and its clinical manifestations are mainly fatigue and low immunity.If leukocytes continue to decrease will not only affect the continuous treatment of cancer patients,but even worse,it may endanger life.The clinical observation have found that Lvjiao Buxue granules can significantly increase the number of white blood cells in patients undergoing radiotherapy and chemotherapy,but they have not conducted in-depth research on modern pharmacology and mechanism of action.And it also were unclear for the main active ingredients,targets,and pathways.The clinical applications of Lvjiao Buxue granules are limited by all those.In this study,leucopenia experimental animal models induced by cyclophosphamide(CY)were adopted,we observe the effect of increase WBC of the Lvjiao Buxue Granules,and use the metabolomics,network pharmacology and other technical means to investigate the pharmacological mechanism.Objective:To study the pharmacodynamic effect of Lvjiao Buxue Granules on leukopenia,and screen the dose for the main pharmacodynamics test;To investigate the differences in the metabolites and pathway related to the therapeutic effect of Lvjiao Buxue granules on leukopenia;Predicting the active ingredients and their targets for promoting the increase WBC effect of Lvjiao Buxue granules,and exploring the possible mechanism of its action.Methods:In this study,we first used cyclophosphamide-induced leucopenia mice/rat model to study the effect of Lvjiao Buxue Granules through body weight,hematological routine data,and bone marrow DNA content.Then,the serum was subjected to ~1H NMR metabolomic analysis,after material identification and normalization.etc.,the data was imported into SIMCA-P 14.0 for multivariate statistical analysis.Combined with VIP values and t-test results,finding differences metabolites associated with Lvjiao Buxue granules interfere.Differential metabolites were substituted into MetPA for pathway analysis to elucidate their biological significance.Finally,the pharmacological methods of network were used to screen out the relevant active ingredients and targets of Lvjiao Buxue Granules,and biological analysis was performed on the relevant targets.Results:1.Lvjiao Buxue granules can significantly increase body weight(P<0.05),WBC content(P<0.05),spleen index(P<0.01),and bone marrow DNA content(P<0.05)in a mouse model of cyclophosphamide-induced leukopenia,and its optimal dose for major pharmacodynamic studies is 3g/kg,6 g/kg,12 g/kg,and 6 g/kg dose is the best.However,there was no significant improvement in cyclophosphamide-induced rat models and further studies are needed.2.~1H NMR metabolomic analysis was used to analyze the serum samples of leukopenic mice treated with Lvjiao Buxue granules.It was found that Lvjiao Buxue granules could reduce the interference of cyclophosphamide on serum metabolites in mice and significantly regulate metabolites level(P<0.01),compared with the control group.And through adjusting the serine,leucine and isoleucine synthesis,glutamate metabolism,alanine,glutamate and aspartate metabolism,glutathione metabolism as well as arginine and proline metabolism play a role of increasing WBC.3.Using network pharmacology,32 potential chemical components were screened out based on the OB and DL values of the drug,and combined with the activity studies of the related literature,a total of 49 potentially active components of Lvjiao Buxue Granules were screened out through match with the target of leukopenia disease.We predicted 89 potential targets,among which EFGR,IL4,and ALB may play a major role.Potential targets are mainly involved in multiple pathways including metabolic pathways,cancer proteoglycans,actin cytoskeleton,PI3K-Akt,Rap1,chemokines,and MAPKs,etc.4.By combining the results of metabolomics and network pharmacology,it was found that the genes involved in the metabolic pathway are the same as the 8 genes in the network pharmacology potential target genes,and thus 8target genes are used as the connecting points to act as pathways.After constructing a metabolite-metabolism pathway-pathway gene target-active ingredient network,29 kinds of active ingredients were found to act on target pathway gene targets,mainly involving glutathione metabolism,alanine,arginine and glutamate metabolism arginine and valine metabolism.It maybe play a role in increase WBC through regulation the expression of GSS,GSTT1,GSTP1,GSTM1,G6PD,ASS1,NOS2,OAT,and increasing the activity of antioxidationConclusion:Lvjiao Buxue granules can significantly improve cyclophosphamide-induced mice leukopenia,and its main pharmacodynamic dose should be set at 3 g/kg,6 g/kg,12 g/kg,and 6 g/kg dose effect is best.Lvjiao Buxue Granules mainly through 29 kinds of active ingredients,regulated glutathione metabolism pathways relevant genes(GSS,GSTT1,GSTP1,GSTM1,G6PD.etc),and increasing the activity of antioxidation,play a role in increase WBC. |
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