Transcriptome Profiling Analysis Reveals The Potential Mechanisms Of Three Bioactive Ingredients Of Fufang E’jiao Jiang During Chemotherapy-induced Myelosuppression In Mice

Fufang E’jiao Jiang(FEJ)is a traditional Chinese medicine(TCM)formula that contains Asini Corii Colla,Ginseng Radix et Rhizoma Rubra,Rehmanniae Radix Praeparata,Codonopsis Radix,and Crataegi fructus.It has pronounced functions of replenish qi and nourish blood(so called ’bu qi yang xue’),therefore is widely used for patients with qi and blood deficiency,dizziness,palpitations,insomnia,loss of appetite,leukopenia,and anemia,etc.As a common problem faced by the majority of hebal medicines,it is not clear for FEJ about it’s complex chemical composition,bioactive ingredients,and mechanism of action due to its multi-component and multi-target interacting.The intergrative application of analytical chemistry,pharmacology and bioinformatics is an effective way to study the mechanism of TCM.In a previous study,we identified 72 phytochemical constituents of FEJ by utilizing high-performance liquid chromatography-mass spectrometry(HPLC-MS),together with high-resolution mass spectrometry(HR-MS).We provided evidences showing that martynoside,R-notoginsenoside R2,and ginsenoside 20(S)-Rg2 stimulates the hematopoiesis of bone marrow cells in mice with 5-fluorouracil(5FU)-induced blood deficiency,respectively.In the current study,we investigated the hematopoietic effects of FEJ,FEJ without E’jiao(designated WFEJ),E’jiao(designated EJ)and the three bioactive ingredients including martynoside,R-notoginsenoside R2(designated R2),ginsenoside 20(S)-Rg2(designated Rg2)in a 5-FU-induced blood deficiency mouse model.In addition,the transcriptome profiling of bone marrow cells was analyzed.Our study confirmed the hematopoietic effects of FEJ and its bioactive ingredients in vivo.In addition,the functional transcriptome maps of martynoside,R-notoginsenoside R2,and ginsenoside 20(S)-Rg2 provide valuable information concerning their underlying mechanisms and provide a framework for the continued study of the complex mode of action of FEJ.The main contents of the thesis are as follows:1.Different dosages of FEJ,WFEJ,EJ,martynoside,R-notoginsenoside R2,or ginsenoside 20(S)-Rg2 were administrated to 5-FU-induced blood deficiency mice.General conditions were observed and body weight was measured daily.Peripheral blood counting was performed on 3,7,10,15 and 20 days post-exposure,respectively.In addition,bone marrow cell counts,flow cytometry staning of hematopoietic stem cell(HSC),colony formation assays for the hematopoietic progenitor cells(HPC)were performed.The results showed that body weight and general conditions of the blood deficiency mice were improved due to the treatments.Besides,the treatment stimulated BMNCs and HSC growth,as well as burst-forming unit-erythroid(BFU-E)and colony-forming unit granulocyte-monocyte(CFU-GM)colony expansion.Furthermore,peripheral blood counting showed that with the treatment of WFEJ and EJ for 15 days,white blood cells and platelets in the peripheral blood was significantly reduced.2.Blood deficiency mice were treated with martynoside,R-notoginsenoside R2,or ginsenoside 20(S)-Rg2 for 15 days and bone marrow cells were subjected to RNA sequencing(RNA-seq).The transcriptome data were processed based on the differentially expressed genes(DEG).The results showed that martynoside,R-notoginsenoside R2 and ginsenoside 20(S)-Rg2 returned 109,1027 and 631 DEG,respectively,some of which were realated to hematopoiesis.The most relevant mechanism appeared to be involved in regulation of immune system processes,hematopoietic or lymphoid organ development and hematopoiesis.Notably,the individual compounds shared similar but specified transcriptome profiles.R-notoginsenoside R2 and ginsenoside 20(S)-Rg2 could simultaneously modulate IL-17 signaling pathway and Toll-like receptor signaling pathway,which were not regulated by martynoside.In summary,we confirmed the hematopoietic effects of FEJ and analyzed its transcriptome profiles in the aspects of whole drug,the drug component and three bioactive ingredients.Our study provided a strategy,that might be used to explain complex therapeutic mechanism of TCM characterized by multi-component and multi-target interacting.