Study On The Hematopoietic Effect And Mechanism Of Ginsenoside Rg2 During Chemotherapy-induced Myelosuppression In Mice At Single-cell Level

Myelosuppression,as the most serious side effect of chemotherapy,directly affects the efficacy,prognosis and regression of patients who accept clinical treatment for malignant tumors.Reducing the toxicity of myelosuppression is essential,and traditional Chinese medicines（TCM）for nourishing blood and tonifying qi have proven their efficacy in clinical practice.However,these TCM formula still suffer from unclear substances and unclear mechanisms of action that need to be addressed.In our previous work,we have conducted a preliminary screening of substances with anti-myelosuppressive activity from a“Liangyi Ointment”formula with blood nourishing and qi tonifying effects.The results suggest that the active ingredients,such as ginsenoside Rg2,may have protective effects against hematopoietic damage.Faced with the complex composition of the bone marrow microenvironment,it is challenging to comprehensively evaluate the effect of drugs on the hematopoietic function.In addition,the heterogeneity of hematopoietic cells has limited our research on the mechanism of action of drugs.The functional identification of the bone marrow microenvironment at the single cell level has become an essential tool for the study of hematopoietic function.If the“drug-target-effect”link between active ingredients and bone marrow hematopoiesis at the single-cell level can be established,it is expected to provide a scientific basis for the clinical application of blood-nourishing and qi-tonifying formulas.In this thesis,based on the previous studies,ginsenoside Rg2 was selected to study its effects and mechanisms of action on chemotherapy-induced myelosuppressed mice in terms of both bone marrow stromal cells（BMSCs）and bone marrow hematopoietic cells.The main work and innovative results are as follows:1.The mice model of myelosuppression was established by a single intraperitoneal injection of 200 mg/kg 5-fluorouracil（5-FU）.When 20 mg/kg Rg2 was administered by gavage on Day 5,Day 10 and Day 14,the following indicators were dynamically measured to evaluate the hematopoiesis:femoral histopathology,the number of bone marrow nucleated cell,the proportion of hematopoietic stem cells（Lin^-Sca1⁺Kit⁺,LSK）and hematopoietic progenitor cell colony count.The results showed that Rg2 had no significant protective effect against myelosuppression on Day 5,while on Day 10 and Day 14,Rg2 ameliorated 5-FU induced-bone marrow injury,including a significant increase in the number of bone marrow nucleated cells,granulocyte/monocyte hematopoietic progenitors and pre-B cells.2.The effect of Rg2 on BMSCs was evaluated using long-term cell culture observation of bone marrow cells.Meanwhile,BMSCs were isolated for RNA-seq and for further bioinformatic analysis.The study showed that Rg2 promoted the differentiation of damaged BMSCs.IL-6 acted as the key effect factor regulated by Rg2in damaged BMSCs according to RNA-seq.In our study,IL-6 was significantly inhibited both on Day 5 and Day 10 after treatment,suggesting that Rg2 has a protective effect against IL-6-induced inflammation injury on BMSCs in a myelosuppressive state.3.Myelosuppressive mice were treated with Rg2 for 10 days and bone marrow cells were extracted for single-cell isolation,labelling and subjected to single-cell RNA-seq（sc RNA-seq）using 10x Genomics Chromium^TM system and Illumina platform.Bioinformatics analysis was used to investigate the effects of Rg2 on each lineage of hematopoietic cells and find its target cells.Firstly,the heterogeneous cell population of bone marrow hematopoietic cells was reduced using dimensionality reduction,clustering and marker annotation,which was classified into hematopoietic precursor cells,myeloid cells and lymphocytes,etc..The results showed that on Day 10 after 5-FU exposure,there was an imbalanced recovery of hematopoietic terminal cells,as evidenced by a large decrease in the number of myeloid（granulocytes,monocytes/macrophages）and B-lineage cells,and a large increase in the number of T,NK,dendritic cells and plasma cells.In contrast,Rg2 contributed to the restoration of the numbers of each cell population to near normal levels,suggesting that Rg2 could correct the 5-FU-induced restoration of the imbalance in numbers of each lineage of hematopoietic cells.Subsequently,the target cells of Rg2 were found to be upstream hematopoietic precursor cells using pseudo-temporal analysis.Based on the intergroup difference analysis between 5-FU vs Ctrl and5-FU+Rg2 vs 5-FU,the differentially expressed genes screening and biological process scoring methods were used to investigate the gene expression and functional expression of hematopoietic precursor cells:1)5-FU up-regulated the expression of spectrum-specific genes related to dendritic cells,plasma cells and NK cells,such as Cd74,Igkc and Ccl5,and down-regulated the expression of granulocyte-related genes,such as Camp,S100a8 and S100a9,while Rg2 directly regressed the expression of some spectrum-related high-variable genes.2)Rg2 increased myeloid lineage differentiation of hematopoietic precursor cells while decreasing their dendritic and lymphoid lineage differentiation.The results suggested that Rg2 could correct the tendency of lymphoid lineage differentiation initiated by 5-FU damage recovery by stabilizing the expression of related lineage genes in hematopoietic precursor cells.Finally,dimensionality reduction,clustering and annotation analysis of hematopoietic precursor cells revealed the existence of different functional subpopulations at a steady state,under 5-FU injury and after Rg2 treatment.In this study,it also first demonstrated that multipotent progenitors（Lin^-Sca1⁺Kit⁺Flt3^-Cd150⁺Cd48⁺Vwf⁺,MPPs）were target cells of Rg2.Rg2accelerated self-recovery process by correcting the bias in cell lineage differentiation of MPP initiated after 5-FU injury.In conclusion,this thesis comprehensively evaluated the anti-myelosuppressive effect of ginsenoside Rg2 and confirmed its definite protective effect against myelosuppression.MPPs were identified as the target cells of Rg2,which may regulate the differentiation ability of damaged MPPs by stabilizing the expression of related genealogy genes to accelerate the recovery from hematopoietic injury as well as to maintaining the homeostasis of hematopoiesis.