Pharmacological and transcriptome profiling analyses of Fufang E'jiao Jiang during chemotherapy-induced myelosuppression in mice.

Fufang E'jiao Jiang (FEJ), a famous traditional Chinese medicine formula from "Liangyi Ointment", consists of five crude drugs, Colla corii asini, Radix Ginseng Rubra, Radix Rehmanniae Preparata, Codonopsis pilosula, and Crataegus pinnatifida Bge. It has pronounced functions of qi-nourishing and blood-activating. Recently, it has been widely used in China as a medication against myelosuppression in cancer treatment. We aimed to investigate the complex mode of action and underlying mechanisms of Fufang E'jiao Jiang (FEJ) regarding its hematopoietic effect. Mice were divided into 5 groups of control, model, high dose FEJ (HFEJ), medium dose FEJ (MFEJ) and low dose FEJ (LFEJ). After 10 days from the administration, bone marrow cells (BMCs) were extracted for nucleated cells counts, flow cytometry analysis of hematopoietic stem cells (HSCs) population, as well as hematopoietic progenitor cells (HPCs) colony-forming unit (CFU) assay. A portion of bone marrow nucleated cells (BMNCs) of MFEJ group were prepared for RNA sequencing (RNA-Seq). The transcriptome data were analyzed based on the differentially expressed genes (DEGs). The molecular mechanisms of FEJ were deducted based on the biological processes and protein-protein interaction (PPI) network. FEJ could significantly increase the percentage of HSCs and the quantities of BFU-E and CFU-GM in BMSCs. FEJ could stimulate the proliferation of HSC and the differentiation of HPC to all lineages, which may thereby accelerate the recovery of hematopoietic function in myelosuppressive mice. By providing transcriptome profile we highlighted several genes and biological processes that might be applicable for FEJ to treat chemotherapy-induced myelosuppression. GO analysis showed that the co-expressed DEGs in FEJ vs model and model vs control group were involved in biological processes including ossification, osteoblast differentiation, bone mineralization and bone development. The KEGG pathway analysis pointed out ECM-receptor interaction and PI3K-AKT signaling pathway as the most relevant pathways to the function of FEJ on myelosuppression. PPI network showed MMP2 and COL1A1 were the relatively large nodes. FEJ has the hematopoietic effect in chemotherapy-induced myelosuppression mice. It might be achieved by improving the proliferative capacity of HSCs and the differentiation ability of HPCs. The molecular mode of action of FEJ might be the improvement of the bone marrow microenvironment via ECM-receptor interaction, the promoted proliferation of HSC through regulation of PI3K-AKT signaling pathway, and the involvement of osteoblasts and osteoclasts. MMP2 and COL1A1 appear to be the key relevant regulatory molecules. These results provide significant insight into the hematopoietic effects of FEJ in myelosuppression and point out novel targets for future validating analyses.

Zhang Y ，Ye T ，Hong Z ，Gong S ，Zhou X ，Liu H ，Qian J ，Qu H ... -  《-》

Hematopoietic effects and mechanisms of Fufang e׳jiao jiang on radiotherapy and chemotherapy-induced myelosuppressed mice.

Fufang e׳jiao jiang (FEJ), which has been widely used in clinic to replenish qi (vital energy) and nourish blood, is a famous traditional Chinese medicine formula made up of Colla corii asini (donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus.), Radix codonopsis pilosulae (the root of Codonopsis pilosula (Franch.) Nannf.), Radix ginseng rubra (the steamed and dried root of Panax ginseng C.A. Mey.), Fructus crataegi (the fruit of Crataegus pinnatifida Bunge) and Radix rehmanniae preparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey.). The present study aimed to investigate the hematopoietic effects of FEJ on myelosuppressed mice induced by radiotherapy and chemotherapy systematically and to explore the underlying hematopoietic regulation mechanisms. The myelosuppressed mouse model was induced by (60)Co radiation, cyclophosphamide and chloramphenicol. FEJ was then administered by i.g. at the dosages of 5, 10, or 20 mL/kg·d for 10d. The numbers of blood cells from peripheral blood and bone marrow nucleated cells (BMNC) were counted. Body weight and the thymus and spleen indices were also measured. The numbers of hemopoietic progenitor cells and colony-forming unit-fibroblast (CFU-F) were measured in vitro. The ratio of hematopoietic stem cells (HSC) in BMNC, cell cycle and apoptosis of BMNC were determined by flow cytometry. The histology of femoral bone was examined by H&E staining. The levels of transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), erythropoietin (EPO), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6) in serum were measured by ELISA. IL-1β, IL-3, IL-6 mRNA levels in spleen were detected by real-time quantitative PCR (RT-qPCR). In addition, bone marrow stromal cells (BMSC) were cultured in vitro followed by treatment with different doses of FEJ (2.5, 5, 10 μL/mL) for 48 h. Then the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were determined by ELISA and RT-qPCR, respectively. FEJ could significantly increase the numbers of peripheral blood cells and BMNC, and reverse the loss of body weight and the atrophy of thymus and spleen in a dose-dependent manner. The quantities of hemopoietic progenitor cells and CFU-F in bone marrow were also significantly increased in a dose-dependent manner after FEJ administration. A high-dose FEJ of 20 mL/kg·d could significantly increase the ratio of HSC in BMNC, promote bone marrow cells entering the proliferative cycle phase (S+G2/M) and prevent cells from proceeding to the apoptotic phase. FEJ could also improve the femoral bone marrow morphology. Furthermore, FEJ could increase the levels of GM-CSF and IL-3 and reduce the level of TGF-β in serum, and enhance the expressions of IL-1β and IL-3 mRNA in spleen. Lastly, the levels of cytokines (IL-6, SCF, GM-CSF) in the conditioned media and their mRNA levels in BMSC were elevated after treatment with FEJ. FEJ was clearly confirmed to promote the recovery of bone marrow hemopoietic function in a myelosuppressed mouse model, which may be attributed to (i) improving bone marrow hematopoietic microenvironment; (ii) facilitating the cell proliferation and preventing BMNC from apoptosis; (iii) stimulating the expressions of IL-1β, IL-3, IL-6, SCF and GM-CSF and inhibiting the expression of TGF-β.

Liu M ，Tan H ，Zhang X ，Liu Z ，Cheng Y ，Wang D ，Wang F ... -  《-》

RNA-sequencing based bone marrow cell transcriptome analysis reveals the potential mechanisms of E'jiao against blood-deficiency in mice.

As a health-care food and traditional Chinese medicine, E'jiao, from the skin of Equus animus L, has been used to nourish blood in China for more than 2000 years. In modern medicine, there are also evidences indicate it has a beneficial effect on chemotherapy-caused blood deficiency. However, its mechanism of action for blood invigoration remains unclear. In the present study, we investigated the hematopoietic effect of E'jiao in 5-Fluorouracil-treated mice. In addition to the counting of bone marrow nucleated cells (BMNCs), flow cytometry was used to detect the population of hematopoietic stem cells (HSCs), and colony-forming unit (CFU) was used to assay the differentiation ability of hematopoietic progenitor cells (HPCs). Gene expression profiles of bone marrow cells were obtained from RNA sequencing (RNA-seq) and differentially expressed genes (DEGs) were analyzed with an emphasis on hematopoiesis-related pathways. The results show that E'jiao promotes the proliferation of both BMNCs and HSCs, as well as the differentiation of HPCs. By providing a hematopoiesis-related molecular regulatory network of E'jiao, we point out that the mechanism of E'jiao is associated with pathways including ECM-receptor interaction, Wnt signaling pathway, PI3K-Akt signaling pathway, TGF-beta signaling pathway, Hematopoietic cell lineage and Osteoclast differentiation, in which Ibsp, Col1a1, Col1a2, Notum, Sost, Dkk1, Irx5, Irx3 and Dcn are the key regulatory molecules. These findings provide valuable molecular basis for the mechanism of action of E'jiao.

Zhang Y ，Ye T ，Gong S ，Hong Z ，Zhou X ，Liu H ，Qu H ，Qian J ... -  《-》

Study on the mechanism of Fufang E'jiao Jiang on precancerous lesions of gastric cancer based on network pharmacology and metabolomics.

Fufang E'jiao Jiang (FEJ) is a prominent traditional Chinese medicine prescription, which consists of Asini Corii Colla (Donkey-hide gelatin prepared by stewing and concentrating from the hide of Equus asinus Linnaeus., ACC), Codonopsis Radix (the dried roots of Codonopsis pilosula (Franch.) Nannf., CR), Ginseng Radix et Rhizoma Rubra (the steamed and dried root of Panax ginseng C.A. Mey., GRR), Crataegi Fructus (the mature fruits of Crataegus pinnatifida Bunge., CF), and Rehmanniae Radix Praeparata (the steamed and sun dried tuber of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C.A. Mey., RRP). It is a popularly used prescription for "nourishing Qi and nourishing blood". To explore the potential mechanism of FEJ on precancerous lesion of gastric cancer in rats by combining network pharmacology and metabolomics. Traditional Chinese Medicine Systems Pharmacology and Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine were used to identify the ingredients and potential targets of FEJ. GeneCards database was used to define PLGC-associated targets. We built a herb-component-disease-target network and analyzed the protein-protein interaction network. Underlying mechanisms were identified using Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. In addition, 40% ethanol, N-methyl-N'-nitro-N-nitroguanidine and irregular eating were used to establish PLGC rats model. We also evaluated the efficacy of FEJ on MNNG-induced PLGC rats by body weight, histopathology, blood routine and cytokine levels, while the predicted pathway was determined by the Western blot. Ultra-performance liquid chromatography-tandem mass spectrometry-based serum non-targeted metabolomics was used to select potential biomarkers and relevant pathways for FEJ in the treatment of PLGC. Network pharmacology showed that FEJ exhibited anti-PLGC effects through regulating ALB, TNF, VEGFA, TP53, AKT1 and other targets, and the potential pathways mainly involved cancer-related, TNF, PI3K-AKT, HIF-1, and other signaling pathways. Animal experiments illustrated that FEJ could suppress inflammation, regulate gastrointestinal hormones, and inhibit the expression of PI3K/AKT/HIF-1α pathway-related proteins. Based on serum non-targeted metabolomics analysis, 12 differential metabolites responding to FEJ treatment were identified, and metabolic pathway analysis showed that the role of FEJ was concentrated in 6 metabolic pathways. Based on network pharmacology, animal experiments and metabolomics, we found that FEJ might ameliorate gastric mucosal injury in PLGC rats by regulating gastrointestinal hormones and inhibiting inflammation, and its mechanism of action is related to the inhibition of excessive activation of PI3K/AKT/HIF-1α signaling pathway and regulation of disorders of body energy metabolism. This comprehensive strategy also provided a reasonable way for unveiling the pharmacodynamic mechanisms of multi-components, multi-targets, and multi-pathways in Traditional Chinese Medicine.

Shi WB ，Wang ZX ，Liu HB ，Jia YJ ，Wang YP ，Xu X ，Zhang Y ，Qi XD ，Hu FD ... -  《-》

Transcriptome Profiling Analysis Reveals the Potential Mechanisms of Three Bioactive Ingredients of Fufang E'jiao Jiang During Chemotherapy-Induced Myelosuppression in Mice.

Li X ，Zhang Y ，Hong Z ，Gong S ，Liu W ，Zhou X ，Sun Y ，Qian J ，Qu H ... -  《Frontiers in Pharmacology》