Mechanism and Molecular Targets of Ejiao Siwu Decoction for Treating Primary Immune Thrombocytopenia Based on High-Performance Liquid Chromatograph, Network Pharmacology, Molecular Docking and Cytokines Validation.

We explored the mechanisms and molecular targets of Ejiao Siwu Decoction (EJSW) for treating primary immune thrombocytopenia (ITP) using network pharmacology and molecular docking. Active compounds of EJSW were identified by high-performance liquid chromatography-diode array detector (HPLC-DAD) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) and their targets were obtained from HERB and SwissTargetPrediction, and ITP targets were obtained from Comparative Toxicogenomics Database (CTD) and GeneCards. STRING and Cytoscape were used for protein-protein interaction (PPI) network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by WebGestalt yielded a gene-pathway network, Autodock molecular docking was applied to screen targets and active compounds, and cytokines were detected using a cytometric bead array (CBA) human inflammation kit. We identified 14 compounds and 129 targets, and 1,726 ITP targets. RAC-alpha serine/threonine-protein kinase (AKT1), tumour necrosis factor (TNF), interleukin-6 (IL6), caspase-3 (CASP3) and tumour suppressor protein (TP53) were core targets (nodes and edges). Functional annotation identified cofactor binding and coenzyme binding, and 20 significantly enriched pathways. Active compounds of EJSW were successfully docked with ITP targets. Tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) were upregulated in ITP patients, vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor D (VEGF-D) were downregulated, and EJSW treatment reversed these trends. EJSW may regulate key ITP targets based on the in silico analyses, and protect vascular integrity through AGE-RAGE signalling, complement and coagulation cascades, and VEGF signalling by downregulating TNF-α, IL-1ß and other inflammatory factors.

Effects of arsenic disulfide on proliferation, cytokine production, and frequencies of CD4(+), CD8(+), and regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

Influence of arsenic disulfide (As2S2) on human immune cells has little been investigated. Effects of As2S2 on proliferation, cytokine production, and frequencies of CD4(+) T, CD8(+) T and CD4(+)CD25(+)Foxp3(+) regulatory T cells in mitogen-activated human peripheral blood mononuclear cells were examined. Anti-proliferative effects of As2S2 on peripheral blood mononuclear cells activated by T-cell mitogen were assessed by a colorimetric assay. Cytokine concentrations in the culture medium were measured with beads-array procedures followed by flow cytometry. CD4(+) T cells, CD8(+) T cells and CD4(+)CD25(+)Foxp3(+) regulatory T cells were stained with fluorescence-labeled specific antibodies followed by flow cytometry analysis. As2S2 at 1-10µM significantly suppressed mitogen-activated proliferation of peripheral blood mononuclear cells (p<0.05). As2S2 at 10µM inhibited production of IL-6, -10, -17A, tumor necrosis factor-α, and interferon-γ from the activated peripheral blood mononuclear cells, though the effects were not statistically significant. As2S2 at 10µM significantly suppressed the frequencies of CD4(+) T and CD8(+) T cells (p<0.05), whereas significantly enhanced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (p<0.05). The data suggest that As2S2 attenuates T cell-mediated immunity by not only suppressing the proliferation of T cells and cytokine release but also increasing the frequency of regulatory T cells. T cell-mediated autoimmunity contributes to bone marrow failure in myelodysplastic syndrome (MDS), and thus the above As2S2 effects are beneficial for the treatment of MDS patients.

Arsenic disulfide-triggered apoptosis and erythroid differentiation in myelodysplastic syndrome and acute myeloid leukemia cell lines.

OBJECTIVES: Effects of arsenic disulfide (As2S2) were investigated by focusing on growth inhibition, apoptosis induction, and erythroid differentiation in MDS-L, F-36p and HL-60 cells, derived from myelodysplastic syndrome (MDS), MDS/acute myeloid leukemia (AML), and de novo AML, respectively. METHODS: Cell viability was determined by MTT assay. Apoptosis induction was analyzed using Annexin V/propidium iodide staining. Erythroid differentiation was assessed by the expression level of CD235a, a marker for detection of the erythroid cell lineage. The activation of p38 MAPK and the expression profile of apoptosis-related proteins Bcl-2 and Bid were analyzed using western blot. RESULTS: As2S2 inhibited cell growth of these cell lines. Of note, the IC50 value of As2S2 in MDS-L cells was comparable to that in F-36p cells, and was half of that in HL-60 cells. A dose-dependent decrease in cell viability and concomitant increase in the percentage of apoptotic cells were observed in F-36p cells treated with 8 and 16 µM As2S2 for 72 hours. However, similar phenomena were only observed in HL-60 cells when treated with as high as 16 µM As2S2. Furthermore, As2S2 exerted more potent erythroid differentiation-inducing activity on F-36p cells than HL-60 cells. Interestingly, negative correlation between p38 MAPK signaling pathway and As2S2-induced erythroid differentiation was observed in HL-60 cells. Treatment with relatively high concentration of As2S2 resulted in the downregulation of Bcl-2 and Bid proteins in HL-60 cells. DISCUSSION: These results suggest that compared to AML cell line, MDS and MDS/AML cell lines are more sensitive to not only the erythroid differentiation-inducing activity of As2S2, but also its cytotoxicity associated with apoptosis induction. These findings further provide novel insight into As2S2 action toward its use for clinical application in patients with hematological disorders.