Angelica sinensis Polysaccharides Ameliorate Stress-Induced Premature Senescence of Hematopoietic Cell via Protecting Bone Marrow Stromal Cells from Oxidative Injuries Caused by 5-Fluorouracil

Abstract

Myelosuppression is the most common complication of chemotherapy. Decline of self-renewal capacity and stress-induced premature senescence (SIPS) of hematopoietic stem cells (HSCs) induced by chemotherapeutic agents may be the cause of long-term myelosuppression after chemotherapy. Whether the mechanism of SIPS of hematopoietic cells relates to chemotherapeutic injury occurred in hematopoietic microenvironment (HM) is still not well elucidated. This study explored the protective effect of Angelica sinensis polysaccharide (ASP), an acetone extract polysaccharide found as the major effective ingredients of a traditional Chinese medicinal herb named Chinese Angelica (Dong Quai), on oxidative damage of homo sapiens bone marrow/stroma cell line (HS-5) caused by 5-fluorouracil (5-FU), and the effect of ASP relieving oxidative stress in HM on SIPS of hematopoietic cells. Tumor-suppressive doses of 5-FU inhibited the growth of HS-5 in a dose-dependent and time-dependent manner. 5-FU induced HS-5 apoptosis and also accumulated cellular hallmarks of senescence including cell cycle arrest and typical senescence-associated β-galactosidase positive staining. The intracellular reactive oxygen species (ROS) was increased in 5-FU treated HS-5 cells and coinstantaneous with attenuated antioxidant capacity marked by superoxide dismutase and glutathione peroxidase. Oxidative stress initiated DNA damage indicated by increased γH2AX and 8-OHdG. Oxidative damage of HS-5 cells resulted in declined hematopoietic stimulating factors including stem cell factor (SCF), stromal cell-derived factor (SDF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), however, elevated inflammatory chemokines such as RANTES. In addition, gap junction channel protein expression and mediated intercellular communications were attenuated after 5-FU treatment. Significantly, co-culture on 5-FU treated HS-5 feeder layer resulted in less quantity of human umbilical cord blood-derived hematopoietic cells and CD34⁺ hematopoietic stem/progenitor cells (HSPCs), and SIPS of hematopoietic cells. However, it is noteworthy that ASP ameliorated SIPS of hematopoietic cells by the mechanism of protecting bone marrow stromal cells from chemotherapeutic injury via mitigating oxidative damage of stromal cells and improving their hematopoietic function. This study provides a new strategy to alleviate the complication of conventional cancer therapy using chemotherapeutic agents.

Keywords: 5-fluorouracil; Angelica sinensis polysaccharide; aging; bone marrow stromal cell; hematopoietic cell; oxidative stress.

Figure 1

5-fluorouracil (5-FU) inhibits the growth of both tumor cells and bone marrow stromal cells in a dose and time-dependent manner. Cell proliferation assay was performed via the Cell Counting Kit-8 kit. (a) Cell inhibition rates are presented as (OD_{control group} − OD_{experimental group})/OD_{control group} × 100%. MCF-7 treated without 5-FU was used as a control; (b) The inhibitory effects of 5-FU on HCT-16 cells are presented as inhibition rates: (OD_{control group} − OD_{experimental group})/OD_{control group} × 100%. HCT-16 treated without 5-FU was used as a control; (c) The cell viabilities of HS-5 were almost entirely decreased with tumor-suppressive doses of 5-FU. Viability rate = OD_{experimental group}/OD_{control group} ×100%. The cell viabilities of HS-5 without 5-FU treatment on each day were set as 100%. Results of HS-5 cell viability were normalized to the OD value of control HS-5; (d) Representative images of Fibroblast-colony forming unit (CFU-F) formed from 5-FU-treated and control HS-5 cells by phase contrast microscopy. CFU-F frequency decreased with increasing 5-FU doses (Scale bar = 50 μm). * p < 0.01 vs. control. CTL: Control; OD: optical density.

Figure 2

Angelica sinensis polysaccharide (ASP) rescues HS-5 cell growth inhibition after 5-FU treatment via anti-apoptosis and anti-senescence effects. (a) HS-5 cells treated with 5-FU, ASP, or a combination of both were cultured for 12 days, then stained by 0.5% crystal violet. CFU-F clusters are blue stained in dishes; (b) CFU-F frequency in different groups is presented as means ± SD; (c) Representative flow cytometry graphs of cell cycle analysis of HS-5 cells are shown; (d) The results of cell cycle distribution of HS-5 cells are presented as means ±SD; (e) Annexin V/Propidium iodide (PI) staining was employed to detect apoptotic HS-5 cells by flow cytometry. In each chart, upper left represents necrotic cells; bottom left represents vital cells; upper right represents intermediate-stage and late-stage apoptotic cells; and bottom right represents early-stage apoptotic cells; (f) The percentage of apoptotic HS-5 cells in different groups is presented as means ± SD. The left panel represents the ratio of intermediate-stage and late-stage apoptotic cells positively stained by PI. The right panel represents the ratio of early-stage apoptotic cells positively stained by Annexin V; (g) Senescence-related SA-β-gal staining was employed to detect senescent HS-5 cells. Senescent cells are blue-green stained (Scale bar = 50 μm); (h) The positive ratio of SA-β-gal staining is presented as means ± SD. * p < 0.01 vs. control group, # p < 0.01 vs. 5-FU group.

Figure 3

ASP alleviates oxidative stress caused by 5-FU in BMSCs. (a) The levels of reactive oxygen species (ROS) in HS-5 cells were measured by dichlorodihydrofluorescein diacetate (DCF-DA) staining and fluorescence microscopy (Scale bar = 50 μm); (b) The mean fluorescence intensity of ROS was quantified and is presented as means ± SD; (c) DSB of DNA was determined by γH2AX flow cytometry. Representative flow cytometric images of γH2AX in HS-5 cells are presented. The green line represents γH2AX negative cells and the purple line represents γH2AX positive cells; (d) The results of γH2AX content in HS-5 cells determined by flow cytometry are presented as means ± SD; (e) DNA damage response was detected by 8-OHdG supernatant Enzyme-linked immuno sorbent assay (ELISA). The results of 8-OHdG content in HS-5 cells are presented as means ± SD. * p < 0.01 vs. control group, # p < 0.01 vs. 5-FU group.

Figure 4

ASP reverses gap junction intercellular communication between bone marrow stromal cells after 5-FU injury. (a) Cx43 protein expression in HS-5 cells was detected by confocal laser scanning microscopy and is shown from a merged image of FITC-conjugated Cx43 and 4',6-diamidino-2-phenylindole (DAPI) staining of the nuclei in control (top), ASP-treated (the second panel), 5-FU-treated (the third panel) and 5-FU + ASP-treated (bottom) cells (Scale bar = 50 μm); (b) Scrape-loading and dye transfer assay was performed. The Lucifer Yellow transmission layers represent the capacity of intercellular communication; (c) Mean fluorescence intensity of Cx43 in HS-5 cells represented as means ± SD; (d) The cell layers of fluorescence transfer indicating the capacity of intercellular communication represented as means ± SD. * p < 0.01 vs. control group, # p < 0.01 vs. 5-FU group.

Figure 5

ASP recovers cytokines production from 5-FU injured bone marrow stromal cells: (a) SDF-1 (also named CXCL12) protein expression in HS-5 cells is detected via confocal laser scanning microscopy and shown from a merged image of FITC-conjugated SDF-1 and DAPI staining of the nuclei in control (top), ASP-treated (the second panel), 5-FU-treated (the third panel) and 5-FU + ASP-treated (bottom) cells (Scale bar = 50 μm); (b) Mean fluorescence intensity of SDF-1 was quantified and is presented as means ± SD; (c) (ELISA) assay was employed to detect the levels of cytokines produced by HS-5 cells. The results are presented as means ± SD. * p < 0.01 vs. control group, # p < 0.01 vs. 5-FU group.

Figure 6

ASP-treated HS-5 feeder layer protects co-cultured hematopoietic cells from oxidative stress-induced premature senescence. (a) Survival CD34⁺ HSPCs co-cultured on HS-5 stromal cell feeder layers were examined via flow cytometry. The green line represents CD34⁻ cells and the purple line represents CD34⁺ cells; (b) The percentage of co-cultured CD34⁺ HSPCs in human umbilical cord blood-derived hematopoietic cells is presented as means ± SD; (c) The senescent hematopoietic cells were positively stained by SA-β-gal to be blue-green. Co-cultured on the 5-FU-treated feeder layer, the frequency of SA-β-gal positive hematopoietic cells increased. However, co-cultured on the ASP-treated feeder layer, the number of senescent hematopoietic cells reduced (Scale bar = 50 μm); (d) The percentage of SA-β-gal positive co-cultured hematopoietic cells is presented as means ± SD; (e) Representative flow cytometry graphs of cell cycle analysis of co-cultured hematopoietic cells; (f) Results of cell cycle distribution of co-cultured hematopoietic cells are presented as means ± SD; (g) Levels of ROS in co-cultured hematopoietic cells were measured by DCF-DA staining and flow cytometry; (h) Mean fluorescence of ROS was quantified and is presented as means ± SD. * p < 0.01 vs. control group, # p < 0.05 vs. 5-FU group, ## p < 0.01 vs. 5-FU group.