The Protective Effect Of Angelica Sinensis Polysaccharides On Bone Marrow Stromal Cells Damaged By 5-fluorouracil

As one of the comprehensive treatment of malignant tumor,chemotherapy also brings various adverse reactions.Bone marrow suppression refers to the decrease of the activity of blood cell precursors in bone marrow.It often occurs in patients with malignant tumor after chemotherapy and radiation therapy,which seriously affects the prognosis and life of patients.The clinical development of recombinant hematopoietic growth factor injection,hematopoietic stem/progenitor cells transplantation,although can obviously shorten the tumor patient's period of bone marrow suppression,it still can not completely restore hematopoietic function and immune function in patients.It suggests that chemotherapy may damage the bone marrow hematopoietic microenvironment,and thus indirectly affect the hematopoietic stem / progenitor cells proliferation,differentiation and maturation,resulting in hematopoietic dysfunction.The basis of experimental and clinical studies have confirmed that in vitro amplification of human bone marrow stromal cells combined with hematopoietic stem cells transfusion is an effective method for damaged bone marrow hematopoietic reconstruction,but the mechanism of chemotherapy-induced bone marrow hematopoietic microenvironment damage and its effect on the function of hematopoietic cells is not clear.Therefore,further study on the biological mechanism of chemotherapy induced bone marrow hematopoietic function decline will has important practical significance to alleviate the side effects of chemotherapy in clinic.In this study,human bone marrow stromal cell line HS-5 was taken to explore whether there is damage effect of 5-fluorouracil(5-fluorouracil,5-FU)on artificial bone marrow hematopoietic microenvironment;And using HS-5 cells as feeder layer,human umbilical cord blood mononuclear cells and HS-5 cells co-culture model was set up to further explore the effects of bone marrow stromal cells after chemotherapy on hematopoietic cells and its underline mechanism.Angelica sinensis polysaccharides(ASP),effective medicinal ingredients isolated from the traditional Chinese medicine angelica,have the function of hematopoietic regulation,immune regulation and anti-radiation damage and anti-tumor.The previous studies in our group found that ASP has a protective effect on aging hematopoietic stem cells in radiated mouse;and ASP can delay the bone marrow stromal cells physiological senescence imitated by D-galactose,thus promote hematopoietic cell proliferation and differentiation.In this study,we further investigated the protective effect of ASP on HS-5 damaged by 5-FU and its possible biological mechanisms,as well as the effect of hematopoietic microenvironment after chemotherapy ameliorated by ASP on the hematopoietic function improvement.The purpose of this study was to provide experimental basis and new ideas for alleviating the side effects of chemotherapy.Method:1.The human bone marrow stromal cell line HS-5 was treated with 5-FU in a tumor suppressive dose in vitro to investigate the effect of 5-FU on human bone marrow stromal cells: Human breast cancer cell line MCF-7,human colon cancer cell line HCT-116 and human bone marrow stromal cell line HS-5 were cultured in vitro.The Cell Counting Kit-8 for determining the sensitivity of MCF-7,HCT-116 and HS-5 in different doses of 5-fluorouracil in vitro was utilized.Experimental groups: Control group(routine culture of 48 h);12.5 ?g/ml groups(Conventional culture with 5-FU 12.5 ?g/ml for 48 h);25 ?g/ml group(Conventional culture with 5-FU 25 ?g/ml for 48 h);50 ?g/ml group(Conventional culture with 5-FU 50 ?g/ml for 48 h);100 ?g/ml group(Conventional culture with 5-FU 100 ?g/ml for 48 h.After HS-5 treated with 5-fluorouracil in different concentration for 48 h,the crystal violet staining assay was used to count the number of colonies forming unit-fibroblast;the distribution of cell cycle was analyzed by Flow Cytometry;apoptosis was assessed by Annexin V/PI double-stained method and Hoechest staining assay.2.Regulatory effects of Angelica sinensis polysaccharide on 5-FU damaged bone marrow stromal cells and its related mechanisms: 25 ?g/ml 5-FU treated for 48 h were selected for the subsequent experiments with HS-5 cells.The experiments were divided into control group ? ASP group?5-FU group and ASP+5-FU group.control group: HS-5 cells were cultured as routine for 48 h;ASP group:the conventional medium with ASP 100 ?g/ml for 48 h;5-FU group:the conventional medium with 5-FU 25 ?g/ml for 48 h;ASP+5-FU group: after pre-treated with ASP 100 ?g/mL for 6 h,the 5-FU 25 ?g/ml were added to the conventional medium for 48 h.crystal violet staining assay was used to count the number of colonies forming unit-fibroblast;the distribution of cell cycle was analyzed by Flow Cytometry(FCM);apoptosis was assessed by Annexin V/PI double-stained method;the senescence associated-?-galactosidase(SA-?-Gal)staining was used to detect the senescent cell;DCFH-DA staining analyzed the level of reactive oxygen species;the content of superoxide dismutase and Glutathione peroxidase were determined by enzyme method;the expression of ?H2AX was analysed by flow cytometry;the content of 8-hydroxydeoxyguanosine(8-OHd G)was dectected by ELISA,and expression of cytokines SCF,GM-CSF,SDF,RANTS were detected by immunofluorescence and ELISA;expression of Connexin CX43 was tested by immunofluorescence and the function of CX43 was tested by fluorescent yellow scratch transmission technology.3.HS-5 and human umbilical cord blood mononuclear cell(hUCB-MNC)co-culture model was established in vitro to observe the effect of damaged bone marrow stromal cells on hematopoietic cells and the regulating effect of ASP: The HS-5 feeder layer was established,the experiment was divided into control group?ASP group?5-FU group and ASP+5-FU group.control group: HS-5 was culture as routine for 48 h;ASP group:the conventional medium with ASP 100 ?g/ml for 48 h;5-FU group:the conventional medium with 5-FU 25 ?g/ml for 48 h;ASP+5-FU group: after pre-treated with ASP 100 ?g/mL for 6 h,the 25 ?g/ml 5-FU was added to the conventional medium for 48 h.The hUCB-MNC was separated by Ficoll density gradient centrifugation.After adherent culture for 6 h,the umbilical cord blood stromal cells were removed from hUCB-MNC,then the suspended hematopoietic cells were collected and cell concentration was adjusted to 1×106/ml respectively,then co-cultured on the four groups of HS-5 feeder layers for 48 h.After that hUCB-MNC were collected and counted by trypan blue staining.FCM was used to detect the cell cycle distribution,ROS level and the ratio of CD34+ cell.The level of glutathione peroxidase(GSH-Px)and total superoxide dismutase(T-SOD)were measured by enzymatic assay.The senescence associated-?-galactosidase(SA-?-Gal)staining was used to detect the senescent hUCB-MNC.Result:1.5-FU from 12.5 ?g/ml~100 ?g/ml inhibited the proliferation of MCF-7,HCT-116 and HS-5 cells in a dose-dependent and time-dependent manner,among them HS-5 is more sensitive to 5-FU.After the treatment of 5-FU in different concentration;The number of fibroblasts colony were decreased;HS-5 cell cycle was blocked,the proportion of cells in G0/G1 phase was increased,while the proportion of S phase was decreased;The apoptosis rate of HS-5 was significantly increased.2.HS-5 was damaged by 5-FU,The number of fibroblasts colony was decreased;cell cycle was blocked in G1 phase,the proportion of cells in S phase decreased;the apoptosis rate was significantly increased;the positive rate of aging cell was significantly increased;the antioxidant capacity of HS-5 also attenuated;the intracellular ROS content of HS-5 were significantly increased;the expression level of ?H2AX and 8-OHd G were increased;the expression of gap junction protein CX43 was decreased and the transmission capacity of fluorescent dye was decreased;the contents of positive regulatory factor of SCF,GM-CSF and SDF-1 were significantly decreased,while the secretion of inflammatory factor RANTES was significantly increased.Pretreatment of HS-5 cells with ASP before 5-FU treatment,the number of fibroblasts colony was significantly increased;cell cycle arrest was decreased;the apoptosis rate was significantly decreased;the positive rate of specific SA-?-Gal staining was significantly decreased;the antioxidant capacity also enhanced;the intracellular ROS content of HS-5 were significantly decreased;the expression level of H2 AX and 8-OHdG were decreased;the expression of gap junction protein Cx43 was increased and the transmission capacity of BMSC fluorescent dye was increased;the contents of positive regulatory factor of SCF,GM-CSF and SDF-1 were significantly increased,while the secretion of inflammatory factor RANTES was significantly decreased.3.Compared with control group,after co-cultured with HS-5 treated with 5-fluorouracil,the number of hUCB-MNC and the ratio of CD34+ cells were decreased.hUCB-MNC cell cycle was blocked in G1 phase.The antioxidant capacity also attenuated and the intracellular ROS content increased significantly.The ratio of senescent hUCB-MNC increased.Compared with the hUCB-MNC in 5-FU group,the number of hUCB-MNC was increased in the group of ASP+5-FU;the cell proportion of G1 phase decreased and the cell proportion of S phase increased;the positive rate of aging cell was decreased;the antioxidant capacity boosted up,the content of ROS was decreased.Conclusion:1.5-FU in tumor suppressing concentration also inhibited the proliferation of bone marrow stromal cells.Bone marrow stromal cells presented apoptosis and senescence.2.Bone marrow microenvironment was changed by 5-FU: 5-FU can cause oxidative damage to bone marrow stromal cells and the expression of intercellular junction protein was down regulated,the intercellular communication function was reduced,and change the function of bone marrow stromal cells to secrete bioactivator.3.5-FU injured bone marrow stromal cell initiates stress-induced premature senescence of hematopoietic cell.4.ASP can delay the oxidative stress induced premature senescence of hematopoietic cells by reducing the damage of 5-FU to hematopoietic microenvironment,the mechanism may be relevant to the reduction of oxidative stress,up-regulation of intercellular communication function,and promotion of hematopoietic positive factor secretion.