| Cell senescence is the basis of body aging, somatic stem cells play a pivotal role in the senescent progression. Literature reported that the mechanism of physiological aging decreased bone marrow function and tumor pathological bone marrow inhibition caused by radiation and chemotherapy are related to the hematopoietic stem cells(HSCs) aging. Bone marrow hematopoietic inductive microenvironment (HIM) regulates the self-renewal、proliferation and differentiation of HSCs. Bone marrow stromal cells(BMSCs)is the core components of HIM, It’s function is closely related to the physiological state of HSC,there is few experimental date confirmed that the alteration of biology aging of BMSCs caused by normal aging and tumor radiation and chemotherapy; the influence and mechanism on HSCs aging caused by senescence of HIM is unclear. This study used D-galactose copying aging rats models to observe the biological alteration of BMSCs in aging rats. D-galactose induced BMSCs senescence in vitro established BMSCs and hematopoietic cells co-culture system to observe the proliferation and differentiation of hematopoietic cells influenced by senescent BMSCs.Angelica sinensis polysaccharide(ASP) is the effective medicinal ingredients which separated and extracted from traditional Chinese medicine-angelica,ASP can regulate hematopoiesis, resistance to radiation damage, anti-tumor and immune regulation and so on. Past studies shows that ASP can promote normal hematopoiesis, and has an positive regulation on hematopoietic cells and BMSCs.The experiment was to further explore the affects and its possible biological mechanisms that ASP regulates BMSCs aging in vivo and vitro aging model, to provide experimental evidence and theoretical basis of aging that ASP regulates HSCs aging from bone marrow microenvironment degree.Methods:1.D-Gal induced to copying aging rats model,observe the senescent biological alteration of BMSCs and ASP regulating biological effect on BMSCs senescence.The experiment was divided into 4 groups:the control group:rats were given 2mL/kg NS by daily neck subcutaneous injection for 6 weeks..aging group:The aging model rats were given 120mg/kg D-galactose (D-Gal) by daily neck subcutaneous injection for 6 weeks. ASP group:rats were given 2mL/kg NS by daily neck subcutaneous injection for 6 weeks, and given 200mg/kg ASP by daily intraperitoneal injection from 3rd week. ASP treatment group:rats were given 120mg/kg D-galactose (D-Gal) by daily neck subcutaneous injection for 6 weeks, and given 200mg/kg ASP by daily intraperitoneal injection from 3rd week, killed the rats on the second day after the model established, the bone marrow stromal cells (BMSCs) were isolated by whole bone marrow adherent culture. Cell Counting Kit-8(CCK-8)、flow cytometry (FCM). senescence-associated (3-galactosidase (SA-β-Gal) and Western Blotting were used to observing the change of senescent biological index; MDA (malonaldehyde) content and total SOD (superoxide dismutase) activity were analyzed as well using enzymatic assay; DCFH-DA fluorescent staining analyzed the level of ROS (reactive oxygen species); the amount of IL-6 and SCF in BMSCs culture supernatant were detected by ELISA.2. BMSCs aging model in. vitro was copied by D-Gal, to research the mechanism of alteration that ASP regulating the senescence of BMSCs: femurs and tibias of rat were taken sterility, and preparation of bone marrow cell suspension was adherent cultured for F3 in vitro For subsequent related experimental testing, the control group was cultured as usual for 96h; aging group:the aging group was cultured with D-galactose (D-Gal) 30mg/mL for 96 hours; the ASP group was cultured with ASP 100μL/mL for 96h;the ASP therapy aging group was cultured with D-galactose (D-Gal) 30mg/mL for 48 hours, and then was cultured with ASP 100μL/mL for 48h; ASP delaying senescence group was cultured with ASP 100μL/mL for 48h, and then was cultured with D-galactose (D-Gal) 30mg/mL for 48 hours. Cell Counting Kit-8(CCK-8)、flow cytometry (FCM)、senescence-associated P-galactosidase (SA-P-Gal) and Western Blotting were used to observing the change of senescent biological index; MDA (malonaldehyde) content and total SOD (superoxide dismutase) activity were analyzed as well using enzymatic assay; DCFH-DA fluorescent staining analyzed the level of ROS (reactive oxygen species); the amount of SCF、IL-1β and GM-CSF in BMSCs culture supernatant were detected by ELISA.3.The model of BMSCs and BMNCs co-cultured was established to observe the influence of ASP regulating BMSCs senescence on the proliferation and differentiation of hematopoietic cells:Preparation of BMSCs feeder layer was devided into control group、aging group、ASP group and ASP anti-aging group. BMNCs were extracted and cultured upon the BMSCs feeder layer for 48h. CFU-Mix、CCK-8 and FCM were used to observing the proliferation and differentiation of hematopoietic cells.Results:1. The proliferation of BMSCs dropped obviously in aging rats;cell cycle blocked in G1 phase;the positive ratio of SA-(3-Gal stained BMSCs also significantly increased;the expression of senescence-related proteins including P16、P21 and P53 were obviously up-regulated.The enzyme activity of SOD dropped,while the level of MDA and ROS raised;secretion of SCF、IL-6 decreased.2. Compared with BMSCs in aging rats,aging rats which were injected ASP,the positive ratio of SA-β-Gal stained BMSCs reduced;the G1 phase decreased,S phase increased;the expression of senescence-related proteins including P16、P21 and P53 were obviously down-regulated;the enzyme activity of SOD increased,while the level of MDA and ROS d^opped;secretion of SCF、IL-6 increased.3. ASP affected aging BMSCs in vitro improved the proliferation of BMSCs;the positive ratio of SA-β-Gal stained BMSCs reduced; the G1 phase decreased, S phase increased; he expression of senescence-related proteins including P16% P21 and P53 were obviously down-regulated;the enzyme activity of SOD increased, while the level of MDA and ROS dropped. ASP enhanced BMSCs secreting SCF、IL-1β and GM-CSF.4. The proliferation of BMNCs that co-cultured with aging BMSCs dropped;the cell cycle distribution changed,the cells in S phase reduced, and G1 phase increased; the CFU-Mix forming number of multipotential progenitor cell reduced obviously; it had contrary results in which co-cultured with ASP anti-aging group.Conclusion:1. BMSCs occurring senescent biological alteration in aging rats.2ASP can defer BMSCs aging in rats.3. ASP defers BMSCs aging in vitro, may be related to ASP increases antioxidant capacity, reduces the level of ROS,inhibits the senescence-associate protein expression.4. ASP promotes proliferation and differentiation of hematopoietic cells by delaying BMSCs senescence,may be related to ASP reduces the oxidant damage of BMSCs, promotes secrete hematopoietic related factors. |
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