Effects Of Astragalus Polysaccharides On The Phagocytosis Of Mycobacterium Tuberculosis By Macrophages And On AC133～+ Cells Of Human Cord Blood In Vitro Culture.

Aim: The herb Astragalus membranaceus is used in traditional Chinese medicine to boost immunity. This study investigated the effects of Astragalus polysaccharides (APS) and astragalosides (AS) on the phagocytosis of Mycobacterium tuberculosis (Mtb) by macrophages.Methods: (1) Generation and identification of peritoneal macrophages: The mice were injected with starch gravy culture medium continuously for macrophages generation and macrophages were collected by peritoneal lavage after 3 day. The viability and purity of macrophages were evaluated by trypanblue and non-specific esterase stain respectively. Obtained macrophages were suspended with RPMI-FCS at the concentration of 1×10~9 cells per liter. (2) Cell culture: 48-well cell-cultured plastic plate was selected for cells culture, and 500μl RPMI-FCS medium, 500μl attenuated Mtb and 100μl APS or AS at the end concentrations of 0.05, 0.2, 0.6, 1.5, 4 and 8 mg/ml respectively together were added into each well. Six well were selected for each group and 0mg/mL APS or AS groups were used for control and Wells without macrophages were prepared for background levels. (3) Evaluations of the phagocytosis of Mtb by macrophages: After 5 hours, the cultures were collected and an enzyme-linked immunosorbent assay (ELISA) was performed on the supernatant to measure the levels of the cytokines interleukins (IL)-6, IL-1βand tumor necrosis factor-α(TNF-α). Real-time polymerase chain reaction (PCR) assay was used to detect Mtb DNA phagocytized by macrophages. Also, the morphologic character was observed using original microscope. (4) Data process: SPSS10.0 software was used to perform a one-way ANOVA.Results: (1) Morphologic observation: In groups of 0.05,4, 8mg/mL APS and AS concentrations, the macrophages were in the situation of static; at groups of 0.2 and 1.5mg/mL APS and AS concentrations, phagocytotic activity was enhanced and Mtb can be found in macrophages; at the point of 0.6mg/mL concentration APS and AS, phagocytosis was significantly activated and a large mount of Mtb can be found in macrophages. (2) PCR quantitative analysis: Phagocytotic activity initially increased gradually with increasing concentrations of APS and AS. It was highest at a concentration of 0.6mg/mL for both APS and AS, with maximum values of 7.04±0.67and 7.40±0.87 respectively (P<0.01 vs. control group) for the copies of Mtb DNA. The increase in phagocytotic was greater after treatment with AS compared with APS, but this difference was not statistically significant. After peaking at 0.6mg/mL, phagocytotic activity began to decrease with further increases of concentration of APS and AS, falling to levels comparable with those of the control group at 8mg/mL. (3) Cytokines secreted by macrophages: APS and AS both promoted the cytokines secretion, but present study revealed optimum concentration point of APS and AS was existed. APS produced the greatest secretion at a concentration of 4mg/mL for IL-1β(20.5 + 1.55) and TNTF-α(68.8±5.73) and 1.5mg/mL for IL-6 (30.7±2.95) respectively (P＜0.01 vs. control group). AS produced the greatest secretion at a concentration of 1.5mg/mL for IL-6 (46.85±4.12) and TNF-α(76.2±6.66) and 0.6mg/mL for IL-1β(29.7±5.46) respectively (P<0.01 vs. control group).Conclusions: The present study provides evidence that APS and AS have strong promoting effects on the phagocytosis of Mtb by macrophages and the secretion of the IL-1β, IL-6 and TNF-αby activated macrophages. The concentration of APS or AS is a critical factor in determining the effect on macrophage function. Aim: This is first study, using human cord blood (HCB)-derived AC133~+ hematopoietic stem cells (HSCs) as objects and cultured with different concentrations Astragalus polysaccharides (APS) in vitro, to investigate: (1) the possibility of APS promoted the cultured AC133~+ HSCs proliferation, differentiation and maturation; (2) the relationship between APS concentrations and survival, proliferation and differentiation.Methods: (1) HCB, used for mononuclear cells (MNCs) isolation, was obtained with the consent of the pregnant mothers who gave birth to health babies within the expected date and admitted to The Second Hospital of Lanzhou University. Total 20 cord blood (CB) samples were collected and each for 60mL. (2) Density gradient centrifugation was performed to isolate MNCs from HCB, and magnetic activated cell sorting (MACS) was used for AC133~+ cells purification. The percentage of AC133~+ cells in HCB-MNCs and purity of isolated AC133~+ cells from HCB were analyzed by Flow cytometry (FCM). (3) Purified cells were cultured in 24-well plates contained total 2mL cells suspension consisted of 2><106 cells and different APS concentrations from 0.1mg/mL to 10mg/mL respectively and RPMI-1640 "cocktail" medium each well. Zero APS concentration group was set as control. (4) Observation and analysis of cultured cells were performed with following technology and methods: such as morphologic characteristics, FCM counts analysis for fluorescence labeled monoclonal antibody (MoAb) and cell apoptosis test, immunofluorescent (IF) microscope for IF double stains and cells functional IF stains inspection. Macintosh CELLQuest software was used for FCM data process and SPSS for Windows XP for statistics.Results: (1) 0.1mg/mL APS has no effects on proliferation, differentiation and protection of cultured cells in vitro (P>0.05 vs. control). (2) 0.5-1.0mg/mL APS protected the cultured cells from apoptosis and necrosis (P＜0.05), but limited effect on proliferation and differentiation (P>0.05). (3) 2.0-5.0mg/mL APS expressed strong effects to advance proliferation, differentiation and maturation of cultured HCB-derived AC133~+ HSCs in vitro (P<0.01), whatever the concentrations is higher or lower, no this effects. (4) Despite of 10mg/mL APS display a significant protection on cultured cells (P<0.05), the proliferation and maturation and activity were inhibited obviously.Conclusions: (1) Optimum APS concentrations show not only evident function to stabilize the cell membrane, diminish the apoptosis, prevent the necrosis and death, but also promote the proliferation, differentiation and maturation of cultured HCB-AC133~+ HSCs. (2) Linear-related ratio between APS concentrations and proliferation, differentiation, maturation, apoptosis and necrosis of cultured cells were not existed in this study. (3) Over high APS concentrations caused cells activity inhibited clearly and made the cultured cells under the situation of static.