Preliminary Study On Immune Regulation And Antitumor Effects Of Astragalus Polysachairns With Different Relative Molecule Masses Extracted From Annual Astragalus Membra-neaceus

The biological activity of Astragalus Polysacharin (APS) has been widely recognizedby many scholars and experts. In immunosuppression, such as the invasion of foreignpathogens, injury or tumorigenesis, APS can significantly enhance the body immunity;enable immune status to tend to balance.Studies have shown there are certain relations between the biological activity ofastragalus polysacharin and its relative molecule mass and growth years, but studies on therelationship of relative molecule mass and the biological activity of astragalus polysacharinwas less. Based on previous studies, we extracted from the annual astragalusmembra-neaceus and isolated into astragalus polysacharins (APSⅠ) with three kinds ofrelative molecule masses including APSⅠ-A、APSⅠ-B and APSⅠ-C and then explored thebioactivity of APS Ⅰ in the aspect of immune regulation and anti-tumor: to detectquantitative changes of inflammatory cytokine secretion after APSⅠacting to macrophagecell line RAW264.7cells and LPS-induced RAW264.7cells and to observe changes of NF-κB p65protein expression of macrophages. Meanwhile, the effect of APSⅠon humanneuroblastoma IMR-32cell proliferation and apoptosis was observed. We preliminarilystudy the function of APSⅠ on immune regulation and anti-tumor and provide sometheoretical basis for the research and development of annual astragalus membranaceuspolysacharins.1. Effect of APSⅠ on macrophage RAW264.7cells and LPS-induced RAW264.7cells1.1Effect of APSⅠ on proliferation activity of RAW264.7cells in respective groupCell proliferation activity was detected using CCK-8method and the results show thatthe APSⅠinhibits the proliferation of RAW264.7cells without LPS stimulation, and theinhibition is enhanced as the dose of APSⅠincreasing (P<0.05) in certain range ofconcentrations; The proliferative activity of the LPS-induced RAW264.7cells is enhanced asincreasing doses of APSⅠ (P<0.05,P<0.01), and the effect of APSⅠ with the smallestrelative molecular mass is most pronounced. This suggests that APSⅠenhances the viability of the inflammatory injured cell, but exhibits the inhibition action on the proliferation ofRAW264.7cells.1.2Effect of the APSⅠ on NO secretion of RAW264.7cellsThe results show that APSⅠcould significantly promote RAW2647cells to secrete NO,and it has statistically significant compared with blank control group (P<0.05,P<0.01). whenthe final concentration of APSⅠis in the range of12.5-50mg/L, the amount of NO secretedby RAW2647cells increases as drug final concentration increases. At the final concentrationof100mg/L, the promotion effect of APS-I has some decreased. However, the NOproduction can be significantly inhibited when APSⅠacting to the LPS-induced RAW264.7cells (P<0.05,P<0.01), and the inhibition effect increased as increasing drug concentration.This suggests that the APSⅠ can reduce the inflammatory mediators NO production inLPS-stimulated cells, and reduce the excessive inflammatory response. The inhibition effectof the APSⅠ with the smallest relative molecular mass on NO secretion of inflammatorycell is the most obvious among APSⅠ of different relative molecular masses, indicating thatthere is a certain relationship between the biological activity of APS and its molecular mass.1.3Effect of APSⅠon TNF-α secretion of RAW264.7cellsThe TNF-α secretion of RAW264.7cells of each group was measured by ELISAmethod. The experimental results show that three kinds of APSⅠ with different relativemolecular masses are able to reduce the TNF-α secretion of RAW264.7cells, the differencewas statistically significant (P<0.05,P<0.01), but the effect of APSⅠ on the TNF-α secretionis not obvious dependent on its final concentration. When APSⅠintervening in LPS-inducedRAW264.7cells, the results show that it can significantly inhibit TNF-α release (P<0.05,P<0.01) and its inhibition ability is dose-dependent. The inhibition effect of APSⅠwith thesmallest relative molecular mass on TNF-α release of LPS-induced RAW264.7cells is themost pronounced among three kinds APSⅠof different relative molecular masses, indicatingthat there is a certain relationship between the biological activity of APS and its molecularmass. This indicates that APSⅠcan lower TNF-α over-expression of activated macrophage,slow down the development and progression of inflammation, and contribute to therestoration of normal body function.1.4Effect of APSⅠon IL-12secretion of RAW264.7cellsAPSⅠcan reduce the IL-12secretion of RAW2647cells. At APSⅠconcentration of 50.0mg/L, the content of IL-12is the lowest (P<0.05,P<0.01), and there is significantdifference. This suggests APSⅠcan inhibit RAW2647cells to produce IL-12, but there is noconcentration dependence. In the inflammation model group, the LPS-induced cells can leadIL-12over-secretion, however, APS Ⅰ can inhibit IL-12secretion of LPS-inducedRAW264.7cells (P<0.05,P<0.01), the two was statistically significant. This indicates thatAPSⅠcan antagonize the inflammatory response by inhibition of IL-12over-expression.APSⅠwith the smallest relative molecular mass has best inhibition effect among three kindsAPSⅠof different relative molecular masses.1.5Effect of APSⅠon IL-10secretion of RAW264.7cellsWhen APSⅠacting to LPS-induced RAW264.7cells, IL-10expression level of thecells is significantly higher than the LPS-induced RAW264.7cells (P<0.05,P<0.01), butthere is no exact dose-dependence. The promotion effect of APSⅠwith the smallest relativemolecular mass on IL-10release of LPS-induced RAW264.7cells is the most pronouncedamong three kinds APSⅠof different relative molecular masses. When APSⅠacting toRAW264.7cells, IL-10secretion also significantly higher than the blank control group witha significant difference (P<0.05,P<0.01). The APS Ⅰ is speculated to play animmunosuppressive effect by regulating the body’s anti-inflammatory cytokine secretionlevels.In summary, the changes of RAW264.7cells anti-inflammatory cytokine secretion byAPSⅠaction can perform at increasing the release of NO and reducing TNF-α and IL-12,and then make the body in the immune state of equilibrium; however, for LPS-inducedRAW264.7cells, APSⅠcan inhibit the anti-inflammatory cytokine release and slow downthe development of inflammation. This indicates that APSⅠcan realize two-way adjustmentof RAW264.7cells, and can antagonize the inflammatory response. Combined with theexperimental results of IL-10, this indicates that the APSⅠis able to increase the bodyanti-inflammatory cytokine levels and reduce the level of proinflammatory cytokines, andhelp promote the development of the organism to a positive health direction. APSⅠwith thesmallest relative molecular mass performs best among three kinds APSⅠof different relativemolecular masses, indicating there is a certain relationship between the biological role of theAPS and its molecular mass. 1.6Effect of APSⅠon NF-κB p65protein expression of RAW264.7cellsThe NF-kappa B p65protein expression levels of cell nucleus are measured usingWestern blot analysis. The results show that the blank control RAW264.7cells group hasweak NF-κB p65band. LPS-control group has significantly higher relative optical density of NF-κBp65. There is significant differences (P<0.01) between the above two. In the APSⅠ+LPS group, therelative optical density values of p65of RAW264.7cells reduce to varying degrees. There is statisticallydifference (P<0.05) compared APSⅠ-B and APSⅠ-C intervening groups with LPS group. This showsthat the APSⅠcan reduced NF-κB p65protein expression of LPS-induced RAW264.7cells, thus inhibitthe anti-inflammatory factors and promote anti-inflammatory cytokine expression, play therole of slowing down the process of inflammation, suggesting that NF-κB pathway is oneof the mechanisms of APSⅠimmunomodulatory.2. Effect of APSⅠon human neuroblastoma IMR-32cells2.1Effect of APSⅠon IMR-32cell proliferationExperimental results of CCK-8show that when treating the human neuroblastoma celltumor IMR-32cells with APSⅠfor48h and72h, IMR-32cell growth is inhibited to varyingdegrees, and with the increasing of APSⅠconcentration in medium and time extending, theIMR-32cell proliferation inhibition rate shows a obvious rising trend, suggesting that theAPSⅠinhibition effect on IMR-32cells proliferation is proportional to drug concentrationand drug action time (P<0.05,P<0.01).The cell proliferation inhibitory effect of APSⅠwithsmallest relative molecular mass is most obvious among three kinds of APSⅠ.2.2Effect of APSⅠon IMR-32cell apoptosisIMR-32cell apoptosis is measured using FCM method. The experimental results showthat APSⅠis able to effectively induce IMR-32cell apoptosis (P<0.05,P<0.01) after48htreating with100mg/L, and APSⅠwith smallest relative molecular mass has best effect oninducing the cell apoptosis. The number of early apoptosis cell and necrotic cells increasesas the relative molecular mass of APSⅠdecreases.In short, APSⅠcan inhibit the growth of human neuroblastoma IMR-32cells, and caninduce cell apoptosis, and play a certain anti-tumor role, this anti-tumor effect enhances asthe relative molecular mass of APSⅠdecreases.The experimental results show that APS Ⅰ can regulate the expression of manyinflammatory genes through the NF-κB signal transduction pathways, thus regulate the anti-inflammatory factors and proinflammatory cytokine secretion, this make the body tendto the state of the immune balance and play a protective role on the body immune; At thesame time, APSⅠcan inhibit tumor cell proliferation and induce cell apoptosis and haveanti-tumor effect. Comparing the performance of three kinds APSⅠof different relativemolecular masses, It suggests that the immunomodulatory effects and anti-tumor effect ofAPS has relationship with its relative molecular mass.