| Objective: In recent decades, botanical polysaccharides isolated from botanical sources (mushrooms, algae, lichens and higher plants) have also attracted a great deal of attention in the biomedical arena because of their broad spectrum of therapeutic properties and relatively low toxicity, and do not cause significant side effects. Thus, botanical polysaccharides are ideal candidates for therapeutics with immunomodulatory, anti-tumor action. At present, in medicine one of important tasks was to find the plant polysaccharides from thousands plants having better function of anti-tumor and immunomodulatory.Acanthopanax senticosus, also called the"Siberian Ginseng"or"Eleutherococcus senticosus", which belongs to the Araliaceae family. It is a kind of precious herb. As the ingredients of folk medicine, it has long been used to treat a variety of human diseases, such as cerebrovascular disease, diabetes, tumor, isochemic heart diseases, hypertension and rheumatic arthritis. Acanthopanax senticosus is also known to be effective for reducing many kinds of stress or fatigue. The herb included various compounds such as acanthosides, senticoside, triterpenic saponin, flavon, polysaccharides ,vitamins and minerals, and they are related to its diverse biological activities. Acanthopanax senticosus polysaccharide ( ASPS ) was bioactive matter extracted from root and stem of A. senticosus. There were previously shown to increase lymphocyte proliferation and macrophage phagocytosis. Furthermore, this polysaccharide significantly inhibited the growth of Sarcoma 180 and prolonged the survival time of tumor-bearing mice. But, it is still unclear how it regulates these immune responses and anti-tumor.In this study, we compared ASPS with Astragalus polysaccharides(APS), Ganoderma lucidum polysaccharides(GLP), Pholiota nameko Polysacharides(PNP)in growth inhibition on K562 and effects on proliferation splenocytes by MTT assay. APS, GLP, PNP that previously showed to take on immunomodulatory and anti-tumor action. In order to choice fit lines we compared growth inhibition of ASPS on K562, H446, SMMC-7721, BGC, HeLa cells.Then particular studies were carried out in cellular and molecular level: The effect of ASPS induced apoptosis of cells and the expression of apoptosis-related gene, the effect of ASPS induced cell cycle arrest and correlative signaling pathways and the effect of ASPS activate the mouse peritoneal macrophages and the production of cytokines, by MTT assay, Hoechst 33258 fluorescent staining, flow cytometry (FCM), RT-PCR, western blot, fluorescence immunocytochemical (SABC-Cy3) method. These studies can provide academic base for exploitation and using of ASPS.Material and methods: 1. Comparison of the effect among four polysaccharides on anti-tumor and immunomodulatory in vitro: Four polysaccharides were provided by the laboratory biochemistry and molecular biology, life science College, Hebei normal university. BALB/c mice were provided by laboratory animal center, Hebei medical university. 1.1 cell culture: K562, H446, SMMC-7721, BGC, HeLa cells were cultured under standard conditions 37℃, 5%CO2, fully humidified atmosphere and use RPMI-1640 which supplemented with 10% new born bovine serum medium, 100 IU/ml penicillin and 100 ug/mL streptomycin. 1.2 Methyl thiazolyl tetrazolium (MTT) assay: K562 cells (1×105/mL) in their exponential growth phase were seeded into 96-well plate each well 100μL. After incubation for 24 hours, 100μL different concentrations of polysaccharides (ending concentration were 25, 50, 100, 200, 400μg/mL, respectively) were added in each well and control group added into RPMI-1640 non-serum medium each well, each concentration was repeated in eight wells. After incubation 24, 48, 72 hours, respectively, 20μL MTT was added to the medium in eight wells at every dose and incubated for 4 hours at 37℃. Culture media were discarded followed by adding 150μL DMSO and vibrating for 10 minutes. The absorbance (A) was measured at 490 nm using a microplate reader. The cell growth inhibitory rate was calculated as follows: [( A of control group - A of experimental group) / ( A of control group)]×100 %. Based on the cell growth inhibitory rate, 50% inhibitory rate (IC50) was calculated though the software of 82798- IC50. 1.3 Making growth curve:K562 cells in exponential growth phase 8×104 cells/well were added in a 24-well plate each well 1 mL, after incubation for 24 hours different concentrations of polysaccharides (ending concentration were 50, 100, 200, 400μg/mL, respectively) were added in and control group added in RPMI-1640 non-serum medium for 24, 48, 72, 96, 120 hours, respectively. Cells number was counted with blood corpuscle every day. Growth curve was made according to cell number. 1.4 Preparation of mouse splenocytes:BALB/c mice were Sacrificed. Spleens were taken out in the condition of asepsis and were gently smashed by pressing with the flat surface of a syringe plunger against stainless steel sieve (200 mesh). Splenocytes were separated with lymphocyte separate liquid. The splenocytes were washed twice with Hanks liquid and then resuspended in complete RPMI-1640. 1.5 Effects of polysaccharides on proliferation splenocytes: Splenocytes(6×106/ mL)were seeded into 96-well plate 100μL each well. 100μL various concentrations of polysaccharides were added into each well and control group added into 100μL RPMI-1640 non-serum medium, each concentration was repeated in eight wells. ConA (5 g/mL) were added into four wells among eight wells. After 48 h incubation, the absorbance was detected by MTT assay. The stimulate index was calculated as follows: (A of experimental group / A of control group)×100 %. 1.6 Comparison the effect of ASPS on different stains: with MTT assay.2. ASPS-induce apoptosis of H446 cells: 2.1 Proliferation inhibitory of ASPS on H446: with MTT assay. 2.2 Hoechst 33258 staining: H446 cells (1×105/mL) in their exponential growth phase were seeded into 24-well plate 1 mL each well. After incubation for 24 hours were treated with different concentrations of ASPS (240, 480, 960μg/mL) and control group added into 1 mL RPMI-1640 non-serum medium. Each concentration was repeated in three wells. After incubation 48 hours, cells were washed two times with PBS(pH7.2)then fixed with polyformaldehyde for 30 min at room temperature. Cells were stained with Hoechst 33258 for 30 min after washed three times with distilled water, then taking photos under fluorescence microscope. 2.3 FCM analysis: The cells were detached with EDTA solution. Through washed with cold PBS three times and fixed by 70% alcohol. After stained with 1mL (50μg/mL) propidium iodide for 10 minutes at room temperature and filtered with nylon mesh, the cells were analyzed by FACSTARCalibur flow cytometer (FCM). Apoptosis rates were calculated through ModFit LT2.0. 2.4 Western blot analysis the effect of ASPS on expression of p53,bcl-2,bax: Proteins were extracted and detected protein concentration with Coomassie brilliant blue G250 kit. 100μg proteins were added in each comb. Protein strap were separated by SDS-PAGE gel electrophoresis, Electrophoresis was stopped when bromophenol blue reach the bottom of separation gel. According to the standard protein bands, cut off PAGE gel. Transfer to nitrocellulose membrane, after blocking in 5% bovine serum albumin (BSA), the membrane was incubated with the primary antibody at 4℃overnight, washed the membrane three times with TBS, then add secondary antibody labeled with horseradish peroxidase (HRP) incube at 37℃1 hour, washed the membrane three times again. Chemiluminescenc was used to detect protein expression. UVP lab work 4.60 software was used to analysis the photodensity. 2.5 Fluorescence immunocytochemical method detected the effect of ASPS on expression of p53,bcl-2,bax: Cells treated were the same as 2.2. After ending culture, medium was put off and washed three times with PBS. Cells were fixed with 4% papraformaldehyde for 30 minutes, and then incubated with 0.3% Triton-X 100 for 20 minutes, washed the cells three times with PBS. Then cells were incubated with 3% H2O2 for 5 minutes. After several washes with distilled water, operated as SABC-Cy3 kit request then took photos under fluorescence microscope.3. ASPS-induce G2/M arrest of H446 cells involves activation of ERK and p38 MAP kinase pathways: 3.1 Flow cytometry analysis cell cycle distributions: Method was the same as described in same as part 2.3. 3.2 Westen blot analysis the effect of ASPS on proteins of ERK, p-ERK, P38, p-P38: same as part 2.4. 3.3 Fluorescence immunocytochemical method detected the effect of ASPS on proteins of p-ERK, p-P38: same as part 2.5.4. Effect of ASPS on immunoregulation: 4.1 Preparation of mouse peritoneal macrophages: 1 mL starch solution was injected into the peritoneal cavity of mouse, waited for 3 days. Sacrifice the mouse, injected 5 mL of PBS into the peritoneal cavity and gently pressed the abdomen to bring the cells into suspension. Opened the abdomen skin and hold up the center of the peritoneum with forceps. Made a small hole in the peritoneum and remove the medium with the pipette. Peritoneal macrophages were harvested and washed twice by D-Hanks'balanced salt solution. Then resuspended in complete RPMI-1640 and seeded in 96-well cell culture cluster at a cell density of 106 /mL. After settlement for 2–4 h, non-adherent cells were washed by PBS. Almost all of adherent cells were macrophages as assessed. 4.2 Phagocytosis of peritoneal macrophages: Phagocytosis of macrophages was measured by neutral red uptake method. Peritoneal macrophages were treated with ASPS. After another 48 h culture, the cells were washed and neutral red (50 ng/mL) was added. The plates were incubated for 3 h and then cells were washed to remove excess dye and blotted dry. The incorporated dye was resuspended in ethanol (50%) containing glacial acetic acid (1%) and the absorbance was measured at 540 nm in a microplate reader. Phagocytosis ratio = test A/normal control A×100%. 4.3 Nitric oxide mensuration: Peritoneal macrophages were treated with ASPS. After another 48 h culture, isolated supernatants were harvested then operated as claim of kit. Absorbance at 540 nm was determined in a microplate reader. The concentration of NO was calculated by formulate as follow:Nitric oxide content=[(testA-blankA)/standardA-blankA)]×standard concentration(100μmol/L)4.4 RT-PCR for cytokine gene expression: Total splenocyte population and peritoneal macrophages were treated with ASPS up to 48 h. Total cellular RNA was extracted from cells with the TRIzol reagent. The reverse transcription reaction was performed by M-MLV and Random primer. The special primers were used to amplify the target gene. After agarose gel electrophoresis, the photodensity of bands were acquired by image analysis system. Expression level of mRNA was calculated by compared the densitometry of each item withβ-actin.Results: 1. Comparison of the effect among four polysaccharides on anti-tumor and immunomodulatory in vitro: 1)Effect of polysaccharides on K562 cells: Inhibitions on K562 cells proliferation by four polysaccharides were in concentration-dependent and time-dependent manner. In same concentration, the inhibition rate of ASPS on K562 cells was biggest. After 24 h cells treated with ASPS, the inhibition rate of ASPS on K562 cells was biggest. After 48 h and 72 hours cells treated with ASPS, inhibition rate was no difference with that of APS but was bigger than that of GLP and PNP. IC50 of K562 cell to ASPS, APS, GLP, PNP were 118.84, 162.42, 270.26, 302.79μg/mL, respectively. IC50 of K562 cells to ASPS was lowest. 2) Effects of four polysaccharides on proliferation of spleen lymphocyte in vitro: ASPS, GLP, PNP could stimulate proliferation of mouse spleen lymphocyte with ConA or not with series of concentration(Stimulate index>1). Stimulate index of ASPS and PNP were in concentration-dependent manner. But stimulate index of GLP increased with concentration increasing at concentration lower than 100μg/mL, decreased with concentration increasing at concentration bigger than 100μg/mL. Stimulate index on mouse spleen lymphocyte, ASPS and PNP were bigger. For stimulation function of each polysaccharide, there was no difference between active and no-active spleen lymphocyte. APS could not stimulate mouse spleen lymphocyte proliferation in vitro. 3)The proliferation inhibitory of ASPS on different strains: Inhibitory rate of ASPS on K562 and H446 cells were bigger than that of BGC and HeLa cells in all concentration. Inhibitory rate on K562 were bigger than that of H446 and SMMC-7721 when ASPS concentration being 100, 200, 400 μg/mL. Inhibitory rate on H446 was bigger than that of SMMC-7721 when ASPS concentration being 200, 400μg/mL.2. Induces apoptosis of ASPS on H446 cells: 1) The proliferation inhibitory of ASPS on H446: The inhibition of H446 cells proliferation by ASPS in concentration-dependent manner. IC50 was 476.36μg/mL. 2) ASPS-induce H446 cells apoptosis: The results of Hoechst 33258 fluorescence staining: The border of the living nuclei stained into steady blue by Hoechst 33258 and could be clearly distinguished under fluorescent microscopy. In apoptotic cells chromatin distributed asymmetry or gathered,nuclei shrunk or broke into fragments. FCM analysis revealed the apoptotic rates of H446 cells were(5.02±0.4)%,(11.12±1.8)%,(19.89±2.5)%,(22. 54±1.8)% , respectively, when treated by ASPS (0, 240, 480, 960μg/mL). 3)The effect of ASPS on expression of p53, bcl-2, bax: Western blot results revealed expression of p53 increase when treated with ASPS, but expression level had not related to concentration. With concentration increase the expression of Bax increased and Bcl-2 decreased, the ratio of Bcl-2 to Bax was decreased. Fluorescence immunocytochemical results also revealed that P53 and Bax protein red fluorescence were stronger than that of control group, but Bcl-2 was reverse.3. ASPS-induce G2/M arrest of H446 cells involves activation of ERK and p38 MAP kinase pathways: 1)Distribution of cell cycle: Compared with control group, when H446 cells treated with ASPS, the G0/G1 phase of cells was no difference in the distribution, S phase decreased with ASPS concentration increase. But G2/M cells increased with ASPS concentration increase. 2) The effect of ASPS on ptoteins of ERK, p-ERK, P38, p-P38: Western blot results revealed that compared with control group when treated by ASPS expression of ERK, p38 were no difference,but the proteins of p-ERK, p-P38 were increased. Fluorescence immunocytochemical results also revealed that red fluorescence of p-ERK, p-P38 proteins were stronger than that of control group.4. Effect of ASPS on immunoregulation: 1) Effects of ASPS on the phagocytosis of peritoneal macrophages: Phagocyteic capacity was 100%, (152.38±10.42) %, (190.45±11.20) %, (261.90±18.32) %, respectively, when peritoneal macrophages treated by ASPS (0, 100, 200, 400μg/ml). Compared to control group, phagocyteic capacity were significantly enhanced. The increase of phagocyteic capacity was caused with dose-dependent manner. 2) In vitro effect of ASPS on nitric oxide production in peritoneal macrophages: The concentration of NO2- in control group was 6.90±0.89, when treatment of the cells with ASPS (100, 200, 400μg/ml) caused a significant increase in the production of nitrite by peritoneal macrophages (10.15±1.57, 13.68±1.89, 17.65±2.10, respectively). ASPS induced nitrite generation in a dose-related manner in mouse peritoneal macrophage. 3) Effects of ASPS on mRNA expression of TNF-α, IL-1, IL-2 in peritoneal macrophages: After cells were directly exposed to ASPS, the expression level of gene was monitored by RT-PCR. The result showed that the transcription of TNF-α, IL-1 mRNA was activated and IL-2 was not expressed. Control groupβ-actin was constitutively expressed and was not affected by the treatment of ASPS. 4)Effects of ASPS on mRNA expression of TNF-α, IL-1, IL-2 in spleen lymphocyte in vitro: After spleen lymphocytes were directly exposed to ASPS, the expression level of gene was monitored by RT-PCR. The result showed that the transcription of TNF-α, IL-2 mRNA was dose-dependently activated and IL-1 was not expressed. Control groupβ-actin was constitutively expressed and was not affected by the treatment of ASPS.Conclusion:1. Inhibition of polysaccharides on proliferation of K562 cells caused with concentration and time-dependent manner in vitro. In same concentration and time the inhibition of ASPS was strongest among ASPS, PNP, GLP and APS.2. ASPS, GLP, PNP could induce proliferation of mouse spleen lymphocyte with ConA or not with series of concentration. The function of ASPS and PNP were better. The stimulation function of each polysaccharide did not cooperate with ConA.3. The proliferation inhibitory of ASPS on K562 and H446 cells were stronger than that of SMMC-7721, BGC and HeLa cells.4. ASPS may inhibit the growth and induce H446 cells apoptosis. The activation of bax and p53 and the suppression of bc1-2 may contribute to the apoptosis mechanism.5. ASPS-induce G2/M arrest of H446 cells involves activation of ERK and p38 MAP kinase pathways.6. ASPS can activate the mouse peritoneal macrophages and increase the production of cytokines.
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