灵芝多糖肽对小鼠巨噬细胞自由基的清除作用

目的 :研究灵芝多糖肽对小鼠腹腔巨噬细胞自由基的清除作用。方法 :用四氧嘧啶、叔丁基氢过氧化物 (tBOOH)为氧化剂 ,分别在小鼠体内、体外损伤小鼠腹腔巨噬细胞 ,以DCHF DA为荧光指示剂 ,用共聚焦显微镜观察巨噬细胞的荧光变化 ,并用共聚焦显微镜作时间系列扫描 ,观察巨噬细胞荧光的动态变化。结果 :四氧嘧啶 (75mg·kg-1,iv)、叔丁基氢过氧化物 (7.76× 10 -5mol·L-1)可造成巨噬细胞的氧化损伤 ,使荧光密度增加 ,灵芝多糖肽可减轻其损伤 ,使荧光密度减少。时间系列扫描显示 :随时间改变 ,灵芝多糖肽可减少静息状态下小鼠腹腔巨噬细胞荧光密度 ,也可减少由PMA(5 0nmol·L-1)诱导的呼吸爆发状态下小鼠腹腔巨噬细胞荧光密度。结论 :灵芝多糖肽具有抗氧化作用 ,对小鼠腹腔巨噬细胞自由基有清除作用     

灵芝多糖肽对氧自由基损伤巨噬细胞的保护作用

目的:研究灵芝多糖肽(GLPP)在离体和整体水平对氧自由基(ROS)(tBOOH为氧化剂)损伤巨噬细胞的保护作用.方法:以tBOOH为氧化剂损伤小鼠腹腔巨噬细胞,以MTT法分析小鼠巨噬细胞存活率,在光镜和电子显微镜下观察细胞的形态改变.结果:GLPP50,100,200 mg/kg腹腔注射5天,能抑制巨噬细胞膜样变性和坏死,细胞存活率提高.在培养的巨噬细胞中加入 GLPP 3.125,12.5,50,200 mg/L,产生相似的保护作用.电镜观察发现,GLPP(100mg/kg)腹腔注射5天可保护细胞器如线粒体免受tBOOH的损伤.结论:GLPP有显著的清除氧自由基和抗氧化作用.     

灵芝多糖肽对小鼠腹腔巨噬细胞一氧化氮产生的影响

目的　研究灵芝多糖肽 (GLPP)对小鼠腹腔巨噬细胞一氧化氮产生的影响并探讨其作用机制。方法　以Griess法 ,观察GLPP对LPS诱导小鼠腹腔巨噬细胞一氧化氮(NO)产生的影响 ;以免疫组化法检测诱导型一氧化氮合成酶 (iNOS)的表达 ,观察GLPP对iNOS的影响。结果　GLPP(2 5～ 2 0 0mg·kg-1)灌胃给药 5d或体外给药 (3 12 5～ 2 0 0mg·L-1)均可促进巨噬细胞NO释放 ,但对LPS刺激NO的释放影响不大 ;GLPP(10 0mg·kg-1)灌胃给药 5d或体外给药 (10mg·L-1)均可使巨噬细胞iNOS含量增加。结论　GLPP可增加小鼠腹腔巨噬细胞NO产生 ,其机制可能与其促进巨噬细胞iNOS合成有关。     

灵芝多糖对小鼠腹腔巨噬细胞活性氧自由基的影响

为进一步探讨灵芝多糖 ( GLP)免疫调节作用机理 ,采用激光扫描共聚焦显微镜技术 ,动态监测灵芝多糖均一体 GLB7对小鼠腹腔巨噬细胞 ( M )活性氧自由基含量的影响 .以 GLB7( 2 0 mg· L-1)刺激 M ,发现探针 DCHF- DA的荧光强度明显下降 ,与静息水平相比 ,平均下降 ( 36± 6) % ( n=6,P<0 .0 5) ;同时还能拮抗 PMA引起的 M 呼吸爆发 ,荧光强度下降幅度为 ( 1 9± 4) % ( n=6,P<0 .0 5) .结果证明 :GLB7能减少 M 内活性氧自由基的生成 ,具有清除活性氧的作用 ,这也是 GLB7产生免疫增强和抗衰老作用的重要途径 .     

灵芝多糖肽对ECV304细胞氧化损伤的保护作用

目的研究灵芝多糖肽对ECV304氧化损伤的保护作用。方法培养ECV304细胞,以叔丁基氢过氧化物(tBOOH)为氧化剂损伤细胞,造成氧化损伤模型,培养液中加入不同浓度灵芝多糖(12.5、25、50、100mg.L-1),以MTT法测细胞存活率;以光镜、电镜检测细胞形态学改变及线粒体损伤;用AnnexinV/PI双标记细胞,流式细胞检测细胞凋亡的百分率。结果灵芝多糖(12.5、25、50、100mg.L-1)可减少叔丁基氢过氧化物对ECV304的氧化损伤,MTT检测灵芝多糖给药组,ECV304细胞存活率增加。光镜下可见细胞损伤减少,电镜可见灵芝多糖(50mg.L-1,温育24h)减轻细胞器如线粒体氧化损伤,减少细胞凋亡。细胞流式检测表明:对照组、给药组、损伤组总凋亡百分率分别2.24%±0.43%、24%±6.4%(P<0.01)、82.1%±7.9%。结论灵芝多糖肽(GLPP)对ECV304细胞氧化损伤具有保护作用。     

灵芝多糖对小鼠腹腔巨噬细胞cGMP含量的影响

目的 :研究灵芝多糖 (GLB7)对巨噬细胞 (MΦ)内cGMP含量的影响 ,为其免疫增强作用机制提供依据。方法 :采用竞争性蛋白结合分析法测定培养的小鼠腹腔MΦ中cGMP含量。结果 :GLB7 能剂量依赖性引起小鼠腹腔中cGMP浓度快速升高 ,10min达峰值 ,之后缓慢下降 ,至30min时基本恢复至原来水平。结论 :GLB7 引起小鼠MΦ内cGMP浓度快速升高 ,可能与其增强小鼠免疫功能有关     

灵芝多糖对小鼠腹腔巨噬细胞蛋白激酶C活性的影响

目的　观察灵芝多糖在体外对小鼠腹腔巨噬细胞(MΦ)蛋白激酶C(PKC)活性是否有影响。方法　采用反相离子对高效液相色谱法测定PKC活力。结果　灵芝多糖GLB7( 40mg·L-1)能引起小鼠腹腔MΦ中PKC活性明显升高 ,30min达峰值 ,2h恢复到基础水平 ;GLB7还可引起MΦ中PKC发生质膜转位 ,并拮抗Staurosporine对MΦ中PKC的抑制作用。结论　灵芝多糖的免疫增强作用与其增强PKC活性有关     

灵芝多糖肽的抗氧化作用

目的 研究灵芝多糖肽(GLPP)的抗氧化作用及其机制。 方法 Cu²⁺诱导人血清低密度脂蛋白(LDL)氧化及iv四氧嘧啶(75 mg·kg^-1)引发小鼠氧自由基损伤。结果GLPP使LDL氧化修饰减少,氧化产物REM降低。GLPP 50,100,200和400 mg·kg^-1 ip 20 d,使血清和心肌匀浆的丙二醛(MDA)水平下降, 谷胱甘肽过氧化物酶(GSHpx)升高,超氧化物歧化酶(SOD)减少,过氧化氢酶(CAT)不变。结论GLPP具有体内外抗氧化作用,其抗氧化作用与清除氧自由基或提高GSHpx水平有关。     

用Folin-酚法测定灵芝产品中多糖肽含量

目的 建立灵芝和灵芝产品中多糖肽的含量测定方法.方法 采用紫外分光光度法,检测波长为750nm,用灵芝活性多糖肽GL-PP-T4为对照品,测定灵芝与灵芝产品中多糖肽的含量.结果 多糖肽GL-PP-T4在25μg·mL-1～250μg·mL-1的范围内呈现良好的线性,r为0.9994,平均回收率是99.45%,RSD为0.98%(n=5).且本法能排除制剂中淀粉添加剂的干扰.结论 本法稳定可靠、简便快速,可作为灵芝及其产品质量控制指标.     

RP-HPLC和UV-VIS法测定灵芝不同收获期的多糖肽和灵芝酸

目的研究灵芝不同栽培方式和不同收获期中多糖肽和灵芝酸A、B的变化,为灵芝规范化栽培及制定产品质量标准提供依据。方法 RP-HPLC测定菌草灵芝和仿野生栽培的灵芝提取物的灵芝酸A、B的变化。色谱柱为XB-C18柱(150mm×4.6mm,5μm),流动相为0.05%磷酸-乙腈(65:35),检测波长254nm,常温,进样量50μL。以灵芝多糖肽为对照品,采用Folin-酚试剂法,通过紫外-可见吸收光谱法检测菌草灵芝和仿野生栽培灵芝不同收获期提取物的多糖肽。结果灵芝酸A、B线性范围分别为14.8～371.0和16.4～409.0μg/mL,平均回收率为95.0%、100.5%,RSD分别为1.18%、3.37%(n=6)。灵芝多糖肽在100～600μg/mL与吸光度值呈线性关系,r=0.9995,回收率95%～100%。结论该方法简便、快速,可用于灵芝及其产品中多糖肽和灵芝酸A、B的质量控制。菌草灵芝沸水提取能将多糖肽和灵芝酸分离,既简化了灵芝水、醇提取工艺,又提高经济效益。     

Hypoglycemic effect of Ganoderma lucidum polysaccharides

AIM: To investigate the hypoglycemic effect of Ganoderma lucidum polysaccharides (Gl-PS) in the normal fasted mice and its possible mechanism. METHODS: Normal fasted mice were given a single dose of Gl-PS 25, 50, and 100mg/kg by ip and the serum glucose was measured at 0, 3, and 6h after administration. Gl-PS 100mg/kg were also given by ip and the serum glucose and insulin levels were measured at 0min, 30min, 1h, 3h, 6h, and 12h Pancreatic islets were isolated and incubated with glucose 5.6mmol/L and different concentration of Gl-PS, the insulin content of islets and insulin release were examined. The islets fluorescent intensity of [Ca^2 ]i was also studied with a confocal microscope. Verapamil and egtazic acid were used to testify whether the insulin-releasing effect of Gl-PS was mediated by its ability to raise the Ca^2 influx. RESULTS: Gl-PS dose-dependently lowered the serum glucose levels at 3h and 6h after administration. Gl-PS 100mg/kg raised the circulating insulin levels at 1h after administration. In vitro, Gl-PS had no effect on islets insulin content, but it stimulated the insulin release after incubation with glucose 5.6mmol/L. Confocal microscope showed that Gl-PS 100mg/L had the capacity toraise the [Ca^2 ]i. The insulin-releasing effect of Gl-PS was inhibited by verapamil/egtazic acid. CONCLUSION:Gl-PS possesses the hypoglycemic effect on normal mice; one mechanism is through its insulin-releasing activity due to a facilitation of Ca^2 inflow to the pancreatic β cells.     

灵芝免疫活性多糖的研究

从云头状灵芝[Ganoderma Lucidum(Leyss.ex Fr.)Karst.]中分离得到三种灵芝多糖BN_3A,BN_3B 及 BN_3C,它们均表现明显免疫调节作用。从灵芝多糖 BN_3B 及 BN_3C 中各分离得到四个多糖均一体,对其中主要成分 BN_3C_1,BN_3C_3,BN_3B_1及 BN_3B_3进行了物理及化学研究。它们的平均分子量依次为1.6×10~4,2.5×10~4,3.5×10~4及4.0×10~4。经完全酸水解、红外光谱测定、过碘酸氧化、甲酸生成、Smith 降解等证明,BN_3B_1及 BN_3C_1均为β-(1→6)(1→3)甙键相连的葡聚糖。BN_3B_3为阿拉伯半乳聚糖。BN_3C_3为由葡萄糖和阿拉伯糖组成的肽多糖。它们均为β-(1→6)(1→3)甙键相连。     

Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide

AIM: To investigate the antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide (GLPP). METHODS: Antitumor effect of GLPP was observed in tumor-bearing mice in vivo. At the same time,the effects of GLPP on proliferation of tumor cells and human umbilical cord vascular endothelial cell (HUVEC) were detected by MTr assay in vitro. Subsequently, spleen lymphocytes proliferation of nude mice was stimulated by LPS or ConA. To investigate the anti-angiogenic effect of GLPP, GLPP 80 μg per disc and GLPP-treated serum 10μL per disc were added to the chick chorioallantoic membrane (CAM) respectively in vivo. RESULTS: GLPP 50, 100, and 200 mg/kg inhibited growth of Sarcoma 180 in BALB/c mice markedly by 35.2 %, 45.2 %, and 61.9 %,respectively. GLPP which was directly added to the cultured medium did not inhibit PG cell proliferation in vitro;but GLPP-treated serum 50, 100, 200 mg/kg potently inhibited PG cell proliferation by 22.5 %, 26.8 %, and 30.3 %,respectively; and reduced the xenograft (human lung carcinoma cell PG) in BALB/c nude mice greatly in vivo by 55.5 %, 46.0 %, and 46.8 %, respectively. Lymphocytes proliferation of nude mice could be stimulated by LPS 5 mg/L but not by ConA 2.5 mg/L, indicating that GLPP could not promote the T lymphocyte proliferation and neutral red phagocytosis of peritoneal macrophages of nude mice. The CAM assay showed that GLPP and GLPP-treated serum had anti-angiogenic effect. GLPP (1, 10, and 100 mg/L) inhibited HUVEC proliferation in vitro with the inhibitory rate of 9.4 %, 15.6 %, and 40.4 %, respectively. CONCLUSION: GLPP has antitumor and antiangiogenic activity. The anti-angiogenesis of GLPP may be a new mechanism underlying its anti-tumor effects.     

Effects of Ganoderma lucidum polysaccharides on proliferation and cytotoxicity of cytokine-induced killer cells

AIM: To study the effects (and the mechanisms thereof) of Ganoderma lucidum polysaccharides (Gl-PS) on the proliferation and the anti-tumor activity of cytokine-induced killer (CIK) cells, and to make use of CIK cells as a means to investigate the interactions between Gl-PS and cytokines. METHODS: CIK cells were prepared by using the standard protocol as a positive control. Experimental groups also underwent the standard protocol, except that Gl-PS (400 mg/L or 100 mg/L) was added and the dose of anti-CD3 and interleukin-2 they received was reduced by 50% and 75%, respectively. For negative controls, Gl-PS in the experimental protocol was replaced with soluble starch or methylcellulose (400 mg/L or 100 mg/L). CIK cell proliferation, cytotoxicity, and phenotype were determined by using the Trypan blue exclusion method, MTT assay, and flow cytometry. RESULTS: By synergizing cytokines, Gl-PS (400 mg/L or 100 mg/L) could decrease the amount of cytokine in lymphokine activated killer (LAK) cells and CIK cells culture, but had no significant effect on the proliferation, cytotoxicity, or phenotype of LAK cells, or CIK cells induced by cytokines at higher doses alone, in which CIK cells expanded about 80-fold and the main effectors, CD3+NK1.1+ cells, expanded by more than 15%. The cytotoxicity of CIK cells in experimental groups was 79.3%+/-4.7%, 76.9%+/-6.8% versus the positive control 80.7%+/-6.8% against P815 (P>0.05) and 88.9%+/-5.5%, 84.7%+/-7.9% versus the positive control 89.8%+/-4.5% against YAC-1 (P>0.05). The activity of Gl-PS could mostly be blocked by anti-CR3. CONCLUSION: Gl-PS was shown to be a promising biological response modifier and immune potentiator. The effect of Gl-PS on CIK cells is possibly mediated primarily through complement receptor type 3.     

灵芝多糖对体外树突状细胞诱导细胞毒性T-淋巴细胞的调节作用(英文)

目的:研究灵芝多糖(Gl-PS)在抗原提呈阶段对体外培养树突状细胞(DC)诱导特异性细胞毒性T-淋巴细胞(CTL)功能的调节及其机制。方法:体外培养小鼠骨髓来源DC经P815肿瘤细胞冻融抗原冲击致敏,并与不同浓度Gl-PS(0.8,3.2,或12.8 mg/L)共培养。成熟DC与脾淋巴细胞共培养诱导P815特异性CTL生成。第5天收集各组悬浮细胞及培养上清,采用乳酸脱氢酶法比较CTL特异性杀伤活性;RT-PCR法测定T扰素γ(IFNγ)及颗粒酶B mRNA在CTL的表达;ELISA或Western blot法检测IFNγ或颗粒酶B蛋白的表达。结果:3种浓度的Gl-PS均可增高培养上清中释放的LDH活性(P<0.01);增加CTL表达IFN7及颗粒酶B mRNA(IFNγ:Gl-PS 12.8 mg/L组与RPMI-1640组,P<0.05;颗粒酶B:P<0.01);促进CTL培养上清中IFNγ蛋白生成(P<0.05)以及CTL表达颗粒酶B蛋白(Gl-PS 12.8 mg/L组与RPMI-1640组,P<0.05)。结论:Gl-PS可在抗原提呈阶段促进P815肿瘤冻融抗原冲击致敏DC所诱导的特异性CTL的杀伤活性,其机制可能是通过IFNγ及颗粒酶B途径的调节。     

灵芝孢子多糖两纯组分的单糖组成研究

目的：对灵芝孢子多糖的2个纯多糖组分GLPSl和GLPS3的单糖组成进行分析。方法：将灵芝孢子多糖原料精制，SephacrylS一400SF型凝胶分离纯化得2个主要组分GLPSl和GLPS3，经酸水解后TLC法和HPLC法鉴别单糖组成，改良的1一苯基一3一甲基一5一吡唑啉酮（PMP）柱前衍生化HPLC法分析单糖组成比例，同时采用LC—MS联用技术鉴定单糖PMP衍生化产物。结果：GLPSl的单糖组成为D一甘露糖：D一葡萄糖：D一半乳糖=1：9．228：1．474；GLPS3的单糖组成为D一甘露糖：D．葡萄糖：D．半乳糖=1：15．900：3．686。结论：灵芝孢子多糖二纯组分均含D一甘露糖，D．葡萄糖和D一半乳糖，仅各单糖的比例不同。     

灵芝多糖对糖尿病大鼠心肌纤维化的保护作用

目的:研究灵芝多糖对2型糖尿病大鼠心肌纤维化的保护作用。方法:SD大鼠高脂饮食4周后,注射STZ(30 mg.kg-1)复制2型糖尿病大鼠模型,造模成功后,将大鼠分为正常对照组、糖尿病组、灵芝多糖低、中、高剂量组(分别灌胃给予灵芝多糖200,400,800 mg.kg-1)及小檗碱对照组(灌胃小檗碱30 mg.kg-1)。治疗8周后,测量大鼠空腹血糖,VG染色观察心肌纤维化程度,荧光分光光度计法检测大鼠血清晚期糖基化终末产物(AGE)的含量,碱水解法测定心肌羟脯氨酸含量,胃蛋白酶限制性降解法测定胶原交联程度,并测量心肌和血清中过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)等氧化应激指标。结果:中高剂量灵芝多糖在降低2型糖尿病大鼠血糖水平的同时,可使VG染色血管周围和间质胶原明显减少,心肌组织中羟脯氨酸含量下降,心肌交联程度得到明显改善,血清中AGE含量降低,心肌和血清中CAT和GSH-PX活力得到显著增强。结论:灵芝多糖可降低2型糖尿病大鼠心肌胶原纤维总量和交联程度,其机制可能与灵芝多糖降低血清AGE含量,增强机体内CAT和GSH-PX活性有关。