摘要
食用真菌被认为是目前最具开发前景的保健食品和药品的新资源,对食用真菌的药理活性研究也成为当今炽热的研究热点。多糖及其复合物是真菌中的重要活性成分，是真菌发挥药理活性的物质基础，研究表明真菌多糖具有抗肿瘤、抗氧化、降血脂以及免疫调节功能等。糙皮侧耳是世界范围内最重要的人工培育食用真菌之一。水提糙皮侧耳多糖及蛋白聚糖成分具有显著的抗肿瘤作用，而碱提多糖的构象分析与活性报道较少。为了充分发掘糙皮侧耳的药用价值，以及在糙皮侧耳中筛选新型抗癌药物的先导化合物，我们利用碱提法分离纯化糙皮侧耳多糖，并鉴定其生物活性，揭示其抗肿瘤的效果及可能的作用机制。研究内容如下：
通过碱液提取、乙醇沉淀、反复冻融、酶法与Sevag法联合脱蛋白，以及离子交换与分子排阻柱层析方法，分离得到糙皮侧耳碱提水溶性多糖WPOP-N1；利用经典化学方法（部分酸水解、高碘酸氧化、Smith降解、甲基化和乙酰化衍生）及仪器分析手段（高效液相色谱、气相色谱、红外光谱、气-质联机）对糙皮侧耳多糖级份的理化性质及结构特征进行描述；利用BalB/c小鼠前肢皮下注射S-180肉瘤细胞建立肿瘤小鼠模型以及体外MTT实验，在整体及细胞水平上评价WPOP-N1的抗肿瘤作用效果；通过检测WPOP-N1对淋巴细胞增殖、巨噬细胞吞噬能力、以及对细胞因子（TNF-α）及抗肿瘤效应物（NO）释放水平的影响，从细胞免疫与体液免疫两个层面揭示WPOP-N1的免疫调节活性；利用RT-PCR及Western Blot，在mRNA水平及蛋白水平上，检测了iNOS和TNF-α的表达情况，以及NF-κBp-65亚基磷酸化、IκB蛋白降解情况，揭示WPOP-N1刺激巨噬细胞相关抗肿瘤活性因子（NO和TNF-α）的表达分子机制。
综合上述实验结果，阐明WPOP-N1在体内具有较强抗肿瘤作用，而在体外不具有细胞毒效应；WPOP-N1可以提高荷瘤小鼠的胸腺指数、脾指数及血清中的TNF-α含量；WPOP-N1可以协同ConA及LPS增加淋巴细胞增殖能力，增强活化巨噬细胞吞噬能力，并通过增强NF-κB转录因子活性使其分泌相应的抗肿瘤活性物质（NO和TNF-α），推测WPOP-N1的抗肿瘤活性可能是通过增强机体免疫功能实现的。同时，为糙皮侧耳这一重要资源的深入开发利用提供理论基础。
Polysaccharide is an important active ingredient with a wide range ofpharmacological activities in fungi. The immunomodulatory and anti-tumor activitiesof polysaccharides from fungi have been widely recognized, but the study on thestructure-activity relationship of the fungal polysaccharide is still insufficient now.Pleurotus ostreatus, a tricholomataceae fungus of basidiomycetes, is one of importantmanmade edible fungi with high nutritional value and medicinal value, which is alsocalled oyster mushroom, oyster-cap fugus, green mushroom, jatropha bacteria and etc.It is well known that fungal polysaccharides are usually extracted with waterextraction and there has been no report on the extraction of polysaccharide componentfrom pleurotus ostreatus with alkaline extraction so far. In the present study, in orderto fully explore the medicinal value of pleurotus ostreatus and screen a leadingcompound for anti-tumor agents from pleurotus ostreatu, the pleurotus ostreatuspolysaccharide was separated and purified with alkaline extraction, the structuralcharacterization of the pleurotus ostreatus polysaccharide, WPOP-N1, was analyzedby classical chemical methods and instrumental means, and its immunomodulatoryand anti-tumor effects were investigated with in vitro cytological and in vivo methodsto reveal the structure-activity relationship of anti-tumor and immunomodulatoryeffects of the pleurotus ostreatus polysaccharide.
Methods
By using alkaline extraction, repeated freezing and thawing, deproteinization,ion-exchange chromatography and gel filtration chromatography, WPOP-N1, analkali-extracted and water-soluble polysaccharide, was separated and prepared frompleurotus ostreatus. Phenol-sulfuric acid, Bradford and hydroxyl biphenyls methodswere used to detect total sugar content, protein content, and uronic acid content ofWPOP-N1respectively. High performance liquid chromatography (HPLC) was used to determine the homogeneous degree and the average molecular weight of WPOP-N1.After derivatization by acetylation, the monomer composition of WPOP-N1wasdetermined by gas chromatography (GC). WPOP-N1polysaccharide structure wasanalyzed with combined chemical method such as partial acid hydrolysis, periodateoxidation, Smith degradation, and methylation, as well as analytical measures such asgas chromatography, infrared spectroscopy, and gas chromatography-massspectrometry. In vivo transplanted mouse sarcoma S-180models, as well as the mousespleen lymphocyte transformation experiments, mouse peritoneal macrophagephagocytic capacity, macrophage TNF-a and NO emission analysis by RT-PCR andWestern Blot, and NF-κB activity were applied to evaluate anti-tumor effects ofWPOP-N1and the possible mechanisms.
Results
Firstly, the water-soluble polysaccharide components from the fruiting body ofpleurotus ostreatus were isolated and removed with hot water boiling extraction, andthe residue was extracted by alkaline extraction and ethanol precipitation to obtain thecrude pleurotus ostreatus polysaccharides, from which the total polysaccharides,WPOP, were achieved using the combined deproteinization methods including therepeated freezing and thawing, enzyme methods and Sevag method, then, they wereeluted in a gradient way with DEAE-cellulose ion exchange chromatography indifferent NaCl solutions, and finally, they were graded with Sepharose CL-6B gelfiltration chromatography based on the molecular distribution of the samples to getthe primary polysaccharide fraction, namely WPOP-N1. The HPLC detectionindicated that WPOP-N1showed a simple peak and the average molecular weight was48.2kDa. The phenol sulfuric acid method verified that the total polysaccharidecontent was97.6%. The GC analysis demonstrated that WPOP-N1was composed ofmannose and a small amount of gloucose, and the molar ratio was5.6:3.3. Them-hydroxyl diphenyl method was negative, indicating that WPOP-N1did not containuronic acid, which was in consistent with the result from GC analysis. Moreover,chemical analytical methods such as partial acid hydrolysis, periodate oxidation,Smith degradation and methylation, as well as instrumental analytical methods suchas IR, GC and GC-MS were applied to analyze the chemical structure of WPOP-N1meticulously and finally determine the repetitive unit of WPOP-N1. The resultsshowed that the basic chemical structure of WPOP-N1should include a backbone composed of β-(1→3)-mannose and (1→3,6)-β-D-mannose, and the molar ratio was1.57:1. In addition, WPOP-N1presented a low branching configuration and there wasa trace of terminated glucose residues on the branch chain. In this study, BALB/cmice were used to establish S-180solid tumor model for the in vivo experiment toexamine the anti-tumor activity. The results showed that WPOP-N1significantlyinhibited the growth of the S-180, an in vivo transplanted tumor, in mice, theinhibition rate reached up to63.1%at the dose of400mg/kg, and the anti-tumoreffect of WPOP-N1was close to that of cyclophosphamide in the positive controlgroup. In the in vitro anti-tumor activity experiments, the results from MTT showedthat WPOP-N1could not inhibit the proliferation of S-180sarcoma cells significantly,suggesting that the anti-tumor activity of WPOP-N1might be due to itsimmunomodulating activity. Our further studies confirmed that WPOP-N1treatmentsignificantly increased the spleen index and thymus index of tumor-bearing mice, andraised the content of tumor necrosis factor-α (TNF-α) in serum. The treatment withWPOP-N1effectively stimulated lymphocyte proliferation, enhanced thephagocytosis of peritoneal macrophages, and increased the secretion of NO andTNF-α by WPOP-N1-treated macrophages. The results from RT-PCR and WesternBlot showed that WPOP-N1could increase the expression of TNF-α and iNOS bothin gene and protein levels, and in the meanwhile, upregulate the phosphorylation levelof p-65of the regulatory subunit of NF-κB, as well as promote the degradation of IκBofthe inhibition subunit of NF-κB, suggesting that WPOP-N1can enhance thetranscriptional activity of NF-κB to promote the secretion of NO and TNFα.
Conclusions
1.WPOP-N1, the alkali-extracted polysaccharide fraction from pleurotusostreatus, could be isolated and obtained by the systemic grading and purifying withalkaline extraction, freezing and thawing, deproteinization, and ionic and molecularexclusion chromatography.
2.Using various chemical methods and instrumental analytical means, WPOP-N1could be identified as a mannose glucan, in which there is a polysaccharide backbonethat is composed of (1→3)-β-D-mannose, the branching point sugar residue is (1→3,6)-β-D-mannose, and the non-reducing end residue of the main chain is glucose.
3.The in vivo experiment demonstrates that WPOP-N1could produce aneffective anti-tumor activity, but the in vitro experiment could not show the cytotoxic effect.
4.WPOP-N1can increase the lymphocyte transformation and simultaneouslyinduce the activation of macrophages, improve their phagocytic ability, and promotethe secretion of the corresponding anti-tumor substances through stimulating thetranscriptional activity of NF-κB, suggesting WPOP-N1may exert its anti-tumoractivity by enhancing the immune function.
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