【作者】 王雪；

【导师】 滕利荣；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2019， 博士

【摘要】 黑虎掌菌（Sarcrodon imbricatus（L.ex Fr.）Karst.）学名枣翘鳞肉齿菌,隶属于担子菌纲多孔菌科多孔菌属,是一类高营养、多活性的药食同源大型真菌资源,主要分布于中欧、北美和亚洲地区,在我国四川、云南、西藏、甘肃和新疆产量最高,已实现大规模人工栽培。黑虎掌菌作为传统药材和健康食品使用已有几千年的历史。祖国医学认为,该菌具有驱风散寒、舒筋活络、降血脂、排毒益气、治风破血等功效。现代医学研究表明,黑虎掌菌能抗癌、解毒、缓解肝硬化,是理想的保健食品之一。国内外对黑虎掌菌的研究相对较少,主要侧重于化学成分分析、多糖提取、菌株液体发酵培养条件优化、抑菌及抗肿瘤活性,缺乏对黑虎掌菌药理药效及作用机制的深入挖掘。迄今为止,有关黑虎掌菌在免疫调节和提升造血功能方面的研究还未见报道。本论文以黑虎掌菌子实体为实验原料,系统分析了黑虎掌菌子实体干品的活性成分及含量分布。在此基础上,我们以黑虎掌菌子实体多糖（SIPS）为研究对象,采用细胞和分子生物学等技术手段,结合体内和体外实验探究SIPS的免疫调节和恢复骨髓造血的能力,初步阐明SIPS发挥相应药理活性的分子学机制,主要结论归纳如下:黑虎掌菌子实体的总糖含量占干重的35.22%,其次是粗纤维23.28%,总蛋白18.33%,甘露醇9.41%,总灰分9.30%,总甾醇3.16%,维生素0.44%,脂肪仅占3.02%;总黄酮(2.05×10^-2%)、总多酚（0.41%）和总三萜(4.12×10^-2%)类化合物含量略少;在矿物质元素中,钾含量最高,为39.57×10^-22 mg/100 g子实体,其次是铁（78.42 mg/100 g子实体）和钙（68.04 mg/100 g子实体）,且重金属含量均未超标;检测结果显示,黑虎掌菌子实体由17种氨基酸（12.44%）、24种脂肪酸组成,涵盖8种人体必需氨基酸,占总氨基酸的4.35%。本论文利用脱色、热水浸提、除蛋白和醇沉的方法,从黑虎掌菌子实体中提取粗多糖,经DEAE-52离子交换柱层析分级纯化,获得纯度为86.8%的SIPS。高效液相色谱（HPLC）显示,SIPS为非均一性多糖,其相对分子质量在56331.8Da⁹3963.6 Da;傅里叶红外光谱（FT-IR）显示,SIPS在3365.8 cm^–1处存在O-H伸缩振动吸收峰,2922.2 cm^–1处存在C-H伸缩振动吸收峰,1597.1 cm^–1存在一较强的糖环吸收峰,1398.4 cm^–1处存在C-H变角振动吸收峰和1028.4 cm^–1处由C-O-H和C-O-C拉伸引起的C-O弯曲振动,1200¹000 cm^–1之间出现三个较弱吸收峰,说明存在吡喃环骨架结构,这些都是多糖的典型特征吸收峰。本论文首先考察了SIPS对环磷酰胺（CTX）诱导的免疫抑制模型小鼠免疫系统功能的影响。SIPS灌胃治疗免疫抑制小鼠28天后,检测小鼠的体重、脾脏指数、胸腺指数、NK细胞杀伤活性、T淋巴细胞转化能力以及免疫相关细胞因子水平,分析Nrf2蛋白及下游抗氧化酶系统的表达情况,揭示SIPS发挥免疫调节作用的分子学机制。结果显示,相比于模型组,经SIPS给药治疗后,免疫抑制小鼠的体重和脾脏指数恢复正常,NK细胞杀伤活性及T淋巴细胞转化能力显著上升。同时SIPS能够促进免疫抑制小鼠血清和脾脏中免疫球蛋白A（IgA）、IgG、IgM、白介素2（IL-2）、IL-6、IL-10、IL-12和干扰素γ（IFN-γ）的分泌,显著降低免疫抑制小鼠脾和胸腺内活性氧（ROS）的积累,提高超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的水平以及酸性磷酸酶（ACP）和溶菌酶（LZM）的活性。病理切片证实SIPS减轻了免疫抑制小鼠脾脏内多核巨细胞和炎症浸润现象,且western-blot分析结果显示,经SIPS治疗后的免疫抑制小鼠脾脏中Nrf2及下游抗氧化酶SOD1、SOD2、血红素氧化酶1（HO-1）、CAT和II型解毒酶（NQO1）的蛋白表达水平均显著上调,表明SIPS可能是通过调控Nrf2介导的氧化应激信号通路改善由CTX诱导的免疫抑制,提示SIPS具有潜在的抗氧化活性。为探究SIPS能否恢复重建骨髓造血功能,我们首先通过体外实验,利用XTT法、联苯胺染色以及流式细胞术（FCM）考察SIPS对K562和CHRF两种造血细胞的细胞活力、细胞分化和凋亡水平的影响。结果表明,SIPS可显著促进两种造血细胞的细胞活力,诱导K562细胞向红系和巨核系的分化,同时上调两种造血细胞中与造血功能相关转录蛋白RSK1p90、c-Myc和ELK1的表达,且不存在细胞毒性,提示SIPS可能是通过上调转录因子的表达进而促进造血细胞的增殖与分化。随后,我们通过建立体内骨髓抑制小鼠模型,探究SIPS对CTX诱导的骨髓抑制小鼠造血系统功能的影响,揭示SIPS提升和重建骨髓造血活性的分子学机制。结果显示,经SIPS灌胃给药28天,骨髓抑制小鼠体重和胸腺指数逐渐回升,脾/肝肿胀现象消退,外周血血象的淋巴细胞（LY）数、中性粒细胞（NE）数以及血红蛋白（HGB）水平明显升高。此外,SIPS能够有效改善骨髓抑制小鼠受损骨髓内的形态结构,缓解脾/肝肿胀程度,诱导骨髓B淋巴细胞的合成,提高造血干/祖细胞（HSCs/HPCs）的增殖能力。抗体芯片和ELISA验证分析结果显示,SIPS显著提高了骨髓抑制小鼠血清和脾脏中IL-2、IL-3、IL-4、IL-5、IL-6、粒细胞集落刺激因子（G-CSF）和巨噬细胞集落刺激因子（M-CSF）的水平,抑制了肿瘤坏死因子（TNF-α）、IFN-γ、C-C基序趋化因子1（CCL1）和单核细胞趋化蛋白-1（MCP1）的分泌,显著上调了骨髓抑制小鼠脾脏中G-CSF、粒细胞集落刺激因子受体（G-CSFR）、M-CSF、酪氨酸激酶（JAK2）和信号传导及转录激活因子3（STAT3）的蛋白表达水平,该结果在体外培养的K562,CHRF和原代骨髓单核细胞（BMMNCs）中均得以证实,提示G-CSF调控的JAK2/STAT3信号通路可能是SIPS促进化疗损伤小鼠造血功能重建的主要分子学机制。总之,以上实验研究表明:（1）SIPS对免疫抑制小鼠的免疫功能有明显的促进作用,其机制可能与Nrf2介导的氧化应激途径有关;（2）SIPS对骨髓抑制小鼠的造血功能有明显的改善作用,其机制可能与G-CSF介导的JAK2/STAT3信号转导通路有关。本课题的提出有助于推动黑虎掌菌从健康食品向生物医药研究领域的转变,也将为筛选和研发具有缓解由长期化疗诱发骨髓免疫抑制的候选药物提供新的思路和方向。更多还原

【Abstract】 Sarcodon imbricatus（SI）,belonging to the family of Basidiomycotina and Aphyllophorales,is an edible and medicinal fungus with rich nutritional value and various pharmacological activities,widely distributed in Central Europe,North America and Asia.Large-scale artificial cultivation has been achieved in Sichuan,Yunnan,Tibet,Gansu and Xinjiang with the highest yield.SI has been used for thousands of years as a traditional medicine and healthy food.Traditional Chinese medicine pointed that it has the functions of chasing wind and dispelling cold,relaxing tendons and activating collaterals,lowering blood lipids,detoxifying qi,curing wind and breaking blood.Modern medicine indicated that SI,an ideal health food,has antitumor,detoxification and relieving hepatocirrhosis effection.Research about SI at home and aboard was not very extensive,mainly focusing on chemical composition,polysaccharide structure and fungi liquid fermentation culture optimization,antibacterial and antitumor activity,lacking of in-depth explore on pharmacological effects and mechanisms.Recently,immunoregulation and hematopoietic activity of SI have not been reported.In the present study,activity ingredient composition and content of SI dried products were systematically analyzed.On this basis,taking Sarcodon imbricatus polysaccharides（SIPS）as the research object,to explore the immunomodulation and hematopoietic recovery ability using cell and molecular biology techniques,combined with in vivo and in vitro experiments,and the corresponding pharmacological mechanisms of SIPS were preliminarily elucidated.Conclusions were summarized as follows:The total sugar content of SI accounted for 35.22%of dry weight,followed by crude fiber content 23.28%,total protein 18.33%,mannitol content 9.41%,total ash content 9.30%,total sterol content 3.16%,Vitamins 0.44%and fat accounted for only3.02%,total flavonoids(2.05×10^-2%),total polyphenols（0.41%）and total triterpenes(4.12×10^-2%)were less.Among the mineral elements,potassium was the highest element,about 39.57×10^-2 mg/100 g dry weight,followed by iron（78.42 mg/100 g dry weight）and calcium（68.04 mg/100 g dry weight）,and heavy metals were not detected.Additionly,SI was composed of 17 amino acids（12.44%）and 24 fatty acids,including 8 essential amino acids,accounting for 4.35%of the total amino acids.In this paper,crude polysaccharides were isolated from SI by decolorization,hot water extraction,protein removal and alcohol precipitation,and fractional purification by DEAE-52 ion exchange column chromatography to obtain SIPS with purity of86.8%.High performance liquid chromatography（HPLC）showed that SIPS was a heterogeneous polysaccharide with molecular weight from 56331.8 to 93963.6 Da.Fourier infrared spectroscopy（FT-IR）showed that SIPS has O-H stretching vibration absorption peak at 3365.8 cm^-1,C-H stretching vibration at 2922.2 cm^-1,stronger sugar ring absorption peak at 1597.1 cm^-1,C-H angular vibration absorption peak at1398.4 cm^-1,C-O bending vibration at 1028.4 cm^-1 caused by C-O-H and C-O-C stretching,and three weak absorption peaks between 1200 and 1000 cm^-1 indicated the presence of pyranoid ring skeleton structure,which were typical characteristics of polysaccharidesWe investigated the effect of SIPS on the immune system function in cyclophosphamide（CTX）-induced immunosuppression mice.After 28 days of SIPS administration,body weight,spleen/thymus index,NK cell cytotoxicity,T lymphocyte transformation capacity and immune-related cytokines in CTX-induced immunosuppression mice were detected,and the expression of Nrf2 and downstream antioxidant enzymes were analyzed to interrogate the molecular mechanisms that SIPS exerted immunoregulation.The results demonstrated that body weight and spleen index returned to normal,NK cell cytotoxicity and lymphocyte transformation capacity were increased significantly compared with the model group.Moreover,SIPS promoted the secretion of immunoglobulin A（IgA）,IgG,IgM,IL-2,IL-6,IL-10,IL-12 and IFN-gamma（IFN-γ）in serum and spleen of immunosuppression mice.Notably,SIPS could reduce the accumulation of reactive oxygen species（ROS）in the spleen and thymus of immunosuppression mice,increase the total antioxidant capacity（SOD,GSH-Px and CAT）,and promote the synthesis of acid phosphatase（ACP）and lysozyme（LZM）in the thymus.Histopathological analysis of spleen revealed the protective effect of SIPS against CTX-induced immunosuppression.Western-blot showed that SIPS upregulated the expression of nuclear factor-erythroid 2-related factor 2（Nrf2）and its downstream target genes encoding antioxidant enzymes:SOD1,SOD2,haem oxygenase-1（HO-1）,CAT and quinone oxidoreductase 1（NQO1）,indicated that SIPS reversed CTX-induced immunosuppression might through Nrf2-mediated oxidative stress pathway,accompany with potential antioxidant activity.In order to explore the hematopoietic function of SIPS,effects of SIPS on cell viability,differentiation and apoptosis activity of two hematopoietic cell（K562 and CHRF）were investigated by XTT method,benzidine staining and flow cytometry（FCM）in vitro.The results showed that SIPS significantly increased hematopoietic cells proliferation,promoted K562 differentiation into erythroid and megakaryocyte lines,and upregulated the expression of RSK1p90,c-Myc and ELK1 in both hematopoietic cells,and without cytotoxicity,suggesting that SIPS promoted the proliferation and differentiation of hematopoietic cells might via activating the expression of transcription factors.Subsequently,we explored the effect of SIPS on bone marrow hematopoietic system function,and the underlying mechanisms of SIPS reconstructing hematopoietic function was revealed in CTX-induced myelosuppression mice model in vivo.After 28 days of administration,SIPS enhanced the body weight and thymus index,alleviated enlargement of the spleen and liver,and contributed to the recovery of lymphocyte（LY）,neutrophil（NE）and hemoglobin（HGB）in peripheral blood to normal levels.Moreover,SIPS improved the morphological structure of femur and organs,promoted the production of B lymphocyte and the proliferation capability of hematopoietic stem/progenitor cells（HSCs/HPCs）significantly in bone marrow.The results of antibody chip screening and ELISA validation analysis showed that SIPS significantly increased the levels of IL-2,IL-3,IL-4,IL-5,IL-6,granulocyte colony-stimulating factor（G-CSF）and macrophage colony-stimulating factor（M-CSF）in serum and spleen of myelosuppression mice,and inhibited the secretion of TNF-alpha（TNF-α）,IFN-γ,C-C motif chemokine-1（CCL1）and monocyte chemotactic protein-1（MCP1）.Meanwhile,SIPS upregulated the protein expression levels of G-CSF,granulocyte colony stimulating factor receptor（G-CSFR）,M-CSF,phosphotyrosine kinase（JAK2）and phosphorylation signal transduction and transcription activator（STAT3）in the spleen of myelosuppression mice,and the results were further confirmed in CHRF,K562,and bone marrow mononuclear cells（BMMNCs）cultured in vitro,suggesting that JAK2/STAT3 signal transduction pathway regulated by G-CSF might be the main mechanism of SIPS promoting hematopoietic function repair in mice with chemotherapy-induced injury.In conclusion,the above experimental results manifested that:（1）SIPS can significantly promote the immune function of CTX-induced immunosuppression mice,and its mechanism might be related to Nrf2-mediated oxidative stress pathway;（2）SIPS can significantly enhance the hematopoietic function of CTX-induced myelosuppression mice,and its mechanism might be related to G-CSF-mediated JAK2/STAT3 pathway.The proposal of this research topic will help to promote the transformation of SI from health food to biomedical field.And will also provide new ideas and new directions for screening and developing candidate drugs that can alleviate bone marrow immunosuppression induced by long-term chemotherapy.更多还原