五味子多糖的结构、生物活性及免疫机制研究
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摘要
五味子为木兰科五味子(Schisandra chinensis (Turcz.) Baill)的干燥成熟果实,具有食用和药用价值,已有2000多年的应用历史,列入可用于保健食品的物品名单。五味子中含有木脂素、多糖、挥发油等多种活性成分,其中木脂素已得到广泛应用,而多糖未被利用。研究发现,多糖作为一类天然大分子化合物,具有多种生物活性,如机体免疫调节、抗肿瘤、抗衰老等,且对机体正常细胞和组织的毒性极小,因此,已成为天然产物研究的热点之一。目前,已有多种多糖作为保健食品和食品添加剂、广谱的免疫促进剂和肿瘤综合治疗中的辅助药物得到了广泛的应用。本课题组的前期研究发现,五味子粗多糖具有免疫调节,对环磷酰胺(CTX)减毒和抗肿瘤等作用,具有广泛的应用前景。但目前关于五味子多糖的研究多集中于粗多糖,而对于五味子多糖组分结构的全面深入研究、免疫调节作用、对5-Fu减毒作用、抗肿瘤作用及其免疫机制等方面都鲜有报道。因此,本文以一种低分子量五味子多糖为研究对象,运用现代分析方法(GC-MS,NMR,TEM, AFM, CD等)对该多糖组分的结构进行全面分析；通过免疫抑制小鼠模型,对该组分多糖的免疫调节作用进行研究,发现其对机体有较好的免疫调节作用,同时前期研究结果表明该多糖组分对肿瘤细胞无直接杀伤作用,因此进一步研究其对5-Fu减毒作用和体内抗肝癌活性；通过活性研究结果,推测该多糖组分主要是通过调节机体免疫来发挥其生物活性的,由此进一步探讨其免疫调节的机制。本研究揭示该五味子多糖组分的结构特征,并阐明其免疫调节机制,为五味子多糖功效成分的研究与应用提供基础数据,为五味子多糖类保健食品和食品添加剂的研究开发提供科学依据,并为五味子资源的高效利用提供新途径,该研究对人类健康和社会经济发展具有重要意义。
主要研究内容如下：
(1)以低分子量五味子多糖组分(SCPP11)为研究对象,通过甲基化、GC-MS、1H NMR、13C NMR、1H-1H COSY谱、HSQC谱及HMBC谱等化学分析手段和物理技术对SCPP11的一级化学结构进行推断和解析,确定了低分子量五味子多糖组分SCPP11的一级结构的重复单元为：采用CD、AFM、TEM和SEM等方法对SCPP11的构象和分予形貌进行研究。发现在不同的外界条件下(处理温度、pH值、加入Ca2+离子、6mol/L的变性剂Urea和等量的80μM的刚果红溶液),SCPP11的CD谱图发生显著变化,表明外界条件的改变可影响其立体构象和非对称性。用激光光散射粒度仪测定了SCPP11的粒度,结果表明SCPP11的粒径为270nm,分散度为0.129,粒度分布较均匀。通过多角度激光光散射得到SCPP11的绝对重均分子量为1.793×104g/mol,多分散系数为1.543,且其构型介于球形和随机线团之间。通过原子力显微镜观察SCPP11分子的形貌,结果表明SCPP11分子间相互缠绕,呈规则团聚状；经加热处理后,SCPP11高度团聚状结构大量减少,链延展开来。通过TEM观察SCPP11的形貌,发现其呈团聚状态,粒子的直径在几百纳米不等。SCPP11粉末呈粗糙的小片状聚集体,表面紧密,规整性不强,说明SCPP11固体为无定型结构。
(2)采用CTX抵制小鼠模型,考察SCPP11对小鼠免疫的调节作用。结果表明,SCPP11对CTX造成的体重增长抑制有较好的改善作用,可不同程度提高胸腺指数和脾脏指数,显著提高免疫抑制小鼠腹腔巨噬细胞的吞噬能力和血清溶血素水平，且对CTX致血清中免疫球蛋白水平、FNF-α和IL-2水平下降有不同程度的恢复作用。因此此,SCPP11对可激活机体免疫,具有免疫调节功能。
(3)采用Heps荷瘤小鼠模型,考察SCPP11对化药物5-氟尿嘧啶(5-Fu)的减毒作用和其体内抗肝癌活性。结果表明,与5-Fu组相比,SCPP11可对抗5-Fu所致的小鼠白细胞减少和免疫器官指数减小,且对5-Fu所致的机体TNF-α和IL-2水平降低具有较好的恢复作用,此外SCPP11对5-Fu所致的肝损伤有定的保护作用。可见,SCPP11对5-Fu具有较好的减毒作用；休内抗肝癌结果表明,在剂量为50mg/kg时,SCPP11可显著抵制肿瘤的增长,肿瘤抑制率达到68.54%SCPP11可显著提高胸腺指数和小鼠血清中IL-2和TNF-α水平；且其对小鼠体重、肝脏指数、肾脏指数和心脏指数无显著影响,可提高荷瘤小鼠外周血白细胞的数量,使荷瘤小鼠血清AST水平恢复正常；同时提高小鼠肝脏的SOD活力,降低MDA水平。可见, SCOO11具有较好的抗肿瘤活性,且对机体无毒。SCPP11主要通过调节机体免疫来抑制肿瘤的增长。(4)以腹腔巨噬细胞RAW264.7为研究对象,考察SCPP11对腹腔巨噬细胞的免疫调节和作用机制。MTT实验结果表明,SCPP11浓度高达1mg/mL, SCPP11对RAW264.7细胞没有明显的毒性作用；经SCPP11刺激的RAW264.7细胞和其上清液均可显著抑制HepG-2细胞的增殖；SCP11可显著提高RAW264.7细胞中性红吞噬活力和分泌NO, TNF-a和IL-1β的水平；通过western blot法和RT-PCR法研究发现SCPP11可显著上调iNO蛋白和mRNA的表达,上调TNF-β mRNA表达,进一步证实SCPP11直接影响蛋白质代谢和基因表达；TLR4/TLR-2/CR3抗体阻断实验结果表明,抗TLR4抗体可显著阻断SCPP11刺激RAW264.7细胞释放NO和TNF-α的作用,说明TLR4受体是SCPP11发挥巨噬细胞免疫调节作用的关键途径。上述结果表明SCPP11通过TLR4受体激活巨噬细胞,并通过促进细胞因子的蛋白表达和基因表达来促进机体的免疫调节功能是其免疫激活机理之一
Schisandra, fruit of a kind of Magnoliaceae plant, is a famous traditional Chinese Medicine. Schisandra has been used for treating illness in China for more than2,000years. Today it could be used as health food and listed in the Catalogue of Health Foods. It has different active components, such as the lignans, volatile oils and polysaccharides, but only lignans are utilized while polysaccharides were seldom researched. Recent studies revealed that polysaccharides possess various activities such as antitumor and immunomodulatory activity and exhibited low toxicity, so it has been a hot topic in the study of natural product. Currently, there are a variety of polysaccharides widly used as health foods, food additives, broad-spectrum immunopotentiator and so on. In our previous study, it was found that crude polysaccharides from Schisandra possess immunoregulation effect, the attenuate effect to CTX and anti-tumour effects, which indicated that polysaccharides from Schisandra have good prospects in the field of health food and food additives. I lowever, at present, the investigation in polysaccharides from Schisandra was focus on the crude one. the water-soluble low molecular weight polysaccharide fraction from Schisandra (SCPP11) has not been comprehensively studied. Aspects such as its primary structure and conformation, toxicity attenuation effect to5-Fu, the anti-hepatoma activities and its immune mechanism have not been well researched. In this investigation, the structure of SCPP11was comprehensively analyzed by GC-MS, NMR, TEM, AFM, CD and other advanced analytical methods. Its immunomodulatory activities, toxicity attenuation effect to5-Fu and anli-hepatoma activities were also investigated systematically. Additionally, its immunomodulatory mechanism was further studied. As a result the comprehensive structural information of SCPP11was obtained and also elucidated. The immunomodulatorv mechanism was also verified. The study provides a basic data for research and application of polysaccharide functional components and a scientific basis for study and development of an anti-hepatpma polysaccharide drug, and offers a new approach to utilize Schisandra resources. It possesses vast importance to human health and socio-economic development.
The step experimental procedures and results of the study are given below:
(1) To study the structure of SCPP11by methylation, GC-MS,'H NMR,13C NMR,'H-'l-I COSY correlation spectroscopy, hetero nuclear singular-quantum correlation spectroscopy (HSQC) and hetero nuclear multiple-quantum correlation (HMBC). The predicted primary structure of SCPP11was established as shown below: To study the conformation of SCPP11by CD, AFM, TEM and SEM. The results showed that the CD spetrum of SCPP11changed significantly with external conditions (temperature, the pH value, ion intensity etc). It indicated that the conformation and asymmetry of the SCPP11was changed with the change of the external conditions. The particle diameter of SCPP11was270nm with a polydisoersity of0.129which indicated that the particals are distributed uniformly. The absolute weight average molecular weight is1.793X104g/mol, the polydisoersity was1.543and its configuration is between the sphere and random lines. The results of TEM and AFM indicated that the molecule of SCPP11twisted each other, which formed the regular aggregates at the room temperature. However, after heat treatment, the aggregates of SCPP11were decreased significantly and the aggregates were turned into extented molecular chains. The results of SEM indicated that the state structure of SCPP11was noncrystal.
(2) To study the immunomodulatory activity of SCPP11to immunosuppressed mouse. The immunosuppressed mouse model was induced by cyclophosphamide. The results indicated that SCPP11could improve the weight suppression which was caused by CTX and increased the thymus and spleen index to varying degrees, as well as the pinocytic activity of peritoneal macrophages in immunosuppressed mice. Moreover, the polysaccharide promoted hemolysin formation. ELISA assay showed that SCPP11could significant elevate the immunoglobulin-A (IgA), interleukin-2(IL-2) and tumor necrosis factor-α (TNF-α) level in serum, but elevate the immunoglobulin-G (IgG) and immunoglobulin-M (IgM) level in different degrees. The results suggested that SCPP11was involved in immunomodulatory effects leading to the exploration of SCPP11as a potential immunostimulant.
(3) To study the attenuation of SCPP11to HepG-2tumor bearing mouse treated by5-Fu and the antitumor activity of SCPP11. HepG-2mouse tumor model was established. The model mice were divided randomly into10groups, including model control group, negative control group, SCPP11(i.g.200,50mg/kg) groups, SCPP11(i.p.40,10mg/kg) groups,5-Fu plus SCPP11200mg/kg group and5-Fu plus SCPP1150mg/kg group,5-Fu plus SCPP1140mg/kg group and5-Fu plus SCPP1110mg/kg group. Ten other mice were used as normal control group. The inhibitory rates of tumor growth, indexes of organs were measured. The blood morphology, hematology parameters, concentrations of interleukin-2, tumor necrosis factor-alpha, and SOD and MDA in liver, kidney and heart were also determined. The results indicated that SCPP11could attenuated the immunological inhibition caused by5-Fu. Moreover. SCPP11could ameliorate the hematological and biochemical parameters. SCPP11also could improve the liver injury induced by5-Fu. It was obvious that SCPP11had significant attenuation effect on Heps tumor bearing mouse treated with5-Fu. The results of antitumor acvitiy indicated that SCPP11could significantly inhibit the growth of Heps cells in vivo at dose of50mg/Kg, and its inhibition rate is68.54%. Moreover, SCPP11could significant elevate the thymus indexes and the IL-2and TNF-α level in serum. The results indicated that SCPP11may indirectly play the role of antitumor activity through improving immunologic functions. SCPP11indicated no toxicity to body weight, liver, kidney and heart simultaneously, however it could ameliorate the hematological and biochemical parameters to almost normal, reduce the formation of MDA and enhance the activities of SOD in liver. This therefore indicates that SCPP11had potent anti-tumor properties, which could be explored as a potential adjuvant against cancer used in the health foods and pharmaceutical therapy.
(4) To study the immunomodulatory effects and action mechanisms of SCPP11on peritoneal macrophages. The effects of SCPP11on murinc macrophage cell line R.AW264.7mediated cytotoxicity towards HepG-2cells and modulating effects of SCPP11on murine macrophage cell line RAW264.7were evaluated. The results indicated that SCPP11had no apparent toxic effects on RAW264.7cells. When RAW264.7cells were treated with SCPP11, cytotoxic activity against HepG-2cells was significantly induced. In addition, phagocytic activity was enhanced in SCPP11-treated RAW264.7cells compared to the control. The levels of cytokines, including TNF-a and interleukin-1(IL-1β) were increased and the production of nitric oxide (NO) was enhanced in a dose-dependent manner. Western blot analysis suggested SCPP11induced a significant increase in the protein expression of iNOS and the RT-PCR analysis demonstrated that SCPP11could significant increase the mRNA expression of iNOS and TNF-a. The function blocks antibodies to TLR-4, but not TLR-2and CR3, markedly suppressed SCPP11-mediated TNF-a and NO production. Therefore, the results suggest that SCPP11possess a potent antitumor activity and immunomodulatory activity by stimulating macrophage and exert its immunostimulating potency via TLR-4activation of signaling pathway. It could be used as an immunotherapeutic adjuvant.
主要研究内容如下：
(1)以低分子量五味子多糖组分(SCPP11)为研究对象,通过甲基化、GC-MS、1H NMR、13C NMR、1H-1H COSY谱、HSQC谱及HMBC谱等化学分析手段和物理技术对SCPP11的一级化学结构进行推断和解析,确定了低分子量五味子多糖组分SCPP11的一级结构的重复单元为：采用CD、AFM、TEM和SEM等方法对SCPP11的构象和分予形貌进行研究。发现在不同的外界条件下(处理温度、pH值、加入Ca2+离子、6mol/L的变性剂Urea和等量的80μM的刚果红溶液),SCPP11的CD谱图发生显著变化,表明外界条件的改变可影响其立体构象和非对称性。用激光光散射粒度仪测定了SCPP11的粒度,结果表明SCPP11的粒径为270nm,分散度为0.129,粒度分布较均匀。通过多角度激光光散射得到SCPP11的绝对重均分子量为1.793×104g/mol,多分散系数为1.543,且其构型介于球形和随机线团之间。通过原子力显微镜观察SCPP11分子的形貌,结果表明SCPP11分子间相互缠绕,呈规则团聚状；经加热处理后,SCPP11高度团聚状结构大量减少,链延展开来。通过TEM观察SCPP11的形貌,发现其呈团聚状态,粒子的直径在几百纳米不等。SCPP11粉末呈粗糙的小片状聚集体,表面紧密,规整性不强,说明SCPP11固体为无定型结构。
(2)采用CTX抵制小鼠模型,考察SCPP11对小鼠免疫的调节作用。结果表明,SCPP11对CTX造成的体重增长抑制有较好的改善作用,可不同程度提高胸腺指数和脾脏指数,显著提高免疫抑制小鼠腹腔巨噬细胞的吞噬能力和血清溶血素水平，且对CTX致血清中免疫球蛋白水平、FNF-α和IL-2水平下降有不同程度的恢复作用。因此此,SCPP11对可激活机体免疫,具有免疫调节功能。
(3)采用Heps荷瘤小鼠模型,考察SCPP11对化药物5-氟尿嘧啶(5-Fu)的减毒作用和其体内抗肝癌活性。结果表明,与5-Fu组相比,SCPP11可对抗5-Fu所致的小鼠白细胞减少和免疫器官指数减小,且对5-Fu所致的机体TNF-α和IL-2水平降低具有较好的恢复作用,此外SCPP11对5-Fu所致的肝损伤有定的保护作用。可见,SCPP11对5-Fu具有较好的减毒作用；休内抗肝癌结果表明,在剂量为50mg/kg时,SCPP11可显著抵制肿瘤的增长,肿瘤抑制率达到68.54%SCPP11可显著提高胸腺指数和小鼠血清中IL-2和TNF-α水平；且其对小鼠体重、肝脏指数、肾脏指数和心脏指数无显著影响,可提高荷瘤小鼠外周血白细胞的数量,使荷瘤小鼠血清AST水平恢复正常；同时提高小鼠肝脏的SOD活力,降低MDA水平。可见, SCOO11具有较好的抗肿瘤活性,且对机体无毒。SCPP11主要通过调节机体免疫来抑制肿瘤的增长。(4)以腹腔巨噬细胞RAW264.7为研究对象,考察SCPP11对腹腔巨噬细胞的免疫调节和作用机制。MTT实验结果表明,SCPP11浓度高达1mg/mL, SCPP11对RAW264.7细胞没有明显的毒性作用；经SCPP11刺激的RAW264.7细胞和其上清液均可显著抑制HepG-2细胞的增殖；SCP11可显著提高RAW264.7细胞中性红吞噬活力和分泌NO, TNF-a和IL-1β的水平；通过western blot法和RT-PCR法研究发现SCPP11可显著上调iNO蛋白和mRNA的表达,上调TNF-β mRNA表达,进一步证实SCPP11直接影响蛋白质代谢和基因表达；TLR4/TLR-2/CR3抗体阻断实验结果表明,抗TLR4抗体可显著阻断SCPP11刺激RAW264.7细胞释放NO和TNF-α的作用,说明TLR4受体是SCPP11发挥巨噬细胞免疫调节作用的关键途径。上述结果表明SCPP11通过TLR4受体激活巨噬细胞,并通过促进细胞因子的蛋白表达和基因表达来促进机体的免疫调节功能是其免疫激活机理之一
Schisandra, fruit of a kind of Magnoliaceae plant, is a famous traditional Chinese Medicine. Schisandra has been used for treating illness in China for more than2,000years. Today it could be used as health food and listed in the Catalogue of Health Foods. It has different active components, such as the lignans, volatile oils and polysaccharides, but only lignans are utilized while polysaccharides were seldom researched. Recent studies revealed that polysaccharides possess various activities such as antitumor and immunomodulatory activity and exhibited low toxicity, so it has been a hot topic in the study of natural product. Currently, there are a variety of polysaccharides widly used as health foods, food additives, broad-spectrum immunopotentiator and so on. In our previous study, it was found that crude polysaccharides from Schisandra possess immunoregulation effect, the attenuate effect to CTX and anti-tumour effects, which indicated that polysaccharides from Schisandra have good prospects in the field of health food and food additives. I lowever, at present, the investigation in polysaccharides from Schisandra was focus on the crude one. the water-soluble low molecular weight polysaccharide fraction from Schisandra (SCPP11) has not been comprehensively studied. Aspects such as its primary structure and conformation, toxicity attenuation effect to5-Fu, the anti-hepatoma activities and its immune mechanism have not been well researched. In this investigation, the structure of SCPP11was comprehensively analyzed by GC-MS, NMR, TEM, AFM, CD and other advanced analytical methods. Its immunomodulatory activities, toxicity attenuation effect to5-Fu and anli-hepatoma activities were also investigated systematically. Additionally, its immunomodulatory mechanism was further studied. As a result the comprehensive structural information of SCPP11was obtained and also elucidated. The immunomodulatorv mechanism was also verified. The study provides a basic data for research and application of polysaccharide functional components and a scientific basis for study and development of an anti-hepatpma polysaccharide drug, and offers a new approach to utilize Schisandra resources. It possesses vast importance to human health and socio-economic development.
The step experimental procedures and results of the study are given below:
(1) To study the structure of SCPP11by methylation, GC-MS,'H NMR,13C NMR,'H-'l-I COSY correlation spectroscopy, hetero nuclear singular-quantum correlation spectroscopy (HSQC) and hetero nuclear multiple-quantum correlation (HMBC). The predicted primary structure of SCPP11was established as shown below: To study the conformation of SCPP11by CD, AFM, TEM and SEM. The results showed that the CD spetrum of SCPP11changed significantly with external conditions (temperature, the pH value, ion intensity etc). It indicated that the conformation and asymmetry of the SCPP11was changed with the change of the external conditions. The particle diameter of SCPP11was270nm with a polydisoersity of0.129which indicated that the particals are distributed uniformly. The absolute weight average molecular weight is1.793X104g/mol, the polydisoersity was1.543and its configuration is between the sphere and random lines. The results of TEM and AFM indicated that the molecule of SCPP11twisted each other, which formed the regular aggregates at the room temperature. However, after heat treatment, the aggregates of SCPP11were decreased significantly and the aggregates were turned into extented molecular chains. The results of SEM indicated that the state structure of SCPP11was noncrystal.
(2) To study the immunomodulatory activity of SCPP11to immunosuppressed mouse. The immunosuppressed mouse model was induced by cyclophosphamide. The results indicated that SCPP11could improve the weight suppression which was caused by CTX and increased the thymus and spleen index to varying degrees, as well as the pinocytic activity of peritoneal macrophages in immunosuppressed mice. Moreover, the polysaccharide promoted hemolysin formation. ELISA assay showed that SCPP11could significant elevate the immunoglobulin-A (IgA), interleukin-2(IL-2) and tumor necrosis factor-α (TNF-α) level in serum, but elevate the immunoglobulin-G (IgG) and immunoglobulin-M (IgM) level in different degrees. The results suggested that SCPP11was involved in immunomodulatory effects leading to the exploration of SCPP11as a potential immunostimulant.
(3) To study the attenuation of SCPP11to HepG-2tumor bearing mouse treated by5-Fu and the antitumor activity of SCPP11. HepG-2mouse tumor model was established. The model mice were divided randomly into10groups, including model control group, negative control group, SCPP11(i.g.200,50mg/kg) groups, SCPP11(i.p.40,10mg/kg) groups,5-Fu plus SCPP11200mg/kg group and5-Fu plus SCPP1150mg/kg group,5-Fu plus SCPP1140mg/kg group and5-Fu plus SCPP1110mg/kg group. Ten other mice were used as normal control group. The inhibitory rates of tumor growth, indexes of organs were measured. The blood morphology, hematology parameters, concentrations of interleukin-2, tumor necrosis factor-alpha, and SOD and MDA in liver, kidney and heart were also determined. The results indicated that SCPP11could attenuated the immunological inhibition caused by5-Fu. Moreover. SCPP11could ameliorate the hematological and biochemical parameters. SCPP11also could improve the liver injury induced by5-Fu. It was obvious that SCPP11had significant attenuation effect on Heps tumor bearing mouse treated with5-Fu. The results of antitumor acvitiy indicated that SCPP11could significantly inhibit the growth of Heps cells in vivo at dose of50mg/Kg, and its inhibition rate is68.54%. Moreover, SCPP11could significant elevate the thymus indexes and the IL-2and TNF-α level in serum. The results indicated that SCPP11may indirectly play the role of antitumor activity through improving immunologic functions. SCPP11indicated no toxicity to body weight, liver, kidney and heart simultaneously, however it could ameliorate the hematological and biochemical parameters to almost normal, reduce the formation of MDA and enhance the activities of SOD in liver. This therefore indicates that SCPP11had potent anti-tumor properties, which could be explored as a potential adjuvant against cancer used in the health foods and pharmaceutical therapy.
(4) To study the immunomodulatory effects and action mechanisms of SCPP11on peritoneal macrophages. The effects of SCPP11on murinc macrophage cell line R.AW264.7mediated cytotoxicity towards HepG-2cells and modulating effects of SCPP11on murine macrophage cell line RAW264.7were evaluated. The results indicated that SCPP11had no apparent toxic effects on RAW264.7cells. When RAW264.7cells were treated with SCPP11, cytotoxic activity against HepG-2cells was significantly induced. In addition, phagocytic activity was enhanced in SCPP11-treated RAW264.7cells compared to the control. The levels of cytokines, including TNF-a and interleukin-1(IL-1β) were increased and the production of nitric oxide (NO) was enhanced in a dose-dependent manner. Western blot analysis suggested SCPP11induced a significant increase in the protein expression of iNOS and the RT-PCR analysis demonstrated that SCPP11could significant increase the mRNA expression of iNOS and TNF-a. The function blocks antibodies to TLR-4, but not TLR-2and CR3, markedly suppressed SCPP11-mediated TNF-a and NO production. Therefore, the results suggest that SCPP11possess a potent antitumor activity and immunomodulatory activity by stimulating macrophage and exert its immunostimulating potency via TLR-4activation of signaling pathway. It could be used as an immunotherapeutic adjuvant.
引文
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