摘要
本论文在课题组前期筛选、驯化得到一株耐硒高产多糖Enterobacter cloacaeZ0206菌株,深层发酵获得Z0206细菌多糖,并初步探索了其免疫调节、抗氧化、抗病毒等生物学功能的基础上,采用离子交换层析、凝胶渗透层析等现代分离纯化技术,得到Z0206细菌多糖的主要组分；建立了高效液相色谱(HPLC)定量分析多糖中丙酮酸含量和气相色谱-质谱联用同步分析多糖中单糖和糖醛酸组成的方法,同时结合核磁共振波谱、光谱等技术对Z0206细菌多糖主要组分的结构进行了鉴定；然后以自发性Ⅱ型糖尿病KKAy小鼠为模型,研究了Z0206细菌多糖及其纯化多糖对KKAy小鼠糖脂代谢的调节作用,并在基因和蛋白水平上揭示了Z0206细菌多糖对糖脂代谢影响的分子机制。主要研究内容及结果如下：
1.反相高效液相色谱(RP-HPLC)定量分析丙酮酸含量方法的建立及Z0206细菌多糖中丙酮酸单元的测定
结合Sevage去和酶法脱蛋白、H2O2脱色、水透析、冷冻干燥以及柱层析等方法对Z0206细菌多糖(CEPS)进行纯化后获得其酸性多糖EPS.该多糖EPS经高效凝胶色谱仪(HPGPC)检测,其分子量约为110KDa.
在此基础上,本试验采用核磁共振波谱(NMR)、电喷雾-四极杆-飞行时间串联质谱(ESI-Q-TOF-MS)等技术确定了EPS结构中含有丙酮酸单元；采用反相高效液相色谱(RP-HPLC)考察不同的酸水解条件以及不同的定容体积等样品前处理条件下的丙酮酸出峰情况,最终确定多糖完全酸水解条件为3M三氟乙酸(TFA)、120℃下水解5h。水解液定容体积为100mL；定容后的酸水解物经过滤后采用RP-HPLC直接分析其中的丙酮酸,酸水解物经C18色谱柱分离、紫外检测器检测(λ=215nm)、外标法定量。结果表明：EPS中丙酮酸含量为7.64%,该方法线性范围0.0025～0.5g/L,标准曲线相关系数r>0.999；检出限(3倍信噪比)达0.05mg/L；加标回收率为100.2%～101.6%。该方法用于分析细菌多糖、黄芪多糖等多糖类物质的丙酮酸含量专属性良好,为多糖类物质中丙酮酸的定量分析提供了新方法。
2.气相色谱-质谱联用(GC-MS)同时测定单糖和糖醛酸含量方法的建立及Z0206细菌多糖的单糖组成和糖醛酸含量分析
在采用反相高效液相色谱确定了EPS中丙酮酸含量的基础上,本试验进一步建立了GC-MS同时测定多糖中单糖和糖醛酸含量的方法,并对EPS的单糖组成和糖醛酸含量进行了分析。将EPS的完全酸水解物进行氮气吹干,采用乙硫醇-三氟乙酸和醋酐-吡啶体系先后对其中的单糖和葡萄糖醛酸进行衍生；电子轰击电离源质谱(EI-MS)解析葡萄糖醛酸酸衍生物质谱裂解途径,并证实葡萄糖醛酸在该体系下得到了有效地衍生化；以木糖为内标,采用GC-MS定量分析该多糖酸水解物中单糖和糖醛酸衍生物发现：该多糖糖链由岩藻糖、葡萄糖、葡萄糖醛酸和半乳糖组成,其相对物质的量比为1.95：1.0：1.13：2.82；中性糖比例与糖醇乙酸酯化分析岩藻糖、葡萄糖和半乳糖的相对物质的量比(2.02：1.0：2.84)接近；糖醛酸咔唑法与该方法分析糖醛酸含量分别为15.23%和16.51%。结果表明新建立的衍生方法及GC-MS同时定量分析多糖酸水解物中单糖和糖醛酸的方法快速、方便。
3.Z0206细菌多糖的结构鉴定及其重复单元的分析
在分析测定了EPS的丙酮酸含量、单糖组成和糖醛酸含量的基础上,进一步开展了Z0206细菌多糖的确切化学结构鉴定及重复单元分析的研究。EPS完全酸水解物衍生物测定结果表明：该EPS由L-岩藻糖、D-葡萄糖、D-半乳糖、D-葡萄糖醛酸和丙酮酸组成,其相对物质的量近似为2：1：3：1：1。以上结果结合高碘酸氧化、Smith降解和甲基化等化学分析,发现该多糖由1,4-连接的岩藻糖、1,3,4-连接的岩藻糖、1,3-连接的葡萄糖,1,3-连接的半乳糖,1,4-连接的半乳糖和1,4,6-连接的端基半乳糖组成,其中丙酮酸取代了该端基半乳糖4,6位羟基。
采用Bio-Gel P2凝胶色谱柱分离、电喷雾电离质谱(ESI-MS)检测馏分,纯化多糖EPS部分酸水解产物,获得一个寡糖—六糖；ESI-MS分析六糖分子量和系列寡糖可能的线性连接；结合多糖组成成分分析、单糖构型分析、糖苷键的键型分析和核磁共振一维、二维波谱分析,推断出该六糖结构。并确定了多糖的结构是以1,3-β-D-半乳糖残基、1,4-β-D-半乳糖残基、2个1,4-a-L-岩藻糖残基为主链,并在其中的一个1,4-连接的岩藻糖残基的O-3位有p-D-葡萄糖残基取代,而1,4-p-D-葡萄糖醛酸残基又取代了该p-D-葡萄糖残基O-3位羟基,同时丙酮酸取代了4,6位羟基的1,4,6-a-D-半乳糖残基取代了1,4-β-D-葡萄糖醛糖残基O-4位羟基。多糖以上述结构为重复单元组成。
4.Z0206细菌多糖对Ⅱ型糖尿病KKAy小鼠糖脂代谢的影响研究
在建立了HPLC测定多糖中丙酮酸含量,GC-MS同时测定多糖中单糖和糖醛酸含量的方法,解析了Z0206细菌多糖主要组分的化学结构的基础上,本实验进一步以自发性Ⅱ型糖尿病KKAy小鼠为模型,从表观变化、组织形态、血液生化指标等方面探讨了Z0206细菌多糖(CEPS)及其纯化多糖(PEPS)对糖脂代谢的影响。试验分为正常对照组(C57/BL)、糖尿病模型组(Model)、Z0206细菌多糖组(CEPS)和Z0206纯化多糖组(PEPS)组,多糖组以10mg/mL分别连续灌胃42天,糖尿病模型组连续灌胃等量的去离子水。研究结果表明：与糖尿病模型组相比,在给药后的14天、28天及42天,CEPS和PEPS均可显著降低KKAy小鼠的体重(P<0.05),提高KKAy小鼠的葡萄糖耐量(P<0.05),并降低小鼠的空腹血糖浓度和血清胰岛素水平(P<0.05),从而抑制小鼠的肥胖、缓解胰岛素抵抗和提高小鼠的糖耐受能力；组织形态观察结果显示：CEPS和PEPS均能通过缓解肝脏细胞和胰岛细胞的肿胀、变性,减少脂滴形成和增加肝脏糖原含量,从而保护肝脏和胰腺组织结构的完整性,并有效缓解肝脏的脂肪变性；生化指标结果表明：CEPS和PEPS均能通过提高小鼠肝脏已糖激酶活力(P<0.05),降低血清总胆固醇、甘油三酯、低密度脂蛋白水平(P<0.05)和游离脂肪酸含量(P<0.05),同时降低小鼠谷丙转氨酶和谷草转氨酶的含量(P<0.05),从而增加葡萄糖在体内的酵解,改善脂代谢紊乱和修复肝脏损伤。以上结果综合表明两种多糖能改善Ⅱ型糖尿病小鼠的胰岛素抵抗症状,保护胰腺功能,增加体内糖原合成和葡萄糖的酵解起到降血糖作用；同时能缓解肝脏损伤,减少脂肪变性,改善机体脂代谢紊乱起到降血脂作用。但是,两种多糖对于小鼠的糖脂代谢影响没有差异。
5.Z0206细菌多糖对Ⅱ型糖尿病KKAy小鼠胰岛素分泌及其肝脏糖脂代谢影响的作用机制研究
在研究了Z0206细菌多糖对糖尿病KKAy小鼠表型、组织形态及生理生化指标影响的基础上,本试验进一步从分子水平开展了Z0206细菌多糖(CEPS)及其纯化多糖(PEPS)对KKAy小鼠胰岛功能和肝脏糖脂代谢影响的作用机制研究。试验重点分析了两种多糖CEPS和PEPS对KKAy小鼠胰腺功能相关基因,肝脏中糖代谢相关酶类和脂肪代谢相关酶类的基因或蛋白表达的影响。胰岛功能结果表明：两种多糖均能通过提高KKAy小鼠胰脏中沉默信息调节因子1(Sirtl)和腺苷酸活化蛋白激酶(AMPK)的蛋白表达,降低线粒体解偶联蛋白2(UCP2)的蛋白表达,改善KKAy小鼠胰腺β细胞功能,抑制因胰岛素抵抗引起的胰岛素代偿分泌增加；糖转运和糖代谢结果表明：细菌多糖能显著提高肝脏中葡萄糖转运蛋白2(Glut2)的蛋白表达水平,增加糖酵解关键酶葡萄糖激酶(GK)的mRNA表达量,同时降低糖异生关键酶葡萄糖6磷酸酶(G6PC)的mRNA表达量,从而增加肝脏对血糖的摄取,促进葡萄糖在肝脏的酵解并减少糖异生作用；脂肪代谢结果表明：多糖能提高肝脏中脂肪分解相关基因激素敏感性脂肪酶(HSL).脂肪甘油三酯水解酶(ATGL)及脂肪酸p氧化限速酶CPT-1α的mRNA表达水平,同时降低脂肪合成相关基因脂肪酸合成酶(FAS)的mRNA表达水平,从而促进肝脏中脂肪的分解并抑制脂肪在肝脏的蓄积;以上结果综合表明,Z0206细菌多糖可以通过增加肝脏对血糖的摄取和利用并抑制糖异生过程起到降血糖作用；同时可以通过AMPK和Sirtl途径促进脂肪的氧化分解并抑制脂肪沉积,从而起到脂代谢调节作用。但是,两种多糖对胰岛素分泌及其肝脏糖脂代谢影响没有差异。
In our previous study, Enterobacter cloacae Z0206, a selenium-tolerant bacterial strain, was screened and acclimated. It was found to produce large amounts of exopolysaccharides (Z0206exopolysaccharides; CEPS) through fermentation, and the Z0206exopolysaccharides were found to possess immunostimulatory properties and antioxidation as well as growth improvement effects when applied to animals. In the present study, another acid exopolysaccharide (EPS) was prepared through deproteinization, decolorization and dialysis as well as column chromatography. The HPLC and GC-MS methods were established to analyze constituents in the EPS. On basis of the above analyses, a combination of chemical and ESI-MS as well as NMR analyses was established to elucidate the stuctrue of the EPS. Besides, KKAy mouse was used as experimental animal model. The diabetic mice were received CEPS and the purified exopolysaccarides (PPES) through salting out method in aqueous suspension daily by oral administration for a period of42days. In addition, the glucose and lipid metabolism in the animals was also studied. Lastly, the effects of CEPS and PEPS on glucose and fat metabolisms in liver and insulin secretion were studied. The primary results are as follows:
1. Establishment a RP-HPLC method to quantify the pyruvate in a Z0206polysaccharide EPS
A novel high molecular weight (110KDa) exopolysaccharide (EPS) produced by Z0206strain was isolated by column chromatography. The pyruvyl groups of the polysaccharide were identified by ultraviolet (UV) spectra, nuclear magnetic resonance (1H NMR,13C NMR) spectra, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and reversed-phase high-performance liquid chromatography (RP-HPLC). To measure the pyruvyl groups, the microbial polysaccharide was hydrolyzed using3M TFA at120℃and subsequently analyzed by isocratic RP-HPLC and UV detection at215nm. Results showed that linear range for the pyruvate was from0.0025g/L to0.5g/L with the detection limit (signal-to-noise ratio of3) of0.05mg/L and the recovery rate ranged from100.2%to101.6%. The new method provided an option to analyze pyruvate content in acid hydrolysatcs of crude polysaccharides without complex pre-treatment.
2. Establishment a GC-MS method to determine the neutral sugars and uronic acid constituents in a Z0206polysaccharide EPS
The EPS was acid hydrolyzed followed by the subsequent derivatization using ethanethiol-trifluoroacetic acid and acetic anhydride-pyridine systems sequentially. Our findings differ from the previous reports that the glucuronic acid was obtained through effective derivatization. The neutral sugars and glucuronic acid were analyzed using gas chromatography-mass spectrometry (GC-MS) with xylose as an internal standard. The EPS was found to be composed of fucose, glucose, glucuronic acid and galactose, with the relative molar ratio of1.95:1.0:1.13:2.82. The neutral sugars ratio was similar to the relative molar ratio for fucose, glucose and galactose of2.02:1.0:2.84through alditol acetates determined by GC. The percentages of glucuronic acid analyzed using either the carbazole and sulfuric acid method or the above method were15.23%and16.51%, respectively. These results indicated that it was practicable to use the derivatization method and GC-MS to quantitatively analyze neutral sugars and glucuronic acid simultaneously in that polysaccharide. For GC-MS analysis, the procedure was developed for the simultaneous determination of the derivatives in25min, and was performed using an HP-5MS column. Molecular ion peaks were observed in the electron ionization (El) mass spectra. The fragmentation mechanism for glucuronic acid derivative was discussed in detail.
3. Stuctural elucidation of a Z0206polysaccharide EPS and the repeat unit
Complete hydrolysis of the EPS followed by gas chromatography mass spectrometry (GC-MS) and high performance liquid chromatography (HPLC) analyses showed that the EPS is composed of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid in the approximate molar ratio of2:1:3:1:1. Partial acid hydrolysis of the purified EPS followed by gel permeation chromatography (GPC) yielded a hexasaccharide. A combination of chemical analysis coupled with mass spectrometry and1D and2D NMR spectroscopy applied to the oligosaccharide showed that the EPS comprised a heptasaccharide repeating unit.
4. Effect of Z0206polysaccharides on the glucose and lipid metabolism in KKAy diabetic mice
With the established RP-HPLC and GC-MS methods for pyruvate, and neutral sugars, uronic acid constituents and chemical structure, respectively, the effects of CEPS and PEPS on the glucose and fat metabolisms were studied to addrss changes, the morphology and blood biochemical paramenters using the spontaneously type II diabetes KKAy mice. Experiment groups were allocated to normal control group (C57/BL), diabetes group (Model), Z0206bacterial polysaccharide group (CEPS) and Z0206purified polysaccharide group (PEPS). As CEPS were administered to KKAy diabetic mice for a period of42days, the body weight, fasting blood glucose (FBG), oral glucose tolerance test (OGTT), serum insulin, blood lipds, liver function and morphology of liver, pancreas and kidney were observed. The results showed that the body weight significantly (P<0.05) decreased in CEPS and PEPS treated animals after14,28and42days of treatment. The administration of CEPS and PEPS in KKAy mice caused a significant enhancement (P<0.05) in the OGTT, whereas a significant reducement (P<0.05) was shown in the FBG level and serum insulin concentration, indicating that EPS could suppress the obesity phenomenon and alleviate insulin resistance; Morphology observation shows that EPS possessed significantly protective effects in the integrity of the organizational structure of the liver and pancreas and formation of steatosis in liver by decreasing the swelling and degeneration of the liver cells and pancreatic islet cells as well as ameliorating the liver glycogen; The biochemical parameter results showed that EPS could enhance the activity of hexokinase (HK) in liver, reduce the total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and FFA concentration, at the same time significantly decrease (P<0.05) the AST and ALT levels in liver, thus increaseing glycolysis of glucose in the body, improving lipid metabolism disorders and repairing liver damages. The above results suggested that CEPS could improve insulin resistance and protect the pancreatic function, increasing the glycogen synthesis and glycolysis in type Ⅱ diabetic mice; it also could alleviate not only liver damages but also steatosis and further improve fat metabolic disorders. But there is no significant difference between CEPS and PESP in its effect on glucose and lipid metabolisms.
5. The effects of Z0206polysaccharides on insulin secretion, glucose and fat metabolisms in KKAy diabetic mice liver
On the basis of the previous four experiments, this experiment was conducted further to study the effects of CEPS and PEPS on glucose and fat metabolisms in liver and insulin secretion. RT-PCR was used to analyse mRNA expression of G6PC, GK, HSL, ATGL, FAS and CPT-la in the liver, and Western blot was used to analyse protein expression of AMPK, Sirtl and Glut2in the liver and AMPK, Sirtl and UCP2in the pancreas. The results of pancreas showed that after supplementation of CEPS or PEPS, SirTl and AMPK protein expressions were increased, as UCP2protein expression was reduced, which means that function of β cell was improved and the compensatory secretion of insulin caused by insulin resistance was also increased. The results of glucose transportation and metabolism showed that with CEPS or PEPS supplementation, Glut2protein and GK mRNA expressions were improved while G6PC gene expression was significantly (P<0.05) decreased, which demonstrated that the glucose uptake and glycolysis in the liver was increased while the gluconeogenesis was reduced; The results of fat metabolism showed that HSL, ATGL and CPT-la gene expression and AMPK, Sirtl protein expressions were increased in the liver of KKAy mice, while FAS gene expression were significantly decreased. Our results clearly demonstrated that with CEPS or PEPS supplementation, glucose transportation and gluconeogenesis were reduced in the liver of diabetic KKAy mice, which could lower the blood glucose level; lipolysis and fatty acid (3oxidation were increased while lipogenesis was reduced through AMPK and Sirtl pathway, which could alleviate the fat accumulation in the liver of diabetic KKAy mice. But there is no significant difference between CEPS and PESP in its effect on glucose and lipid metabolisms.
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