摘要
白术为菊科多年生植物白术(Atractylodis macrocephalae Koidz.)的干燥根茎,是临床常用的一种补益类中药。前期研究发现,口服白术水煎剂后注射免疫口蹄疫(FMD)疫苗可增强动物的免疫反应。本文在此基础上主要研究白术不同提部分—多糖、挥发油和内酯对口蹄疫疫苗的口服佐剂作用,筛选出具有显著佐剂作用的活性成分,并探讨其增强小鼠免疫反应的作用机理,为进一步研究开发白术提供参考。
1.白术多糖和白术挥发油的提取分离
目的从生药白术中提取白术多糖和白术挥发油,测定其得率,并分析白术多糖的单糖组成情况。方法一白术多糖的提取和含量测定。取生药白术100g,粉碎后加25倍量水,煎煮2次,每次煎煮2h,合并煎煮液,过滤。滤液浓缩至200ml左右,边搅拌边缓缓加入4倍体积95%乙醇,4℃冰箱中静置24h,布氏漏斗抽滤,沉淀加热复溶于1000ml蒸馏水中,布氏漏斗抽滤,滤液上D101大孔树脂柱,用蒸馏水进行洗脱。洗脱液浓缩至200m1,加入4倍体积95%乙醇,4℃冰箱中静置24h,沉淀加200ml蒸馏水溶解,得多糖溶液。Sevage法除蛋白后分别对自来水和蒸馏水透析48h(cut-off Mw7000Da)。袋内糖溶液减压浓缩至适量体积,冷冻干燥得白术多糖,即为RAMPS.用苯酚硫酸法-测定多糖含量；方法二白术多糖成分分析。称取白术多糖20mg,加入2mol/L三氟乙酸15mL,室温浸渍过夜,110℃密闭水解6h,水解液于50℃减压蒸发至干,乙酰化后进行气象色谱分析其单糖组成；方法三白术挥发油的提取。取生药白术200g,加入12倍量的石油醚,70℃回流提取70min,抽滤后,同等条件下将滤渣再提取一次,合并滤液,加入适量无水硫酸钠干燥,抽滤,将滤液用旋转蒸发仪浓缩,至无溶剂滴出,即得白术挥发油。结果与结论白术多糖得率为4.2%,多糖含量为86.2%,由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、半乳糖组成,物质的量比为：1.00：2.49：2.07：4.94：11.33：1.35；白术挥发油,油显金黄色,粘稠、有香气,得率为1.185%。
2.口服白术多糖、挥发油和内酯Ⅰ对口蹄疫疫苗佐剂作用的评价
目的通过研究白术多糖、白术挥发油和白术内酯Ⅰ对口蹄疫疫苗的免疫增强作用,筛选出具有免疫佐剂作用的提取物。方法一将42只ICR小鼠随机分成7组,每组6只,分别设生理盐水对照组、白术水煎剂组和5个不同剂量的白术多糖组。每只小鼠每天灌胃给药,连续4d,于灌胃结束后24h皮下注射O型FMD疫苗免疫,0.2mL/只,2周后采取同样方法加强免疫一次。2免后3周采血,检测血清抗FMDV特异性抗体水平；方法二将48只ICR小鼠随机分成8组,每组6只,分别设生理盐水对照组、吐温-80组、白术水煎剂组和5个不同剂量的白术挥发油组。每只小鼠每天灌胃给药,连续4d,于灌胃结束后24h皮下注射O型FMD疫苗免疫,0.2mL/只,2周后采取同样方法加强免疫一次。2免后3周采血,检测血清抗FMDV特异性抗体水平；方法三将48只ICR小鼠随机分成8组,每组6只,分别设生理盐水对照组、DMSO组、白术水煎剂组和5个不同剂量的白术内酯Ⅰ组。每只小鼠每天灌胃给药,连续4d,于灌胃结束后24h皮下注射O型FMD疫苗免疫,0.2mL/只,2周后采取同样方法加强免疫一次。2免后3周采血,检测血清抗FMDV特异性抗体水平。结果与结论口服白术多糖可明显提高小鼠产生的抗FMDV特异性抗体的水平,而口服白术挥发油和白术内酯Ⅰ并不能提高小鼠血清中特异性抗体水平,表明小鼠口服白术多糖对FMDV疫苗免疫佐剂作用。3.口服白术多糖对口蹄疫疫苗免疫佐剂作用的研究
目的观察白术多糖口服给药对小鼠皮下注射口蹄疫疫苗的免疫反应。方法将35只ICR小鼠随机分成5组,每组7只。分别设生理盐水对照组、白术水煎剂组和3个不同剂量的白术多糖组。每只小鼠每天灌胃给药,连续4d,于灌胃结束后24h皮下注射O型FMD疫苗免疫,0.2mL/只,2周后采取同样方法加强免疫一次。2免后3周眼球采血,间接ELISA法检测血清抗FMDV抗体效价及抗体亚类IgG1、IgG2a、IgG2b和IgG3水平,western blot检测血清中IFN-γ和IL-5水平；无菌分离脾淋巴细胞,用MTT法检测淋巴细胞增殖和RT-PCR法检测FMDV抗原刺激后脾脏淋巴细胞细胞因子和转录因子mRNA表达水平。结果0.025g/d、0.05g/d和0.01g/d的白术多糖连续灌胃4d后,小鼠血清抗FMDV特异性抗体效价、抗体亚类水平、脾淋巴细胞刺激指数、脾淋巴细胞Th1/Th2类细胞因子IFN-γ/IL-4以及转录因子T-bet/GATA-3mRNA的表达水平都显著高于对照组(P<0.05),其中以0.05g／d白术多糖灌胃的提高幅度最大；0.05g／d白术多糖组小鼠血清中IFN-γ和IL-5的水平也得到明显提高(P<0.05)。结论口服白术多糖能够显著提高小鼠对口蹄疫疫苗免疫产生的免疫应答,促进T、B淋巴细胞活化,并激活Th1和Th2型免疫应答反应,对FMD疫苗具有佐剂作用。
4.口服白术多糖对小鼠肠道黏膜免疫的影响
目的观察口服白术多糖对小鼠肠道黏膜免疫系统的影响。方法将56只雌性ICR小鼠随机分成4组,每组14只。分别设白术多糖和生理盐水对照免疫组以及白术多糖和生理盐水对照非免疫组。每只小鼠每天灌胃给药0.25mL,连续4d,免疫组小鼠于灌胃给药后24h腹股沟皮下注射O型FMD疫苗免疫,0.2mL/只,2周后采取同样方法加强免疫一次。首免后1周和2免后2周,每组取7只小鼠眼球采血,间接ELISA法检测血清抗FMDV抗体水平；采集十二指肠,用于肠道内容物的收集、肠道组织mRNA的提取以及石蜡切片的制备,采用ELISA法测定肠道内容物中总IgA的水平,RT-PCR检测肠道组织细胞因子(?)nRNA表达水平、HE染色计数肠上皮淋巴细胞(IEL)数量、免疫组化法计数IgA+细胞数量。结果口服白术多糖可显著提高小鼠血清中抗FMDV特异性抗体水平。口服白术多糖后1周后,小鼠十二指肠内容物中IgA水平、肠道IgA+浆细胞面积、肠组织TGF-β、IL-6、TNF-α mRNA表达水平以及小肠IEL细胞数目均显著高于对照组；灌胃后2周,以上指标有升高趋势,但差异不明显。结论口服白术多糖可提高小鼠肠道黏膜免疫水平,灌胃后1周的效果要高于灌胃后2周。
5.白术多糖体外对小鼠巨噬细胞模式识别受体的作用
目的观察白术多糖体外对小鼠巨噬细胞模式识别受体有无作用。方法以Raw-Blue细胞(稳转pNifty-2质粒的RAW264.7细胞)为刺激对象,分别向细胞培养液中加入不同终浓度的白术多糖和黄芪多糖刺激细胞,使最终浓度为0、1μg/ml、4μg/ml、8μg/ml、16μg/ml、25μg/ml、50μg/ml、100μg/ml和200μg/ml。37℃,5%C02培养箱中培养18h后,取40μl细胞培养上清,加入到160μl显色液中,37℃培养2h后,检测报告基因SEAP (分泌型碱性磷酸酶)的表达水平。结果与结论白术多糖对细胞上清中SEAP的水平没有影响,表明白术多糖并不能刺激Raw-Blue细胞NF-κB的表达,对TLRs、NLRs或RLRs等PRRs无作用；
综上所述,口服白术挥发油和白术内酯Ⅰ对FMD疫苗无免疫增强作用,而口服白术多糖可显著提高小鼠血清的抗FMDV特异性抗体水平,促进T、B淋巴细胞的增殖,提高Thl和Th2型免疫应答,增强小鼠肠道黏膜免疫水平,表明白术水煎剂的口服佐剂作用主要是由白术多糖所引起；白术多糖的佐剂作用并不通过TLRs、NLRs或RLRs信号途径所介导,具体的作用机制还有待进一步研究。
Atractylodis macrocephalae Koidz. is a natural plant in family of Compositae, The rhizome of the plant (RAM) has been utilized as a digestive stimulator in traditional Chinese medicine. Our previous study has demonstrated that oral administration of a decoction made from RAM has significantly increased immune responses to vaccines against FMD in mice. In the present thesis, adjuvant activity of different extracts from RAM were studied in order to screening for the adjuvant fractions, and then its effect on the systemic and intestinal mucosal immune response to FMD vaccine in mice was investigated.
1. Extraction and purification of polysaccharide and volatile oil from the rhizome of Atractylodis macrocephalae Koidz.(RAM)
Objective To extract and purify the polysaccharide and volatile oil from RAM. Methods Three experiments were designed and described as follows. In experiment A, the rhizome of RAM (100g) was ground into powder and then extracted with boiling water two times under reflux for2h each time. The aqueous portion was filtered through filter paper. The filtrate was concentrated under reduced pressure. Four volumes of95%ethanol were added to the supernatant, and kept overnight at4℃. The resulting precipitate was dissolved in distilled water, subjected to Macroporous Adsorption Resin column D101, and then washed with water.The collected elute was concentrated, dialyzed against distilled water (cut-off Mw7000Da) and lyophilized to afford a total RAM polysaccharide (RAMPS). Total sugar content was estimated by the phenol-sulfuric acid analysis using glucose as a standard. In experiment B, RAMPS (20mg) were hydrolyzed with15ml of2M TFA at110℃for6h to release component monosaccharides. The hydrolyzed monosaccharides (inositol as the internal standard) were derivatized to acetylated aldononitriles and isothermally separated by gas chromatography (GC) in an Agilent6890N system equipped with a flame-ionization detector (FID) and a DB-5capillary column (30.0m×0.32mm× 0.25μm). In experiment C, the rhizome of RAM (200g) was ground into powder and then extracted with petroleum ether at70℃two times under reflux for70min each time. The filtrate was concentrated under reduced pressure until no solvent left to obtain volatile oil of RAM. Results and Conclusions The yield of RAMPS in this experiment was4.2%, The polysaccharide contained in RAMPS was86.2%. GC quantitative analysis with derivatization revealed that RAMPS was composed of rhamnose, arabinose, xylose, mannose, glucose, and galactose with the molar ratio of1.00:2.49:2.07:4.94:11.33:1.35.The extraction rates of volatile oil was1.185%.
2. Adjuvant effects of oral administration of RAMPS、volatile oil and atractylenolide I against FMD vaccine in mice
Objective To screen for the ingredient(s) contributing the adjuvant activity of RAM. Methods In experiment A, Forty-two female ICR mice were randomly divided into seven groups with six mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or RAMPS (6.25,12.5,25,50or100mg) or RAM decoction (250mg). Blood samples were collected3weeks after the booster immunization for detection of of FMDV-specific antibody response. In experiment B, Forty-eight female ICR mice were randomly divided into eight groups with six mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or tween-80(2%) or volatile oil of RAM (1,2,4,8or16mg) or RAM decoction (250mg). Blood samples were collected3weeks after the booster immunization for detection of of FMDV-specific antibody response. In experiment C, Forty-eight female ICR mice were randomly divided into eight groups with six mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or DMSO (0.5%) or atractylenolide I (0.05,0.1,0.2,0.4or0.8mg) or RAM decoction (250mg). Blood samples were collected3weeks after the booster immunization for detection of of FMDV-specific antibody response. Results and Conclusions Oral administration of RAMPS tended to enhance serum sepcific IgG response against FMDV immunization, neither volatile oil nor atractylenolide I have been found adjuvant properties in our experiments. Therefore, the adjuvant activities of RAM may be attributed to RAMPS.
3. Adjuvant effects of oral administration of RAMPS to FMD vaccine in mice
Objective To investigate the effect of RAMPS on the immune responses to a commercial FMD vaccine in mice. Methods Thirty-five female ICR mice were randomly divided into five groups with7mice in each. The animals were subcutaneously injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or RAMPS (0.025,0.05or0.1g) or RAM decoction (0.25g). Blood samples were collected3weeks after the booster immunization for detection of IgG titers, the IgG subclasses and western blot analysis. Splenocytes were harvested for determination of lymphocyte proliferation and cytokines mRNA expression. Results After oral administration for4days of RAMPS, immunization of a commercial FMD vaccine induced significantly higher serum specific IgG and the IgG isotype responses in association with up-regulated serum IFN-y and IL-5. In addition, RAMPS significantly increased splenocyte proliferative responses to ConA, LPS and FMDV, as well as mRNA expression of Thl/Th2cytokines (IFN-y/IL-4) and transcription factors (T-bet/GATA-3) by splenocytes. Conclusions Oral administration of RAMPS can enhance the activities of T and B cells and promote a balanced Th1/Th2immune responses against FMD in mice. Considering its natural origin and low side effects, RAMPS could be used as an effective adjuvant to FMD vaccine.
4. Effects of oral administration of RAMPS on the intestinal mucosal immunity of mice
Objective To investigate the effect of RAMPS on the intestinal mucosal immune syetem of mice. Methods Fifty-six female ICR mice were randomly divided into four groups with14mice in each. Two groups of the animals were subcutaneous!y injected twice with200μl of FMDV type O vaccine with2-week intervals. One day before each immunization, the mice had already been orally administered for4days with0.25ml of0.89%saline solution, or RAMPS0.05g.The other two groups were not immunized with vaccine, but had been orally administered at the same time.One week after first immunization and two weeks after the booster immunization, half mice of every group were sacrificed. Blood samples were collected for detection of antigen-specific antibody response. Intestine duodenum of mice were seperated. Fecal samples were collected from individual mice and extracted by making a1:10suspension (wt/vol) with stool diluent. The suspension was vortexed and centrifuged for20min at12000g. The supernatant was collected for detection of Total IgA levels. Part of the duodenum was grinded with liquid nitrogen for determination of cytokine mRNA expression. Tissue paraffin sections were prepared and stained with haematoxylin and eosin (HE) for analysis of intraepithelial lymphocytes (IEL) and immunohistochemical analysis were also performed for analysis of IgA+plasma cells. Results Oral administration of RAMPS at the dose of0.05g/d for4days could improve serum antigen-specific antibody responses as well as total IgA levels, mRNA expression of TGF-β, IL-6, TNF-α, the area of IgA+plasma cells and the number of IELs in the duodenum of mice. Conclusions Oral administration of RAMPS significantly increased systemic as well as gut mucosal immunity in mice immunized with FMD vaccine.
5. Effect on PRRS of a macrophage cell line of mice by RAMPS
Objective To observe the effect of RAMPS on the activation of PRRs in a macrophage cell line of mice. Methods Raw-Blue is a macrophage cell line (Raw264.7), which was stably transfected with plasmid (pNifty-2), which can be induced by activated NF-κB to secrete SEAP protein. Raw-Blue cells were stimulated with RAMPS and APS at different final concentration (0、1、48、16、25、50、100、200μg/ml) for18h, then the SEAP in supernate were detected by substrate. Results and Conlusions There was no significantly ascensus of SEAP in the supernate, thus RAMPS and APS could not activate the signal pathways of NF-κB, showed no effects on PRRs(TLRs or NLRs or RLRs).
All together, neither volatile oil nor atractylenolide I have been found adjuvant properties in our experiments.But oral administration of RAMPS tended to enhance serum sepcific IgG response against FMDV immunization, the activity of T and B cells of the mice and a balanced Th1/Th2immune responses against FMD, as well as the intestinal mucosal immune responses. Therefore, the oral adjuvant activities of RAM may be attributed to the fraction of polysaccharide (RAMPS). But the adjuvant activity of RAMPS was not mediated by TLRs or NLRs or RLRs, the mechanisms need to be further investigated.
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