甘草多糖的提取及对小鼠腹腔巨噬细胞的免疫调节
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摘要
几千年来,甘草因具有广泛的医用药用价值,被历代名医尊称为“众药之王”。甘草多糖(glycyrrhiza polysaccharid,GP)是甘草中重要活性成分之一,近些年来的药理学研究表明,GP具有增强免疫、噬菌、抗病毒、抗补体等作用,且没有细胞毒副作用,但目前对GP的研究相对较少,并且多以粗多糖为试验材料。本论文以提取纯化GP为原料,详细研究了GP对巨噬细胞的免疫调节机理。
首先,对影响GP提取率的主要因素进行了研究,先采用单因素试验确定出合适的水平,然后利用响应面程序进行优化,最佳提取条件为液固比10.27∶1,浸提温度88.53℃,浸提时间140.97 min,提取次数为2次,理论上粗多糖提取率可达4.25%,其中浸提时间对提取率的影响最大。通过对多糖析出条件的研究,证明浓缩后体积为提取液总体积的1/4-1/5,浓缩液与无水乙醇的体积比在1∶3-1∶4,在4℃条件多糖提取率较高,GP经过DEAE-52和G-200层析柱的洗脱分离,冷冻干燥后为纯度约为90.7%,多糖得率约为0.87%。
通过高效液相色谱法确定GP的相对分子量约为11 kDa。利用原子力显微镜观察,GP是具有空间结构的复杂大分子,是以葡萄糖为主链,通过α-(1,4)键连接的单一吡喃型葡聚糖结构。同时发现GP分子链呈现出单个的分子链及其多个侧枝的结构,并且还有些大大小小的环状和螺旋状结构,GP分子间存在螺旋(有序)和线圈(无序)间的可逆热转换,这两种状态具有共存现象。
通过研究GP对巨噬细胞化学形态学影响及进行化学定量分析,初步探讨了GP对巨噬细胞激活的作用机理。研究表明巨噬细胞受到GP刺激进一步分化成熟,成为活化巨噬细胞,使细胞体积增大,活力增强,糖氧化作用和吞噬功能增加,促使细胞内溶菌酶大量增多,细胞内DNA、RNA、糖原体、ACPase、ANAE、ATPase和SDH等多种酶活性显著增强,并能明显增强SOD和O2-的活性。巨噬细胞膜上的非特异性受体通过其终端的-OH基与多糖-H基结合,启动了细胞内环化酶系统,使细胞的cGMP增加,致细胞内酶的分泌及活性增加,细胞代谢能力提高,奠定了巨噬细胞活化的基础。细胞因子是免疫细胞中重要的免疫因子,进一步从细胞因子的角度考察了GP对巨噬细胞的激活机理。结果证明当巨噬细胞培养48 h,在GP添加0-400μg/mL范围内,能剂量依赖地增加巨噬细胞中IL-1、IL-6、IL-12、TNF-α和淋巴细胞中IL-2的分泌量,其中IL-6的分泌量最大。同样培养时间(0-72 h)也能明显影响细胞因子的分泌量,在72 h时分泌量最大。
NO作为信息分子和细胞毒分子的双重因子,通过研究GP对NO、iNOS和核蛋白的影响证明:GP添加浓度和细胞培养时间对NO、iNOS和核蛋白的生成量都有明显的影响,随着GP添加浓度的增加和细胞培养时间延长,NO、iNOS和核蛋白的生成量明显增加。GP与LPS/IFN-γ协同实验证明:GP与LPS/IFN-γ协同增加NO和iNOS分泌量,并明显高于相应的单独添加GP组。用LPS预刺激巨噬细胞,再加入GP产生的NO、iNOS和核蛋白量以及增加趋势明显高于同时添加LPS和GP产生的量。巨噬细胞抗肿瘤、抗病毒、抑制胞内病原体增殖等作用主要通过NO的释放完成的。
mRNA是联结基因与表达产物之间的桥梁,基因活化的直接结果是mRNA转录的增加,而蛋白质的合成依赖于mRNA的翻译。采用RT-PCR方法研究GP对巨噬细胞IL-1α、IL-1β、IL-6、IL-12p35、iNOS、TNF-α的mRNA表达情况,进一步证实GP直接影响蛋白质代谢,可在转录水平和翻译水平调节蛋白质合成,验证GP通过促进细胞因子的基因表达来促进机体的免疫调节功能可能是其免疫激活的机理之一。
Glycyrrhiza uralensis Fisch has been known as“the king of herbs”due to its widely medicinal and officinal properties for thousands of years. Glycyrrhiza polysaccharide (GP), one of the main active ingredients of glycyrrhiza is attributed to many healing properties of the herb. Recently, it has been reported that GP has many functions such as immunity regulation, phagocytosis, anti-complement, anti-virus, anti-tumor, and it has low cellular toxicity. At present, there were not many studies of GP, and the experiment materials were crude polysaccharides. In the present study, the immunomodulatory effects of GP on mice macrophages were demonstrated by using the purified polysaccharides.
The main factors on the yield of GP were evaluated, and the optimized conditions are a water/root mass ratio of 10.27:1, extraction temperature 88.53℃, time 140.97 min, frequency 2, the maximal yield of GP was 4.25% in theory, and extraction time was the most impact factor. The separated factors of GP were also studied, the optimized conditions were the volume of condense to the pre-condense of 1/4-1/5, the volume condense fluid to ethanol 1:3-1:4, temperature 4℃. Total polysaccharide obtained from the elution that was fractionated by DEAE-52 and G-200 columns. Percentage and the yield of purified polysaccharide was approximately 90.7% and 0.87% respectively.
The molecular weight of purified GP measured by HPLC was approximately 11 kDa. GP is the complex large molecule which has space structure equally to protein and enzyme. glycosidic units were composed ofα(1→4) linked D-glucan. Molecule backbone composed of single chain and many side chains, big and small rings and helix structures. GP exists in the reversible thermal transition of helix (order) to loop (disorder), and has a concomitant phenomenon.
The mechanism of GP on mice macrophages was primarily discussed by investigating of cytochemistry shape and quantitative analysis. The results showed that macrophages were stimulated and maturated by GP and become activated cells, and which make the cell volume augment, energy enhancement, oxidation effect and phagocytosis increase, intracellular carbohydrate, DNA, RNA, ACPase, ANAE, ATPase, SDH and lysozyme were largely enhanced, the level of SOD and O2- was increased. The non-specific acceptor of cell wall in macrophages was linked by -OH of terminal non-specific acceptor with -H radicel of polysaccharide, and inspired the system of intracellular cyclic enzyme, make cGMP enhanced, resulted in the increase of enzyme level, the improvement of metabolism level of macrophages, which is the basic of macrophages activity.
Cytokines are the important of immune factors of immune cells. The activation mechanism of GP on the macrophages was further investigated from the point of cytokines. The results showed that the production of IL-1、IL-6、IL-12、TNF-αand IL-2 was increased in a dose-depended manner for 48 h at the concentration of 0-400μg/mL GP, and maximal at the dose of 400μg/mL. Likely, the production of IL-1、IL-6、IL-12、TNF-αand IL-2 was increased in a time-depended manner for 0-72 h and peaked at the 72 h time point.
NO is the principal messenger and effector molecule produced by macrophages for cytotoxic activity and can be used as a quantitative index of macrophage activation. The level of NO, iNOS and nuclein in macrophages stimulated by GP was studied. The results showed that the production of NO, iNOS and nuclein was significantly increased in dose and time-depended manners. Macrophages were used to study the synergistic effects of GP plus LPS on production of NO, iNOS and nuclein. The results showed that production of NO, iNOS and nuclein of macrophage treated with GP and LPS/IFN-γwas increased as compared with those of GP alone after 48 h incubation. Increases of NO and iNOS production exposed to combined GP with LPS were lower than those of pretreated with LPS at the same concentrations of GP. NO is involved in anti-tumor, anti-virus, anti-microbial defenses in macrophages.
mRNA is the bridge which linked gene with expression, the gene activation is directed to the increase of mRNA, and synthesization of protein relies on mRNA transcription. The gene expressions of IL-1α、IL-1β、IL-6、IL-12p35、iNOS、TNF-αmRNA were assayed by RT-PCR. The levels of those above cytokines in macrophages were increased when cultured with GP. It was suggested that the immunomodulating effect of GP may be associated with cytokine mRNA expression stimulation at the transcriptive level.
首先,对影响GP提取率的主要因素进行了研究,先采用单因素试验确定出合适的水平,然后利用响应面程序进行优化,最佳提取条件为液固比10.27∶1,浸提温度88.53℃,浸提时间140.97 min,提取次数为2次,理论上粗多糖提取率可达4.25%,其中浸提时间对提取率的影响最大。通过对多糖析出条件的研究,证明浓缩后体积为提取液总体积的1/4-1/5,浓缩液与无水乙醇的体积比在1∶3-1∶4,在4℃条件多糖提取率较高,GP经过DEAE-52和G-200层析柱的洗脱分离,冷冻干燥后为纯度约为90.7%,多糖得率约为0.87%。
通过高效液相色谱法确定GP的相对分子量约为11 kDa。利用原子力显微镜观察,GP是具有空间结构的复杂大分子,是以葡萄糖为主链,通过α-(1,4)键连接的单一吡喃型葡聚糖结构。同时发现GP分子链呈现出单个的分子链及其多个侧枝的结构,并且还有些大大小小的环状和螺旋状结构,GP分子间存在螺旋(有序)和线圈(无序)间的可逆热转换,这两种状态具有共存现象。
通过研究GP对巨噬细胞化学形态学影响及进行化学定量分析,初步探讨了GP对巨噬细胞激活的作用机理。研究表明巨噬细胞受到GP刺激进一步分化成熟,成为活化巨噬细胞,使细胞体积增大,活力增强,糖氧化作用和吞噬功能增加,促使细胞内溶菌酶大量增多,细胞内DNA、RNA、糖原体、ACPase、ANAE、ATPase和SDH等多种酶活性显著增强,并能明显增强SOD和O2-的活性。巨噬细胞膜上的非特异性受体通过其终端的-OH基与多糖-H基结合,启动了细胞内环化酶系统,使细胞的cGMP增加,致细胞内酶的分泌及活性增加,细胞代谢能力提高,奠定了巨噬细胞活化的基础。细胞因子是免疫细胞中重要的免疫因子,进一步从细胞因子的角度考察了GP对巨噬细胞的激活机理。结果证明当巨噬细胞培养48 h,在GP添加0-400μg/mL范围内,能剂量依赖地增加巨噬细胞中IL-1、IL-6、IL-12、TNF-α和淋巴细胞中IL-2的分泌量,其中IL-6的分泌量最大。同样培养时间(0-72 h)也能明显影响细胞因子的分泌量,在72 h时分泌量最大。
NO作为信息分子和细胞毒分子的双重因子,通过研究GP对NO、iNOS和核蛋白的影响证明:GP添加浓度和细胞培养时间对NO、iNOS和核蛋白的生成量都有明显的影响,随着GP添加浓度的增加和细胞培养时间延长,NO、iNOS和核蛋白的生成量明显增加。GP与LPS/IFN-γ协同实验证明:GP与LPS/IFN-γ协同增加NO和iNOS分泌量,并明显高于相应的单独添加GP组。用LPS预刺激巨噬细胞,再加入GP产生的NO、iNOS和核蛋白量以及增加趋势明显高于同时添加LPS和GP产生的量。巨噬细胞抗肿瘤、抗病毒、抑制胞内病原体增殖等作用主要通过NO的释放完成的。
mRNA是联结基因与表达产物之间的桥梁,基因活化的直接结果是mRNA转录的增加,而蛋白质的合成依赖于mRNA的翻译。采用RT-PCR方法研究GP对巨噬细胞IL-1α、IL-1β、IL-6、IL-12p35、iNOS、TNF-α的mRNA表达情况,进一步证实GP直接影响蛋白质代谢,可在转录水平和翻译水平调节蛋白质合成,验证GP通过促进细胞因子的基因表达来促进机体的免疫调节功能可能是其免疫激活的机理之一。
Glycyrrhiza uralensis Fisch has been known as“the king of herbs”due to its widely medicinal and officinal properties for thousands of years. Glycyrrhiza polysaccharide (GP), one of the main active ingredients of glycyrrhiza is attributed to many healing properties of the herb. Recently, it has been reported that GP has many functions such as immunity regulation, phagocytosis, anti-complement, anti-virus, anti-tumor, and it has low cellular toxicity. At present, there were not many studies of GP, and the experiment materials were crude polysaccharides. In the present study, the immunomodulatory effects of GP on mice macrophages were demonstrated by using the purified polysaccharides.
The main factors on the yield of GP were evaluated, and the optimized conditions are a water/root mass ratio of 10.27:1, extraction temperature 88.53℃, time 140.97 min, frequency 2, the maximal yield of GP was 4.25% in theory, and extraction time was the most impact factor. The separated factors of GP were also studied, the optimized conditions were the volume of condense to the pre-condense of 1/4-1/5, the volume condense fluid to ethanol 1:3-1:4, temperature 4℃. Total polysaccharide obtained from the elution that was fractionated by DEAE-52 and G-200 columns. Percentage and the yield of purified polysaccharide was approximately 90.7% and 0.87% respectively.
The molecular weight of purified GP measured by HPLC was approximately 11 kDa. GP is the complex large molecule which has space structure equally to protein and enzyme. glycosidic units were composed ofα(1→4) linked D-glucan. Molecule backbone composed of single chain and many side chains, big and small rings and helix structures. GP exists in the reversible thermal transition of helix (order) to loop (disorder), and has a concomitant phenomenon.
The mechanism of GP on mice macrophages was primarily discussed by investigating of cytochemistry shape and quantitative analysis. The results showed that macrophages were stimulated and maturated by GP and become activated cells, and which make the cell volume augment, energy enhancement, oxidation effect and phagocytosis increase, intracellular carbohydrate, DNA, RNA, ACPase, ANAE, ATPase, SDH and lysozyme were largely enhanced, the level of SOD and O2- was increased. The non-specific acceptor of cell wall in macrophages was linked by -OH of terminal non-specific acceptor with -H radicel of polysaccharide, and inspired the system of intracellular cyclic enzyme, make cGMP enhanced, resulted in the increase of enzyme level, the improvement of metabolism level of macrophages, which is the basic of macrophages activity.
Cytokines are the important of immune factors of immune cells. The activation mechanism of GP on the macrophages was further investigated from the point of cytokines. The results showed that the production of IL-1、IL-6、IL-12、TNF-αand IL-2 was increased in a dose-depended manner for 48 h at the concentration of 0-400μg/mL GP, and maximal at the dose of 400μg/mL. Likely, the production of IL-1、IL-6、IL-12、TNF-αand IL-2 was increased in a time-depended manner for 0-72 h and peaked at the 72 h time point.
NO is the principal messenger and effector molecule produced by macrophages for cytotoxic activity and can be used as a quantitative index of macrophage activation. The level of NO, iNOS and nuclein in macrophages stimulated by GP was studied. The results showed that the production of NO, iNOS and nuclein was significantly increased in dose and time-depended manners. Macrophages were used to study the synergistic effects of GP plus LPS on production of NO, iNOS and nuclein. The results showed that production of NO, iNOS and nuclein of macrophage treated with GP and LPS/IFN-γwas increased as compared with those of GP alone after 48 h incubation. Increases of NO and iNOS production exposed to combined GP with LPS were lower than those of pretreated with LPS at the same concentrations of GP. NO is involved in anti-tumor, anti-virus, anti-microbial defenses in macrophages.
mRNA is the bridge which linked gene with expression, the gene activation is directed to the increase of mRNA, and synthesization of protein relies on mRNA transcription. The gene expressions of IL-1α、IL-1β、IL-6、IL-12p35、iNOS、TNF-αmRNA were assayed by RT-PCR. The levels of those above cytokines in macrophages were increased when cultured with GP. It was suggested that the immunomodulating effect of GP may be associated with cytokine mRNA expression stimulation at the transcriptive level.
引文
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