海带岩藻聚糖分级纯化及对肉仔鸡巨噬细胞免疫调节的研究
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摘要
本研究从海带中提取岩藻聚糖并进行分级纯化,通过肉鸡饲喂试验及细胞培养试验,系统地探讨了海带岩藻聚糖(FLJ)及其级分(Fraction)对肉鸡巨噬细胞免疫功能的调控作用,为研究开发海带岩藻聚糖作为饲料添加剂提供了理论依据。
1、海带岩藻聚糖的提取和分级纯化
经过正交试验获得了海带岩藻聚糖的最佳提取条件,分别为物料比1:25,浸提时间6h,浸提温度100℃。在此条件下,海带岩藻聚糖提取物的得率为16.21%,海带岩藻聚糖得率为5.47%。通过DEAE纤维柱和Sephacryl凝胶柱对海带岩藻聚糖进行分级纯化,得到5个级分,分子量分别为40KD、74KD、163KD、240KD、360KD,各级分中海带岩藻聚糖的纯度大于90%,通过薄层层析分析表明,各个级分均以岩藻糖为主,且含有比例不同的葡萄糖、木糖和鼠李糖,通过紫外光谱和红外光谱分析,表明5个级分均不含有蛋白质和核酸,而且具有多糖特有的振动峰。
2、海带岩藻聚糖对肉鸡生产性能和免疫功能的影响
试验采用单因子设计,将600只1日龄AA肉鸡随机分为4个处理组,每组设6个重复,分别为添加0(对照组)、100 mg/kg(低剂量组)、500 mg/kg(中剂量组)和1000mg/kg(高剂量组)的海带岩藻聚糖,测定肉鸡生产性能和免疫功能。研究结果显示,日粮中添加海带岩藻聚糖对肉鸡生产性能无显著影响,可提高42日龄肉鸡脾脏和胸腺指数,促进ConA诱导的T淋巴细胞转化,提高CD4+T淋巴细胞亚群比例,提高腹腔巨噬细胞iNOS活性和NO产生量,促进呼吸爆发。表明海带岩藻聚糖可提高肉鸡的免疫机能,同时提示肉鸡腹腔巨噬细胞和T淋巴细胞可能是海带岩藻聚糖发挥作用的靶细胞。
3、海带岩藻聚糖及其级分对体外培养肉鸡巨噬细胞免疫功能的影响
通过三个独立试验分别研究不同剂量海带岩藻聚糖及其级分对体外培养肉鸡巨噬细胞功能的影响。试验采用单因子设计,每个试验分为7个处理组,分别为岩藻聚糖组、5个级分组和空白对照组,每个处理组设8个重复。研究结果表明,中、高剂量海带岩藻聚糖对体外培养肉鸡巨噬细胞的免疫功能具有促进作用,能够提高巨噬细胞吞噬活性,促进巨噬细胞呼吸爆发、NO的产生量和iNOS的酶活,促进巨噬细胞分泌细胞因子IL-1;不同级分对肉鸡巨噬细胞免疫功能的调控作用不同,其中FractionⅢ和FractionⅣ的促进作用最强,而FractionⅤ仅在中剂量条件下提高了巨噬细胞NO和iNOS的活性,对其他免疫指标无显著作用。
4、海带岩藻聚糖及其级分对氧化应激条件下巨噬细胞功能的影响
试验采用单因子设计,分为8个处理组,分别为空白对照组、氧化损伤组、氧化损伤+岩藻聚糖组、氧化损伤+5个级分组,每个处理组设8个重复。本试验结果表明,tBOOH诱导肉鸡巨噬细胞氧化损伤,同时显著抑制巨噬细胞的免疫功能,此时添加海带岩藻聚糖及其级分可缓解巨噬细胞的氧化损伤,并提高巨噬细胞的免疫功能。海带岩藻聚糖不同级分的抗氧化活性不同,以FractionⅢ和FractionⅣ的作用最强,同时FractionⅢ和FractionⅣ对巨噬细胞免疫功能的促进作用也最强,由此表明,海带岩藻聚糖对巨噬细胞的免疫促进作用与其抗氧化活性有关。
5、海带岩藻聚糖及其级分对肉鸡巨噬细胞钙信号传导的影响
由两个单独的试验组成,采用单因子完全随机设计。第一个试验共设6个处理,分别为海带岩藻聚糖及其5个级分,测定细胞培养液中加入多糖后的钙信号变化。试验结果显示,海带岩藻聚糖及其级分影响肉鸡巨噬细胞胞内钙离子浓度,其中岩藻聚糖、FractionⅢ、FractionⅣ显著升高胞内钙离子浓度,FractionⅠ、FractionⅡ和FractionⅤ显著降低胞内钙离子浓度。第二个试验共设8个处理,分别为A23187组、EGTA组、岩藻聚糖、岩藻聚糖+EGTA、FractionⅢ、FractionⅢ+EGTA、FractionⅣ、FractionⅣ+EGTA,测定不同处理组的钙离子浓度和巨噬细胞存活率、吞噬活性、NO、iNOS、细胞因子等免疫指标。用A23187升高胞内钙离子浓度,巨噬细胞免疫功能加强,用EGTA螯合细胞外钙,巨噬细胞免疫功能降低,同时岩藻聚糖、FractionⅢ、FractionⅣ加入EGTA后,细胞钙离子浓度显著降低,巨噬细胞的吞噬功能、呼吸爆发、NO、iNOS、细胞因子IL-1均显著降低。结果表明,海带岩藻聚糖、FractionⅢ、FractionⅣ促进巨噬细胞免疫功能与其促进细胞钙离子浓度的升高有关。
综合上述体内、体外试验结果,可以得出:海带岩藻聚糖可显著提高肉鸡腹腔巨噬细胞的吞噬活性、呼吸爆发、NO、iNOS活性和细胞因子的分泌,调节肉鸡的免疫功能。不同岩藻聚糖级分对肉鸡巨噬细胞免疫功能的促进作用不同,以FractionⅢ、Ⅳ的作用最为显著。海带岩藻聚糖及其级分对肉鸡巨噬细胞免疫功能的影响与其抗氧化能力和调节细胞内钙离子浓度有关。
1、Extracted and purified Fucoidan from Laminaria japonica
The fucoidans was extracted and isolated from Laminaria japonica by the method of water boiling and ethanol precipitation. The optimized condition were as follows: radio of samples to water,1:25; time, 6h; temperature,100℃.Under the conditions shown above, the resulted content of Fucoidan was 5.47%. Fucoidan was further purified by ion exchange chromatography DEAE and gel filtration chromatography Sephacryl into five different Fractions, FractionⅠto FractionⅤ. Their molecular weights were 40KD、74KD、163KD、240KD、360KD respectively. The polysaccharide was composed of fucose, glucose, xylose, rhamnose according to thin-layer chromatography. All five Fractions exhibited character vibration peak of polysaccharide and had no protein and nucleic acid.
2、Effects of Fucoidan isolated from Laminaria japonica on growth performance and immunity of broilers
The trial was conducted to study the effects of Fucoidan extracted from Laminaria japonica on the immunity and growth performance of broilers. Total of 600 broilers were randomly allotted to one of four dietary treatments, with six replicate pens per treatment and twenty-five chickens per pens. Dietary treatments included a control corn-soybean meal (Control), a diet with 100mg/kg Fucoidan (Low level), a diet with 500mg/kg Fucoidan (Middle level), a diet with 1000mg/kg Fucoidan (High level). Birds were raised from hatch to 42 d. At the age 42d, the growth performance, the immune organ indexes, the proliferation of blood lymphocyte, the proportions of T cell subsets in blood were measured. The macrophages were harvested and the phagocytic activity, NO, iNOS and respiratory burst production of macrophages were detected. Feeding Fucoidan had no significant effects on growth performance of broilers relative to the control. Compared to the control, feeding Fucoidan significantly increased spleen index and thymus index (P<0.05), enhanced the proliferation of T lymphocyte induced by ConA (P<0.05).The percentage of CD4+ T cell was higher in 42d old chickens maintained on Fucoidan feed than in chichens receiving the control corn-soybean meal. Fucoidan had significantly increased macrophages to relesee NO, significantly promoted iNOS activity and stimulated the respiratory burst activity. The Fucoidan isolated from Laminaria japonica possess potent immunomodulatory activity on T lymphocyte and macrophages on broilers.
3、Effects of Fucoidan and its Fraction isolated from Laminaria japonica on immunity of broiler macrophages in vitro
The immunomodulatory activities of Fucoidan (FLJ) and its Fraction were investigated in vitro in relation to broiler peritoneal macrophages. The broiler macrophages were cultured with Fucoidan (200μg/ml) and Fraction fromⅠtoⅤ(100μg/ml) for 24h, the cell vialibility were detected by using the mothod of MTT and the phagocytic activity were detected. The induced nitric oxide synthase (iNOS), NO, IL-1 and TNF-αin culture supernatants were measured. The respiratory burst capacity in macrophages was measured by luminal dependent chemiluminescence.Cell vialibility results showed that the proliferation of macrophages was not influenced by FLJ and its Fractions. The treatment of FLJ and its Fraction increased the phagocytic activity, enhanced the production of IL-1, NO, iNOS and the respiratory burst capacity (P<0.05). The Fucoidan and its Fraction had no significantly effect on TNF-α(P>0.05). The different Fraction had different effect on broiler macrophage immunity function. FractionⅢand FractionⅣhad the most effective promoting action. Their promotion effects were higher than FractionⅤ.The FractionⅤhad no effect on broilers macrophages immunity function. The Fucoidan isolated from Laminaria japonica had promoting effect on broiler macrophage immunity at 200μg/ml.The FractionⅢand FractionⅣpurified from Fucoidan were the effective Fraction among five Fraction.
4、Effect of Fucoidan and its Fraction isolated from Laminaria japonica on immunity of broiler macrophages under oxidation stress condition
The immunomodulatory activities of a Fucoidan (FLJ) and its Fraction were investigated in vitro under oxidation stress in broiler macrophages. After the broiler macrophages were cultured with FLJ and its Fraction for 24h, the tBOOH was added to the culture as oxygen reagent. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and induced nitric oxide synthase (iNOS) were detected. The content of lactate dehydrogenase (LDH), malonic dialdehyde (MDA), NO, IL-1 and TNF-αwere measured. The respiratory burst capacity of broiler macrophages was measured by luminal dependent chemiluminescence. The elevated MDA and LDH, decreased cell vialibility, increased SOD and GSH-Px levels indicated that adding tBOOH in culture caused the oxidation stress in broiler macrophages. The decreased of phagocytic activity, NO, iNOS, IL-1 and respiratory burst capacity indicated that the immune function of broiler macrophages were influenced by tBOOH. When the FLJ and its Fraction were present in culture, the oxidation stress were eliminated. The cell vialibility, SOD and GSH-Px were significantly increased (P<0.05), LDH and MDA were significantly decreased (P<0.05). The immune function of macrophages was enhanced. The different Fraction had different antioxidant activities. FractionⅢand FractionⅣhad the most effective antioxidant activities. These data suggest that the FLJ and its Fraction had antioxidant activities, and the antioxidant activities of FLJ and its Fraction were related to their immunomodulatory activities.
5、Macrophage Immunomodulatory mechanism of Fucoidan and Fraction isolated from Laminaria japonica: Role of calcium
The effect of Fucoidan (FLJ) and its Fraction on Ca~(2+) content of broiler macrophages were investigated in vitro to conform if FLJ and its Fraction can activize Ca~(2+) signal transmit system in immune cell. Using the highly sensitive Ca~(2+) fluorescent dye, Fura-2/AM, the kinetic changes of Ca~(2+) in single intact living cells were measured, before and after FLJ and its Fraction directly treatment. Using A23187 and EGTA to change Ca~(2+) content of macrophages, the immunity of cell were detected in vitro. The intracellular Ca~(2+) content increased after FLJ treatment (P<0.05). The different Fraction had different action to the Ca~(2+) content. The FractionⅢand FractionⅣinduced intracellular Ca~(2+) content increase (P<0.05), while the FractionⅠ, FractionⅡand FractionⅤinduced intracellular Ca~(2+) content decreased (P<0.05). Using A23187 to increase the Ca~(2+) content of lived cell, the immune function of broiler macrophages were enhanced, while using EGTA to decrease the Ca~(2+) content, the immune function of lived cell were reduced. After adding EGTA and Fraction into the culture at the same time, the phagocytic activity, the respiratory burst capacity, the production of IL-1, NO, iNOS of broiler macrophages were decreased significantly (P<0.05). The immunomodulatory activities of a Fucoidan and its Fraction had a relationship with increasing the intracellular Ca~(2+) content.
1、海带岩藻聚糖的提取和分级纯化
经过正交试验获得了海带岩藻聚糖的最佳提取条件,分别为物料比1:25,浸提时间6h,浸提温度100℃。在此条件下,海带岩藻聚糖提取物的得率为16.21%,海带岩藻聚糖得率为5.47%。通过DEAE纤维柱和Sephacryl凝胶柱对海带岩藻聚糖进行分级纯化,得到5个级分,分子量分别为40KD、74KD、163KD、240KD、360KD,各级分中海带岩藻聚糖的纯度大于90%,通过薄层层析分析表明,各个级分均以岩藻糖为主,且含有比例不同的葡萄糖、木糖和鼠李糖,通过紫外光谱和红外光谱分析,表明5个级分均不含有蛋白质和核酸,而且具有多糖特有的振动峰。
2、海带岩藻聚糖对肉鸡生产性能和免疫功能的影响
试验采用单因子设计,将600只1日龄AA肉鸡随机分为4个处理组,每组设6个重复,分别为添加0(对照组)、100 mg/kg(低剂量组)、500 mg/kg(中剂量组)和1000mg/kg(高剂量组)的海带岩藻聚糖,测定肉鸡生产性能和免疫功能。研究结果显示,日粮中添加海带岩藻聚糖对肉鸡生产性能无显著影响,可提高42日龄肉鸡脾脏和胸腺指数,促进ConA诱导的T淋巴细胞转化,提高CD4+T淋巴细胞亚群比例,提高腹腔巨噬细胞iNOS活性和NO产生量,促进呼吸爆发。表明海带岩藻聚糖可提高肉鸡的免疫机能,同时提示肉鸡腹腔巨噬细胞和T淋巴细胞可能是海带岩藻聚糖发挥作用的靶细胞。
3、海带岩藻聚糖及其级分对体外培养肉鸡巨噬细胞免疫功能的影响
通过三个独立试验分别研究不同剂量海带岩藻聚糖及其级分对体外培养肉鸡巨噬细胞功能的影响。试验采用单因子设计,每个试验分为7个处理组,分别为岩藻聚糖组、5个级分组和空白对照组,每个处理组设8个重复。研究结果表明,中、高剂量海带岩藻聚糖对体外培养肉鸡巨噬细胞的免疫功能具有促进作用,能够提高巨噬细胞吞噬活性,促进巨噬细胞呼吸爆发、NO的产生量和iNOS的酶活,促进巨噬细胞分泌细胞因子IL-1;不同级分对肉鸡巨噬细胞免疫功能的调控作用不同,其中FractionⅢ和FractionⅣ的促进作用最强,而FractionⅤ仅在中剂量条件下提高了巨噬细胞NO和iNOS的活性,对其他免疫指标无显著作用。
4、海带岩藻聚糖及其级分对氧化应激条件下巨噬细胞功能的影响
试验采用单因子设计,分为8个处理组,分别为空白对照组、氧化损伤组、氧化损伤+岩藻聚糖组、氧化损伤+5个级分组,每个处理组设8个重复。本试验结果表明,tBOOH诱导肉鸡巨噬细胞氧化损伤,同时显著抑制巨噬细胞的免疫功能,此时添加海带岩藻聚糖及其级分可缓解巨噬细胞的氧化损伤,并提高巨噬细胞的免疫功能。海带岩藻聚糖不同级分的抗氧化活性不同,以FractionⅢ和FractionⅣ的作用最强,同时FractionⅢ和FractionⅣ对巨噬细胞免疫功能的促进作用也最强,由此表明,海带岩藻聚糖对巨噬细胞的免疫促进作用与其抗氧化活性有关。
5、海带岩藻聚糖及其级分对肉鸡巨噬细胞钙信号传导的影响
由两个单独的试验组成,采用单因子完全随机设计。第一个试验共设6个处理,分别为海带岩藻聚糖及其5个级分,测定细胞培养液中加入多糖后的钙信号变化。试验结果显示,海带岩藻聚糖及其级分影响肉鸡巨噬细胞胞内钙离子浓度,其中岩藻聚糖、FractionⅢ、FractionⅣ显著升高胞内钙离子浓度,FractionⅠ、FractionⅡ和FractionⅤ显著降低胞内钙离子浓度。第二个试验共设8个处理,分别为A23187组、EGTA组、岩藻聚糖、岩藻聚糖+EGTA、FractionⅢ、FractionⅢ+EGTA、FractionⅣ、FractionⅣ+EGTA,测定不同处理组的钙离子浓度和巨噬细胞存活率、吞噬活性、NO、iNOS、细胞因子等免疫指标。用A23187升高胞内钙离子浓度,巨噬细胞免疫功能加强,用EGTA螯合细胞外钙,巨噬细胞免疫功能降低,同时岩藻聚糖、FractionⅢ、FractionⅣ加入EGTA后,细胞钙离子浓度显著降低,巨噬细胞的吞噬功能、呼吸爆发、NO、iNOS、细胞因子IL-1均显著降低。结果表明,海带岩藻聚糖、FractionⅢ、FractionⅣ促进巨噬细胞免疫功能与其促进细胞钙离子浓度的升高有关。
综合上述体内、体外试验结果,可以得出:海带岩藻聚糖可显著提高肉鸡腹腔巨噬细胞的吞噬活性、呼吸爆发、NO、iNOS活性和细胞因子的分泌,调节肉鸡的免疫功能。不同岩藻聚糖级分对肉鸡巨噬细胞免疫功能的促进作用不同,以FractionⅢ、Ⅳ的作用最为显著。海带岩藻聚糖及其级分对肉鸡巨噬细胞免疫功能的影响与其抗氧化能力和调节细胞内钙离子浓度有关。
1、Extracted and purified Fucoidan from Laminaria japonica
The fucoidans was extracted and isolated from Laminaria japonica by the method of water boiling and ethanol precipitation. The optimized condition were as follows: radio of samples to water,1:25; time, 6h; temperature,100℃.Under the conditions shown above, the resulted content of Fucoidan was 5.47%. Fucoidan was further purified by ion exchange chromatography DEAE and gel filtration chromatography Sephacryl into five different Fractions, FractionⅠto FractionⅤ. Their molecular weights were 40KD、74KD、163KD、240KD、360KD respectively. The polysaccharide was composed of fucose, glucose, xylose, rhamnose according to thin-layer chromatography. All five Fractions exhibited character vibration peak of polysaccharide and had no protein and nucleic acid.
2、Effects of Fucoidan isolated from Laminaria japonica on growth performance and immunity of broilers
The trial was conducted to study the effects of Fucoidan extracted from Laminaria japonica on the immunity and growth performance of broilers. Total of 600 broilers were randomly allotted to one of four dietary treatments, with six replicate pens per treatment and twenty-five chickens per pens. Dietary treatments included a control corn-soybean meal (Control), a diet with 100mg/kg Fucoidan (Low level), a diet with 500mg/kg Fucoidan (Middle level), a diet with 1000mg/kg Fucoidan (High level). Birds were raised from hatch to 42 d. At the age 42d, the growth performance, the immune organ indexes, the proliferation of blood lymphocyte, the proportions of T cell subsets in blood were measured. The macrophages were harvested and the phagocytic activity, NO, iNOS and respiratory burst production of macrophages were detected. Feeding Fucoidan had no significant effects on growth performance of broilers relative to the control. Compared to the control, feeding Fucoidan significantly increased spleen index and thymus index (P<0.05), enhanced the proliferation of T lymphocyte induced by ConA (P<0.05).The percentage of CD4+ T cell was higher in 42d old chickens maintained on Fucoidan feed than in chichens receiving the control corn-soybean meal. Fucoidan had significantly increased macrophages to relesee NO, significantly promoted iNOS activity and stimulated the respiratory burst activity. The Fucoidan isolated from Laminaria japonica possess potent immunomodulatory activity on T lymphocyte and macrophages on broilers.
3、Effects of Fucoidan and its Fraction isolated from Laminaria japonica on immunity of broiler macrophages in vitro
The immunomodulatory activities of Fucoidan (FLJ) and its Fraction were investigated in vitro in relation to broiler peritoneal macrophages. The broiler macrophages were cultured with Fucoidan (200μg/ml) and Fraction fromⅠtoⅤ(100μg/ml) for 24h, the cell vialibility were detected by using the mothod of MTT and the phagocytic activity were detected. The induced nitric oxide synthase (iNOS), NO, IL-1 and TNF-αin culture supernatants were measured. The respiratory burst capacity in macrophages was measured by luminal dependent chemiluminescence.Cell vialibility results showed that the proliferation of macrophages was not influenced by FLJ and its Fractions. The treatment of FLJ and its Fraction increased the phagocytic activity, enhanced the production of IL-1, NO, iNOS and the respiratory burst capacity (P<0.05). The Fucoidan and its Fraction had no significantly effect on TNF-α(P>0.05). The different Fraction had different effect on broiler macrophage immunity function. FractionⅢand FractionⅣhad the most effective promoting action. Their promotion effects were higher than FractionⅤ.The FractionⅤhad no effect on broilers macrophages immunity function. The Fucoidan isolated from Laminaria japonica had promoting effect on broiler macrophage immunity at 200μg/ml.The FractionⅢand FractionⅣpurified from Fucoidan were the effective Fraction among five Fraction.
4、Effect of Fucoidan and its Fraction isolated from Laminaria japonica on immunity of broiler macrophages under oxidation stress condition
The immunomodulatory activities of a Fucoidan (FLJ) and its Fraction were investigated in vitro under oxidation stress in broiler macrophages. After the broiler macrophages were cultured with FLJ and its Fraction for 24h, the tBOOH was added to the culture as oxygen reagent. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and induced nitric oxide synthase (iNOS) were detected. The content of lactate dehydrogenase (LDH), malonic dialdehyde (MDA), NO, IL-1 and TNF-αwere measured. The respiratory burst capacity of broiler macrophages was measured by luminal dependent chemiluminescence. The elevated MDA and LDH, decreased cell vialibility, increased SOD and GSH-Px levels indicated that adding tBOOH in culture caused the oxidation stress in broiler macrophages. The decreased of phagocytic activity, NO, iNOS, IL-1 and respiratory burst capacity indicated that the immune function of broiler macrophages were influenced by tBOOH. When the FLJ and its Fraction were present in culture, the oxidation stress were eliminated. The cell vialibility, SOD and GSH-Px were significantly increased (P<0.05), LDH and MDA were significantly decreased (P<0.05). The immune function of macrophages was enhanced. The different Fraction had different antioxidant activities. FractionⅢand FractionⅣhad the most effective antioxidant activities. These data suggest that the FLJ and its Fraction had antioxidant activities, and the antioxidant activities of FLJ and its Fraction were related to their immunomodulatory activities.
5、Macrophage Immunomodulatory mechanism of Fucoidan and Fraction isolated from Laminaria japonica: Role of calcium
The effect of Fucoidan (FLJ) and its Fraction on Ca~(2+) content of broiler macrophages were investigated in vitro to conform if FLJ and its Fraction can activize Ca~(2+) signal transmit system in immune cell. Using the highly sensitive Ca~(2+) fluorescent dye, Fura-2/AM, the kinetic changes of Ca~(2+) in single intact living cells were measured, before and after FLJ and its Fraction directly treatment. Using A23187 and EGTA to change Ca~(2+) content of macrophages, the immunity of cell were detected in vitro. The intracellular Ca~(2+) content increased after FLJ treatment (P<0.05). The different Fraction had different action to the Ca~(2+) content. The FractionⅢand FractionⅣinduced intracellular Ca~(2+) content increase (P<0.05), while the FractionⅠ, FractionⅡand FractionⅤinduced intracellular Ca~(2+) content decreased (P<0.05). Using A23187 to increase the Ca~(2+) content of lived cell, the immune function of broiler macrophages were enhanced, while using EGTA to decrease the Ca~(2+) content, the immune function of lived cell were reduced. After adding EGTA and Fraction into the culture at the same time, the phagocytic activity, the respiratory burst capacity, the production of IL-1, NO, iNOS of broiler macrophages were decreased significantly (P<0.05). The immunomodulatory activities of a Fucoidan and its Fraction had a relationship with increasing the intracellular Ca~(2+) content.
引文
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