麒麟菜天然海藻色素糖蛋白的制备及其对H_(22)肝癌的抑制作用研究
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摘要
目的研究从麒麟菜中提取天然海藻色素糖蛋白(Natural Seaweed Pigment Glycoprotein,英文简称NSPG)的方法并制备实验样品。应用红外、紫外分光法、液相-质谱法、凝胶色谱法等技术手段测定天然海藻色素糖蛋白样品的分子量及结构特征等指标,采用体外细胞培养和体内实验方法,观察麒麟菜天然海藻色素糖蛋白对H22肝癌的抑制作用。
方法包括麒麟菜天然海藻色素糖蛋白的制备与分析,及其对肝癌抑制作用等实验方法：
1NSPG的制备与分析
1.1NSPG制备：将洗净的麒麟菜切为0.5-1.0cm的长条,置于pH3.0的盐酸溶液中浸提,将上述酸性浸提液进行减压抽滤,收集滤液调节pH至4.5,再加入3倍体积的95%的乙醇,搅拌,放置过夜,析出沉淀,真空抽滤得到滤纸上的沉淀部分,将沉淀分别部分用无水乙醇、丙酮洗涤,室温干燥,得NSPG。
1.2NSPG组成与结构分析
1.2.1NSPG多糖和蛋白质含量测定：以葡萄糖为标准物质,用硫酸-苯酚法测定NSPG中多糖的含量。以牛血清白蛋白为标准物质,用考马斯亮蓝法测定NSPG中蛋白质的含量。
1.2.2NSPG结构分析：用紫外和红外光谱法测定多糖和蛋白质的特征吸收峰。根据β-消除反应和液-质谱法确定糖苷键的类型。
1.2.3NSPG分子量测定：用凝胶色谱分析法测定NSPG的分子量。
1.2.4感官等理化指标：包括形态、色泽与水溶性等指标测定。
2NSPG对肝癌的抑制作用研究
2.1体外实验：对肝癌细胞增殖活性的抑制作用进行研究。
将小鼠H22肝癌细胞和HepG2人肝癌细胞分别与不同剂量的NSPG(浓度分别为10mg/L、50mg/L、100mg/L)共同培养,另设不加NSPG的作为对照,用MTT法测定肝癌细胞增殖活性。
2.2体内实验：对小鼠H22肝癌的抑制作用进行研究
无菌条件下抽取接种H22肝癌细胞第6d的小鼠腹水,接种在50只体重为18-20g的健康昆明种小鼠的右侧腋窝处,每只皮下注射0.2ml。将接种的小鼠随机分5个组：肿瘤对照组、低、中、高剂量NSPG组和环磷酰胺组,每组十只,雌雄各一半。其中三个NSPG剂量组每日经口灌胃浓度分别为10、50、100mg/kg的水溶液,给对照组经口灌胃等量的生理盐水,环磷酰胺组小鼠隔日1次腹腔注射20mg/kg的环磷酰胺,连续10d。最后一次给药后第二天将小鼠称重→摘小鼠眼球取血,分离血清→取瘤组织、取脾脏和胸腺,用电子天平称量重量→剥取瘤组织备检。
2.2.1瘤体比和抑瘤率：用电子天平称取小鼠体重、瘤重并计算瘤重与体重比值得出瘤体比、并计算抑瘤率。
2.2.2胸腺指数和脾脏指数：取动物脾脏和胸腺,用电子天平称量重量,根据体重计算脾脏指数和胸腺指数。
2.2.3血清肿瘤坏死因子(tumor necrosis factor,TNF)-α水平：摘小鼠眼球取血,分离血清后用ELISA法检测血清的TNF-α水平。
2.2.4瘤细胞增殖活性：用四甲基偶氮唑(MTT)法检测各组小鼠肿瘤细胞的增殖活性。
2.2.5瘤组织中PCNA、Caspase-3、Bax、Bcl-2蛋白表达水平：用免疫组化法检测。
结果麒麟菜天然海藻色素糖蛋白的制备与分析以及对肝癌抑制作用的研究结果如下：
1NSPG的制备与分析
1.1NSPG制备：建立了等电点沉淀法从麒麟菜提取天然海藻色素糖蛋白的方法并制备了足量的实验样品,已申请并获得国家发明专利证书。NSPG外观为棕红色粉末,溶于水,不溶于乙醇乙醚等有机溶剂,其提取得率为8.3%。
1.2NSPG多糖和蛋白质含量测定：NSPG中多糖的含量为93.3%,蛋白质的含量为2.04%。
1.3NSPG紫外光谱分析：在紫外光谱195nm出现多糖的特征吸收峰,其吸收值为1.834,在280nm处蛋白质的吸收值为0.417,但吸收峰不明显,说明蛋白质的含量较低。
1.4NSPG红外光谱分析：从红外光谱可见1651cm-1有酰胺键的特征吸收。在1034cm-1附近有吸收峰,表明NSPG的糖苷类型为吡喃型。
1.5NSPG糖苷键测定：NSPG经β-消除反应前后有紫外吸收的变化,说明NSPG中有O-连接糖苷键的存在。液-质谱图中各峰之间的差异为115,是天冬氨酸的分子量,说明NSPG中同时存在N-连接糖苷键。
1.6NSPG分子量的测定：凝胶色谱分析结果,NSPG相对分子量为213.8KDa。
2NSPG对肝癌的抑制作用研究
2.1体外实验结果：HepG2人肝癌细胞培养48小时、小鼠H22肝癌细胞培养24小时,NSPG高、中、低3个剂量组对增殖活性的抑制作用均依次升高,差异有显著性意义(p<0.05),表明NSPG对小鼠H22肝癌细胞和HepG2人肝癌细胞的增殖活性均有一定的抑制作用。
2.2体内实验结果
2.2.1瘤体比和抑瘤率结果：NSPG高剂量组和对照组的平均瘤体比分别为0.31±0.05g和0.55±0.23g,两者有显著性差异(p<0.05); NSPG高剂量组肿瘤生长抑制率为43.64%,低剂量组肿瘤生长抑制率为9.10%,差异有显著性意义(p<0.05)。
2.2.2胸腺指数和脾脏指数结果：NSPG高、中、低剂量组的胸腺指数和脾脏指数与对照组比较均有所增加,但环磷酰胺组与对照组、高、中、低剂量NSPG组比较,环磷酰胺组胸腺指数和脾脏指数显著降低,并与对照组比较差异有显著的统计意义(p<0.01)。
2.2.3血清TNF-α水平结果：血清中NSPG高剂量组的TNF-α浓度为92.17±4.01pg/ml,TNF-α血清水平明显低于对照组和NSPG中、低剂量组,而且较对照组有显著性差异(p<0.05)。
2.2.4瘤细胞增殖活性结果：用MTT法测定各组瘤细胞增殖活性水平结果高剂量和中剂量NSPG组瘤细胞增殖活性水平分别为0.71±0.03和0.83±0.02,对照组细胞增殖活性为1.14±0.03,高、中剂量NSPG组及其与对照组之间有显著性差异(p<0.05)。
2.2.5PCNA蛋等白表达结果：PCNA蛋白表达水平结果对照组肝癌细胞PCNA蛋白阳性表达率为72.78%,高剂量NSPG组为28.32%,,二者差异有显著性意义(p<0.05)。NSPG高剂量组Bax和Bcl-2蛋白阳性表达率分别为38.10%和16.78%,对照组分别为4.68%和65.16%,两组间差异均存在显著性意义(p<0.05)。对照组的Caspase-3蛋白阳性表达率为5.00%,NSPG高剂量组为40.20%,有显著性差异(p<0.05)。
结论通过上述实验结果对麒麟菜天然海藻色素糖蛋白的实验得出以下结论：
1.建立了从麒麟菜中提取天然海藻色素糖蛋白的方法,得率为8.3%。麒麟菜天然海藻色糖蛋白的相对分子量为213.8KDa。海藻色素糖蛋白的多糖含量为93.3%,蛋白质含量为2.04%。糖苷键有O-连接糖苷键,同时存在N-连接糖苷键。
2.麒麟菜天然海藻色素糖蛋白不仅在体外对HepG2人肝癌细胞增殖和小鼠H22肝癌细胞增殖具有一定的抑制作用,在体内对小鼠肝癌细胞的增殖也有显著的抑制作用,说明麒麟菜天然海藻色素糖蛋白能够抑制肝癌细胞的体内外增殖活性。
3.麒麟菜天然海藻色素糖蛋白降低小鼠H22肝癌组织中Bcl-2和PCNA蛋白表达水平、促进肝癌细胞Bax和Caspase-3蛋白表达水平。证明通过抑制增殖和促进凋亡而抑制H22肝癌细胞的生长。
4.麒麟菜天然海藻色素糖蛋白抑制血清TNF-α水平异常升高,使其维持在一定水平发挥抗肿瘤作用,进一步证明海藻色素糖蛋白对肝癌的抑制作用。
5.环磷酰胺虽然对H22肝癌细胞有抑制作用,但环磷酰胺可损伤荷瘤动物的脾脏和胸腺从而导致免疫功能的衰退,而天然海藻色素糖蛋白不仅对H22肝癌细胞的增长有抑制作用,还对脾脏和胸腺有保护作用。这为麒麟菜的开发利用以及天然海藻色素糖蛋白在预防和治疗肝癌等疾病方面的应用提供科学依据。
Objective In this paper, by isoelectric precipitation method extracted natural seaweed pigment glycoprotein(NSPG) from Eucheuma, determined it's structure by IR、UV、Liquid-mass spectrometry and gel permeation chromatography. By adopting the method of cell culture in vivo and in vitro, observe the effect of NSPG on the inhibition of hepatoma cells.
Methods
1Preparation and analysis of NSPG
1.1NSPG Preparation:Eucheuma was cut into0.5-1.0cm long of strips, placed in a solution of hydrochloric acid pH3.0, the collected filtrate was adjusted to pH4.5, and then adding3volumes of95%ethanol, stirred, allowed to stand overnight, precipitated and filtered to obtain the precipitate on the filter paper part, and the precipitate portion were dried over anhydrous ethanol, washed with acetone, dried at room temperature, obtained NSPG
1.2NSPG Analysis:
1.2.1NSPG polysaccharide and protein content determination:Glucose as the standard material, using sulfuric acid-phenol method for the determination of glucose content. Bovine serum albumin as a standard material, with Coomassie brilliant blue protein content were determined.
1.2.2NSPG structural analysis:UV spectra and IR spectra were determined polysaccharides and proteins characteristic absorption peaks.According to β-elimination reaction and liquid chromatography-mass spectrometry results determined glycosidic bond type.
1.2.3NSPG molecular weight determination:The molecular weight of NSPG was determined by gel chromatography.
1.2.4Sensory indicators:Observed the shape, color and other indicators.
2NSPG on the inhibition of liver cancer
2.1In vitro experiment:Different concentrations of NSPG (100mg/L50mg/L、10mg/L) were cultured with H22hepatocarcinoma cells of mice and HepG2human hepatoma cells. Without NSPG was set as a separate control group, and the cell proliferation was determined by MTT assay.
2.2In vivo experiment:Under sterile conditions, the H22passage section of the mice ascites liver cancer was inoculation to the weighing18-20g50healthy Kunming mice, which were inoculated subcutaneously at right axilla0.2ml. All of the animals were randomly divided into five groups, which were control group、high、medium and low doses of natural seaweed pigment glycoprotein group and cyclophosphamide group with half gender mice in each group. Mice in high, medium and low dose group were given by gavages concentration of100、50and10mg/kg natural seaweed pigment glycoprotein aqueous, the cylophosphamide group were injected20mg/kg every the other day, and control group were treated with saline, once a day for10days. The day after the last dose the mice were weighed→abstract mice eyeball blood, serum was separated→the tumor weights spleen weight and thymus weight were examined by electronic scale→stripping tumor tissue to be seized.
2.2.1Tumor index and inhibitory rate:The tumor weight and body weight were examined by electronic scale and the tumor index and inhibitory rate were calculated.
2.2.2Thymus index and spleen index:The thymus weight and spleen weight were examined by electronic scale and indexes were calculated.
2.2.3Serum levels of TNF-α:The serum TNF-α was measured by enzyme linked immunosorbent assay.
2.2.4The proliferative activity:Determined by MTT proliferation of tumor cells.
2.2.5Protein expression:The level of PCNA、Bcl-2、Caspase-3and Bax protein was measured by immunohistochemistry method.
Results
1Preparation and analysis of NSPG
1.1NSPG Preparation:Yield was8.3%.
1.2NSPG Determination:Polysaccharide content of93.3%, the protein content of2.04%.
1.3NSPG structural analysis:In the UV spectrum, at195nm appears polysaccharide characteristic peaks, Its absorption value is1.834; at280nm appears protein's absorption value is0.417, but the absorption peak is not obvious, indicating that low levels of protein. In the IR spectrum, showing characteristic absorption of an amide bond、sulfate groups and sulfate group substitution positions、pyranose glycosidic bond characteristic absorption peak, these proved that NSPG is a glycoprotein. After β-elimination reaction the UV absorption and amino acid composition have changed, indicating that there is O-connected glycosidic bond. In the mass spectrum, the difference between each peak is115, which is the molecular weight of aspartic acid. Description NSPG has N-linked glycosidic bond too.
1.4NSPG molecular weight determination:GPC analysis showed that NSPG relative molecular weight is213.8kDa.
2NSPG inhibition on H22hepatoma
2.1In vitro experiment results:The proliferative activity of cultured cells in vitro in100mg/LNSPG group were0.40±0.08, which were significantly lower than the tumor control group (P<0.05), and those in10mg/L and50mg/L NSPG group were0.52±0.09and0.45±0.05respectively. Indicates with increasing doses of NSPG, proliferation activity of H22hepatoma cells was decreased (P<0.05),and that a certain dose of NSPG has inhibition effect on proliferation activity of H22hepatoma cells.
2.2In vivo experiment results:
2.2.1Tumor index and inhibitory rate:The average of tumor weight in high dose group and tumor control group were0.31±0.05g and0.55±0.23g respectively, the differences were statistically significant (P<0.05).
2.2.2Thymus index and spleen index:High-, middle-and low-dose group NSPG thymus index and spleen index compared with the control group, no significant differences (P>0.05), but higher than the tumor control group (P<0.01).
2.2.3Serum levels of TNF-α The serum TNF-a concentration of the high dose NSPG group were92.17±4.01(pg/ml), significantly lower than that of the tumor control (p<0.05).
2.2.4The proliferative activity:The proliferation activity of the tumor control group was1.14±0.03, significantly higher than that of the high-dose NSPG group (P<0.05).
2.2.5Expression Results:The PCNA positive expression rate in tumor control group were72.78%, and those in high dose group were28.32%, differences were significant (P<0.05). The positive expression level of Bax and Bcl-2protein in high-dose group were38.10%and16.78%, respectively, and those of the tumor control group were4.68%and65.16%, respectively. There were significant differences between the groups (P<0.05). The A value of Caspase-3of the high-dose NSPG group was0.53±0.09, significantly higher than that of the tumor control group (P<0.05).
Conclusion
1. Established the method of extraction of NSPG from Eucheuma. NSPG relative molecular weight is213.8kDa.NSPG polysaccharide content was93.3%, protein content was2.04%. There was O-connected glycosidic bond, and has N-linked glycosidic bond too. Yield was8.3%.
2. NSPG not only for H22hepatoma cells and human liver cancer cell proliferation with a certain degree of inhibition in vitro, on H22hepatoma cells also inhibited in vivo, indicating NSPG can inhibit liver cancer cell proliferation activity.
3. NSPG could reduce the expression level of Bcl-2and PCNA protein on H22hepatoma tissue; promote the expression of Bax and Caspase-3protein on H22hepatoma tissue. Description by inhibiting the proliferation and promoting the apoptosis inhibit the growth of liver cancer.
4. NSPG suppress abnormal elevation of serum TNF-α, further evidence that NSPG inhibiting the growth of liver cancer.
5. Although cyclophosphamide was inhibited on H22cells, but it can damage the spleen and thymus, which led to the decline of immune function. However, NSPG not only inhibit the growth of H22hepatoma cells, but also has a protective effect on the spleen and thymus. NSPG can inhibit growth of H22hepatoma tumor on mice, which provide theoretical basis on the development of Eucheuma and prevention and treatment of liver diseases.
方法包括麒麟菜天然海藻色素糖蛋白的制备与分析,及其对肝癌抑制作用等实验方法：
1NSPG的制备与分析
1.1NSPG制备：将洗净的麒麟菜切为0.5-1.0cm的长条,置于pH3.0的盐酸溶液中浸提,将上述酸性浸提液进行减压抽滤,收集滤液调节pH至4.5,再加入3倍体积的95%的乙醇,搅拌,放置过夜,析出沉淀,真空抽滤得到滤纸上的沉淀部分,将沉淀分别部分用无水乙醇、丙酮洗涤,室温干燥,得NSPG。
1.2NSPG组成与结构分析
1.2.1NSPG多糖和蛋白质含量测定：以葡萄糖为标准物质,用硫酸-苯酚法测定NSPG中多糖的含量。以牛血清白蛋白为标准物质,用考马斯亮蓝法测定NSPG中蛋白质的含量。
1.2.2NSPG结构分析：用紫外和红外光谱法测定多糖和蛋白质的特征吸收峰。根据β-消除反应和液-质谱法确定糖苷键的类型。
1.2.3NSPG分子量测定：用凝胶色谱分析法测定NSPG的分子量。
1.2.4感官等理化指标：包括形态、色泽与水溶性等指标测定。
2NSPG对肝癌的抑制作用研究
2.1体外实验：对肝癌细胞增殖活性的抑制作用进行研究。
将小鼠H22肝癌细胞和HepG2人肝癌细胞分别与不同剂量的NSPG(浓度分别为10mg/L、50mg/L、100mg/L)共同培养,另设不加NSPG的作为对照,用MTT法测定肝癌细胞增殖活性。
2.2体内实验：对小鼠H22肝癌的抑制作用进行研究
无菌条件下抽取接种H22肝癌细胞第6d的小鼠腹水,接种在50只体重为18-20g的健康昆明种小鼠的右侧腋窝处,每只皮下注射0.2ml。将接种的小鼠随机分5个组：肿瘤对照组、低、中、高剂量NSPG组和环磷酰胺组,每组十只,雌雄各一半。其中三个NSPG剂量组每日经口灌胃浓度分别为10、50、100mg/kg的水溶液,给对照组经口灌胃等量的生理盐水,环磷酰胺组小鼠隔日1次腹腔注射20mg/kg的环磷酰胺,连续10d。最后一次给药后第二天将小鼠称重→摘小鼠眼球取血,分离血清→取瘤组织、取脾脏和胸腺,用电子天平称量重量→剥取瘤组织备检。
2.2.1瘤体比和抑瘤率：用电子天平称取小鼠体重、瘤重并计算瘤重与体重比值得出瘤体比、并计算抑瘤率。
2.2.2胸腺指数和脾脏指数：取动物脾脏和胸腺,用电子天平称量重量,根据体重计算脾脏指数和胸腺指数。
2.2.3血清肿瘤坏死因子(tumor necrosis factor,TNF)-α水平：摘小鼠眼球取血,分离血清后用ELISA法检测血清的TNF-α水平。
2.2.4瘤细胞增殖活性：用四甲基偶氮唑(MTT)法检测各组小鼠肿瘤细胞的增殖活性。
2.2.5瘤组织中PCNA、Caspase-3、Bax、Bcl-2蛋白表达水平：用免疫组化法检测。
结果麒麟菜天然海藻色素糖蛋白的制备与分析以及对肝癌抑制作用的研究结果如下：
1NSPG的制备与分析
1.1NSPG制备：建立了等电点沉淀法从麒麟菜提取天然海藻色素糖蛋白的方法并制备了足量的实验样品,已申请并获得国家发明专利证书。NSPG外观为棕红色粉末,溶于水,不溶于乙醇乙醚等有机溶剂,其提取得率为8.3%。
1.2NSPG多糖和蛋白质含量测定：NSPG中多糖的含量为93.3%,蛋白质的含量为2.04%。
1.3NSPG紫外光谱分析：在紫外光谱195nm出现多糖的特征吸收峰,其吸收值为1.834,在280nm处蛋白质的吸收值为0.417,但吸收峰不明显,说明蛋白质的含量较低。
1.4NSPG红外光谱分析：从红外光谱可见1651cm-1有酰胺键的特征吸收。在1034cm-1附近有吸收峰,表明NSPG的糖苷类型为吡喃型。
1.5NSPG糖苷键测定：NSPG经β-消除反应前后有紫外吸收的变化,说明NSPG中有O-连接糖苷键的存在。液-质谱图中各峰之间的差异为115,是天冬氨酸的分子量,说明NSPG中同时存在N-连接糖苷键。
1.6NSPG分子量的测定：凝胶色谱分析结果,NSPG相对分子量为213.8KDa。
2NSPG对肝癌的抑制作用研究
2.1体外实验结果：HepG2人肝癌细胞培养48小时、小鼠H22肝癌细胞培养24小时,NSPG高、中、低3个剂量组对增殖活性的抑制作用均依次升高,差异有显著性意义(p<0.05),表明NSPG对小鼠H22肝癌细胞和HepG2人肝癌细胞的增殖活性均有一定的抑制作用。
2.2体内实验结果
2.2.1瘤体比和抑瘤率结果：NSPG高剂量组和对照组的平均瘤体比分别为0.31±0.05g和0.55±0.23g,两者有显著性差异(p<0.05); NSPG高剂量组肿瘤生长抑制率为43.64%,低剂量组肿瘤生长抑制率为9.10%,差异有显著性意义(p<0.05)。
2.2.2胸腺指数和脾脏指数结果：NSPG高、中、低剂量组的胸腺指数和脾脏指数与对照组比较均有所增加,但环磷酰胺组与对照组、高、中、低剂量NSPG组比较,环磷酰胺组胸腺指数和脾脏指数显著降低,并与对照组比较差异有显著的统计意义(p<0.01)。
2.2.3血清TNF-α水平结果：血清中NSPG高剂量组的TNF-α浓度为92.17±4.01pg/ml,TNF-α血清水平明显低于对照组和NSPG中、低剂量组,而且较对照组有显著性差异(p<0.05)。
2.2.4瘤细胞增殖活性结果：用MTT法测定各组瘤细胞增殖活性水平结果高剂量和中剂量NSPG组瘤细胞增殖活性水平分别为0.71±0.03和0.83±0.02,对照组细胞增殖活性为1.14±0.03,高、中剂量NSPG组及其与对照组之间有显著性差异(p<0.05)。
2.2.5PCNA蛋等白表达结果：PCNA蛋白表达水平结果对照组肝癌细胞PCNA蛋白阳性表达率为72.78%,高剂量NSPG组为28.32%,,二者差异有显著性意义(p<0.05)。NSPG高剂量组Bax和Bcl-2蛋白阳性表达率分别为38.10%和16.78%,对照组分别为4.68%和65.16%,两组间差异均存在显著性意义(p<0.05)。对照组的Caspase-3蛋白阳性表达率为5.00%,NSPG高剂量组为40.20%,有显著性差异(p<0.05)。
结论通过上述实验结果对麒麟菜天然海藻色素糖蛋白的实验得出以下结论：
1.建立了从麒麟菜中提取天然海藻色素糖蛋白的方法,得率为8.3%。麒麟菜天然海藻色糖蛋白的相对分子量为213.8KDa。海藻色素糖蛋白的多糖含量为93.3%,蛋白质含量为2.04%。糖苷键有O-连接糖苷键,同时存在N-连接糖苷键。
2.麒麟菜天然海藻色素糖蛋白不仅在体外对HepG2人肝癌细胞增殖和小鼠H22肝癌细胞增殖具有一定的抑制作用,在体内对小鼠肝癌细胞的增殖也有显著的抑制作用,说明麒麟菜天然海藻色素糖蛋白能够抑制肝癌细胞的体内外增殖活性。
3.麒麟菜天然海藻色素糖蛋白降低小鼠H22肝癌组织中Bcl-2和PCNA蛋白表达水平、促进肝癌细胞Bax和Caspase-3蛋白表达水平。证明通过抑制增殖和促进凋亡而抑制H22肝癌细胞的生长。
4.麒麟菜天然海藻色素糖蛋白抑制血清TNF-α水平异常升高,使其维持在一定水平发挥抗肿瘤作用,进一步证明海藻色素糖蛋白对肝癌的抑制作用。
5.环磷酰胺虽然对H22肝癌细胞有抑制作用,但环磷酰胺可损伤荷瘤动物的脾脏和胸腺从而导致免疫功能的衰退,而天然海藻色素糖蛋白不仅对H22肝癌细胞的增长有抑制作用,还对脾脏和胸腺有保护作用。这为麒麟菜的开发利用以及天然海藻色素糖蛋白在预防和治疗肝癌等疾病方面的应用提供科学依据。
Objective In this paper, by isoelectric precipitation method extracted natural seaweed pigment glycoprotein(NSPG) from Eucheuma, determined it's structure by IR、UV、Liquid-mass spectrometry and gel permeation chromatography. By adopting the method of cell culture in vivo and in vitro, observe the effect of NSPG on the inhibition of hepatoma cells.
Methods
1Preparation and analysis of NSPG
1.1NSPG Preparation:Eucheuma was cut into0.5-1.0cm long of strips, placed in a solution of hydrochloric acid pH3.0, the collected filtrate was adjusted to pH4.5, and then adding3volumes of95%ethanol, stirred, allowed to stand overnight, precipitated and filtered to obtain the precipitate on the filter paper part, and the precipitate portion were dried over anhydrous ethanol, washed with acetone, dried at room temperature, obtained NSPG
1.2NSPG Analysis:
1.2.1NSPG polysaccharide and protein content determination:Glucose as the standard material, using sulfuric acid-phenol method for the determination of glucose content. Bovine serum albumin as a standard material, with Coomassie brilliant blue protein content were determined.
1.2.2NSPG structural analysis:UV spectra and IR spectra were determined polysaccharides and proteins characteristic absorption peaks.According to β-elimination reaction and liquid chromatography-mass spectrometry results determined glycosidic bond type.
1.2.3NSPG molecular weight determination:The molecular weight of NSPG was determined by gel chromatography.
1.2.4Sensory indicators:Observed the shape, color and other indicators.
2NSPG on the inhibition of liver cancer
2.1In vitro experiment:Different concentrations of NSPG (100mg/L50mg/L、10mg/L) were cultured with H22hepatocarcinoma cells of mice and HepG2human hepatoma cells. Without NSPG was set as a separate control group, and the cell proliferation was determined by MTT assay.
2.2In vivo experiment:Under sterile conditions, the H22passage section of the mice ascites liver cancer was inoculation to the weighing18-20g50healthy Kunming mice, which were inoculated subcutaneously at right axilla0.2ml. All of the animals were randomly divided into five groups, which were control group、high、medium and low doses of natural seaweed pigment glycoprotein group and cyclophosphamide group with half gender mice in each group. Mice in high, medium and low dose group were given by gavages concentration of100、50and10mg/kg natural seaweed pigment glycoprotein aqueous, the cylophosphamide group were injected20mg/kg every the other day, and control group were treated with saline, once a day for10days. The day after the last dose the mice were weighed→abstract mice eyeball blood, serum was separated→the tumor weights spleen weight and thymus weight were examined by electronic scale→stripping tumor tissue to be seized.
2.2.1Tumor index and inhibitory rate:The tumor weight and body weight were examined by electronic scale and the tumor index and inhibitory rate were calculated.
2.2.2Thymus index and spleen index:The thymus weight and spleen weight were examined by electronic scale and indexes were calculated.
2.2.3Serum levels of TNF-α:The serum TNF-α was measured by enzyme linked immunosorbent assay.
2.2.4The proliferative activity:Determined by MTT proliferation of tumor cells.
2.2.5Protein expression:The level of PCNA、Bcl-2、Caspase-3and Bax protein was measured by immunohistochemistry method.
Results
1Preparation and analysis of NSPG
1.1NSPG Preparation:Yield was8.3%.
1.2NSPG Determination:Polysaccharide content of93.3%, the protein content of2.04%.
1.3NSPG structural analysis:In the UV spectrum, at195nm appears polysaccharide characteristic peaks, Its absorption value is1.834; at280nm appears protein's absorption value is0.417, but the absorption peak is not obvious, indicating that low levels of protein. In the IR spectrum, showing characteristic absorption of an amide bond、sulfate groups and sulfate group substitution positions、pyranose glycosidic bond characteristic absorption peak, these proved that NSPG is a glycoprotein. After β-elimination reaction the UV absorption and amino acid composition have changed, indicating that there is O-connected glycosidic bond. In the mass spectrum, the difference between each peak is115, which is the molecular weight of aspartic acid. Description NSPG has N-linked glycosidic bond too.
1.4NSPG molecular weight determination:GPC analysis showed that NSPG relative molecular weight is213.8kDa.
2NSPG inhibition on H22hepatoma
2.1In vitro experiment results:The proliferative activity of cultured cells in vitro in100mg/LNSPG group were0.40±0.08, which were significantly lower than the tumor control group (P<0.05), and those in10mg/L and50mg/L NSPG group were0.52±0.09and0.45±0.05respectively. Indicates with increasing doses of NSPG, proliferation activity of H22hepatoma cells was decreased (P<0.05),and that a certain dose of NSPG has inhibition effect on proliferation activity of H22hepatoma cells.
2.2In vivo experiment results:
2.2.1Tumor index and inhibitory rate:The average of tumor weight in high dose group and tumor control group were0.31±0.05g and0.55±0.23g respectively, the differences were statistically significant (P<0.05).
2.2.2Thymus index and spleen index:High-, middle-and low-dose group NSPG thymus index and spleen index compared with the control group, no significant differences (P>0.05), but higher than the tumor control group (P<0.01).
2.2.3Serum levels of TNF-α The serum TNF-a concentration of the high dose NSPG group were92.17±4.01(pg/ml), significantly lower than that of the tumor control (p<0.05).
2.2.4The proliferative activity:The proliferation activity of the tumor control group was1.14±0.03, significantly higher than that of the high-dose NSPG group (P<0.05).
2.2.5Expression Results:The PCNA positive expression rate in tumor control group were72.78%, and those in high dose group were28.32%, differences were significant (P<0.05). The positive expression level of Bax and Bcl-2protein in high-dose group were38.10%and16.78%, respectively, and those of the tumor control group were4.68%and65.16%, respectively. There were significant differences between the groups (P<0.05). The A value of Caspase-3of the high-dose NSPG group was0.53±0.09, significantly higher than that of the tumor control group (P<0.05).
Conclusion
1. Established the method of extraction of NSPG from Eucheuma. NSPG relative molecular weight is213.8kDa.NSPG polysaccharide content was93.3%, protein content was2.04%. There was O-connected glycosidic bond, and has N-linked glycosidic bond too. Yield was8.3%.
2. NSPG not only for H22hepatoma cells and human liver cancer cell proliferation with a certain degree of inhibition in vitro, on H22hepatoma cells also inhibited in vivo, indicating NSPG can inhibit liver cancer cell proliferation activity.
3. NSPG could reduce the expression level of Bcl-2and PCNA protein on H22hepatoma tissue; promote the expression of Bax and Caspase-3protein on H22hepatoma tissue. Description by inhibiting the proliferation and promoting the apoptosis inhibit the growth of liver cancer.
4. NSPG suppress abnormal elevation of serum TNF-α, further evidence that NSPG inhibiting the growth of liver cancer.
5. Although cyclophosphamide was inhibited on H22cells, but it can damage the spleen and thymus, which led to the decline of immune function. However, NSPG not only inhibit the growth of H22hepatoma cells, but also has a protective effect on the spleen and thymus. NSPG can inhibit growth of H22hepatoma tumor on mice, which provide theoretical basis on the development of Eucheuma and prevention and treatment of liver diseases.
引文
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