黄精炮制前后多糖的相对分子质量分布和免疫活性比较

Comparison of Relative Molecular Weight Distribution and Immune Activity of Polysaccharides in Polygonati Rhizoma Before and After Processing

目的:研究黄精炮制前后多糖相对分子质量及其分布变化,以及其不同多糖级分对小鼠腹腔巨噬细胞免疫功能和炎症反应的影响。方法:采用高效凝胶渗透色谱法(HPGPC)测定黄精多糖(SC)和酒黄精多糖(JC)相对分子质量及其分布情况,并通过透析法得到不同相对分子质量多糖级分,将上述多糖级分分别作用于正常和经脂多糖(LPS)诱导的小鼠腹腔巨噬细胞,采用细胞增殖与活性检测试剂盒-8(CCK-8)法选取最佳给药浓度,酶联免疫吸附测定法(ELISA)测定细胞上清液中肿瘤坏死因子(TNF)-α,白细胞介素(IL)-1β的浓度,格里斯(Griess)试剂法检测一氧化氮(NO)的浓度。结果:SC和JC按相对分子质量及其分布范围可分为4个部分,第Ⅰ部分(14 800~2 273 kDa),第Ⅱ部分(2 148~296 kDa),第Ⅲ部分(12~1 kDa),第Ⅳ部分(818~362 Da)。而黄精经酒制后,第Ⅰ部分和第Ⅲ部分的多糖级分发生了改变,因此,选择将SC和JC按重均相对分子质量(M_(W))>50 kDa和<50 kDa各分成2个部分。对于正常小鼠腹腔巨噬细胞,JC能显著促进细胞分泌TNF-α(P<0.01),但SC无明显作用;4个多糖级分SD(M_(W)>50 kDa的SC级分),JD(M_(W)>50 kDa的JC级分),SX(M_(W)<50 kDa的SC级分),JX(M_(W)<50 kDa的JC级分)均能显著促进细胞分泌TNF-α(P<0.01),但仅JX能明显促进细胞分泌NO(P<0.05);另外,JX组促进细胞分泌TNF-α作用显著强于JD组(P<0.01)。对于LPS诱导巨噬细胞模型,JC和SC均能显著抑制细胞分泌TNF-α和IL-1β(P<0.01),且JC作用更强;不同多糖级分间,JX抑制细胞分泌TNF-α和IL-1β的作用显著强于JD(P<0.01),SX抑制细胞分泌TNF-α作用显著强于SD(P<0.01)。结论:黄精酒制前后多糖相对分子质量及其分布发生了改变,JC和SC增强免疫调节作用主要是通过抑制炎症反应,M_(W)<50 kDa级分是其主要有效部位,且黄精在酒制后其多糖成分抑制炎症反应的作用增强。

Objective: To study the relative molecular weight and distribution of polysaccharides in Polygonati Rhizoma(PR)before and after processing,as well as the effects of different polysaccharide fractions on immune function and inflammatory response of mouse peritoneal macrophages. Method: High-performance gel permeation chromatography(HPGPC)was used to determine the relative molecular weight and distribution of polysaccharides in PR(named SC)and polysaccharides in PR processed with wine(named JC),and polysaccharide fractions with different relative molecular weights were obtained by dialysis. Different polysaccharide fractions were applied to mouse peritoneal macrophages,which was normal or induced by lipopolysaccharide(LPS). Cell counting kit-8(CCK-8)method was used to select the optimal administration concentration. Enzyme linked immunosorbent assay(ELISA)was used to determine the concentrations of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the cell supernatant. The Griess method was used to detect the concentration of nitric oxide(NO). Result: SC and JC could be divided into four parts according to relative molecular weight and its distribution range,including part Ⅰ(14 800-2 273 kDa),part Ⅱ(2 148-296 kDa),part Ⅲ(12-1 kDa)and part Ⅳ(818-362 Da). Based on the differences of part Ⅰ and part Ⅲ after processing,the SC and JC were divided into two-part according to the weight-average relative molecular weight(M_(W)). For normal mouse peritoneal macrophages,JC could significantly promote the secretion of TNF-α(P<0.01),while SC had no significant effect. Four polysaccharide fractions,named SD(SC fraction with M_(W)>50 kDa),JD(JC fraction with M_(W)>50 kDa),SX(SC fraction with M_(W)<50 kDa)and JX(JC fraction with M_(W)<50 kDa),also could significantly promote the secretion of TNF-α(P<0.01),but only JX could significantly promote the secretion of NO(P<0.05). In addition,the effect of JX group stimulated secretion of TNF-α was better than the JD group(P<0.01). For the LPS-induced macrophage model, JC and SC group could significantly inhibit the secretion of TNF-α and IL-1β(P<0.01),and the effect of JC was stronger. To compare different polysaccharide fractions,the impact of JX on inhibiting the secretion of TNF-α and IL-1β was significantly stronger than JD(P<0.01),and SX inhibited the secretion of TNF-α was significantly stronger than SD(P<0.01). Conclusion: The relative molecular weight and distribution of polysaccharides in PR before and after processing have changed. JC and SC improve the immune regulation mainly by inhibiting the inflammatory response,the fraction of M_(W)<50 kDa is the main effective part,and the effect of PR polysaccharides in inhibiting the inflammatory is enhanced after processing with wine.