远志糖蛋白的结构特征及其对小鼠学习记忆障碍功能的影响

Structural Characterization of Radix Polygala Glycoprotein and Effect on Learning and Memory Impairment in Mice

目的研究远志糖蛋白的结构特征及其对小鼠学习记忆障碍功能的影响。方法采用苯酚-硫酸法、BCA法、液相色谱分析、氨基酸分析、SDS-PAGE、UV、IR、β-消去反应、圆二色性(CD)光谱分析、液质分析等分析方法对远志糖蛋白进行结构表征。采用腹腔注射氢溴酸东莨菪碱(2 mg·kg^(-1))建立学习记忆障碍模型,通过旷场、新物体识别、Morris水迷宫等实验手段考察小鼠行为能力;ELISA检测小鼠脑组织中GSH、GSH-PX、GLU、ROS、CAMKⅡ等指标。结果分离的远志糖蛋白PG-2具有蛋白质与糖类的特征吸收峰,其蛋白质、糖类质量分数分别为56.9%、26.8%,相对分子质量约为26 kDa;含有16种氨基酸;含有葡萄糖、半乳糖、阿拉伯糖、鼠李糖等单糖;蛋白质主链二级结构以α-螺旋为主;PG-2中筛选出的5条多肽为(EVIADIAK、ISSLESLR、NLSADIISK、PRVKMADK、LYPAVPVDAK)。动物实验表明,与空白组相比,模型组小鼠自主活动水平与垂直得分均显著性降低(P<0.01,P<0.01);新物体识别指数显著减小(P<0.01);穿越平台次数与平台区域停留时间均显著减少(P<0.01,P<0.01);GSH、GSH-PX、CAMKII含量均显著降低(P<0.01,P<0.01,P<0.01),GLU、ROS含量显著增加(P<0.01,P<0.01)。与模型组动物相比,低剂量组小鼠的自主活动水平与垂直得分均增加(P<0.05,P<0.05);新物体识别指数显著增加(P<0.05);GSH、GSH-PX、CAMKII含量均显著增加(P<0.05,P<0.05,P<0.05),GLU、ROS含量显著增加(P<0.01,P<0.01);高剂量组小鼠的自主活动水平与垂直得分均增加(P<0.01,P<0.01);新物体识别指数显著增加(P<0.01);穿越平台次数增加与平台区域停留时间显著增加(P<0.05,P<0.05);GSH、GSH-PX、CAMKⅡ含量均显著增加(P<0.01,P<0.01,P<0.05),GLU、ROS含量显著降低(P<0.01,P<0.01)。结论PG-2含大量人体所需氨基酸,为优质植物蛋白,远志糖蛋白可对学习记忆障碍小鼠的学习记忆能力起到改善作用,为远志的进一步研究提供参考。

Objective To extract,purify and characterize the structure of Radix Polygala glycoprotein.Methods Radix Polygala was handled by Tris-Hcl buffer,salting separation,sevage method,then purified by anion.Physicochemical properties and composition of PG-2 were analyzed by phenol-sulfuric acid method,BCA method,Molish test,ninhydrin reaction,gas chromatography,amino acid analysis,SDS-PAGE,UV,IR andβ-elimination reaction,CD,LC-MS spectra.The learning and memory impairment model was established by intraperitoneal injection of scopolamine hydrobromide(2 mg·kg^(-1)).The behavior changes of mice were investigated by Open field、New object recognition、Morris water maze test,and GSH,GSH-PX,GLU,ROS,CAMKⅡet al levels in mouse brain tissue were detected by ELISA.Results PG-2 has the characteristic absorption peak of protein and sugar,its protein and sugar mass scores are 56.9%and 26.8%respectively,and the relative molecular mass is about 26 kDa;contains 16 amino acids;contains glucose,galactose,arabinose,liose;the protein backbone secondary structure is mainlyα-helix;the polypeptides loading amount of five selected polypeptides(EVIADIAK、ISSLESLR、NLSADIISK、PRVKMADK、LYPAVPVDAK).Compared with the model group,the autonomous activity level compared with vertical scores were significantly reduced(P<0.01,P<0.01);the new object recognition index decreased(P<0.01),the number of mouse platform crossings and residence time decreased(P<0.01,P<0.01),the biochemical index GSH,GSH-PX,CAMKⅡcontent in brain tissue(P<0.01,P<0.01,P<0.01)and the GLU,ROS content increased(P<0.01,P<0.01).In contrast to the model group of animals,The levels of autonomous activity were increased compared with vertical scores in medium-dose mice(P<0.05,P<0.05);The new object recognition index was significantly increased(P<0.05);Significant increase of biochemical index GSH,GSH-PX,CAMKⅡin brain tissue(P<0.05,P<0.05,P<0.05),The GLU,ROS content was significantly reduced(P<0.01,P<0.01);Autonomous activity levels were increased compared with vertical scores in high-dose mice(P<0.01,P<0.01);The new object recognition index was significantly increased(P<0.01);Increased mouse travers with significantly increased residence time in the platform regions(P<0.05,P<0.05);Significant increase of biochemical index GSH,GSH-PX,CAMKⅡin brain tissue(P<0.01,P<0.01,P<0.05),The GLU,ROS content was significantly increased(P<0.01,P<0.01).Conclusion PG-2 contains a large amount of amino acids needed by human body and belongs to high quality protein.Radix Polygala glycoprotein can improve the learning memory ability inmice with learning memory impairment,and provide reference for further research.