CN117510662A - 一种肉苁蓉多糖和制备方法及其在慢传输型便秘中的应用 - Google Patents
一种肉苁蓉多糖和制备方法及其在慢传输型便秘中的应用 Download PDFInfo
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Abstract
本发明涉及一种肉苁蓉多糖和制备方法及其在慢传输型便秘中的应用,荒漠肉苁蓉药材采集于甘肃省汇勤荒漠肉苁蓉种植基地,将荒漠肉苁蓉干燥药材处理后,经水提醇沉、透析处理后,制备得到肉苁蓉粗多糖。制备得到的肉苁蓉粗多糖CDPs对洛哌丁胺(loperamide)诱导的STC的模型小鼠的结肠肌间神经具有保护作用,能够用于制备治疗或预防慢传输型便秘药物,肉苁蓉粗多糖CDPs为具有生物活性的天然化合物,其无副作用且药物依赖性低。
Description
技术领域
本发明属于生物制药领域,尤其是涉及一种肉苁蓉多糖和制备方法及其在慢传输型便秘中的应用。
背景技术
功能性便秘(Functional constipation,FC)是常见的功能性肠道疾病之一,影响着全球14%的人口。近年来由于饮食结构改变、生活节奏加快以及复杂的社会和心理因素的影响,FC的患病率持续增加。研究表明,FC增加了心血管事件和精神障碍发生的风险,对各年龄段患者的身心健康和生活质量产生严重的负面影响。慢传输型便秘(Slow transitconstipation,STC)是FC的一种常见类型,其主要的临床特点是结肠动力功能障碍导致结肠传输时间延长。目前STC病因及发病机制的研究仍处在探索阶段,治疗主要停留在长期应用药物缓解症状,不能从本质上治疗,严重患者最后往往需要进行外科手术干预。因此,探究STC的发病机制并寻找更安全有效的治疗策略一直是医学领域的热点和难点。
肠神经系统(enteric nervous system,ENS)是由肠神经元以及肠神经胶质细胞组成的独立神经系统,在调节肠道生理功能方面发挥着重要的作用,包括控制肠蠕动、液体的吸收和分泌、免疫和血流调控。研究发现STC的发生通常伴随着ENS的改变。氧化应激是生物体内氧化反应产生的自由基与抗氧化系统之间的失衡状态。活性氧(Reactive oxygenspecies,ROS)是主要的内源性氧自由基。当ROS产生超过机体抗氧化能力时,可造成脂质过氧化、蛋白质结构和功能破坏、DNA损伤,最终导致不可逆的细胞死亡。研究发现,氧化应激可诱导高迁移率族蛋白1(high-mobility group box,HMGB1)在肌间神经元内的转位,引起结肠神经病变。然而,通过抑制APE1/Ref-1氧化还原信号通路可以抑制HMGB1转位,从而缓解小鼠的肌间神经元损伤及肠道功能障碍。目前,关于结肠肌间神经元氧化应激损伤与STC关系的研究尚属空白。
发明内容
为解决上述技术问题,本发明提供一种肉苁蓉多糖和制备方法及其在慢传输型便秘中的应用。
本发明采用的技术方案是:一种肉苁蓉多糖的制备方法,包括如下步骤:
步骤一:取荒漠肉苁蓉干燥药材,粉碎后加入70%乙醇溶液回流提取三次,脱脂处理;
步骤二:脱脂后的药材加水回流提取三次;
步骤三:合并三次水提取液,加入乙醇进行醇沉,取醇沉物;
步骤四:醇沉物加水溶解,透析浓缩后冻干,制备得到荒漠肉苁蓉粗多糖CDPs。
优选地,步骤三中,将三次水提取合并液浓缩,加入95%乙醇至终浓度为80%,4℃过夜醇沉。
优选地,步骤四中,透析中分子截留3500Da。
优选地,制备得到的苁蓉粗多糖CDPs包括4个分子量段的多糖,分子量分布为11.2kD的多糖占主要部分,其他还包括45.5kD、4.0kD和1.8kD的多糖。
优选地,制备得到的苁蓉粗多糖CDPs主要由Glc残基组成,另外含有少量的Rha、GalA、Gal和Ara残基
优选地,制备得到的苁蓉粗多糖CDPs主要包括(1→4)-Glcp,另外还有(1→4,6)-Glcp、(1→4)-Galp和t-Rhap残基。
一种肉苁蓉多糖,主要由Glc残基组成,另外含有少量的Rha、GalA、Gal和Ara残基;主要包括(1→4)-Glcp,另外还有(1→4,6)-Glcp、t-Glcp、(1→4)-Galp和t-Rhap残基;分子量分布为11.2kD的多糖占主要部分,其他还包括45.5kD、4.0kD和1.8kD的多糖。
优选地,由肉苁蓉多糖的制备方法制备得到。
肉苁蓉多糖在制备治疗或预防慢传输型便秘药物中的应用。
本发明具有的优点和积极效果是:提供一种荒漠肉苁蓉多糖CDPs及其制备方法,该类结构的肉苁蓉粗多糖CDPs能够用于制备治疗或预防慢传输型便秘药物,与传统泻药比较,具有生物活性的天然化合物其无副作用且药物依赖性低;并能够有效缓解便秘症状。
附图说明
图1肉苁蓉粗多糖CDPs的结构特征表征,(A)HPGPC色谱图;(B)红外光谱图;(C)单糖组成色谱图;(D)甲基化分析总离子流图;
图2慢传输型便秘相关参数变化。(A)粪便颗粒数;(B)粪便含水量;(C)首颗黑便排出时间;(D,E)血清中胃肠动力激素(SP和VIP)水平。数据以平均值±sem(SEM)。*P<0.05,**P<0.01,***P<0.001,and****P<0.0001。CON:对照组;LOP:洛哌丁胺诱导的便秘模型组;LOP+PL:低剂量组(100mg/kg);LOP+PM:中剂量组(200mg/kg);LOP+PH:高剂量组(400mg/kg);
图3肉苁蓉多糖给药后小鼠结肠形态学变化。(A)结肠切片HE染色(箭头指示结肠肌层厚度;比例尺:400或100μm);(B)结肠切片alcian blue染色(箭头指示杯状细胞;比例尺:400或100μm);(C)统计分析结肠肌肉层厚度;(D);统计分析结肠隐窝中杯状细胞数目。数据以平均值±sem(SEM)。*P<0.05,**P<0.01,***P<0.001,and****P<0.0001。CON:对照组;LOP:洛哌丁胺诱导的便秘模型组;LOP+PL:低剂量组(100mg/kg);LOP+PM:中剂量组(200mg/kg);LOP+PH:高剂量组(400mg/kg);
图4肉苁蓉多糖给药后小鼠肌间神经元及nNOS+神经元数目变化。(A)结肠LMMP组织肌间神经元(箭头指示神经元;比例尺:300μm);(B)结肠LMMP组织肌间神经元及nNOS+神经元双染(箭头指示nNOS+神经元;比例尺:100μm);(C)统计分析结肠肌间神经元数目;(D)统计分析结肠肌间nNOS+神经元数目;(E)统计分析结肠肌间nNOS+神经元占总神经元比例。数据以平均值±sem(SEM)。表示*P<0.05,**P<0.01,***P<0.001,and****P<0.0001。CON:对照组;LOP:洛哌丁胺诱导的便秘模型组;LOP+PL:低剂量组(100mg/kg);LOP+PM:中剂量组(200mg/kg);LOP+PH:高剂量组(400mg/kg);
图5小鼠结肠组织中抗氧化相关参数。结肠组织中(A)SOD活性,(B)GSH含量变化;(C)脂质过氧化(MDA)水平;(D-F)LMMP组织中iNOS及Cox2蛋白表达水平;(G-H)LMMP组织中IL-1β和TNF-α的mRNA表达水平。数据以平均值±sem(SEM)。表示*P<0.05,**P<0.01,***P<0.001,and****P<0.0001。CON:对照组;LOP:洛哌丁胺诱导的便秘模型组;LOP+PL:低剂量组(100mg/kg);LOP+PM:中剂量组(200mg/kg);LOP+PH:高剂量组(400mg/kg);
图6肉苁蓉多糖对便秘小鼠结肠肌间神经元中线粒体的氧化应激和功能障碍的影响。(A)Mitosox(红色)标记线粒体中超氧化物产生水平(比例尺:100μm)。(B)JC-1(绿色)标记线粒体膜电位(比例尺:100μm)。(C,D)统计分析荧光强度来评估Mitosox及JC-1产生水平。数据以平均值±sem(SEM)。表示*P<0.05,**P<0.01,***P<0.001,and****P<0.0001。CON:对照组;LOP:洛哌丁胺诱导的便秘模型组;LOP+PL:低剂量组(100mg/kg);LOP+PM:中剂量组(200mg/kg);LOP+PH:高剂量组(400mg/kg)。
具体实施方式
下面结合附图对本发明的实施例做出说明。
本发明涉及一种肉苁蓉多糖和制备方法及其在慢传输型便秘中的应用。荒漠肉苁蓉药材采集于甘肃省汇勤荒漠肉苁蓉种植基地,将荒漠肉苁蓉干燥药材处理后,经水提醇沉法处理后,纯化后制备得到肉苁蓉粗多糖。制备得到的肉苁蓉粗多糖CDPs对洛哌丁胺(loperamide)诱导的STC小鼠结肠肌间神经的保护作用,能够用于制备治疗或预防慢传输型便秘药物。
肉苁蓉多糖的制备方法,具体包括如下步骤:
步骤一:取荒漠肉苁蓉干燥药材,粉碎后加入70%乙醇溶液回流提取三次,脱脂处理,三次提取时间分别为2小时、1小时、1小时;
步骤二:脱脂后的药材加水回流提取三次,提取时间分别为2小时、1小时、1小时;
步骤三:合并三次水提取液,减压浓缩,加入95%乙醇至终浓度为80%,4℃过夜醇沉,离心弃去上清后收集沉淀得醇沉物;
步骤四:醇沉物加入适量去离子水,于室温下充分搅拌使其溶解完全后再次离心,上清液透析,分子截留3500Da,透析内液浓缩后冻干,制备得到荒漠肉苁蓉粗多糖CDPs。
对制备得到的肉苁蓉粗多糖CDPs进行分析,检测器分子量,及单糖组成。CDPs包括4个分子量段的多糖,分子量分布为11.2kD的多糖占主要部分,其他还包括45.5kD、4.0kD和1.8kD的多糖;主要由Glc残基组成,另外含有少量的Rha、GalA、Gal和Ara残基,具体为主要包括(1→4)-Glcp,另外还有(1→4,6)-Glcp、t-Glcp、(1→4)-Galp和t-Rhap残基。
肉苁蓉粗多糖CDPs能够用于制备治疗或预防慢传输型便秘药物,与传统泻药相比,具有生物活性的天然化合物由于其副作用小且药物依赖性低而受到重视,CDPs治疗提高了便秘小鼠血清中的抗氧化酶水平并降低了脂质过氧化。基于CDPs抗氧化特性,CDPs能够对结肠肌间神经元产生保护作用。
CDPs治疗显著降低了LMMP组织中的氧化应激相关生物标志物水平和结肠肌间丛内线粒体超氧化物产生。在生理条件下,线粒体负责大部分ROS的产生并调节细胞氧化还原平衡。过量的ROS产生与各种细胞成分的损伤相关,特别是线粒体DNA,因为它缺乏内含子并具有高转录速率。ROS诱导的氧化应激损伤导致线粒体DNA突变,影响线粒体功能和细胞过程。此外,过量的ROS产生可能导致ATP产生不足,增加线粒体通透性并通过打开通透性转换孔而降低ΔΨ,导致细胞功能障碍甚至细胞死亡。结果显示,CDPs治疗以剂量依赖方式减少了结肠肌间神经元中的ΔΨ耗散,推测CDPs在洛哌丁胺诱导的STC小鼠中提供了神经保护作用。
nNOS神经元在肠神经系统中起着调节肠道蠕动的作用,它产生的一氧化氮(NO),是一种抑制性神经递质,参与肠道平滑肌的松弛。先前的研究观察氧化应激可导致nNOS表达的增加,这种上调可能与神经元无法维持Ca2+稳态有关。细胞应激期间,会出现细胞内Ca2 +的内流,细胞质Ca2+水平的升高会激活nNOS。随后,由nNOS激活介导的NO释放导致STC患者结肠蠕动障碍。此外,大量NO产生可通过与超氧阴离子反应生成过氧亚硝酸盐,过氧亚硝酸盐是一种高度活性和氧化性的化合物,能够引起氧化损伤并修饰蛋白质、脂质和DNA等生物分子。结果发现,与正常小鼠相比,STC小鼠中nNOS阳性神经元在总神经元中所占比例较高。然而,CDPs治疗降低了结肠丛中nNOS神经元的比例,这可能有效防止了细胞内氧化应激的升高,进而减轻了细胞结构紊乱并保护了细胞功能。与此同时,还观察到CDPs治疗小鼠的LMMP中iNOS的表达减少,这可能导致NO产生减少,从而缓解结肠肌间神经病变。
CDPs主要由(1→4)-Glucan组成,另外含有少量果胶类多糖。通过小鼠模型实验能够看出,CDPs能够显著影响洛哌丁胺诱导的STC小鼠的排便相关参数、调节肠道调节相关肽的水平并改善结肠病理损伤。此外,CDPs通过减少氧化应激损伤,维持线粒体功能,从而保护结肠肌间神经元。这些数据揭示了CDPs能够通过改善氧化应激引起的结肠肌间神经病变缓解便秘症状,为CDPs应用于预防和治疗STC提供了理论依据。
下面结合附图对本发明方案做出说明,其中,未具体说明操作步骤的实验方法,均按照相应商品说明书进行,实施例中所用到的仪器、试剂、耗材如无特殊说明,均可从商业公司购买得到。
实施例1:肉苁蓉多糖CDPs的制备
荒漠肉苁蓉药材采集于甘肃省汇勤荒漠肉苁蓉种植基地,标本留存于天津医科大学药学院生药教研室。
取荒漠肉苁蓉干燥药材,粉碎后加入70%乙醇溶液回流提取三次,提取时间分别为2小时、1小时、1小时。取脱脂后的药材加水回流提取三次,提取时间分别为2小时、1小时、1小时,合并三次水提取液、减压浓缩,加入95%乙醇至终浓度为80%,于4℃过夜醇沉,离心弃去上清后收集沉淀得醇沉物。于醇沉物中加入适量去离子水,于室温下充分搅拌使其溶解完全后再次离心,上清液透析(分子截留3500Da),透析内液浓缩并冻干,得到荒漠肉苁蓉粗多糖(CDPs)。粗多糖得率(%)=(肉苁蓉粗多糖质量/肉苁蓉药材质量)×100%。对制备得到的产物进行称重,从荒漠肉苁蓉中提取出粗多糖的质量为55.5g,计算得率为2.78%。
在有关肉苁蓉多糖含量的研究中,各个实验室通过水提醇沉法提取多糖的含量范围相差很大,主要原因与提取温度、提取时间、药材产地和计量方法不同有关。本实施例中,最终所得的粗多糖是经过多次离心以及进行透析除杂后的水溶性肉苁蓉多糖质量。
实施例2:CDPs的理化特征研究
采用HPGPC法对CDPs的分子量分布进行了测定。以普鲁兰系列(分子量分别为642、337、194、107、47.1、21.1、9.6和6.1kDa)为标准品,称取标准品和样品各5mg,以超纯水溶解,配置5mg/mL普鲁兰系列标准溶液和样品溶液,过0.22μm滤膜备用。另称取CDPs样品5mg,以纯水溶解,配置成5mg/mL样品溶液,离心,上清液过0.22μm滤膜,制备得样品液。分别取10μL标准溶液和样品液,注入高效凝胶色谱(HPGPC)系统,以CH3COONH4溶液为流动相洗脱。以保留时间A为纵坐标,分子量对数C为横坐标,绘制标准曲,通过标准曲线计算CPCD的分子量分布范围。
由图1A可知,肉苁蓉粗多糖CDPs主要分布在1.1-2743kD范围内,主要由4个分子量段的多糖(P1,45.5kD;P2,11.2kD;P3,4.0kD;P4,1.8kD)组成,其中分子量分布为11.2kD的多糖占主要部分。近年来有学者从荒漠肉苁蓉中分离得到多个多糖,分子量均分布在该范围内,如从冷水浸泡液中提取分离得到的阿拉伯半乳聚糖分子量为201kDa,1,4-链接的葡聚糖(1,4-glucan)平均分子量为10kDa,鼠李半乳糖醛酸聚糖分子量为870kD,通过连续膜过滤从荒漠肉苁蓉中分离纯化出的三种果胶类多糖(CDP-A,CDP-B和CDP-C)分子量分别为400kDa,240kDa,120kDa。可见,分离得到的酸性多糖的分子量分布在较高的范围内,而中性的葡聚糖主要分布在较低的范围内。本实施例分离得到的CDPs具有较低的分子量。
以硫酸苯酚法测定总糖含量。配制1mg/mL半乳糖标准品溶液,并依次稀释成200μg/mL、100μg/mL、50μg/mL、25μg/mL和12.5μg/mL,加入0.2mL5%苯酚溶液,快速加入l.0mL浓硫酸,充分震荡后,静置,在490nm处测定吸光度,以浓度C为横坐标,吸光度A为纵坐标,绘制标准曲线。取干燥样品配制成100μg/mL和50μg/mL溶液,按照上述方法显色后测定吸光度,代入标准曲线并计算肉苁蓉多糖样品总糖的平均含量,结果如表1所示。
以间羟基联苯法测定糖醛酸含量。配制1mg/mL半乳糖醛酸标准品溶液并稀释为100μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL和1.25μg/mL系列标准品溶液,分别取200μL半乳糖醛酸标准品系列溶液,加入硫酸-四硼酸钠溶液,冰浴冷却,然后100℃水浴加热5min,冷却后加入20μL间羟基联苯溶液,充分震荡后,静置5min,在520nm测定吸光度,以浓度C为横坐标,吸光度A为纵坐标,绘制标准曲线。取干燥肉苁蓉多糖样品配制为100μg/mL和50μg/mL溶液,按照上述方法显色后测定吸光度,带入标准曲线,计算肉苁蓉多糖样品糖醛酸的平均含量,结果如表1所示。
表1
通过光谱图初步判定CDPs的结构特征,以FT-IR光谱初步分析CDPs的主要官能团。取多糖CPCD样品2mg,加入100mg干燥KBr,放入干燥箱中烘干水分,转入玛瑙研钵中研磨混合,在液压器中进行压片,将压好的KBr片用红外光谱仪在4000-400cm-1区域内进行红外扫描。图1B为CDPs的红外光谱图,3428cm-1和2927cm-1处的吸收峰分别归属于糖环上的O-H的伸缩振动和糖环上亚甲基C-H伸缩振动,为典型的的多糖吸收峰。1027cm-1为糖环上C-O-C的伸缩振动,提示CDPs中吡喃糖环的存在。另外,在1748cm-1处的信号为羰基的(C=O)振动产生,表明含有一定量的糖醛酸,与化学组成中糖醛酸含量测试结果一致。
实施例3:CDPs单糖组成及糖苷键组成分析
采用PMP衍生结合HPLC测定法分析CDPs的单糖组成。准确称取单糖标准品(Fuc、Xyl、Rha、Ara、Man、Gal、Glc、GlcA、GalA)和样品各1mg,以去离子水溶解(配制浓度为1mg/mL),分别取各样品0.3mL,加入0.3mL 4mol/LTFA溶液,于120℃下加热水解2h,40℃水浴,N2蒸干。再以0.3mL去离子水溶解,转移至螺帽反应管中,加入等体积0.6mol/L NaOH溶液与0.6mL 0.5mol/L PMP甲醇液,混匀后于金属浴70℃避光反应30min,冷却至室温,以HCl调节至中性。以三氯甲烷萃取4-5次,弃去氯仿层。过微孔滤膜(0.22μm),转移至HPLC小瓶中。色谱条件:等度洗脱,流动相A(CH3COONH4)83%,流动相B(乙腈)17%,总流速1mL/min,检测波长245nm,进样量5μL,柱温30℃。
由图1C和表1可以看出,CDPs主要由葡萄糖(Glc)残基组成,另外含有少量的鼠李糖(Rha),半乳糖醛(GalA),半乳糖(Gal)和阿拉伯糖(Ara)残基。结果提示,肉苁蓉多糖可能主要由葡聚糖和果胶类多糖组成。
为了进一步确定糖苷键的类型,根据单糖组成,按照甲基化、水解还原、乙酰化等一系列步骤对CDPs的糖苷键组成进行了分析。结果如图1D和表2所示,总离子流图显示,p2为CDPs的主要糖苷键组成,鉴定为(1→4)-链接的葡萄糖残基((1→4)-Glcp),其占绝对比例(91.7%),另外还有p1((t-Glcp),p3((1→4,6)-Glcp),(1→4)-Galp,和t-Rhap残基。结果提示,CDPs主要由(1→4)-Glcuan组成,且在O-6位有分支。
表2
实施例4:CDPs对小鼠便秘参数的影响
4.1动物及分组
取8周龄,体重在20±2g之间,无特定病原体感染的C57BL/6小鼠,由北京华阜康生物科技股份有限公司提供。所有小鼠均被饲养在12/12小时明暗循环并提供标准商用鼠粮和水的恒温房间内。所有动物饲养及实验方案都严格按照中国动物保护委员会的指南进行,并获得天津医科大学总医院动物伦理及福利委员会的批准。
一周适应期后,将25只小鼠随机分为5组(每组n=5),分别为:正常对照组(CON);洛哌丁胺诱导的STC模型组(LOP);洛哌丁胺+低剂量CDPs组(LOP+PL);洛哌丁胺+中剂量CDPs组(LOP+PM);洛哌丁胺+高剂量CDPs组(LOP+PH)。除了CON组外,其他各组的小鼠口服洛哌丁胺10mg/kg,每天两次,连续14天。在洛哌丁胺灌胃期间,给予不同浓度(100、200和400mg/kg)的CDPs,而CON组和LOP组口服灌胃等体积的生理盐水。
每天监测小鼠的一般生理状态及粪便情况。在动物实验结束时,用乙醚麻醉并处死小鼠,收集血清及结肠组织进行下一步实验分析。
4.2检测排便相关指标
灌胃给药14天后,所有小鼠禁食24小时后给予0.2ml的印度墨水(0.4mg/ml,上海源叶生物科技有限公司),以确定第一颗黑便排出的时间来评估全胃肠道传输时间。在印度墨水给药后的6个小时内,收集每只小鼠的粪便,并记录粪便粒数及湿重。将收集的粪便在60℃电烤箱中干燥直至重量不变,即为干重。根据下面公式计算粪便含水量:粪水含量(%)=[(湿重-干重)/湿重]x 100。
经统计发现,LOP组表现出明显的便秘症状,包括6小时排便颗粒数减少及粪便含水量降低(图2A-B,LOP组与CON组,P<0.0001)。经过CDPs治疗可有效减轻上述症状,并且与LOP+PL组相比,LOP+PH组的缓解效果更为显著(P<0.05)。此外,LOP组小鼠首粒黑便排出时间延长,CDPs呈剂量依赖性缩短排便时间(图2C,LOP+PH组与LOP+PL组,P<0.01)。
为了更好研究CDPs对小鼠便秘症状的缓解效果,检测血清中2种与肠动力调节相关肽水平。结果表明LOP组小鼠SP水平最低,而VIP水平最高。然而,CDPs治疗后,相关肽水平变化得到了明显的恢复(图2D-E,P<0.01),其中LOP+PH组的疗效最为显著,SP含量增加了16.01ng/L,VIP含量降低了12.18ng/L。CDPs可显著缓解STC小鼠的便秘症状。
4.3组织病理学检测
收集小鼠远端结肠组织,用10%多聚甲醛溶液固定2天并石蜡包埋,然后将结肠组织切成5μm厚的切片,遵照标准组织学染色步骤,样本用苏木精和伊红(hematoxylin andeosin,H&E,Solarbio,北京,中国)或阿利新蓝(Alcian blue,Solarbio,北京,中国)染色。并在光学显微镜下评估组织形态学变化。
对远端结肠组织切片进行了HE和阿利新蓝染色以评估不同剂量CDPs对结肠形态以及杯状细胞数量变化的影响。结果如图3A-B所示,LOP组上皮细胞排列紊乱,结肠黏膜层较不完整,而CDPs治疗可恢复结肠黏膜的完整性。此外,还观察到随着CDPs剂量的增加,治疗效果明显提高,主要表现在相较于其他组,400mg/kg剂量给药可显著恢复结肠肌层厚度图(图2C,LOP+PH组与LOP+PL组,P<0.05)并增加杯状细胞数量(图2D,LOP+PH组与LOP+PL组,P<0.0001)。4.4CDPs对结肠肌间神经元病变的作用
小鼠眼眶取血,在室温下静置30分钟后,3500转/分钟4℃离心10分钟后收集上层血清。使用ELISA试剂盒(48T,南京森贝伽生物科技有限公司,中国南京)按照生产厂家的说明检测血清中物质P(substance P,SP)和血管活性肠肽(vasoactive intestinalpeptide,VIP)的含量。
用含蛋白酶抑制剂的RIPA缓冲液从LMMP中提取总蛋白。BCA蛋白浓度测定试剂盒(Solarbio,Beijing,China)估计样品中蛋白浓度。从每个样品中取等量蛋白质稀释于蛋白上样缓冲液中并加热变性。变性的蛋白质用SDS-PAGE凝胶电泳分离并转移到PVDF膜上。PVDF膜用5%奶粉封闭2小时,然后与一抗4℃孵育过夜:抗兔iNOS(1:1000,Affinity);Cox2(1:1500,Affinity)和GAPDH(1:3000,Servicebio)。第二天,PVDF膜在室温下与二抗(抗兔,1:3000)孵育40分钟。最后,ECL试剂显影蛋白印迹,用Image J软件计算。
洛哌丁胺诱导的STC小鼠结肠肌间神经元丧失,为了探究CDPs是否具有缓解结肠肌间神经元病变的潜力,用β3-微管蛋白抗体标记LMMP制备物中的神经元。
制备纵形肌-肌间神经丛(longitudinal muscle and myenteric plexus,LMMP)制备物。将结肠组织浸泡在含氧磷酸盐缓冲液(PBS)中并冲洗肠腔内残留物,然后将组织拉伸固定在硅板上,用眼科镊在显微镜下将LMMP与结肠其他层分离以暴露肌间神经丛。
用4%多聚甲醛将LMMP制备物4℃固定过夜后在0.5% Triton中孵育2个小时,用5%正常山羊血清在室温下封闭2个小时,随后与一抗:β3-管蛋白(兔,1:300,CellSignalling Technologies),HUC/D(小鼠,1:200,Invitrogen)和nNOS(兔,1:200,CellSignalling Technologies)在4℃孵育过夜,第二天与标有不同荧光团的特异性二抗:羊-抗兔IgG(1:100,Proteintech)和羊-抗小鼠IgG(1:100,Abclonal)在室温下孵育1小时。最后用抗荧光淬灭封片剂封片并在荧光显微镜下观察。随机选取了每个样本的5个区域,计数HuC/D+和nNOS+神经元的数量。
结果如图4A,CDPs改善了洛哌丁胺导致的结肠肌间神经纤维密度稀疏。为了定量分析这种改善效果,用泛神经元抗体HuC/D来标记神经元胞体并计数神经元数量,发现与正常组相比,LOP组中神经元数量显著减少(P<0.0001),而CDPs以剂量依赖方式缓解了结肠肌间神经元丢失(图4B-C,LOP+PH组与LOP+PL组,P<0.01)。
LOP组nNOS+神经元数量减少(图4B,D,LOP组与CON组,P<0.01)但nNOS+神经元占总神经元的比例比CON组高(图4B,E,P<0.0001)。在CDPs治疗后,LOP+PH组nNOS+神经元数量增加(LOP+PH组与LOP组,P<0.01),几乎接近CON组水平。同时,LOP+PL组nNOS+神经元比例显著下降(P<0.05)。
洛哌丁胺用于诱导STC动物模型,其适用性和稳定性已经得到了广泛的验证。LOP组中所有小鼠均表现出典型的便秘表型,而CDPs治疗显示出剂量依赖性的恢复效果。试验结果表明,CDPs在改善洛哌丁胺诱导的STC小鼠中具有显著的作用。
4.5抗氧化酶及脂质过氧化检测
CDPs是一种天然的抗氧化剂,能有效清除多种自由基。本实施例评估了CDPs抵抗洛哌丁胺诱导的结肠及LMMP组织氧化应激的能力。
取适量结肠组织在裂解缓冲液中磨成匀浆,离心获取上清液。用超氧化物歧化酶(Superoxide Dismutase,SOD)活性检测试剂盒(Solarbio,北京,中国)和还原型谷胱甘肽(Reduced Glutathione,GSH)含量检测试剂盒(Solarbio,北京,中国)评估结肠组织中抗氧化酶水平,用丙二醛(Malondialdehyde,MDA)含量检测试剂盒(Solarbio,北京,中国)评估脂质过氧化水平。以上实验步骤均按照制造商说明进行。
定量聚合酶链式反应(q-PCR)分析,根据制造商说明书(简石生物,北京,中国)从LMMP中提取纯化总RNA,并用分光光度计(Thermo Fisher Scientific Inc.)确定RNA的浓度和纯度。用FastKing gDNA Dispelling RT SuperMix(TIANGEN BIOTECH,Beijing,China)进行反转录。随后配制PCR反应体系进行扩增。引物序列如表3所示,用2-ΔΔCT方法计算目标基因相对表达水平。
表3
结果如图5A-C所示,与LOP组相比,CDPs明显提高结肠组织中SOD活性(P<0.001)和GSH含量(P<0.05),降低了MDA含量(P<0.01),并且CDPs抗氧化应激的能力随着给药剂量的增加而增强。近一步探究了CDPs在LMMP组织中抗氧化应激能力。治疗组中氧化应激相关蛋白iNOS(图5E,P<0.01)和Cox2(图4F,P<0.05)水平显著低于LOP组。此外,q-PCR分析显示,CDPs治疗后LMMP组织中炎性细胞因子IL-1β(P<0.05)和TNF-α(P<0.0001)的mRNA表达显著降低(图5G-H),证明CDPs具有抗炎特性。
4.6评估线粒体超氧化物产生
LMMP制备物中线粒体产生的超氧化物用MitoSOX线粒体超氧化物红色荧光探针(YEASEN,中国上海)检测。为了观察并量化结肠肌间神经元线粒体超氧化物的产生,用新型荧光探针MitoSOX Red标记LMMP制备物,MitoSOX Red荧光探针可以被线粒体内的超氧阴离子(O2-)氧化后发出红色荧光。
制备物在5μM的MitoSOX指示剂中37℃恒温孵育40分钟,并用4%多聚甲醛4℃固定过夜,第二天用β3-微管蛋白4℃过夜孵育,加入二抗(山羊抗兔IgG,1:100)在室温下孵育1小时后用荧光显微镜拍照。所有图像均以相同的曝光条件拍摄并用Image J软件计算平均荧光强度。
结果如图6所示,LOP组荧光强度最强(图6A,C,P<0.0001)表明洛哌丁胺加重了线粒体氧化应激水平。CDPs处理后,MitoSOX荧光强度呈现下降的趋势(P<0.05),其中最高剂量的CDPs给药能显著缓解结肠肌间神经元中线粒体氧化应激。
4.7检测线粒体膜电位变化
为了进一步探究CDPs对线粒体损伤的保护作用,用JC-1荧光探针特异性检测线粒体膜电位(ΔΨ)变化情况。用线粒体膜电位检测试剂盒(JC-1,Solarbio,北京,中国)来评估结肠肌间神经元线粒体膜电位变化,这可以反映出线粒体损伤情况。将LMMP制备物解剖出来后立即用JC-1溶液在37℃孵育20分钟。随后用JC-1缓冲液洗涤(3×10分钟)并在荧光显微镜下成像。
结果如图6所示,由于与操作相关的细胞应激会引起线粒体短暂去极化,导致各组JC-1荧光强度增高。然而,LOP组荧光强度增加更为显著(图6B,D,LOP组与CON组,P<0.0001)。与LOP组相比,CDPs给药导致JC-1荧光强度呈剂量依赖性下降(LOP+PH组与LOP+PL组,P<0.05)。
CDPs具有清除多种自由基的能力且呈剂量依赖关系,如羟基自由基、超氧阴离子自由基、DPPH自由基和ABTS自由基。CDPs可消除自由基,抑制脂质过氧化。此外,CDPs增强内源性抗氧化防御机制,特别是通过上调SOD活性和GSH含量。CDPs治疗显著降低了LMMP组织中的氧化应激相关生物标志物水平和结肠肌间丛内线粒体超氧化物产生。CDPs治疗以剂量依赖方式减少了结肠肌间神经元中的ΔΨ耗散,推测CDPs在洛哌丁胺诱导的STC小鼠中提供了神经保护作用。
与正常小鼠相比,STC小鼠中nNOS阳性神经元在总神经元中所占比例较高。然而,CDPs治疗降低了结肠丛中nNOS神经元的比例,这可能有效防止了细胞内氧化应激的升高,进而减轻了细胞结构紊乱并保护了细胞功能。与此同时,通过实验结果还观察到CDPs治疗小鼠的LMMP中iNOS的表达减少,这能够导致NO产生减少,从而缓解结肠肌间神经病变。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (9)
1.一种肉苁蓉多糖的制备方法,其特征在于:包括如下步骤:
步骤一:取荒漠肉苁蓉干燥药材,粉碎后加入70%乙醇溶液回流提取三次,脱脂处理;
步骤二:脱脂后的药材加水回流提取三次;
步骤三:合并三次水提取液,加入乙醇进行醇沉,取醇沉物;
步骤四:醇沉物加水溶解,透析浓缩后冻干,制备得到荒漠肉苁蓉粗多糖CDPs。
2.根据权利要求1所述的肉苁蓉多糖的制备方法,其特征在于:步骤三中,将三次水提取合并液浓缩,加入95%乙醇至终浓度为80%,4℃过夜醇沉。
3.根据权利要求1所述的肉苁蓉多糖的制备方法,其特征在于:步骤四中,透析中分子截留3500Da。
4.根据权利要求1-3中任一所述的肉苁蓉多糖的制备方法,其特征在于:制备得到的苁蓉粗多糖CDPs包括4个分子量段的多糖,分子量分布为11.2kD的多糖占主要部分,其他还包括45.5kD、4.0kD和1.8kD的多糖。
5.根据权利要求4所述的肉苁蓉多糖的制备方法,其特征在于:制备得到的苁蓉粗多糖CDPs主要由Glc残基组成,另外含有少量的Rha、GalA、Gal和Ara残基。
6.根据权利要求5所述的肉苁蓉多糖的制备方法,其特征在于:制备得到的苁蓉粗多糖CDPs主要包括(1→4)-Glcp,另外还有(1→4,6)-Glcp、t-Glcp、(1→4)-Galp和t-Rhap残基。
7.一种肉苁蓉多糖,其特征在于:主要由Glc残基组成,另外含有少量的Rha、GalA、Gal和Ara残基;主要包括(1→4)-Glcp,另外还有(1→4,6)-Glcp、t-Glcp、(1→4)-Galp和t-Rhap残基;分子量分布为11.2kD的多糖占主要部分,其他还包括45.5kD、4.0kD和1.8kD的多糖。
8.根据权利要求7所述的肉苁蓉多糖,其特征在于:由权利要求1-6中任一所述的肉苁蓉多糖的制备方法制备得到。
9.权利要求7或8所述的肉苁蓉多糖在制备治疗或预防慢传输型便秘药物中的应用。
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