CN115501264A - 霸王花水提物在制备治疗结肠炎药物中的应用 - Google Patents
霸王花水提物在制备治疗结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，具体涉及霸王花水取物在制备治疗结肠炎药物中的应用。本发明研究结果显示，霸王花水提物可以显著降低溃疡性结肠炎模型小鼠的主要疾病特征，包括体重下降、疾病活动指数上升、结肠缩短及脾脏指数上升，改善结肠炎模型小鼠的肠道菌群紊乱，调节结肠炎模型小鼠结肠炎症和肠道屏障相关蛋白的表达，改善结肠炎对结肠造成的病理损伤。霸王花疗效确切，安全性高，药物开发基础好。
Description
技术领域
本发明属于生物医药技术领域。更具体地，涉及霸王花水提物在制备治疗结肠炎药物中的应用。
背景技术
溃疡性结肠炎(UC)是一种的慢性非特异性肠道炎症性疾病，其发病机制复杂，主要损害结肠粘膜和粘膜下层结构。UC患者分布人群广，由于复发率高，他们长期备受腹痛，腹泻等病症的折磨，且长期炎症刺激易导致病变部位发生炎症-癌症转化，因此UC患者患结直肠癌的风险显著高于其他患者。目前对UC的治疗不是很理想，临床常规药物治疗包括皮质类激素，氨基水杨酸和抗生素有许多局限性，包括过敏，高烧，骨折和感染风险增加等副反应以及长期使用造成的经济负担。因此，开发一种有效，低毒，经济的治疗UC的药物十分迫切。
霸王花(HUF)为仙人掌科植物量天尺的花，又名剑花。量天尺的果实为火龙果，目前广西种植面积达10万亩，已经成为全国最大的火龙果种植基地。在种植过程中采用“去花保果”，平均保一个果实要去掉6朵花，从而产生大量农业废弃物，而霸王花具有较高的营养价值，因其富含多种人体必需氨基酸、植物纤维及微量元素等营养物质而被人们广泛用作蔬菜食用。霸王花拥有优良的药用价值，其药用记载始载于萧步丹《岭南采药录》：“止气痛，理痰火咳嗽，和猪肉煎汤服之”，又如部分中药典籍记载“清热润肺，止咳，治肺结核......”(广州空军《常用中草药手册》)。霸王花富含营养和药用活性物质，包括多种人体必需氨基酸、微量金属元素、膳食纤维、果胶和黄酮、皂苷、多糖类化合物等，这些丰富的活性物质使得霸王花有着良好的抗炎、抗氧化、抗胃肠病和呼吸疾病等药理作用。对霸王花治疗结肠炎的机制进行探索，有望提供一种价格低廉、药效明确、副作用小的治疗结肠炎的药物。
发明内容
本发明要解决的技术问题是克服现有化药长期服用具有不同程度的副作用，提供一种霸王花水提物在制备治疗结肠炎中的应用，拓宽了霸王花提取物的应用范围，为霸王花提取物的应用提供了新途径。
本发明的目的是提供一种霸王花水提物在制备治疗结肠炎中的应用。
本发明上述目的通过以下技术方案实现：
发明人经动物实验证明，霸王花水提物可以显著降低溃疡性结肠炎模型小鼠的主要疾病特征，包括体重下降、疾病活动指数上升、结肠缩短及脾脏指数上升，改善结肠炎模型小鼠的肠道菌群紊乱，调节结肠炎模型小鼠结肠炎症和肠道屏障相关蛋白的表达，改善结肠炎对结肠造成的病理损伤。基于上述成果，本发明要求保护霸王花水提物在制备治疗哮喘中的应用。
进一步地，所述霸王花水提物的制备方法包括以下步骤：
将风干的霸王花药材剪碎成块状，加入质量体积比为10g/ml的水，煎煮提取两次，每次两小时，过滤，合并滤液，减压浓缩得霸王花水提物。
更进一步地，霸王花水提物的生药量为5～10kg/g。
进一步地，所述霸王花水提物可以降低结肠炎小鼠的主要疾病特征，包括体重下降、疾病活动指数上升、结肠缩短、脾脏指数上升以及肠炎症因子的表达。
TNF-α在UC中是主要的炎症因子之一，可进一步破坏紧密连接结构，升高肠壁通透性，从而影响肠上皮屏障功能。TNF-α还可以增加IL-1β、IL-6的表达，与肠道炎症的发生紧密相关。IL-6含量过高会影响肠上皮的分泌功能，从而导致上皮通透性增加，易造成机体内环境紊乱，诱发或者加重UC。IL-1β也被认为是UC进展过程中的重要致病指标，许多临床研究表明，结肠组织分泌的IL-1β水平与UC的严重程度密切相关。
更进一步地，霸王花水提物可以下调p38MAPK、上调IL-10、Occludin的表达。
p38MAPK是MAPK家族成员之一，可以介导炎症、应激等细胞反应。p38MAPK 通过磷酸化被激活后，调控下游转录因子，促进IL-1、TNF-α等细胞因子的分泌，引起肠黏膜屏障炎症反应。研究表明UC患者炎症部位肠黏膜中p-p38MAPK活性明显上调。IL-10 对控制肠道炎症有关键作用，IL-10可保护宿主免受过度炎症引起的组织损伤，抑制抗原提呈和产生，并抑制抗原表达和促炎趋化因子和细胞因子的产生。Occludin蛋白是跨膜蛋白的主要成员，有负责调控肠道屏障的通透性、封闭相邻细胞间隙和调节信号转导分子聚集等作用。
更进一步地，所述霸王花水提物可以调节结肠炎小鼠肠道菌群的紊乱。
肠道菌群失调已被证明是结肠炎发展的一个重要风险因素。肠道菌群与宿主免疫之间的相互作用在UC发病及调控中扮演重要角色，肠道微生物群的失衡是许多肠道疾病发展的重要危险因素。正常情况下，肠道菌群处于动态平衡，一旦菌群失衡，就会导致条件致病菌群或致病菌群增加，进而导致肠道疾病的发生。相关研究表明，UC 患者的肠道菌群的改变与炎症程度密切相关，肠道菌群致病菌所分泌的肠毒素使肠上皮通透性增高，肠腔内细菌及其产物进入肠黏膜固有层，导致黏膜免疫失调，损害肠黏膜屏障，进一步影响和改变肠道微生物群的多样性和组成。
本发明具有以下有益效果：
本发明研究结果显示，霸王花水提物可以抑制结肠炎模型小鼠体重下降、肠道缩短，降低结肠炎症因子表达水平，改善肠道损伤，下调p38MAPK并上调IL-10、 Occludin的表达，调节结肠炎小鼠肠道菌群，疗效确切，安全性高，药物开发基础好。
附图说明
图1为霸王花水提物对结肠炎小鼠体重及疾病活动指数的影响数据统计柱状图。
图2为霸王花水提物对结肠炎小鼠结肠长度和脾脏指数的影响数据统计柱状图。
图3为霸王花水提物对结肠炎小鼠结肠病理状态影响的HE、AB-PAS染色图。
图4为霸王花水提物对结肠炎小鼠结肠炎症因子的影响数据统计柱状图。
图5为霸王花水提物对结肠炎小鼠肺部p38MAPK、IL-10、Occludin表达印迹图和定量结果的影响统计柱状图。
图6为霸王花水提物对结肠炎小鼠肠道菌群的影响统计图。
图中，*表示与模型组相比，P＜0.05，**表示与模型组相比，P＜0.01；#表示与正常组相比，P＜0.05，##表示与正常组相比，P＜0.01。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
1.实验用主要试剂和仪器。
1.1实验用主要试剂：葡聚糖硫酸钠盐(DSS)(批号：MB5535-2，大连美伦生物技术有限公司)：202112，上海凡科维生物科技有限公司)；一抗IL-10(1∶1000，批号：ab9969，英国Abcam)，p38MAPK(1∶000，批号：3514064-1-AP，中国武汉三鹰)，Occludin(1∶1000，批号：27260-1-AP，中国武汉三鹰)，p-p38MAPK(1∶1000，批号： 28796-1-AP，中国武汉三鹰)，二抗Rabbit IgG H&L(HRP，1∶10000，批号：WJ340126，中国赛默飞世尔)。
1.2实验用主要仪器：酶联免疫检测仪(USBio-Tek公司)；凝胶成像系统(LiCorodyse CLx，美国)；显微摄影系统(DM18，德国徕卡)；Illumina MiSeq平台(美国Illumino sequencer)。
1.3除非特别说明，以下实施例所用试剂和材料均为市购。
1.4所述霸王花水提物的制备方法包括以下步骤：
1.5霸王花(HUF)购自广西玉林中药材市场(批号：201912251)，由谢阳娇研究院(广西中医药大学药学院)鉴定为鉴定为量天尺的花。HUF水提取物的制备如下：将风干的霸王花药材剪碎成块状，加入质量体积比为10g/ml的水，煎煮提取两次，每次两小时，过滤，合并滤液，减压浓缩得霸王花水提物1.5L。
2.溃疡性结肠炎动物模型的建立。
2.1雄性C57BL/6小鼠(7-8周龄)购自北京斯贝福生物科技有限公司(中国北京；编号SCXK2019-0010)，饲养在无病原体啮齿动物设施中，可自由获得食物和水。动物的护理和治疗是根据《公共卫生服务政策》制定的实验动物人道护理和使用指南进行的。所有实验动物方案均经广西医科大学动物护理与使用委员会批准。
2.2小鼠被随机分为5组(每组10只)：(1)正常对照组(NC)，(2)模型组(DSS)，(3)100mg/kg 5-氨基水杨酸组(5-ASA)，(4)10g/kg HUF(HUF-H)， (5)5g/kg HUF(HUF-L)。用3％的DSS代替饮用水7天(从第1天到第7天)，诱导实验性结肠炎。对照组小鼠口服ddH2O。在实验的第8至14天，分别给HUF组和5-ASA组的小鼠分别口服HUF和5-ASA，每天一次。NC组和DSS组给予ddH2O。每天观察并记录各组小鼠的体重和疾病活动指数(DAI)评分。在处死小鼠时，测量结肠长度并称重脾脏。
3.指标观察与检测方法。
3.1疾病活动指数：每天通过3个指标的组合计算每只小鼠的DAI：体重减轻 (0：无；1：减少1-5％；2：减少5-10％；3：减少11-15％；4：减少＞15％)，大便稠度(正常0分，稀便2分，腹泻4分)和大便潜血(正常0点，潜血阳性2分，明显出血4分)。DAI＝(体重减轻评分+粪便稠度评分+粪便潜血评分)/3。
3.2结肠组织学分析：组织学分析和高碘酸-希夫染色处死小鼠后，取结肠组织并用4％多聚甲醛固定。石蜡包埋的结肠组织切片用苏木精和伊红(H.E.)染色进行组织病理学评分，并用阿尔辛蓝-高碘酸-希夫(AB-PAS)评估结肠组织杯状细胞的变化。组织病理学评分是炎性细胞浸润的综合衡量标准，组织损伤的变化由专业病理学分析员进行：固有层中的少量炎性细胞渗透，0；固有层中炎性细胞数量增加，1：炎性细胞延伸到粘膜下层，2：跨壁浸润，3：组织损伤(无粘膜损伤，0；离散性淋巴上皮损伤，1：表面粘膜侵蚀，2：广泛的粘膜损伤和通过肠壁深部结构的延伸)。使用Image Pro Plus 6.0分析系统通过计算积分光密度(IOD)来评估杯状细胞。。
3.3结肠炎症因子分析：从每只小鼠称量一部分结肠，并以1∶9(m∶V)的比例加入PBS，并用自动均质机进行组织均质。在3000×g离心20分钟后，收集上清液。进行酶联免疫吸附试验(ELISA)以确定伤津液中的IL-1β、IL-6、TNF-α水平。
3.4蛋白质印迹分析：将肺组织样品在蛋白质细胞裂解缓冲液中裂解。样品的蛋白质浓度使用BCA蛋白质测定试剂盒测定。将样品煮沸15分钟，使用10％十二烷基硫酸钠聚丙烯酰胺凝胶(SDS-PAGE)进行电泳。将蛋白质转移到聚偏二氟乙烯 (PVDF)膜后，将膜在快速封闭缓冲液中下封闭15分钟。将PVDF膜与各种一级抗体在4℃下孵育过夜。用0.1％tris缓冲盐水吐温(TBST)洗涤膜三次，在室温下用二级抗体在封闭缓冲液中孵育2小时，然后再次用TBST洗涤。凝胶成像系统(Li Corodyse CLx，美国)用于生成灰度强度测量和定量分析。
3.5小鼠粪便肠道菌群16 S核糖体DNA(16 S rDNA)分析：所有粪便样本均进行了粪便细菌DNA提取、16S rDNA基因PCR扩增和测序以及16S rDA基因分析。简而言之，根据制造商的说明，使用QIAamp DNA粪便微型试剂盒(德国希尔登Qiagen) 从粪便样品中提取基因组DNA。提取基因组DNA后，使用Oebiotech(中国上海)，使用Illumina MiSeq平台(美国Illumino sequencer)分析16 S rRNA基因的V3-V4：343 F(5′-TACGGRAGGCAGCAG-3′)和798R(5′-AGGGTATCTAATCCT-3′)高变区的 DNA焦磷酸测序。QIIME软件Vsearch版本1.11.1和1.9用于执行序列数据读取，使用额外的质量脚本选择操作分类单元，包括聚类、去复制和嵌合体检测。
3.6统计分析：所有数据均表示为平均值±标准偏差(SD)。使用SPSS 23.0 软件进行统计分析。采用单因素方差分析进行多重比较，然后采用最小显著性差异 (LSD)检验进行组间比较。小于0.05的p值被认为具有统计学意义。
4.实验结果分析。
4.1体重和DAI：如图1和图2所示，与NC组相比，DSS组在治疗的第五天小鼠体重显著下降，DAI评分显著增加。此外，与NC组相比，DSS组小鼠的结肠长度显著缩短，脾脏指数显著升高。治疗后，HUF-H和5-ASA组的小鼠在第13天体重显著恢复，所有治疗组的DAI评分在第13天后显著下降。与DSS组相比，HUF-H组小鼠的结肠长度显著恢复，而在HUF-L和5-ASA治疗后，脾脏指数显著降低。这些结果表明，HUF有效地改善了UC的症状。
4.2组织学结果分析：如图3所示，NC组小鼠肠道腺体清晰，隐窝结构完整，绒毛排列整齐，无明显破损，无明显炎性细胞浸润。相比之下，DSS组的肠粘膜结构严重受损，隐窝结构不完整，绒毛组织紊乱，有明显的骨折和局部轻度至重度坏死，大量炎性细胞浸润肠壁。组织学评分显著增加。与DSS组相比，各剂量的HUF组和5-ASA组处理的小鼠的结肠粘膜结构显著改善，隐窝破坏明显减少，上皮结构相对完整，炎性细胞浸润迹象减少，组织病理学评分显著降低。NC组结肠杯状细胞的数量完整，DSS组显著减少，HUF-L和HUF-H治疗均显著恢复杯状细胞数量。这些结果表明， HUF可以减少DSS诱导的结肠粘膜损伤以及炎症反应。
4.3结肠炎症因子分析：如图4所示，DSS组的IL-β、TNF-α和IL-6均显著高于NC组。相反，每个剂量的HUF和5-ASA显著降低IL-β分泌水平。在HUF-L组中，升高的TNF-α有显著逆转，而在HUF-H组和5-ASA组中，虽然不显著，但有降低的趋势。每个治疗组都显示出降低IL-6促炎因子水平的趋势，但不幸的是，与模型组相比没有差异。这些结果表明HUF可以降低结肠异常炎症因子水平。
4.4结肠炎相关蛋白表达分析：如图5所示，DSS组紧密连接蛋白表达被抑制，occludin、IL-10蛋白表达显著下调，下调p38MAPK蛋白的表达。HUF和5-ASA治疗后，这些变化被显著逆转，表明HUF可能通过修复紧密连接结构和逆转肠道炎症进展的方式治疗结肠炎。
4.5肠道菌群分析：如图6所示，维恩图可以用来统计多组或多个样品中共同和独特物种(如OTU)的数量，可以直观地显示不同环境样品中物种组成的相似性和重叠性。如图6A所示，图中央的数字表示六组样品的重合度，从各组的物种组成重合度来看，六组环境样品的物种组成数量具有相似性。从图内的数字可以发现，与NC 组相比，DSS组环境样品中的微生物种类数量减少；与模型组相比，HUF各剂量组的微生物种类数量明显增加。肠道微生物群的丰富性和多样性是用α多样性来评价的。与 NC组相比，OVA组的Chao1指数和Shannon指数明显下降(图6E，G)，表明UC小鼠的微生物群落丰富度和多样性均降低。对比DSS组，HUF-L可以明显提高小鼠菌群的Chao1指数和Shannon指数，而HUL-H和5-ASA则无明显改善。另外，Chao1和 shannon指数显示，随着测序的深入，各组样品的曲线趋于平缓，说明测序深度已经覆盖了样品的所有物种，说明各组α-多样性的变化是由药物干预引起的，而不是测序过程引起的系统误差(图6D，F)。此外，采用Principal coordinate’s analysis(PoCA)分析和Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)确定5个实验组的群落组成相似性，不同组别的群落被划分为不同颜色区域。OPLS-DA结果显示(图6C)，DSS组样本曲线图明显偏离NC组，说明DSS诱导改变了肠道微生物群。而经过HUF各剂量组和5-ASA治疗后，群落曲线图显著接近恢复至NC组范围。PoCA结果为图6B，DSS组和各药物治疗组样本曲线图明显偏离NC组曲线图，但DSS组偏离更严重，且HUF-L治疗过后肠道微生物群的结构更接近NC组。这些结果表明了DSS 诱导了小鼠肠道菌群整体结构的破坏，而HUF能在一定程度上有效缓解肠道菌群这些破坏。
进一步分析UC小鼠肠道菌群的组成，如图6H所示，在phylum level上，各组小鼠肠道微生物群样品中，Firmicutes、Bacteroidetes和Proteobacteria占优势；与NC 组相比，经过DSS诱导的各组小鼠Firmicutes相对丰度显著增加而Bacteroidetes显著降低(图6I，J)；DSS组Proteobacteria的相对丰度较高，经过HUF和5-ASA治疗后，其丰度显著降低(图6K)。在genus level上，与NC组相比，DSS组小鼠的Mucispirillum、 Prevotella、[Prevotella]、Oscillospira和Paraprevotella相对丰度显著降低，相反地， Shigella、Bacteroides、和Blautia相对丰度则明显升高(图6L，M)。物种热图顶部的色块分别代表每个分组，物种组成越相近则相似度越高，图6M中HUF-L组和HUF-H 组与NC组相邻，且差异物种数量有所减少，表明HUF一定程度上改善了肠道菌群的紊乱。另外，采用lineardiscriminant analysis effect size(LEfSe)在不同组间定位具有统计学意义的标志菌群。图6O代表了每组中的标志物种，结合图6N来看，DSS组的标志物种明显更远离NC组，HUF-L组大部分与NC组重合。上述结果表明，UC小鼠的肠道菌群结构被破坏了。HUF治疗后，小鼠的微生物组成与NC组接近，说明HUF 对肠道菌群的紊乱有积极的调节作用。
实验总结。
由上述结果可知，中草药HUF通过抑制肠道屏障障碍、降低肠道炎症反应，改善UC相关联的细胞、分子和生物参数的表达，改善肠道损伤，上调结肠occludin、 IL-10并抑制p38MAPK蛋白的表达，调节肠道菌群，起到治疗UC的作用。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (7)
1.霸王花水提物在制备治疗结肠炎药物中的应用。
2.根据权利要求1所述应用，其特征在于，所述霸王花水提物的制备方法包括以下步骤：将风干的霸王花药材剪碎成块状，加入质量体积比为10g/ml的水，煎煮提取两次，每次两小时，过滤，合并滤液，减压浓缩得剑花水提物。
3.根据权利要求2所述应用，其特征在于，所得霸王花水提物的生药量为5～10kg/g。
4.根据权利要求1或2所述应用，其特征在于，所述霸王花水提物可以降低结肠炎小鼠的主要疾病特征，包括体重下降、疾病活动指数上升、结肠缩短及脾脏指数上升。
5.根据权利要求1或2所述应用，其特征在于，所述霸王花水提物可以降低结肠炎症因子表达。
6.根据权利要求1或2所述应用，其特征在于，所述霸王花水提物可以下调p38MAPK、上调IL-10、Occludin的表达。
7.根据权利要求1或2所述应用，其特征在于，所述霸王花水提物可以改善肠道菌群的紊乱。
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