CN114028375A - 衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用 - Google Patents
衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用 Download PDFInfo
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Abstract
本发明涉及衣康酸二甲酯在制备治疗和/或预防肥胖诱导肠道微生态失调的药物中的应用。经过试验验证,衣康酸二甲酯可纠正肥胖小鼠的肠道微生态失调,下调肠道炎症,维持肠道屏障完整性。
Description
技术领域
本发明涉及衣康酸二甲酯在制备治疗和/或预防肥胖诱导肠道微生态失调药物中的应用。
背景技术
肥胖是严重危害人类健康的流行性、全身代谢性疾病。2016年《柳叶刀》报道,目前全球肥胖人口约6亿,大量证据表明,肥胖不仅可显著增加心、脑血管疾病、糖尿病、非酒精性脂肪肝、高血压病、癌症等疾病的风险,而且可引起认知功能的减退,是引发认知功能障碍和阿尔茨海默病的重要诱因。因此,肥胖引起的相关疾病已成为严峻的公共卫生问题。
肠道微生态平衡是维持人类健康的重要因素。长期摄入高脂饮食会造成肠道菌群紊乱,损伤肠粘膜完整性,内毒素(LPS)增多,诱导肠道炎症。LPS等细菌产物可通过损伤肠粘膜进入血液,诱发全身性慢性低度炎症。后者会导致胰岛素抵抗、加剧糖尿病进程;可通过肠-肝/脑轴介导脂肪肝、神经炎症及认知下降等。由此可知,肠道微生态失调在推动肥胖及其引起的相关疾病进展中发挥着极其重要的作用。因此,纠正肠道菌群紊乱,改善肠道微生态,是治疗肥胖及其相关疾病的重要策略。
衣康酸是一种不饱和二元羧酸,是一种重要的化工原料。免疫代谢学的兴起,极大地推动了衣康酸在免疫调节及干预炎性疾病中的作用认识。研究发现,巨噬细胞受到LPS等炎性刺激时会产生大量衣康酸。现已证实,衣康酸是巨噬细胞中免疫与代谢的重要连接点,也是负向调控其炎症反应的一个关键结点。由于天然的衣康酸无法通过细胞膜,限制了其作为抗炎药物的应用。衣康酸二甲酯是重要的衣康酸酯类衍生物,可透过细胞膜,可显著下调巨噬细胞诱导的炎症反应,具有较好的抗氧化应激能力。近期,研究者在银屑病、多发性硬化症等小鼠模型中,证实了给予衣康酸二甲酯可明显改善这些炎性疾病的病情。这些结果提示,衣康酸二甲酯具有作为候选抗炎药物的潜力。但暂未见将衣康酸二甲酯用于预防和/或治疗肥胖诱导肠道微生态失调的报道。
发明内容
为了克服上述现有技术的不足,本发明提供了衣康酸二甲酯用于预防和/或治疗肥胖诱导肠道微生态失调的药物中的应用。
本发明采用的技术方案是:衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述衣康酸二甲酯,包括衣康酸二甲酯及其药学上可接受的盐制备的药物;所述药物用于下列用途中的至少一种:
a)预防和/或治疗肥胖诱导的肠道微生态失调;
b)预防和/或治疗肥胖诱导的粘膜屏障受损;
c)预防和/或治疗肥胖诱导的肠道炎症反应。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述药物适用于增加肠道粘液厚度以及增加紧密连接蛋白的表达。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述紧密连接蛋白包括ZO-1和Occludin。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述药物适于降低肠组织中炎症因子表达。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述炎症因子包括TNF-α、IL-6和IL-1β。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述药物中还含有药学上可接受的辅料。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述药学上可接受的辅料为填充剂、粘合剂、崩解剂、润滑剂、矫味剂、着色剂、掩味剂、pH调节剂、缓冲剂、防腐剂、稳定剂、抗氧化剂、润湿剂、湿度调节剂、表面活性剂、悬浮剂和吸收增强剂中的至少一种。
作为本发明所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用的优选方案:所述药物为口服制剂或注射剂。
本发明的有益效果是:本发明通过具体试验证明了衣康酸二甲酯可有效预防肠道微生态失调,维持肠道屏障完整性,下调肠道炎症,为基于改善肠道微生态的肥胖及相关疾病的预防和治疗策略提供了新思路。
附图说明
图1是衣康酸二甲酯对小鼠肠道菌群OTU的影响;A、正常小鼠;B、高脂饮食诱导肥胖小鼠;
图2是衣康酸二甲酯对小鼠肠道菌群的α多样性的影响;A、Chao1指数;B、Shannon指数;*P<0.05;***P<0.001;
图3是衣康酸二甲酯对小鼠肠道菌群相对丰度的影响;
图4是衣康酸二甲酯对小鼠肠道厚壁菌门、拟杆菌门丰度及二者比例的影响;A、厚壁菌门丰度;B、拟杆菌门丰度;C、厚壁菌门与拟杆菌门丰度比值;*P<0.05,***P<0.001;
图5是衣康酸二甲酯对小鼠肠道厚壁菌门各级细菌丰度的影响;
图6是衣康酸二甲酯对小鼠肠道厚壁菌门各级细菌丰度的影响;
图7是衣康酸二甲酯对小鼠肠道乳杆菌丰度的影响;
图8是衣康酸二甲酯对正常饮食小鼠优势菌群的LDA分析;
图9是衣康酸二甲酯对高脂饮食诱导肥胖小鼠优势菌群的LDA分析;
图10是衣康酸二甲酯影响小鼠肠道菌群的KEGG分析(level 2水平);
图11是衣康酸二甲酯影响小鼠肠道菌群的KEGG分析(level 3水平);
图12是衣康酸二甲酯对肥胖小鼠肠道菌群中与阿尔兹海默病相关菌群的影响;
图13是衣康酸二甲酯对肥胖小鼠肠道菌群中与内毒素产生相关菌群的影响;
图14是阿尔新蓝染色检测衣康酸二甲酯对小鼠结肠粘膜厚度的影响;
图15是免疫荧光染色检测衣康酸二甲酯对小鼠结肠紧密连接蛋白ZO-1表达的影响;A、代表图;B、ZO-1荧光强度的统计结果;**P<0.01;***P<0.001;
图16是RT-PCR检测衣康酸二甲酯对小鼠结肠组织中紧密连接蛋白mRNA表达的影响;A、ZO-1;B、Occludin;*P<0.05,**P<0.01,***P<0.001;
图17是RT-PCR检测衣康酸二甲酯对小鼠结肠组织中炎症因子mRNA表达的影响;A、TNF-α;B、IL-6;C、IL-1β;**P<0.01;***P<0.001。
具体实施方式
下面进一步列举实施例以详细说明本发明。同样应理解,以下实施例只用于对本发明进行进一步说明,不能理解为对本发明保护范围的限制,本领域技术人员根据本发明阐述的原理做出的一些非本质的改进和调整均属于本发明的保护范围。下述示例具体的工艺参数等也仅是合适范围中的一个示例,即本领域技术人员可以通过本文的说明做合适范围内的选择,而并非要限定于下文示例的具体数据。
本发明实施例中使用的试验材料及方法如下:
需要说明的是,以下实验结果中对应的附图中所指:LC+Vehicle为正常饮食小鼠注射PBS对照组、LC+DI为正常饮食小鼠注射衣康酸二甲酯(Dimethyl itaconate,DI,货号:617-52.7,Sigma-Aldrich)对照组、HF+Vehicle为高脂饮食诱导肥胖小鼠注射PBS组、HF+DI为高脂饮食诱导肥胖小鼠注射DI组。
1、小鼠及分组
7周龄雄性C57BL/6J小鼠购自北京维通利华实验动物技术有限公司,适应一周后,随机分成4组,每组10只:①LC+Vehicle组:常规饲料喂养,每鼠每周2次腹腔注射200μl的无菌PBS溶液;②LC+DI组:常规饲料喂养,每鼠每周2次腹腔注射含DI(Dimethyl itaconate,0.5mg/kg,货号:617-52.7)的PBS溶液200μl;③HF+Vehicle组:高脂饲料喂养,每鼠每周2次腹腔注射200μl的无菌PBS溶液;④HF+DI组:高脂饲料喂养,每鼠每周2次腹腔注射含DI(0.5mg/kg)的PBS溶液200μl。上述小鼠于SPF级动物中心饲养12周。每周监测摄食量以及体重变化。
2、16S rRNA测序
提取小鼠粪便的基因组DNA并使用PCR扩增。根据PCR产物进行混样和纯化。构建好文库后,使用NovaSeq6000进行上机测序。根据Barcode序列和PCR扩增引物序列从下机数据中拆分出各样本数据,并对每个样本数据进行拼接质控,去除嵌合体得到有效数据。利用Uparse软件对样本的有效数据进行聚类,默认以97%的一致性将序列聚类成为OTUs(Operational Taxonomic Units),并对每个OTU的代表性序列进行筛选以作进一步注释。使用LEfSe软件进行线性判别分析(linear discriminant analysis,LDA)。基于Greengene数据库和OTU上的基因信息,进行PICRUSt功能预测。
3、阿尔新蓝染色
新鲜结肠用卡氏固定液(乙醇与冰醋酸按体积3:1混合)固定24h后,石蜡包埋,切成5μm的切片。将切片用二甲苯及梯度乙醇脱蜡,放入阿尔新蓝染液A(Servicebio,G1027)中染色10-15分钟,自来水冲洗后放入阿尔新蓝染液B(Servicebio,G1027)浸泡3分钟。洗净染液后梯度乙醇脱水封片。在光学显微镜下拍照并利用Image J统计结肠粘膜厚度。
4、免疫荧光
新鲜结肠用质量分数4%多聚甲醛固定24h后,石蜡包埋,制作石蜡切片(5μm)。切片脱蜡至水,抗原修复,3%BSA封闭,用ZO-1(Servicebio,GB11195,1:200稀释)一抗4℃下孵育过夜,用Cy3标记的山羊抗兔荧光二抗避光室温下孵育50分钟,DAPI(Servicebio,G1012)复染细胞核,避光室温孵育10分钟。滴加自发荧光淬灭剂5分钟,抗荧光淬灭封片剂封片。
使用OLYMPUS IX51倒置荧光显微镜对上述结肠组织样本进行观察,并用ImageJ统计视野内阳性细胞数量。
5、荧光定量RT-PCR
用Trizol法提取结肠组织RNA,并进行逆转录,使用Roche LightCycler 480II荧光定量PCR仪进行检测。用公式2-ΔΔCt计算特定基因的mRNA水平,并用β-actin mRNA水平进行归一化。荧光信号由荧光DNA结合染料(SYBR Green I)产生。荧光定量PCR仪上机程序:①预变性:95℃5分钟。②扩增循环:变性:95℃15秒。退火:60℃15秒。延伸:72℃15秒。共45个循环。③熔解程序:95℃5秒,65℃1分钟,然后加热升温至97℃变性DNA产物。④冷却程序:40℃30秒。引物序列见表1。
表1荧光定量PCR引物序列
6、统计分析
正态分布的计量资料以表示。多组计量资料间差异比较采用单因素方差分析(ANOVA),各组间的两两比较使用Turkey’s法检验,其中多个实验组与对照组比较采用Dunnett法检验。检验水准α=0.05。应用SPSS 21.0软件进行统计学分析。
7、结果
7.1、衣康酸二甲酯能够改善高脂饮食诱导肥胖小鼠的肠道微生态失调
16S rRNA测序结果表明,与正常饮食对照组相比,衣康酸二甲酯干预正常饮食小鼠肠道菌群存在825个独特的OTU;而与高脂饮食组相比,衣康酸二甲酯干预肥胖小鼠存在1080个独特的OTU(图1)。
与高脂饮食诱导肥胖小鼠相比,衣康酸二甲酯干预肥胖小鼠肠道菌群的α多样性(Chao1指数及Sharnnon指数)显著增加(图2)。
与高脂饮食诱导肥胖小鼠相比,衣康酸二甲酯干预肥胖小鼠肠道菌群的菌群相对丰度发生明显改变(图3);尤其厚壁菌门(p-Firmicutes)丰度显著降低,而拟杆菌门(p-Bacteroidetes)丰度显著增加,且二者比例显著降低(图4)。
进一步分析发现,衣康酸二甲酯主要可降低厚壁菌门中Clostridia纲、Clostridiales目、Ruminococcaceae and Lachnospiraceae科、Ruminiclostridium,Lachnospiraceae_NK4A136_group以及Lachnoclostridium属细菌的相对丰度(图5);而增加拟杆菌门中Bacteroidia纲、Bacteroidia目、Prevotellaceae科细菌的相对丰度(图6)。有意思的是,衣康酸二甲酯可增加Bacilli纲、Lactobacillales目、Lactobacillaceae科、Lactobacillus属细菌的相对丰度(图7)。Lactobacillus是一种重要的益生菌,已被证实可改善神经系统功能。
LEfSe分析发现,衣康酸二甲酯干预肥胖小鼠厚壁菌门细菌种类减少,而厚壁菌门细菌种类增多(图8,图9)。
以上结果表明,衣康酸二甲酯可有效改善肥胖小鼠的肠道微生态失调。
7.2、衣康酸二甲酯能够改善肥胖及相关疾病进展相关的肠道菌群
PICRUSt发现衣康酸二甲酯干预改变的小鼠菌群可能影响宿主功能。聚类分析发现,在2级水平及3级水平上,衣康酸二甲酯干预肥胖小鼠肥胖菌群均与未干预肥胖小鼠菌群不同(图10,图11)。
大量研究表明,肥胖会导致认知障碍,是阿尔兹海默病的重要诱因。我们发现,衣康酸二甲酯可明显降低肥胖小鼠中阿尔兹海默病相关细菌的丰度(图12)。
肠道细菌产生的内毒素(LPS)可通过肠-肝/脑轴等途径,促进肥胖相关脂肪肝、胰岛素抵抗及认知障碍。我们发现,衣康酸二甲酯可显著减少肥胖小鼠中能够合成LPS的细菌丰度(图13)。
以上研究结果表明,衣康酸二甲酯能够抑制肥胖小鼠中与疾病进展密切相关的有害细菌丰度。
7.3、衣康酸二甲酯能够改善高脂饮食对小鼠肠道粘膜的损伤作用
阿尔新蓝染色结果显示,相对正常对照组,肥胖小鼠结肠的粘膜厚度明显变薄,但衣康酸二甲酯干预可明显增加肥胖小鼠的粘膜厚度(图14)。
免疫荧光染色结果显示,相对正常对照组,肥胖小鼠结肠中紧密连接蛋白ZO-1+细胞数量减少;而衣康酸二甲酯可明显上调肥胖小鼠结肠中ZO-1+阳性细胞数量(图15)。
RT-PCR结果显示,相对正常对照组,肥胖小鼠结肠中紧密连接蛋白ZO-1及Occludin的mRNA表达明显下调;而衣康酸二甲酯可明显上调肥胖小鼠结肠中ZO-1及Occludin的表达(图16)。
以上研究结果表明,衣康酸二甲酯能够减轻高脂饮食对小鼠肠道粘膜的损伤作用,从而维持肠道粘膜的完整性。
7.4、衣康酸二甲酯能够减轻高脂饮食诱导肥胖小鼠结肠炎症
RT-PCR结果显示,相对对照肥胖小鼠,衣康酸二甲酯干预肥胖小鼠结肠中IL-6、TNF-α及IL-1β均明显下调(图17)。
这些结果表明,衣康酸二甲酯可有效减轻高脂饮食诱导肥胖小鼠结肠炎症。
综上,衣康酸二甲酯能够改善肥胖诱导的肠道微生态失调、增强肠道粘膜完整性以及下调肠道炎症反应。
以上公开的仅为本发明的具体实施例,但是,本发明实施例并非局限于此,任何本领域的技术人员能思之的变化都应落入本发明的保护范围。
Claims (9)
1.衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用。
2.根据权利要求1所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述药物包括衣康酸二甲酯及其药学上可接受的盐制备的药物;所述药物至少用于下列用途中的一种:
a)预防和/或治疗肥胖诱导的肠道微生态失调;
b)预防和/或治疗肥胖诱导的粘膜屏障受损;
c)预防和/或治疗肥胖诱导的肠道炎症反应。
3.根据权利要求2所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述药物用于增加肠道粘液厚度以及增加紧密连接蛋白的表达。
4.根据权利要求3所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述紧密连接蛋白包括ZO-1和Occludin。
5.根据权利要求2所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述药物用于降低肠组织中炎症因子表达。
6.根据权利要求5所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述炎症因子包括TNF-α、IL-6和IL-1β。
7.根据权利要求2所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述药物中还含有药学上可接受的辅料。
8.根据权利要求7所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述药学上可接受的辅料为填充剂、粘合剂、崩解剂、润滑剂、矫味剂、着色剂、掩味剂、pH调节剂、缓冲剂、防腐剂、稳定剂、抗氧化剂、润湿剂、湿度调节剂、表面活性剂、悬浮剂和吸收增强剂中的至少一种。
9.根据权利要求2所述的衣康酸二甲酯在制备用于预防和/或治疗肥胖诱导肠道微生态失调药物中的应用,其特征在于:所述药物为口服制剂或注射剂。
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CN115105463A (zh) * | 2022-07-27 | 2022-09-27 | 上海交通大学医学院附属第九人民医院 | 一种用于修复皮肤损伤的软膏 |
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