CN113402582A - Ank三肽及其应用 - Google Patents
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- CN113402582A CN113402582A CN202110682017.XA CN202110682017A CN113402582A CN 113402582 A CN113402582 A CN 113402582A CN 202110682017 A CN202110682017 A CN 202110682017A CN 113402582 A CN113402582 A CN 113402582A
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Abstract
本发明公开了一种ANK三肽，其氨基酸序列为Ala‑Asn‑Lys。通过试验验证发现本发明的ANK三肽能够有效降低促炎因子mRNA表达水平，具有一定的医学价值，可用于开发预防和/或辅助治疗炎症性肠病的食品或药物。
Description
技术领域
本发明属于生物技术领域，具体涉及生物活性肽领域，特别涉及ANK三肽及其应用。
背景技术
克罗恩病(CD)和溃疡性结肠炎(UC)是最主要的两类炎症性肠病IBD，是世界范围内除心血管疾病外的严重性疾病之一，其发病率及流行率曲线随着时间的推移呈上升趋势。至今为止，IBD的发病机制尚不完全清楚，影响因素众多，如：环境因素，免疫反应因素，遗传因素，微生物因素等(Arora et al.,2015)。虽然IBD的治疗已经取得了很多进展，但它仍然是不治之症。临床上，UC的主要治疗方法是氨基水杨酸盐，严重的UC患者可使用短期的类固醇治疗，若以上治疗方式仍不能缓解UC的肠道炎症则对患者施用免疫调节剂和抗肿瘤坏死因子α(anti-TNFα)抑制剂以缓解其肠道炎症。氨基水杨酸盐在CD中几乎无效，对于远端回肠、回盲肠或右侧结肠疾病的患者，可以使用肠溶布地奈德诱导缓解，严重的CD患者可使用泼尼松龙缓解肠道炎症。若以上治疗方式仍不能缓解CD患者的肠道炎症则对其施用anti-TNFα抑制剂，但大约30％的患者使用抗anti-TNFα无效。在对抗肿瘤坏死因子α抑制剂有效的患者中，每年也有高达10％的患者对该药物失去反应。此外，目前的IBD药物伴随着严重的副作用，包括丧失免疫耐受性和耐药性、感染并发症、内分泌损伤、肾脏肝脏损伤(Nielsen,Bjerrum,Herfarth,&Rogler,2013；Poitras,Gougeon,Binn,&Bouin,2008)。目前的治疗方案疗效低，不良反应升高是临床治疗主要关注的问题。因此，寻求新的具有抗炎活性的食源性天然产物作为治疗IBD的替代药物或营养补充剂成为了研究者关注的重点。
研究表明，RPIV能够抑制TNF-α诱导Caco-2细胞IL-8的分泌，并抑制NF-κB、MAPK信号通路中磷酸化蛋白的表达。CR，FL，HC，LL和MK显著抑制TNF-α诱导de Caco-2细胞促炎细胞因子mRNA的表达，上调抑炎因子IL-10的表达。PAY有效抑制LPS诱导的RAW264.7细胞NO和COX-2的分泌。VPY显著抑制IL-8和TNF-α的分泌。
发明内容
本发明的目的是解决现有技术的不足，提供一种ANK三肽，其氨基酸序列为Ala-Asn-Lys，分子量为331Da。该ANK三肽通过固相合成法获得，能够有效预防并缓解炎症性肠病，可以应用在制备预防和/或缓解炎症性肠病的食品或药物中。
本发明的另一目的是提供一种药物组合物，其活性成分包括ANK三肽，用于预防和/或缓解炎症性肠病。该药物组合物还可以包括溶剂或可药用辅助剂。
本发明采用TNF-α诱导人肠上皮细胞(HT-29)体外炎症模型来研究在2、4、8、16mM的浓度该三肽的抗炎活性。测定典型细胞因子IL-8的含量，同时运用WST-1试剂盒测定细胞活力。本发明采用RT-PCR结合Western-blot，验证其抗炎作用机制。本发明采用DSS诱导Balb/c小鼠急性结肠炎模型，研究该三肽在体内的抗炎活性。
本发明的有益效果为：本发明提供了一种ANK三肽，通过试验验证发现本发明的ANK三肽能够有效降低促炎因子mRNA表达水平，具有一定的医学价值，可用于开发预防和/或辅助治疗炎症性肠病的食品或药物。
附图说明
图1所示为ANK对TNF-α诱导的HT-29细胞IL-8的分泌影响及其对细胞活力的影响情况图；
图2所示为ANK对TNF-α诱导的HT-29细胞因子mRNA表达水平的影响情况图；
图3所示为ANK对TNF-α诱导的HT-29细胞NF-κB通路中关键蛋白表达水平的影响情况图；
图4所示为ANK对小鼠的综合影响情况图，其中，(A)为ANK对DSS诱导的小鼠体重减轻的影响情况图，(B)为第14天各组小鼠体重变化情况图，(C)为ANK对DSS诱导的小鼠临床症状的影响情况图，(D)和(E)为ANK对DSS诱导的结肠长度的影响情况图；
图5所示为ANK对小鼠病变程度的影响情况图和个组小鼠结肠组织评分图，其中，(A)为ANK对DSS诱导的小鼠结肠组织病变程度的影响情况图，(B)为各组小鼠结肠组织评分图；
图6所示为ANK对DSS诱导的小鼠结肠组织中MPO表达水平的影响情况图；
图7所示为ANK对DSS诱导的小鼠结肠组织中促炎细胞因子TNF-α和IL-6含量的影响情况图；
图8所示为ANK对DSS诱导的小鼠结肠组织中促炎细胞因子mRNA表达水平的影响情况图。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、方案和效果。
一、三肽ANK在体外炎症模型中抗炎活性的验证及其作用机制的探讨
1.炎症模型建立
以3×105cells/mL的密度将细胞种于细胞培养板上，每隔24h换液，培养5-7天待细胞融合到85％左右，弃去培养液，用HBSS缓冲液洗2次。首先加入用含5％FBS的DMEM-F12新鲜培养液配置的不同浓度的三肽样品预孵育2h，其次再向培养板中加入TNF-α(使其终浓度为5ng/mL)继续孵育4h。孵育结束后，收集细胞上清液用于ELISA测定，下层细胞可根据不同实验要求进行处理。
2.细胞活力测定
参考Mengya Zhang(Zhang et al.,2018)等人所述方法对处理后细胞进行活力测定。实验细胞经HBSS清洗三次后，每孔加入等量WST-1稀释液均匀铺满细胞培养板，放入细胞培养箱中避光孵育15分钟左右，待溶液颜色变化显著时在450nm处测定OD值，按照如下公式计算细胞活力：
细胞活率(％)＝(As450/Ac450)x100％，其中，As450代表样品处理组，Ac450阴性未处理组。
3.IL-8含量测定
参考史雅凝等人(史雅凝，2015)的方法对IL-8含量进行测定。将样品稀释五倍后用于检测。
4.RNA-PCR检测
实验细胞经冰冷的HBSS清洗三次后，去除表面液体后加入适量TransZol裂解液，充分裂解后根据TransZOL Up Plus RNA Kit试剂盒提取HT-29中总RNA。通过测定A260/A280nm处比率评估RNA的浓度和纯度。采用Easy Script One-Step gDNA Removal andcDNA Synthesis SuperMix试剂盒合成cDNA。RT-PCR使用Perfect StartTM Green qPCRSuperMix试剂盒进行扩增，其条件为94℃变性5s，60℃退火15s和72℃延伸10s。整个反应保持40个扩增循环，测定关键基因的表达变化，使用GAPDH作为内部参考基因，并以POS阳性模型组做对照，计算其他组Fold Change值表达百分比，引物序列如表1。
表1
5.蛋白免疫印迹
对培养好的细胞进行预处理获得变性总蛋白。细胞蛋白经10％SDS-PAG胶分离后转移至用甲醇激活的PVDF膜上，5％脱脂奶粉室温封闭1.5h。经洗涤后，将膜充分浸入一抗稀释液中4℃过夜孵育。经用洗涤后，再将膜充分浸入二抗稀释液中孵育1h。经洗涤后，取出PVDF膜至ChemiDocTMTouch仪器中，用移液器滴入ECL显色液进行显色，并拍照记录。采用Image J软件进行灰度分析，计算各组蛋白相对表达量。
二、采用体内模型对三肽ANK进行体内抗炎活性验证，并探讨其作用机制
1.小鼠急性结肠炎模型
将由湖南斯克莱景达公司提供的80只6-8周龄，平均体重为18-20g的健康雌性Balb/c小鼠圈养并饲养一周后进行试验。圈养温度为25-27℃，湿度为74％，12h周期的光照与黑暗交替，并自由获取食物和水。将小鼠随机分为10组，如下表2所述，其中每组10只小鼠。表2
2.小鼠结肠炎临床症状评估
参照Cooper(Cooper，Murthy,Shah,&Sedergran,1993)提出的疾病活动指数评分标准如表3，每日观察并评估小鼠体重变化，大便黏稠度和大便出血情况。
表3
DAI＝(体重指数评分+大便性状评分+出血情况)/3。
3.结肠组织HE评分
用生理盐水清洗远端结肠并用4％多聚甲醛固定，石蜡包埋并切片，用苏木精和伊红染色液进行染色，在光学显微镜下拍照并评估组织学评分。
4.结肠组织TNF-α、IL-6测定
用生理盐水清洗结肠组织，根据动物组织全蛋白提取试剂盒按照说明书提取结肠组织总蛋白，并用BCA蛋白定量试剂盒测定上清蛋白浓度，按照小鼠ELISA试剂盒测定TNF-α和IL-6促炎因子浓度，结果以POS作为对照计算相对百分比。
5.抗氧化酶MPO测定
用生理盐水清洗结肠组织，并进行组织匀浆，按照MPO试剂盒说明测定酶活，结果以POS作为对照计算相对百分比。
6.结肠组织中基因表达测定
用生理盐水清洗结肠组织，经液氮研磨后加入1mL Trizol裂解液裂解5min，12000rpm 4℃离心10min，根据试剂盒提取组织总RNA。采用Easy Script One-Step gDNARemoval and cDNA Synthesis SuperMix试剂盒合成cDNA。RT-PCR使用Perfect StartTMGreen qPCR SuperMix试剂盒进行扩增，其条件为94℃变性5s，60℃退火15s和72℃延伸10s。整个反应保持40个扩增循环。测定关键基因的表达变化，并使用GAPDH作为内部参考基因，并以POS阳性模型组做对照，计算其他组Fold Change值表达百分比，引物序列如表4所示。
表4
三、体外试验结果
1.三肽ANK的抗炎活性验证
IL-8是一种中性粒细胞引诱剂，可诱导中性粒细胞从外周血迁移到炎症组织，与健康人群相比，IBD患者的组织中IL-8的显著增加。因此选用IL-8作为炎症标志物，初步筛选可能具有抗炎活性的三肽(Mahida,Ceska,Effenberger,Kurlark,Lindley,&Hawkey,1992)。如图1所示，采用TNF-α诱导HT-29细胞炎症模型，通过对炎症标志物IL-8的测定，研究在2、4、8、16mM浓度下三肽ANK的抗炎活性。结合WST-1法测定其细胞活力，发现模型组与空白对照组之间细胞活力无显著性差异。经三肽预处理的HT-29炎症细胞有效抑制IL-8的分泌(p<0.05)且无细胞毒性。与模型组相比，当ANK浓度高达16mM时，IL-8分泌量分别下降了99％。
2.三肽ANK对TNF-α诱导的HT-29细胞细胞因子mRNA表达水平的影响
基于前期研究，发现ANK具有抗炎作用。为了进一步探讨其抗炎机制，采用RT-PCR技术对经三肽ANK(16mM)处理后的HT-29细胞内mRNA表达进行分析。如图2显示，ANK能够有效抑制促炎因子TNF-α、IL-6、IL-8、IL-1β、IL-12和环氧化酶(COX-2)mRNA的表达。
3.三肽ANK对NF-κB信号通路的影响
NF-KB是一种关键的转录因子，调节编码促炎介质的基因的表达。采用Western-blot检测各试验组细胞p-p65、p-IκBα蛋白水平。结果表明(如图3所示)，ANK通过阻断TNF-α诱导HT-29细胞的IκBα的磷酸化和p65的磷酸化发挥其抗炎作用。
四、体内试验结果
1.三肽ANK对DSS诱导的小鼠急性结肠炎临床症状及体重减轻的影响
如图所示4(A)，各处理组初始体重无显著性差异，经DSS诱导炎症模型之前，各组小鼠体重未见显著下降，说明三肽ANK既不影响体重，也不导致腹泻。除空白组外各组小鼠体重在第10天发生显著下降，直至第14天呈持续下降趋势。如图4(B)，仅摄取4.5％DSS的模型组小鼠体重下降了15.56％，与模型组相比，施用50mg/mL和150mg/mL浓度的ANK有效减缓DSS诱导的急性结肠炎小鼠的体重减轻。
众多学者指出，DSS诱导小鼠急性结肠炎模型会出现小鼠体重下降、大便粘稠、直肠出血，精神状态萎靡等症状(Zhao,Yao,Xu,Wang,Wang,&Tu,2017)。我们根据前人建立的DAI评分系统对小鼠状态进行评价。如图4(C)所示，除空白组外，各组DAI值随病程加重而升高。与模型组相比，经ANK灌胃后各组小鼠DAI值均小于模型组，临床症状相对较轻。
在DSS诱导的小鼠结肠炎模型中，小鼠结肠缩短与结肠炎的严重程度密切相关。如图4(D)所示，在本研究中，模型组的结肠长度为(4.56±0.22)cm，而空白组结肠长度为(7.58±1.04)cm。ANK(50mg/mL和150mg/mL)干预能够有效改善结肠缩短程度(P<0.05)。
2.三肽ANK对急性结肠炎小鼠结肠组织病变的影响
为继续探究三肽ANK对DSS诱导的小鼠急性结肠炎组织损伤的影响，对其结肠组织进行HE切片染色分析。与其他学者研究相似的是，对仅用DSS诱导的小鼠结肠组织进行病理组织学评估，其结肠表现为结肠组织结构重度异常，粘膜层隐窝结构几乎全部消失；大量及纤维组织增生，如图5(A)POS中黑色箭头所示；部分粘膜上皮细胞缺失，固有层裸露，如红色箭头所示；粘膜下层水肿并可见炎症细胞浸润，如黄色箭头所示。根据前人所述病理组织学评分方法对各组小鼠结肠组织进行评分，结果如图5(B)所示，与模型组相比，经ANK干预后的小鼠结肠组织组织学评分显著下降。证实了ANK对预防和干预小鼠急性结肠炎发挥有益的作用。
3.三肽ANK对DSS诱导的小鼠结肠组织MPO活性的影响
据报道，MPO含量与炎症的发展息息相关，中性粒细胞的聚集可导致大量MPO的释放(Joh&Kim,2011)。通过对MPO酶活的测定，以探究三肽ANK对DSS诱导的小鼠急性结肠炎结肠组织中中性粒细胞聚集的影响。如图6所示，与空白对照组相比，经DSS诱导后模型组MPO含量显著上升。而与模型组相比，ANK-50下降了58％，ANK-150下降了78％，因此ANK能够有效抑制DSS诱导的急性结肠炎小鼠结肠组织中MPO的分泌。
4.三肽ANK对小鼠结肠组织促炎细胞因子TNF-α、IL-6含量的影响
测定小鼠结肠中TNF-α和IL-6的含量，探究三肽ANK在小鼠体内的抗炎活性。结果以模型组作为对照，观察其他组促炎因子分泌情况。如图7所示，与空白组相比，仅以DSS诱导的小鼠结肠组织中TNF-α和IL-6表达显著上升(p<0.05)。与模型组相比，三肽ANK能够有效抑制TNF-α(p<0.05)和IL-6(p<0.05)的分泌，其中ANK-50分别下降了56.88％和48.23％，ANK-150分别下降了72.63％和66.26％。
5.三肽ANK对促炎细胞因子mRNA表达的影响
为进一步探讨ANK在体内的抗炎活性，对结肠组织中炎症细胞因子mRNA进行测定。结果以模型组做对照，观察其他组促炎因子mRNA表达情况。如图8所示，与模型组相比，三肽ANK呈剂量依赖式有效抑制促炎因子TNF-α(p<0.05)、IL-1β(p<0.05)、IL-6(p<0.05)、IL-17(p<0.05)、MCP-1(p<0.05)、IFN(p<0.05)mRNA的表达水平。
综上，三肽ANK在体内和体外均具有良好的抗炎活性。因此，三肽ANK可以用于预防或减缓炎症性肠病保健品或药品的研发。
以上所述，只是本发明的较佳实施例而已，本发明并不局限于上述实施方式，只要其以相同的手段达到本发明的技术效果，都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。
Claims (6)
1.ANK三肽，其特征在于，其氨基酸序列为Ala-Asn-Lys。
2.根据权利要求1所述的ANK三肽，其特征在于，其分子量为331Da。
3.根据权利要求1所述的ANK三肽，其特征在于，其通过固相合成法获得。
4.权利要求1至3任一项所述的ANK三肽在制备预防和/或缓解炎症性肠病的食品或药物中的应用。
5.一种药物组合物，其特征在于，其活性成分为权利要求1至3任一项所述的ANK三肽，用于预防和/或缓解炎症性肠病。
6.根据权利要求5所述的药物组合物，其特征在于，还包括溶剂或可药用辅助剂。
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