CN115869296A - 一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用 - Google Patents
一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,优选为犬尿氨酸及其代谢物在制备改善结肠炎免疫应答紊乱、保护肠道屏障完整性和保护肠道黏膜免疫屏障的药物中的应用。本发明的犬尿氨酸及其代谢物能显著抑制DSS诱导的结肠炎小鼠体重的下降,显著缓解小鼠结肠缩短,显著抵抗结肠组织炎症细胞浸润和侵蚀;另外,犬尿氨酸可激活AhR信号通路,抑制NLRP3信号通路,通过降低NF‑κB,IKKβ和IKBα的蛋白磷酸化水平抑制NF‑κB通路来缓解小鼠肠道炎症,为结肠炎防治提供新策略。
Description
技术领域
本发明涉及结肠炎药物的技术领域,尤其是涉及一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用。
背景技术
炎症性肠病(Inflammatory bowel disease,IBD)是一种慢性且易复发的自身免疫性疾病,包括溃疡性结肠炎(ulcerative colitis,UC)和克罗恩病(crohn's disease,CD)两种疾病类型。过去一直认为IBD是西方疾病,主要集中在北美、欧洲、澳大利亚和新西兰等发达国家,而近年来IBD在亚洲、中东等新兴工业国家的发病率急剧上升,已发展为全球性的疾病。IBD临床表现为腹泻、便血、体重降低等症状,UC主要影响结肠黏膜,引起血便,CD可在整个胃肠道呈节段性分布,引起瘘管,严重影响人们生活质量,由于治疗周期长且易复发,给病人带来沉重的经济压力。目前,IBD的病因和发病机制尚未完全明确,但随着检测技术的发展,越来越多的证据表明宿主肠道的共生微生物失调引发先天性和适应性免疫反应紊乱进而导致遗传易感宿主出现肠道炎症。现有IBD的治疗方法主要通过药物治疗,虽然效果好,但存在较大的毒副作用,不适合长期使用,因此,寻找安全、有效的IBD治疗方法至关重要。
目前临床上治疗IBD的药物主要包括氨基水杨酸类药物、类固醇激素类药物和免疫抑制剂类药物。目前,氨基水杨酸主要包括5-氨基水杨酸、柳氮磺胺吡啶,主要用于治疗轻中度的IBD,其主要机制是通过激活PPARγ(Peroxisome proliferator-activatedreceptor gamma),降低促炎性因子的分泌,进而抑制NF-κB炎症通路的激活。糖皮质激素主要用于治疗中重度IBD患者,可抑制细胞分化和活化,减少促炎性细胞因子的产生,诱导DC和T细胞程序性凋亡,虽然抗炎效果好,但容易引起机体代谢失调,诱导不良反应,不可用于长期维持治疗的药物,仅可作为应急药物。对于不能使用氨基水杨酸类药物和糖皮质激素类药物进行治疗的IBD患者,免疫抑制类药物是重要的治疗药物,主要有硫唑嘌呤、6-巯基嘌呤和甲氨喋吟;其作用机制为减少淋巴细胞在内的细胞增殖,抑制炎症基因的表达,但会产生特异性的不良反应,例如恶心、发热、关节痛、急性胰腺炎等。在IBD中使用硫嘌呤治疗会使恶性肿瘤、淋巴瘤和非黑色素瘤皮肤癌的相对风险增加三倍。
犬尿氨酸是色氨酸的代谢物,是色氨酸沿犬尿氨酸途径形成的下游代谢物的直接前体,该途径可降解哺乳动物中超过95%的色氨酸。犬尿氨酸可与芳香烃受体AhR结合,AhR在炎症反应中充当调节因子,在保持人体屏障功能稳定方面发挥着关键的作用,这有利于肠道稳态的平衡。一些报告指出,AhR激活可能会在炎症过程和免疫反应期间在体外和体内产生抗炎作用,并对IBD具有保护作用。目前,关于犬尿氨酸在治疗结肠炎方面的应用未见报道。
发明内容
本发明要解决的问题是针对现有技术中所存在的上述不足而提供一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用。
本发明的上述发明目的是通过以下技术方案得以实现的:
一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用。
进一步地,所述犬尿氨酸及其代谢物选自下式(1)~(5)化合物及其在药学上可接受的盐,
更进一步地,所述式(1)~(5)化合物为D型化合物和L型化合物中的一种或两种的混合物。其中,D型化合物和L型化合物是根据甘油醛摆位置,羧基位于上端,R取代基位于下端,若氨基在左则定为L型化合物,若氨基在右则定为D型化合物。
更进一步地,所述药学上可接受的盐为钠盐、钾盐和钙盐中的一种或几种的混合物。
其中,本发明中的“犬尿氨酸及其代谢物”可通过吲哚胺-2,3-二加氧酶1(IDO1)、吲哚胺-2,3-二加氧酶2(IDO2)、色氨酸-2,3-双加氧酶(TDO)、犬尿氨酸甲酰胺酶(A FMID)催化的色氨酸分解得来,或通过色氨酸的光催化分解(Hamdy MS,Scott EL,Carr RH,Sanders JP.ANovel Photocatalytic Conversion ofTryptophan to Kynurenine UsingBlack Lig ht as a Light Source.Catal.Lett.2012,142:338-344)、L-色氨酸的氧化断裂(Maitrani C,He yes DJ,Hay S,Arumugam S,PopikVV,Phillips RS.Preparationandphotophysical propertie s ofa cagedkynurenine.Bioorg.Med.Chem.Lett.2012,22(8):2734-2737)、2-碘苯胺和丝氨酸衍生的有机锌试剂制剂偶联等方法得来(JacksonRFW,Turner D,Block MH.Carbonylati ve coupling ofan amino acid-derivedorganozinc reagent with functionalized aryl iodides:synthesisofkynurenine.J.Chem.Soc.,Chem.Commun.,1995,21:2207-2208)。
进一步地,所述犬尿氨酸及其代谢物在制备改善结肠炎免疫应答紊乱、保护肠道屏障完整性和保护肠道黏膜免疫屏障的药物中的应用。
更进一步地,所述结肠炎免疫应答紊乱的药物为通过抑制结肠组织炎症因子IL-1β、IL-6、TNF-α和IL-18的释放、以治疗结肠炎的药物。
更进一步地,所述保护肠道屏障完整性的药物为通过升高结肠组织中紧密连接蛋白Occludin、ZO-1的基因表达水平和黏液分泌相关蛋白Muc1、Muc2、Muc3、Klf4基因表达水平、以治疗结肠炎的药物。
更进一步地,所述保护肠道黏膜免疫屏障的药物为通过犬尿氨酸及其代谢物作为芳香烃受体AhR的配体参与调控与肠道免疫密切相关的AhR信号通路、以治疗结肠炎的药物。
最进一步地,在所述保护肠道黏膜免疫屏障的药物中,犬尿氨酸及其代谢物用于激活芳香烃受体AhR,以通过AhR抑制细胞中NF-κB的激活,并且NLRP3炎性小体活性因NF-κB的抑制而受到减弱,由此减轻了其下游炎症因子的活化,从而保护肠道黏膜免疫屏障。
更进一步地,所述药物包括犬尿氨酸及其代谢物、和药学上可接受的辅料。
最进一步地,所述药学上可接受的辅料选自溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂和释放阻滞剂中的一种或几种的混合物。
更进一步地,所述药物以口服方式给药,犬尿氨酸及其代谢物每天的口服剂量为20~50mg/kg。其中,给药剂量是不包括药学上可接受的辅料在内的所述犬尿氨酸及其代谢物的重量,药物的给药剂量根据患者的年龄、性别、病情的轻重和个人反应,给药途径、给药次数和给药剂量会有所不同;给药频率可以为,但不限于每天两次、每天一次、每两天一次、每周一次或每月一次给药,或者可以以缓释制剂的形式给予本发明提供的药物,在这种情况下,需要较少的给药频率;给药剂量和频率根据制剂在用药者体内的半衰期而不同,此外,也可以根据是预防性应用还是治疗性应用而有所不同。
更进一步地,所述药物以口服灌胃方式给药。
最进一步地,所述药物的剂型为口服制剂,所述口服制剂选自固体分散体、片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂。其中,片剂使用的药学上可接受的辅料一般包括乳糖和玉米淀粉,另外也可加入润滑剂如硬脂酸镁。胶囊剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。口服混悬剂则通常是将所述犬尿氨酸与适宜的乳化剂和悬浮剂混合制备而成的制剂。
最进一步地,所述口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
综上所述,本发明的有益技术效果为:本发明的犬尿氨酸及其代谢物能显著抑制DSS诱导的结肠炎小鼠体重的下降,显著缓解小鼠结肠缩短,显著抵抗结肠组织炎症细胞浸润和侵蚀;另外,犬尿氨酸可激活AhR信号通路,抑制NLRP3信号通路,通过降低NF-κB,IKKβ和IKBα的蛋白磷酸化水平抑制NF-κB通路来缓解小鼠肠道炎症,为结肠炎防治提供新策略。
附图说明
图1是本发明的各组小鼠的体重变化图;
图2是本发明的各组小鼠的结肠长度图;
图3是本发明的各组小鼠结肠组织H&E染色图;
图4是本发明的各组小鼠结肠组织脂多糖LPS以及炎症因子IL-1β、IL-6、TNF-α和IL-18水平图;
图5是本发明的各组小鼠结肠组织AB-PAS染色图;
图6是本发明的各组小鼠结肠组织结肠黏液以及紧密连接蛋白相关基因的相对mRNA水平图;
图7是本发明的各组小鼠结肠组织与NLRP3激活相关的基因的相对mRNA水平图;
图8是本发明的各组小鼠结肠组织AhR、CYP1A1、NLRP3和Claudin-1的蛋白印迹条带及其定量图;
图9是本发明的利用免疫荧光实验检测AhR,NLRP3和Claudin-1在肠炎小鼠结肠中的表达图;
图10是本发明的各组小鼠结肠组织MyD88、NF-κB和IKKβ的相对mRNA水平图;
图11是本发明的免疫荧光法检测各组小鼠结肠组织NF-κB的表达图;
图12是本发明的各组小鼠结肠组织NF-κB、p-NF-κB、IKKβ、p-IKKβ、IKBα和p-IKBα的蛋白印记条带以及p-NF-κB/NF-κB、p-IKKβ/IKKβ和p-IKBα/IKBα的相对定量图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与作用更加清楚及易于了解,下面结合附图和具体实施方式对本发明作进一步阐述。
实施例
实施例1:为本发明公开的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用。
其中,犬尿氨酸及其代谢物选自下式(1)~(5)化合物及其在药学上可接受的盐,
上式(1)~(5)化合物为D型化合物和L型化合物中的一种或两种的混合物。其中,D型化合物和L型化合物是根据甘油醛摆位置,羧基位于上端,R取代基位于下端,若氨基在左则定为L型化合物,若氨基在右则定为D型化合物。本发明的上式(1)~(5)化合物既可以通过实验室提纯制备得到,也可以通过市售直接购得,只要满足犬尿氨酸及其代谢物的化学结构式即可。
实施例2:为本发明公开的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,与实施例1的不同之处在于,本发明公开了犬尿氨酸及其代谢物在制备改善结肠炎免疫应答紊乱、保护肠道屏障完整性和保护肠道黏膜免疫屏障的药物中的应用。
其中,结肠炎免疫应答紊乱的药物为通过抑制结肠组织炎症因子IL-1β、IL-6、TNF-α和IL-18的释放、以治疗结肠炎的药物。保护肠道屏障完整性的药物为通过升高结肠组织中紧密连接蛋白Occludin、ZO-1的基因表达水平和黏液分泌相关蛋白Muc1、Muc2、Muc3、Klf4基因表达水平、以治疗结肠炎的药物。保护肠道黏膜免疫屏障的药物为通过犬尿氨酸及其代谢物作为芳香烃受体AhR的配体参与调控与肠道免疫密切相关的AhR信号通路、以治疗结肠炎的药物;在保护肠道黏膜免疫屏障的药物中,犬尿氨酸及其代谢物用于激活芳香烃受体Ah R,以通过AhR抑制细胞中NF-κB的激活,并且NLRP3炎性小体活性因NF-κB的抑制而受到减弱,由此减轻了其下游炎症因子的活化,从而保护肠道黏膜免疫屏障。
实施例3:为本发明公开的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,与实施例1的不同之处在于,上述药物包括犬尿氨酸及其代谢物、和药学上可接受的辅料。其中,药学上可接受的辅料选自溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂和释放阻滞剂中的一种或几种的混合物。
此外,上述药物以口服灌胃方式给药,犬尿氨酸及其代谢物每天的口服剂量为20~50mg/kg。其中,给药剂量是不包括药学上可接受的辅料在内的所述犬尿氨酸及其代谢物的重量,药物的给药剂量根据患者的年龄、性别、病情的轻重和个人反应,给药途径、给药次数和给药剂量会有所不同;给药频率可以为,但不限于每天两次、每天一次、每两天一次、每周一次或每月一次给药,或者可以以缓释制剂的形式给予本发明提供的药物,在这种情况下,需要较少的给药频率;给药剂量和频率根据制剂在用药者体内的半衰期而不同,此外,也可以根据是预防性应用还是治疗性应用而有所不同。
同时,上述药物的剂型为口服制剂,口服制剂选自固体分散体、片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂。其中,片剂使用的药学上可接受的辅料一般包括乳糖和玉米淀粉,另外也可加入润滑剂如硬脂酸镁。胶囊剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。口服混悬剂则通常是将所述犬尿氨酸与适宜的乳化剂和悬浮剂混合制备而成的制剂。另外,口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
性能检测试验
一、犬尿氨酸的体内药效学实验
(1)实验试剂
葡聚糖硫酸钠,上海阿拉丁/中国;犬尿氨酸(犬尿氨酸),Sigma/美国;Trizol,TaKaR a/日本;氯仿,CHEMSONIC/中国;异丙醇,CHEMSONIC/中国;无水乙醇,安徽安特生物化工/中国;甲醇,Honeywell/美国;吐温-20,上海生工/中国;TBS缓冲液,上海生工/中国;SDS-PAGE凝胶配置试剂盒,杭州诺扬/中国;蛋白酶抑制剂,Merck/美国;磷酸酶抑制剂,Merck/美国;加强型化学发光显色液,北京四正柏/中国;RIPA裂解液,Merck/美国;抗体稀释液,Seracare/美国;BCA试剂盒,Fisher Scientific/美国;PVDF膜,上海生工/中国;反转录试剂盒,ToYoBo/日本;荧光定量PCR反应试剂,ToYoBo/日本;氯化锂,上海源叶/中国;醋酸钠,Life Technologies/美国;AhR抗体,博士德/中国;CYP1A1抗体,博士德/中国;Claudin-1抗体,Abcam/英国;NLRP3抗体,CST/美国;Caspase1抗体,ABclonal/中国;IL-1β抗体,ABclonal/中国;NF-κB、p-NF-κB抗体,Abcam/英国;IKKβ、p-IKKβ抗体,Abcam/英国;IKBα、p-IKBα抗体,Abcam/英国;HRP二抗,CST/美国。
(2)实验仪器
灭菌锅,日本HIRAYAMA;台式冷冻离心机,德国EppendofCentrifuge 5425R;纯水仪,Merck Millipore明澈D24 UV;蛋白印迹电泳仪,北京DYCP-31DN;蛋白印迹转膜仪,美国WIX-miniBLOT;蛋白胶成像仪,美国ChemIDO1c1708280;小型离心机,北京PMC-060;超低温冰箱,中国DW-HL398;电子天平,德国BSA224S-CW;全自动样品快速研磨仪,上海CBFSTPRP-96;电热恒温水浴锅,上海BLHH-4D;实时荧光定量PCR仪,瑞士LightCyc ler480IIRealplex;荧光显微镜,日本Nikon;酶标仪,美国Powerwave XS;超微量分光光度计,上海Tnano-800;烘箱,上海DHG-9023A
(3)动物分组及给药
实验所选动物为8周C57BL/6雄性小鼠,体重约25克,从中国国家实验动物资源中心(上海松江)获得,先将小鼠安置于标准动物房,使其适应环境一周,再按照体重随机分为4组,每组10只小鼠,组别分别命名为Con、DSS、犬尿氨酸mg/kg/d和犬尿氨酸mg/kg/d组。实验开始后,Con组接受普通饮用水12d,DSS、犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组给予含2%DSS的饮用水9d,之后接受普通饮用水3d,以灌胃形式将20和80mg/kg/d犬尿氨酸分别注入到犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组的小鼠胃肠道中,犬尿氨酸从第0d开始处理,每两天注入0.1mL。
(4)样本采集
在第13d时处死所有小鼠并解剖,收集血清、结肠和肝脏等组织进行后续检测,同时量取每组小鼠结肠长度。从Con组、DSS组、犬尿氨酸20mg/kg/d组、犬尿氨酸80mg/kg/d组中随机选择结肠组织的3个样品用于病理学分析。新鲜组织切块后,用10%福尔马林溶液浸泡24h。其余部分保存在-80℃冰箱待测。
二、犬尿氨酸对小鼠宏观变化的影响
如图1和图2所示,经DSS诱导后,DSS、犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组的小鼠体重自第7天较对照组开始下降,然而犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组的小鼠体重下降的幅度较DSS组平缓,尤其在犬尿氨酸80mg/kg/d组中最为明显;在DSS诱导的结肠炎小鼠中观察到结肠的长度比明显变短,但经犬尿氨酸治疗的小鼠结肠长度要长于DSS组小鼠。
三、犬尿氨酸对结肠炎C57BL/6雄性小鼠结肠病理变化的影响
结肠组织的病理学分析步骤如下:
(1)组织石蜡包埋:分别从Con组、DSS组、犬尿氨酸20mg/kg/d组、犬尿氨酸80mg/kg/d组中随机选择结肠组织的3个样品用于病理学分析。新鲜组织切块后,用10%福尔马林溶液浸泡24h。随后将固定的结肠组织取出,修切平整,依次放入75%乙醇2h,85%乙醇2h,90%乙醇2h,95%乙醇1h,无水乙醇I、II各1h,二甲苯I、II各45min,56℃石蜡I、I I、III各1h进行脱水,结束后放入不锈钢模具包埋。
(2)H&E染色:使用切片机将包埋的石蜡切成4μm厚的薄片,用摊片机将其展平,之后放到载玻片上转移至65℃烘箱中进行烤片。H&E染色按照以下流程进行:
①脱蜡:将切片依次放入二甲苯I、II各20min,无水乙醇I、II各10min,95%酒精5min,90%酒精5min,80%酒精5min,70%酒精5min,用蒸馏水清洗;
②染色:将切片用苏木素染剂浸泡3-8min,然后用自来水洗净并使用促蓝液返蓝,流水洗净后再用伊红染料染色1-3min;
③脱水封片:将切片依次放入95%酒精I、II各5min,无水乙醇I、II各5min,二甲苯I、II各5min,取出晾干,用中性树胶封片。
(3)AB-PAS染色:将包埋好的石蜡块置于切片机上切成4μm厚的切片,在摊片机上将组织展平,用载玻片捞起放入65℃烘箱内拷片。AB-PAS染色按以下流程进行:
①脱蜡:依次用二甲苯I、II浸没切片各20min,随后用无水乙醇I、II浸没各5min,最后95%酒精浸没5min,用蒸馏水清洗;
②染色:切片在高碘酸染液中浸泡15min,用蒸馏水冲洗多次。然后切片放入雪佛染液中避光进行30min的染色,用流水冲洗5min。最后切片用苏木素染剂浸泡1-3min,自来水冲洗后用盐酸水溶液分化数秒,用蒸馏水清洗并氨水返蓝,再用流水冲洗;
③脱水封片:依次用无水乙醇I、II、III浸泡切片各5min,正丁醇浸泡5min,最后用二甲苯I、II浸泡各5min,用中性树胶塑封晾干的切片。
实验结果如图3和图5所示。实验结果显示:在2%DSS处理后,小鼠结肠组织出现明显的隐窝结构改变、基底炎性细胞浸润以及肠壁增厚等典型肠炎病理症状,而犬尿氨酸可使DSS诱导的小鼠结肠炎在隐窝结构、水肿、黏膜损伤和炎症细胞浸润方面产生显著的病理学改善;DSS组与对照组相比,黏液分泌显著减少,而经犬尿氨酸治疗的结肠炎小鼠肠道黏液分泌显著增加。
四、ELISA方法检测结肠组织中的炎症因子
取30mg结肠样品用270μL预冷的PBS匀浆以获得上清液。根据试剂盒的说明书,用相应的小鼠ELISA试剂盒进行检测,同时使用BCA检测试剂盒测定每个结肠组织样品的蛋白含量。
实验结果如图4所示,结肠组织中LPS、IL-1β、TNF-α和IL-18的含量在结肠炎小鼠中的以上指标均升高,而犬尿氨酸80mg/kg/d组中的指标较DSS组有明显回落。
五、Western Blot检测结肠组织中蛋白水平
根据AhR、NLRP3和NF-κB通路,考察犬尿氨酸对DSS诱发的结肠炎小鼠的抗炎作用。
多环芳香烃受体(AhR)属于螺旋环螺旋(bHLH)PAS转录因子家族的一员,是一种依靠配体激活的转录因子,可在许多脊椎动物的各类型细胞中广泛表达。通常,非活性的AhR存在于由伴侣保护的胞质溶胶中,与配体结合后的AhR转移至细胞核并与其伴侣蛋白ARNT结合组装成有活性的异二聚体。这种异二聚体通过与外源性反应元件(XRE)和辅助调节剂结合来调控靶基因的表达,例如CYP1A1、CYP1B1、AhRR、IL-22等。此外,AhR还可以与一些其他的转录调控因子(包括NF-kB,Nrf2等)发生直接或间接的作用从而影响它们的特异性结合位点。AhR的激活会抑制包括IBD在内的炎症性疾病的免疫反应,减少促炎因子的表达减少,如IL-6、IL-12、IL-17、TNF-α等,缓解肠黏膜炎症。
NLRP3炎性小体是一类主要由传感器、衔接蛋白和效应器组成的蛋白复合物,其激活分为两步:首先TLR对PAMP或DAMP的识别启动了NF-κB激活的信号通路。活化的NF-κB由细胞质转移至细胞核内发挥调控作用,即参与了NLRP3、Pro-IL-1β和Pro-IL-18的转录及翻译。其次效应器(Pro-caspase1)被NLRP3和包含CARD结构域的凋亡相关点样蛋白(AS C)组装成一个活跃的炎性小体,在炎性小体作用下,Pro-caspase1、Pro-IL-18和Pro-IL-1β转化为活性因子。通过检测结肠组织中AhR及下游相关的CYP1A1,NLRP3的蛋白表达,考察犬尿氨酸对AhR通路和NLRP3通路的作用;检测p-NF-κB/NF-κB、p-IKKβ/IKKβ和p-IKBα/IKBα蛋白表达,考察犬尿氨酸对NF-κB信号通路的作用;测定紧密连接蛋白claudin-1的表达水平,考察犬尿氨酸对DSS诱发的结肠炎小鼠的肠黏膜保护屏障的保护效果。
结肠组织Western Blot步骤如下:
(1)蛋白样品制备:取一定量结肠组织至1.5mL离心管中,加入适量含有1%磷酸酶抑制剂和1%蛋白酶抑制剂的1×放射免疫沉淀测定裂解缓冲液(RIPA)以及至少2颗磁珠,在65Hz条件下,在组织研磨机中充分研磨270s,吸取上清至新的离心管中,在12,000rpm,4℃条件下离心15min,所得的上清液即为组织蛋白原液;
(2)蛋白印迹分析:依据目标蛋白的分子量配置不同浓度的分离胶,本章中选择10%和8%SDS-PAGE胶来分离不同大小的蛋白质;电泳结束后放入转膜缓冲液中将分离的蛋白质转移至聚偏二氟乙烯(PVDF)膜上,室温下在PVDF膜上切下目标蛋白条带,使用封闭液将其封闭1h。随后将条带放入一抗中,在4℃下孵育过夜;用1×TBST缓冲液清洗条带3次,每次10min,洗涤后的条带在室温下与对应的二抗孵育1h,同样使用1×TBST缓冲液清洗,最后使用超敏ECL化学发光试剂盒在蛋白成像系统中观察并拍照。
实验结果如图8和图12所示,经犬尿氨酸治疗后的结肠炎小鼠中的AhR和CYP1A1蛋白表达较DSS组有明显增强,而NLRP3的蛋白表达较DSS组有明显下降,同时犬尿氨酸治疗后的结肠炎小鼠中的Claudin-1蛋白表达增多,一定程度的说明肠屏障功能得到改善;犬尿氨酸处理后显著下调结肠炎小鼠中p-NF-κB、p-IKKβ和p-IKBα的蛋白水平,同时p-NF-κB/N F-κB、p-IKKβ/IKKβ和p-IKBα/IKBα的含量也有不同程度的下降。
六、Real-time PCR检测结肠组织中基因的相对mRNA水平
结肠组织Real-time PCR步骤如下:
(1)RNA提取及反转录:结肠组织中加入Trizol裂解液充分匀浆,加入氯仿萃取得到上层水相,加入等体积异丙醇混匀;离心弃上清,加入75%乙醇清洗RNA沉淀;冰上干燥后加入超纯水溶解;按照反转录试剂盒(ToYoBo)说明书将RNA反转录成cDNA。
(2)RT-PCR:10μl反应体系:SYBR 5μL、前后引物各0.4μL、H2O 3.2μL以及cDNA1μL。
步骤1:95℃3min
步骤2:(95℃15s,60℃1min)×45个循环
步骤3:GAPDH作为内参基因,通过2-ΔΔCT计算目标基因的相对表达量。
所采用Q-PCR引物及序列如下表1所示:
表1基因名称及引物序列
实验结果如图6所示,黏液分泌相关的基因(包括Muc1、Muc2、Muc3、Klf4)在DSS组小鼠中显著下降,但在犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组中较DSS组有不同程度的回升。图7所示,经犬尿氨酸治疗后的结肠炎小鼠中的AhR、CYP1A1和CYP1B1基因表达较DSS组有明显上升。在NLRP3信号通路中,经犬尿氨酸治疗后的结肠炎小鼠中的NL RP3、ASC和Caspase1基因表达较DSS组有明显的抑制,同时NLRP3所激发的炎症因子(I L-1β、IL-18和TNF-α)的基因表达也在经犬尿氨酸治疗后得到明显的抑制。以上表明经犬尿氨酸治疗后的结肠炎小鼠中的AhR在分子水平上的表达变化与NLRP3以及炎症因子的表达变化呈负相关。图10所示,肠炎小鼠结肠组织中的NF-κB信号中的部分基因被激活,MyD88、NF-κB和IKKβ的基因表达显著升高,而犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组中的NF-κB基因表达较DSS组显著下降,同时也发现该通路的表达与AhR的基因表达呈现相反趋势。
七、免疫荧光检测结肠中蛋白表达
结肠组织免疫荧光步骤如下:
(1)石蜡切片脱蜡至水:按顺序先用二甲苯Ⅰ浸没切片15min,再用二甲苯Ⅱ浸没15min,之后在无水乙醇Ⅰ,Ⅱ里依次浸泡5min,然后无水乙醇浸泡5min,再依次用85%酒精,75%酒精各浸泡5min,最后用蒸馏水清洗;
(2)抗原修复:组织切片放入盛满EDTA抗原修复缓冲液(pH=8.0)。用微波炉中火加热8min直至沸腾,停火8min,再转中低火7min。玻片自然冷却后再置于PBS(pH=7.4)中,用脱色摇床晃动洗涤3次,每次5min;
(3)画圈血清封闭:切片稍甩干后用组化笔在组织周围画圈,甩干PBS,滴加BSA,封闭30min;
(4)加一抗:轻轻甩掉封闭液,在切片上滴加稀释好的一抗,切片平放于湿盒内4℃孵育过夜;
(5)加二抗:玻片置于PBS(pH=7.4)中,用脱色摇床晃动洗涤3次,每次5min。切片稍甩干后用与一抗相对应种属的二抗从圈内滴加并逐渐覆盖组织,避光室温孵育60min;
(6)DAPI复染细胞核:玻片置于PBS(pH=7.4)中,用脱色摇床晃动洗涤3次,每次5min。切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min;
(7)淬灭组织自发荧光:玻片置于PBS(pH=7.4)中,用脱色摇床晃动洗涤3次,每次5min。在圈内加入自发荧光淬灭剂5min,流水冲洗10min;
(8)封片:切片稍甩干后用抗荧光淬灭封片剂封片。
实验结果如图9、图11所示,DSS组的AhR荧光强度较对照组明显减弱,而犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组中的AhR荧光强度较DSS组明显增强。同时,Claudin-1的荧光强度趋势与AhR的趋势相同,但在NLRP3荧光强度中,DSS组的NLRP3荧光强度较对照组明显增强,而犬尿氨酸20mg/kg/d和犬尿氨酸80mg/kg/d组的NLRP3荧光强度较DS S组明显减弱;DSS组的NF-κB荧光强度明显增强,而犬尿氨酸20mg/kg/d和犬尿氨酸80m g/kg/d组的NF-κB荧光强度较DSS组明显减弱,说明犬尿氨酸治疗后抑制了结肠炎小鼠中N F-κB信号的激活。
综上,经犬尿氨酸治疗缓解了结肠炎小鼠出现病理学症状,包括体重下降缓解、结肠变长、肠屏障相关基因和蛋白表达增强等。另外,荧光定量PCR、WB和免疫荧光结果显示经犬尿氨酸治疗的结肠炎小鼠中AhR信号通路得到增强,NLRP3以及NF-κB激活得到抑制,达到缓解肠炎进程的效果。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用。
3.根据权利要求2所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述式(1)~(5)化合物为D型化合物和L型化合物中的一种或两种的混合物。
4.根据权利要求2所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述药学上可接受的盐为钠盐、钾盐和钙盐中的一种或几种的混合物。
5.根据权利要求1所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述犬尿氨酸及其代谢物在制备改善结肠炎免疫应答紊乱、保护肠道屏障完整性和保护肠道黏膜免疫屏障的药物中的应用。
6.根据权利要求5所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述结肠炎免疫应答紊乱的药物为通过抑制结肠组织炎症因子IL-1β、IL-6、TNF-α和IL-18的释放、以治疗结肠炎的药物。
7.根据权利要求5所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述保护肠道屏障完整性的药物为通过升高结肠组织中紧密连接蛋白Occludin、ZO-1的基因表达水平和黏液分泌相关蛋白Muc1、Muc2、Muc3、Klf4基因表达水平、以治疗结肠炎的药物。
8.根据权利要求5所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述保护肠道黏膜免疫屏障的药物为通过犬尿氨酸及其代谢物作为芳香烃受体AhR的配体参与调控与肠道免疫密切相关的AhR信号通路、以治疗结肠炎的药物。
9.根据权利要求5所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述药物包括犬尿氨酸及其代谢物、和药学上可接受的辅料。
10.根据权利要求5所述的一种犬尿氨酸及其代谢物在制备治疗结肠炎药物中的应用,其特征在于:所述药物以口服方式给药,犬尿氨酸及其代谢物每天的口服剂量为20~50mg/kg。
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