CN109999052A - β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途 - Google Patents
β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途 Download PDFInfo
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Abstract
本发明属于医药技术领域,具体涉及β‑烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。本发明要解决的技术问题为制备治疗或缓解呼吸障碍或疾病的药物或保健品提供一种新的替代方案,解决上述技术问题的技术方案为提供了β‑烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。β‑烟酰胺单核苷酸或β‑烟酰胺核糖能有效治疗慢性阻塞性肺疾病等肺部疾病,并能明显缓解可吸入颗粒引起的肺部巨噬细胞老化,具有很好的应用前景。
Description
技术领域
本发明属于医药技术领域,具体涉及β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。
背景技术
烟酰胺核苷酸也叫β-烟酰胺单核苷酸(β-Nicotinamide Mononucleotide,β-NMN或NMN) 或烟酰胺单核苷酸或烟酰胺核苷酸等,分子式见下:
β-烟酰胺单核苷酸(NMN)
烟酰胺核苷酸是烟酰胺磷酸核糖转移酶(NAMPT)调控反应的重要产物,NAD+合成过程的关键中间体,在细胞能量代谢中发挥着重要作用。而β-烟酰胺核糖(NicotinamideRiboside, NR)是β-烟酰胺单核苷酸(NMN)的一种前体物。
β-烟酰胺核糖(NR)
众所周知,烟酰胺腺嘌呤二核苷酸(NAD+)是细胞能量转化的重要辅酶,在细胞能量代谢中发挥着重要作用。烟酰胺核糖(nicotinamide riboside,NR)与β-烟酰胺单核苷酸(β-Nicotinamide Mononucleotide,NMN)是合成NAD+的关键前体,是烟酰胺磷酸核糖转移酶(NAMPT)调控反应的重要产物。在人体细胞中以及多种食物,如西兰花、卷心以及牛奶中都天然存在,但都很微量。多项研究表明:NAD+与一些老年相关性疾病、衰老、以及生殖妊娠有重要关系。但是,就NAD+而言,其本身很难直接给入人体,而作为其前体的NR与 NMN很容易合成NAD+。有研究表明,补充NAD+前体NMN或NR可以提高组织中的NAD+ 含量,延缓由衰老引起的生理功能衰退:包括抑制体重增加,增强能量代谢,提高对胰岛素的敏感性,改善视力等,NMN被发现可以改善衰老引起II型糖尿病葡萄糖耐量异常;预防老年青光眼;NR增加肌肉干细胞数目及运动能力等。
慢性阻塞性肺疾病是由慢性炎症及气道和肺实质破坏所引起的慢性进行性疾病,并且通常与吸烟或长期暴露于其他有害微粒及气体相关,慢性阻塞性肺疾病以气道阻塞和来自肺的最大呼气流量降低为主要特征。该病的特征在于有时可通过给予支气管扩张药而部分逆转的进行性气流阻塞。典型的症状为咳嗽、痰生成过多及呼吸困难。术语慢性阻塞性肺疾病涵盖一系列肺活动过程,是一种具有气流阻塞特征的慢性支气管炎和(或)肺气肿。
在全世界范围内,慢性阻塞性肺疾病无论是发病率还是死亡率仍然在逐年增加,预计到 2030年,它将成为全球第3大死亡原因。在中国40岁以上的居民群体中,慢性阻塞性肺疾病的发病率高达8.2%,居中国因病死因的第二位,因此病致死的人数毎年都超过100万人,并且这个数目呈不断上升的趋势。超过40岁后,慢性阻塞性肺疾病的发生率将开始呈指数级的上升,患者数量庞大,将造成的沉重的卫生医疗负担。
但是,慢性阻塞性肺疾病的治疗选择及疗效却非常有限,多流于对症处理,常使用支气管扩张剂。尚无措施从根本上缓解慢性阻塞性肺疾病进程。因此迫切需要开发用于治疗慢性阻塞性肺疾病的新药。当前慢性阻塞性肺疾病治疗药主要用于对症治疗,并无可改变基础炎症或改变疾病进展的药物或者治疗手段。
颗粒物是指漂浮在空气中的固态和液态颗粒物的总称,其粒径范围约为0.1-100微米。可吸入颗粒物可以被人体吸入,沉积在呼吸道、肺泡等部位从而引发疾病。通常把粒径在10 微米以下的颗粒物称为可吸入颗粒物,又称PM10。颗粒物的直径越小,进入呼吸道的部位越深。10微米直径的颗粒物通常沉积在上呼吸道,5微米直径的可进入呼吸道的深部,2微米以下的可100%深入到细支气管和肺泡。可吸入颗粒物中常见的化学成分有颗粒元素碳 (PEC,有时也称为碳黑)、无机离子、微量元素、和有机化合物,有时可吸入颗粒物上还吸附有病原微生物(病毒和细菌)。可吸入颗粒物主要经呼吸道进入人体,也有一小部分可通过消化道或皮肤进入人体。可吸入颗粒物沉积在人体呼吸道后,它们的清除、滞留和转移与其粒径、沉积地点有关。一般来说,粒径越小、沉积地点越远,所需的清除时间就越长,就越易滞留在人体内,越易使毒性物质转移到身体的其他部位。
碳黑(carbon black,CB)是中国主要的空气污染物之一,吸入后对人体有害,燃烧是这一颗粒物质的主要来源,由于巨大的能源消耗,中国东部和北部是世界上最严重的气溶胶污染地点之一。碳黑也是很多地区的可吸入颗粒物的重要和典型组成成分,CB进入呼吸道后,大多数颗粒通过粘膜纤毛运动被清除。然而,超细颗粒能够穿透血气屏障并移位到肺部和全身循环。体内和体外研究显示CB可以降低肺上皮细胞活力,导致肺部慢性支气管炎、肺气肿等慢性阻塞性肺疾病的改变。此外,在细胞和非细胞中,碳纳米颗粒(Carbonblack nanoparticle,CBNPs)能诱导活性氧(reactive oxygen species,ROS)的生成。
目前,还没有β-烟酰胺单核苷酸(β-Nicotinamide Mononucleotide,NMN)与β-烟酰胺核糖(Nicotinamide Riboside,NR)治疗或缓解慢性阻塞性肺疾病以及治疗或缓解与可吸入颗粒引起的肺部疾病尤其是肺巨噬细胞老化的相关报道。
发明内容
本发明要解决的技术问题为:为制备治疗或缓解呼吸障碍或疾病的药物或保健品提供一种新的替代方案。
本发明解决上述技术问题的技术方案为:提供一种β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。
其中,上述用途中,所述β-烟酰胺单核苷酸前体为β-烟酰胺核糖。
其中,上述用途中,所述的呼吸障碍或疾病为肺疾病。
其中,上述用途中,所述的肺疾病为慢性阻塞性肺疾病。
其中,上述用途中,所述的肺疾病为由空气中可吸入颗粒引起的肺部疾病。
其中,上述用途中,所述的由空气中可吸入颗粒引起的肺部疾病为空气中可吸入颗粒引起的肺损伤。
其中,上述用途中,所述的肺损伤为肺巨噬细胞老化。
进一步的,上述用途中,所述的空气中可吸入颗粒为PM10颗粒、PM2.5颗粒或碳黑颗粒中的至少一种。
其中,上述用途中,所述药物是以β-烟酰胺单核苷酸或其前体为活性成分,加入药学上接受的辅料或辅助性成分,制备而成的制剂。
进一步的,所述制剂为口服制剂。
进一步的,每单位口服制剂含有β-烟酰胺单核苷酸或其前体25-1000mg。
进一步的,所述的口服制剂包括固体制剂、液体制剂或悬浮液制剂。
进一步的,所述的固体制剂包括胶囊剂、片剂、丸剂、散剂或颗粒剂。
进一步的,所述的液体制剂包括乳液、溶液、悬浮液、糖浆或酊剂。
本发明的有益效果在于:
本发明创造性的提供了β-烟酰胺单核苷酸及其前体β-烟酰胺核糖在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。本发明研究发现,β-烟酰胺单核苷酸及其前体可以有效的治疗或缓解呼吸障碍或疾病,如慢性阻塞性肺疾病,并减少以PM10颗粒、PM2.5颗粒或碳黑颗粒引起的肺巨噬细胞的老化,其在以慢性阻塞性肺疾病为代表肺部疾病和以纳米碳颗粒为代表的可吸入颗粒造成的肺部损伤的缓解和恢复等方面为临床用药提供了一种新的有效选择。
附图说明
图1所示为PPE诱导小鼠慢性阻塞性肺疾病计量结果。
图2A和图2B所示为PPE对小鼠慢性阻塞性肺疾病的治疗作用。
图3所示为小鼠肺重结果。
图4所示为肺组织染色结果。
图5所示为NMN对慢性阻塞性肺疾病小鼠肺中性粒细胞的影响。
图6所示为NMN对慢性阻塞性肺疾病小鼠肺单核细胞比例的影响
图7所示为肺组织特异性脂酶染色图。
图8所示为PPE诱导的慢性阻塞性肺疾病模型小鼠肺灌洗细胞和肺组织中p16和p21蛋白的水平。
图9所示为NMN对慢性阻塞性肺疾病小鼠p16和p21蛋白表达的影响;a为肺灌洗细胞蛋白, b为肺组织蛋白。
图10所示为慢性阻塞性肺疾病模型小鼠肺组织免疫组化染色图。
图11所示为慢性阻塞性肺疾病模型小鼠肺组织中qPCR结果。
图12A、图12B所示为慢性阻塞性肺疾病模型小鼠肺功能结果,图12A为准静态肺顺应性 (Cchord)的统计图,图12B中Cfvc50为达到50%肺活量时的肺顺应性,CP0为压力在0时的肺顺应性。
图13所示为慢性阻塞性肺疾病模型组小鼠肺FEV100值。
图14A-图14E所示为慢性阻塞性肺疾病模型组小鼠肺MMEF、PEF、IC、ERV、FVC值结果。
图15A、图15B所示为肺泡巨噬细胞内的γh2ax的表达,图示为染色细胞占的百分比(%) (**p<0.01)。
图16所示为肺细胞内的sa-β-gal的表达,图示为染色细胞占的百分比(%)。(**p<0.01)。
图17A、图17B所示为CBNPs引起巨噬细胞形态发生变化(图为细胞直径um,与对照组相比 *p<0.05,**p<0.01。
图18所示为巨噬细胞经CBNPs处理后,细胞内老化相关蛋白水平的变化。
图19所示为巨噬细胞经不同浓度的CBNPs处理后细胞内ROS水平的变化。为腹腔巨噬细胞处理1周)(****p<0.0001)。
具体实施方式
本发明提供了一种β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。
其中,上述用途中,所述β-烟酰胺单核苷酸前体为β-烟酰胺核糖。
其中,上述用途中,所述的呼吸障碍或疾病为肺疾病;进一步的,所述肺疾病为慢性阻塞性肺疾病。
其中,所述的肺疾病为由空气中可吸入颗粒引起的肺部疾病。进一步的,所述的由空气中可吸入颗粒引起的肺部疾病为空气中可吸入颗粒引起的肺损伤。
其中,上述用途中,所述的肺损伤为肺巨噬细胞老化。
进一步的,上述用途中,所述的空气中可吸入颗粒为PM10颗粒、PM2.5颗粒或碳黑颗粒中的至少一种。
所述PM10颗粒指空气中粒径在10微米以下的颗粒物。所述PM2.5颗粒指空气中粒径在 2.5微米以下的颗粒物。
其中,上述用途中,所述药物是以β-烟酰胺单核苷酸或其前体为活性成分,加入药学上接受的辅料或辅助性成分,制备而成的制剂。
进一步的,所述制剂为口服制剂。
进一步的,每单位口服制剂含有β-烟酰胺单核苷酸或其前体25-1000mg。
进一步的,所述的口服制剂包括固体制剂、液体制剂或悬浮液制剂。
进一步的,所述的固体制剂包括胶囊剂、片剂、丸剂、散剂或颗粒剂。
进一步的,所述的液体制剂包括乳液、溶液、悬浮液、糖浆或酊剂。
本发明中,所述的"治疗"意指治疗性治疗和预防性或防止性措施,其目标是防止或延缓(减轻)不需要的生理病症、紊乱或疾病,或获得有益或所需的临床结果。有益或所需的临床结果包括,但不限于,症状的缓解;病症、紊乱,或疾病的程度的减轻;病症、紊乱,或疾病的状态的稳定(即,不恶化);病症、紊乱,或疾病进展的发生的延迟或放慢;病症、紊乱,或疾病状态的改善或缓解(无论是部分或全部),无论是否为可检测的或不可检测的;至少一个可测量的人体生理参数的改善,所述参数不必是患者可辨别的;或病症、紊乱,或疾病的改善(enhancement)或改进。治疗包括引起临床上显著的反应,而没有过度水平的副作用。
本发明β-烟酰胺单核苷酸或其前体,如β-烟酰胺核糖在治疗或缓解肺部疾病时施用方式没有特别限制,代表性的施用方式包括但并不限于:口服、静脉内、肌肉内或皮下等肠胃外给药方式和局部给药。
用于制备口服制剂的固体制剂包括胶囊剂、片剂、丸剂、散剂或颗粒剂。在这些固体制剂中,β-烟酰胺单核苷酸或其前体作为活性成分,与至少一种常规惰性赋形剂(或载体)混合,如柠檬酸钠或磷酸二钙,或与下述成分混合:(a)填料或增容剂,例如,淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸;(b)粘合剂,例如,羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶;(c)保湿剂,例如,甘油;(d)崩解剂,例如,琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠;(e)缓溶剂,例如石蜡;(f)吸收加速剂,例如,季胺化合物;(g)润湿剂,例如鲸蜡醇和单硬脂酸甘油酯;(h)吸附剂,例如,高岭土;和(i)润滑剂,例如,滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠,或其混合物。胶囊剂、片剂和丸剂中,剂型也可包含缓冲剂。
固体制剂如片剂、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备,如肠衣和其它本领域公知的材料。它们可包含不透明剂,并且,这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。
所述的液体制剂包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外,液体剂型可包含本领域中常规采用的惰性稀释剂,如水或其它溶剂,增溶剂和乳化剂,例知,乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油,特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外,液体剂型也可包含助剂,如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料等。
本发明所述悬浮剂中中还可包含乙氧基化异十八烷醇、聚氧乙烯山梨醇、脱水山梨醇酯、微晶纤维素、甲醇铝或琼脂等。
除上述剂型外,还可制备用于肠胃外注射的组合物。可包含生理上可接受的无菌水或无水溶液、分散液、悬浮液或乳液,和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
本发明所述药学上可接受的辅料或辅助性成分,是指除活性成分以外包含在剂型中的物质。可以是指载体、运载物、稀释剂或辅料种的至少一种,通常在化学上或物理上与构成某药物剂型的其它成分相兼容,并在生理上与受体相兼容。
本发明所述药学上接受的辅助性成分,具有一定生理活性,但该成分的加入不会改变上述药物组合物在疾病治疗过程中的主导地位,而仅仅发挥辅助功效,这些辅助功效仅仅是对该成分已知活性的利用,是医药领域惯用的辅助治疗方式。若将上述辅助性成分与本发明药物组合物配合使用,仍然应属于本发明保护的范围。
下面将通过实施例对本发明的具体实施方式做进一步的解释说明,但不表示将本发明的保护范围限制在实施例所述范围内。
除特别说明的以外,实施例中所用试剂均为普通市售产品。
实施例1 NMN和NR对慢性阻塞性肺疾病的治疗作用研究
(一)小鼠慢性阻塞性肺疾病模型建立
动物:6~8周,C57小鼠;
试剂:猪胰腺弹性蛋白酶溶液PPE(美国,Sigma),稀释于无菌PBS中;
方法:
1)麻醉小鼠(以1.5-2L/min之间的速率加入异氟烷),将其从包装盒中取出并放置仰卧位的斜台,小心地打开它的嘴和橡皮筋附加到上门牙,将橡皮筋从钉子上吊起。
2)使用一小镊子,轻轻抓住老鼠的舌头,把它从嘴里拉出来0.5~1cm,将弹性蛋白酶溶液(50μL)滴在口咽部的远端,即舌头后面。
3)用一只手捏住舌头,用另一只手堵住两个鼻孔,保持5-6秒,随着舌头的伸展和鼻子的堵塞,小鼠会无法吞咽,弹性蛋白酶溶液将会被吸入下呼吸道。
4)释放小鼠舌头,轻轻按摩小鼠胸部,弹性蛋白酶液向下流动并分布在整个肠道气道中,取下橡皮筋,并将小鼠放回。
5)观察5~10min,注意小鼠苏醒状况和呼吸状况。
以不同剂量的PPE(0.68U、1.00U、1.50U)诱导C57小鼠慢性阻塞性肺疾病。结果如图 1所示。由图可知:PBS舌后滴注后,小鼠肺与正常小鼠肺无差别,而PPE舌后滴注后,小鼠肺较PBS组明显增大,且呈计量依赖,当PPE计量达到1.5U时,小鼠肺出现明显坏死。
(二)实验分组及给药
分组:
实验分为对照组(PBS组,以PBS舌后滴注),模型组(PPE组,以PPE舌后滴注建立慢性阻塞性肺疾病模型),治疗组(PPE+NMN组,以NMN治疗慢性阻塞性肺疾病模型小鼠), n=5。
给药:
β-烟酰胺单核苷酸(NMN,美国,Sigma),溶于PBS中,0.10g/mL,灌胃,200μL/只(20mg/ 只*天)。治疗组给按上述口服NMN药物,对照组及模型组给等体积PBS。给药时间为30d。
(三)实验结果检测
1、肺力学测量(Measurement of lung mechanics)
测定肺的机械性能:
小鼠称重,通过腹腔注射戊巴比妥(90mg/kg)和泮库溴铵(0.5mg/kg)深度麻醉。切开气管,将气管插入套管,并用缝合线将气管与套管绑紧,将套管连接到计算机控制的小动物呼吸机上,测定小鼠肺功能相关参数值,每个参数测定三次。测定完成后取下小鼠,取出肺用于石蜡切片、蛋白提取及qPCR。
以β-烟酰胺单核苷酸(NMN)对PPE诱导发生慢性阻塞性肺疾病的小鼠进行治疗。结果如图2A和图2B所示。由图2A和图2B可知:慢性阻塞性肺疾病模型组(PPE组)肺较PBS 组明显增大,PPE诱导慢性阻塞性肺疾病模型建立成功。在PPE诱导慢性阻塞性肺疾病,同时,给以小鼠NMN灌胃20mg/天ORAL(PPE+NMN20mg)显示有效,其小鼠肺较慢性阻塞性肺疾病模型组有明显改善。
肺重统计结果如图3所示。图3显示,慢性阻塞性肺疾病模型PPE组较PBS对照组显著增加,而在给以PPE的同时口服NMN,可显著降低小鼠肺重。*P<0.05,**P<0.01。
2、肺灌洗(BAL)
麻醉后处死小鼠,颈部切开气管,并用0.6mL 0.9%氯化钠将肺灌洗3次。合并灌洗液,将灌洗液离心,1200rpm,5min,收集上清液,分装,在-80℃冷冻保存以备后续分析。将肺灌洗液(BAL)沉淀重悬于1mL 0.9%氯化钠中,通过在血细胞计数器上计数测定总细胞数。用0.9%氯化钠洗涤1次,细胞用于蛋白提取。
3、肺组织和BLA中中性粒细胞检测
肺组织单细胞制备:取出小鼠整肺组织,在0.9%氯化钠中洗去淤血,去掉多余组织,将肺剪碎,以5mL 1mg/mLⅠ型胶原酶,37℃消化1小时后,以70μm筛网过滤,1200rpm离心5min,加入裂红液3mL裂红3分钟,并以PBS洗涤两次。以血细胞计数器计数细胞,加入巨噬、单核细胞、T细胞、中性粒流式检测抗体染色,4℃,30min。并以PBS洗涤2次后上机检测。
对肺组织和BLA中中性粒细胞染色,洗涤两次后上机检测。
肺组织染色如图4所示。HE染色显示对照组支气管和肺泡结构清晰,起到黏膜上皮细胞排列整齐,肺泡大小均匀;慢性阻塞性肺疾病模型组支气管黏膜上皮细胞出现脱落现象,肺泡壁塌陷,肺泡空腔出现不规则扩大现象,气道管壁周围及肺间质内出现不同程度的炎症细胞浸润,支气管黏膜皱缩,突入管腔,管腔变窄或闭塞,腔内可见黏液。使用NMN后小鼠肺以上病理形态学改变明显减轻。
取小鼠肺组织剪碎,并以1mg/mLⅠ型胶原酶消化后染色,进行流式检测后发现(图5和图6),PPE诱导慢性阻塞性肺疾病模型小鼠肺组织中中性粒细胞比例为18.74%,较对照组 (5.04%)显著增加,口服NMN后,中性粒细胞比例显著下降。*P<0.05,**P<0.01,***P<0.001。
4、肺形态学(Lung morphometry)
取出小鼠肺,用4%PFA(四氟乙烯-全氟烷氧基乙烯基醚共聚物)固定。将固定的肺脱水,包埋在石蜡中,并使用旋转切片机切成3μm石蜡切片。对肺切片进行H&E染色,使用Meta Morph软件分析。
小鼠肺组织石蜡切片进行特异性脂酶染色,结果如图7所示。结果显示PPE组肺组织中中性粒数量较PBS对照组增多,给予NMN后,中性粒数量明显下降。
5、全细胞裂解物的制备(Preparation of hole cell lysate protein)
从肺组织和BAL细胞制备全细胞裂解物和核蛋白。
肺组织蛋白制备:取小鼠肺组织0.1g,并加入0.5ml放射免疫沉淀测定(RIPA)缓冲液 (50mmol/l Tris-HCl,150mmol/l NaCl,1mmol/l EDTA,0.25%脱氧胆酸盐,1mmol/lNa3VO4,1mmol/l NaF,1mg/l亮抑蛋白酶肽,1mg/l抑肽酶和1mmol/l PMSF),并用组织匀浆器进行组织匀浆,在冰上保持45min以使细胞裂解完全。裂解完全后离心,13000rpm, 15min,收集上清液作为组织蛋白提取物备用。BCA(聚氰基丙烯酸正丁酯)法测定总蛋白含量,以最低浓度为参照调齐所有样品蛋白浓度。
BAL细胞蛋白制备:BAL细胞中加入RIPA缓冲液50μL,在冰上裂解30min,然后涡旋15秒。以13,000rpm离心5min后,收集上清液作为全细胞裂解物备用。
6、免疫印迹(Immunoblot.)
1)以12.5%SDS-PAGE胶分离来自肺组织匀浆和BAL细胞裂解物的蛋白质样品。
2)将分离的蛋白质电印迹到0.2μm的硝酸纤维素膜上。
3)用5%脱脂牛奶将膜在室温下封闭1小时,TBST洗涤3次,加入抗p21、抗p16抗体,4℃孵育过夜。
4)洗涤3次(每次5min)后,使用辣根过氧化物酶标记的二抗(用含5%脱脂牛奶,0.1% Tween 20[v/v]的PBS1:10,000稀释)孵育,室温孵育1h。
5)洗涤3次(每次10min)后,显影液中浸湿,在曝光机上曝光。
通过定量蛋白质以及β-actin杂交来确定样品等量上样。
结果如图8和9所示。由图可知,PPE诱导的慢性阻塞性肺疾病模型小鼠肺灌洗细胞和肺组织中p16和p21蛋白的水平显著升高,口服NMN后,p16和p21蛋白的水平均有下降。 *P<0.05,**P<0.01,***P<0.001,versus PBS;###P<0.001,versus PPE.
7、冰冻切片免疫组织化学染色(Immunohistochemical staining)
1)冰冻切片(4μm)在4℃预冷的甲醇中固定10min。
2)PBS(PH为7.2~7.4)洗涤3次,每次5min。
3)将切片浸泡在3%过氧化氢溶液中,避光,15min。
4)用5%正常山羊血清孵育切片30分钟,阻断抗体与组织切片的非特异性结合。
5)弃去血清,将肺组织切片与1:100稀释的p21或1:50稀释的P16抗体4℃孵育过夜;
6)PBS洗涤后,切片与生物素标记的抗兔Ig二抗孵育1小时。
7)DAB染色液显色,适时以自来水冲洗切片终止显色。
8)进行苏木精复染,镜检。
冰冻切片(4~6μm,原放大倍数*100)进行免疫组化染色(图10所示)发现,p16和p21在慢性阻塞性肺疾病模型小鼠肺组织中表达水平增加,口服NMN后,p16和p21蛋白的水平均有下降。
qPCR发现(图11所示):p16蛋白在慢性阻塞性肺疾病模型小鼠肺组织中表达水平增加,口服NMN后,p16蛋白表达水平显著下降。*P<0.05,**P<0.01,versus PBS,#P<0.05,versus PPE。
8、对小鼠肺功能的影响
小鼠肺功能检测发现(图12A,图12B),慢性阻塞性肺疾病模型组小鼠肺顺应性较PBS 组显著降低,给以NMN后,治疗组小鼠肺顺应性明显增加。*P<0.05,**P<0.01。
慢性阻塞性肺疾病模型组小鼠肺FEV100值较PBS组显著降低,给以NMN后,治疗组明显增加。*P<0.05,**P<0.01(图13)。
此外,慢性阻塞性肺疾病模型组小鼠肺MMEF、PEF、IC、ERV、FVC等值较PBS组显著降低,给以NMN后,治疗组明显增加。*P<0.05,**P<0.01(图14A-图14E)。
实施例1的结果从多方面表明,NMN能有效治疗慢性阻塞性肺疾病。也发现烟酰胺核糖 (nicotinamide riboside,NR)也有明显减轻小鼠慢性阻塞性肺疾病的病理形态学改变,进而治疗慢性阻塞性肺疾病。
实施例2 β-烟酰胺单核苷酸NMN与β-烟酰胺核糖NR减少碳颗粒等引起的肺巨噬细胞的老化
(一)提取C57BL/6巨噬细胞
C57BL/6肺泡巨噬细胞提取,剪开小鼠胸腔的肋骨,以便于肺脏充分扩张,之后分离出气管,沿环状软骨间剪开小口,将磨平的20ml的空针插入气管,吸1ml的生理盐水,注入气管,持针器固定针头,回抽,反复2次,离心机1000rpm,处理5min,DMEM洗涤一次后,转入培养皿,加入DMEM(含有小牛血清),待2小时贴壁后,丢弃原培养基,所得贴壁细胞即为肺泡巨噬细胞。C57BL/6腹腔巨噬细胞提取,于小鼠腹腔中注射生理盐水10ml,按揉小鼠腹腔两侧,使生理盐水和腹腔充分接触后,抽取生理盐水于BD管中。并将液体离心, 1,000rpm,5min,用DMEM培养基重悬并洗涤1次后,重新悬浮于含10%小牛血清的DMEM 培养基中培养,孵育2h后去除含悬浮的细胞的培养液,并用DMEM培养基洗涤1次后,即得贴壁的巨噬细胞。
(二)试验步骤及结果
1、碳纳米颗粒诱导巨噬细胞老化和NR对其老化作用的抑制的研究
CBNPs诱导肺巨噬细胞的老化和验证NR是否有抑制CBNPs对巨噬细胞的老化作用,采用了γh2ax抗体(1:5000稀释)和Sa-beta-gal抗体(1:50稀释)对三组进行免疫荧光染色,巨噬细胞进行不同的处理一周后,移除原培养基,取出爬片,PBS洗一次后,以-20℃预冷甲醇固定15min。固定后用PBS洗3次,每次5min。随后加入PBS稀释的γh2ax抗体,于湿盒中4℃孵育过夜。PBS洗3次,每次5min,之后分别加入FITC-标记和CY3-标记的荧光二抗 (1:1000稀释),4℃下于湿盒孵育2h。染DAPI并于PBS中漂洗后,置于正置荧光显微镜下观察、拍照。结果发现,C57小鼠的肺泡巨噬细胞(图15A和图15B)与CBNPs孵育1周后,相比于未处理的对照组的细胞和NR组,含有带有荧光的细胞比例更多,并于高倍镜下观测百分比发现CBNPs组的阳性细胞高于对照组和NR组。因此,说明CBNPs可促进巨噬细胞老化,同时NR可抑制CBNPs对巨噬细胞的老化作用。
我们假设碳纳米颗粒会导致巨噬细胞的老化,并且NR具有抑制其老化的作用。为了进一步确认CBNPs对巨噬细胞的作用,将20lCbnps(终浓度为0.5μg/cm2)溶液加入新鲜提取的C57小鼠的巨噬细胞并在24孔板中孵育1周后,直接加入sa-beta-gal工作液进行sa-beta-gal 染色,37摄氏度无二氧化碳的孵箱中孵育1h左右。结果如图16所示,通过sa-beta-gal染色液直接于24孔板中染色,并于倒置荧光显微镜下观察,观测到部分细胞质蓝染,C57BL/6 小鼠的肺巨噬细胞于高倍镜下观测蓝染的阳性细胞的百分比,发现加入CBNPs悬液的实验组,老化细胞比例大于对照组,经NR处理组老化的细胞减少。
为了观测CBNPs对细胞形态的影响,通过使用DMEM培养基配制浓度为0.05mg/ml,在培养皿中稀释成0.5μg/cm2的cbnps悬液,将20μl Cbnps溶液加入新鲜提取的C57小鼠的腹腔巨噬细胞并在24孔板(生长面积2cm2)中孵育1周后观测其细胞形态变化。进一步发现(如图17A和图17B所示),巨噬细胞形态发生变化,巨噬细胞直径增大,细胞突触增多,细胞活化,但经NR处理组的体积变大的细胞减少。
为进一步核实CBNPs和NR对巨噬细胞的作用,我们以Western检测了巨噬细胞处理一周后所得细胞中的与老化相关蛋白,如图18所示:在Western实验中,CBNPs组的P-Src(Y418)/P-Src(Y529)及sa-beta-gal最高,NR组其次,Control组最低。进一步确认CBNPs 可诱导巨噬细胞老化。同时NR可抑制CBNPs的老化作用。
2、碳纳米颗粒导致巨噬细胞ROS升高
前文已经通过免疫荧光,Western和sa-beta-gal染色发现了CBNPs能诱导肺泡巨噬细胞的老化。并且,NR有抑制CBNPs的老化作用。进一步为了检测巨噬细胞经CBNPs处理后氧化应激的情况,以流式细胞术测定了CBNPs处理后的巨噬细胞中细胞内活性氧成分的变化。方法如下:细胞内活性氧浓度由H2DCF-DA测定。以不同浓度CBNPs(浓度分别为0.5μg /cm2,1μg/cm2,2.5μg/cm2,5μg/cm2,10μg/cm2)±NR(浓度0.5mM)处理细胞20min 后。或者以CBNPs(浓度为0.5μg/cm2)±NR(浓度0.5mM)处理细胞1周,去除原培养基,无血清DMEM洗3遍,并加入H2DCF-DA探针(终浓度为0.1μM),于37℃避光孵育30min,胰酶消化收集贴壁细胞后上机检测。
实验结果如图19所示:巨噬细胞经CBNPs处理一周后细胞内活性氧浓度升高,并且, NR抑制细胞内活性氧浓度。说明,CBNPs能刺激ROS的增加,而NR能抑制该作用。
实施例2的结果从多方面表明,烟酰胺核糖(nicotinamide riboside,NR)NR能有效缓解纳米碳颗粒造成的肺部巨噬细胞的老化。也发现β-烟酰胺单核苷酸(β-Nicotinamide Mononucleotide, NMN)也有明显减轻纳米碳颗粒造成的肺部巨噬细胞的老化的作用。
由本发明实施例可知:空气中可吸入颗粒,如PM10颗粒、PM2.5颗粒或碳黑颗粒等会引起的肺巨噬细胞的老化,继而引起肺损伤,导致慢性阻塞性肺疾病或呼吸障碍,研究发现β-烟酰胺单核苷酸或其前体(β-烟酰胺核糖)具有减轻上述肺损伤的作用,因此本发明提供了β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途,为治疗由可吸入颗粒导致的肺损伤类肺部疾病提供了一种新的治疗选择,具有重要的意义。
Claims (14)
1.β-烟酰胺单核苷酸或其前体在制备治疗或缓解呼吸障碍或疾病的药物或保健品中的用途。
2.根据权利要求1所述的用途,其特征在于:所述的β-烟酰胺单核苷酸前体为β-烟酰胺核糖。
3.根据权利要求1或2所述的用途,其特征在于:所述的呼吸障碍或疾病为肺疾病。
4.根据权利要求3所述的用途,其特征在于:所述的肺疾病为慢性阻塞性肺疾病。
5.根据权利要求3或4所述的用途,其特征在于:所述的肺疾病为由可吸入颗粒引起的肺部疾病。
6.根据权利要求5所述的用途,其特征在于:所述的肺部疾病为肺损伤。
7.根据权利要求6所述的用途,其特征在于:所述的肺损伤为肺巨噬细胞老化。
8.根据权利要求5~7任一项所述的用途,其特征在于:所述的可吸入颗粒为PM10颗粒、PM2.5颗粒或碳黑颗粒中的至少一种。
9.根据权利要求1~8任一项所述的用途,其特征在于:所述药物是以β-烟酰胺单核苷酸或其前体为活性成分,加入药学上接受的辅料或辅助性成分,制备而成的制剂。
10.根据权利要求9所述的用途,其特征在于:所述制剂为口服制剂。
11.根据权利要求10所述的用途,其特征在于:每单位口服制剂含有β-烟酰胺单核苷酸或其前体25-1000mg。
12.根据权利要求10或11所述的用途,其特征在于:所述的口服制剂包括固体制剂、液体制剂或悬浮液制剂。
13.根据权利要求12所述的用途,其特征在于:所述的固体制剂包括胶囊剂、片剂、丸剂、散剂或颗粒剂。
14.根据权利要求12所述的用途,其特征在于:所述的液体制剂包括乳液、溶液、悬浮液、糖浆或酊剂。
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CN110548040A (zh) * | 2019-10-17 | 2019-12-10 | 苏州大学 | β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用 |
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CN110483601A (zh) * | 2019-08-12 | 2019-11-22 | 上海龙翔生物医药开发有限公司 | 制备β-烟酸胺单核苷酸的方法及其应用 |
CN110548040A (zh) * | 2019-10-17 | 2019-12-10 | 苏州大学 | β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用 |
WO2021073249A1 (zh) * | 2019-10-17 | 2021-04-22 | 苏州大学 | β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用 |
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