CN118001279A - 犬尿喹啉酸在制备缓解和/或治疗类风湿关节炎的药物中的应用 - Google Patents
犬尿喹啉酸在制备缓解和/或治疗类风湿关节炎的药物中的应用 Download PDFInfo
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Abstract
本发明涉及犬尿喹啉酸在制备缓解和/或治疗类风湿关节炎的药物中的应用。本发明证明了犬尿喹啉酸参与类风湿关节炎免疫调控的重要作用，其通过影响炎症环境下巨噬细胞的代谢重编程、修复炎症状态下的线粒体功能，实现了缓解炎症的效果。并且犬尿喹啉酸可以缓解动物体内由胶原诱导的关节炎，减少炎症因子的产生，对类风湿关节炎患者的治疗、症状缓解、和生活质量提高具有重要意义，在制备缓解和/或治疗类风湿关节炎的产品中具有广阔的应用前景。
Description
技术领域
本发明属于生物医药领域，具体涉及犬尿喹啉酸在制备缓解和/或治疗类风湿关节炎的药物中的应用。
背景技术
类风湿关节炎（rheumatoid arthritis, RA）是一种以炎性滑膜炎为主的慢性全身自身免疫性疾病。患者临床表现为关节肿胀畸形，可伴发关节外炎症损伤，严重时甚至会导致高度致残。RA的病理机制与巨噬细胞、T细胞、B细胞和成纤维样滑膜细胞等各种细胞、以及白介素 1β(IL-1β)和肿瘤坏死因子α(TNF-α)等炎性细胞因子共同作用有关。患者机体内通常会产生针对自身抗原的自身抗体, 最终导致滑膜炎症和软骨破坏。RA发病机制中免疫稳态的失衡一直是研究者广泛关注的热点，代谢和免疫之间的相互作用复杂而密切，代谢紊乱可能导致免疫功能失调。而活性代谢物可表现为协调细胞通讯以及免疫功能的有机配体和底物，其在免疫应答过程中发挥关键作用。因此，针对过度活化的免疫细胞（巨噬细胞、T细胞、B细胞）内的代谢反应, 探寻RA免疫调节的关键代谢物以及相关代谢通路作为药物靶标进行特异性调节, 以达到适度调控机体免疫应答的效果，对于预防和治疗RA至关重要。
色氨酸(TRP)是蛋白质生物合成所需的必需氨基酸，并且也是体内多种生物活性分子的前体。其基本上通过犬尿氨酸途径(KP)代谢，产生至少约一百种的大量代谢物，例如犬尿氨酸(KYN)、犬尿喹啉酸(KynA)、邻氨基苯甲酸(AA)、黄尿酸（XanA）、喹啉酸(QUIN)、吡啶甲酸(PICO)、喹哪啶酸(QL-Dic)或3OH-犬尿氨酸（Widner B等，1997），其余的游离TRP主要用于蛋白质合成以及神经递质，如血清素，和神经调节剂，如色胺的合成。已有研究发现，TRP代谢是细胞和机体沟通策略中重要的一部分，TRP及其代谢物在多种不同的生理过程中起着重要作用，包括作为蛋白质的组成部分维持细胞的生长、增殖，或者作为神经递质和信号分子协调机体对环境和饮食等外部影响的反应等。
犬尿喹啉酸作为色氨酸代谢的产物之一，广泛分布于哺乳动物的体液和中枢神经系统中，能拮抗兴奋性氨基酸(EAA)和烟碱受体，与某些神经系统、眼科、肾脏等疾病都有关联。由于RA的发病机制及治疗方案较复杂，相关科研人员尚未完全研究清楚，目前还未有通过施用施用色氨酸代谢物，例如KynA来缓解或治疗RA的相关报道。
发明内容
为了克服现有技术中存在的上述不足，本发明提供犬尿喹啉酸在制备缓解和/或治疗类风湿关节炎的药物中的应用。
根据本发明所提供的上述应用，其中所述药物以犬尿喹啉酸为活性成分，配以药学上可接受的辅料制备成制剂使用。
根据本发明所提供的上述应用，其中所述制剂选自注射液、片剂、粉剂、颗粒剂、胶囊剂、口服液、丸剂、缓释剂中的一种。
本发明还提供一种缓解和/或治疗类风湿关节炎的药物，其中所述药物中含有犬尿喹啉酸。
进一步的，本发明所提供的药物中，所述药物还含有药学上可接受的辅料。
优选地，所述辅料包含抗黏合剂、渗透促进剂、缓冲剂、增塑剂、表面活性剂、消泡剂、增稠剂、包合剂、吸收剂、保湿剂、溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、pH值调节剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、整合剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、发泡剂、助悬剂、包衣材料、芳香剂、稀释剂、絮凝剂与反絮凝剂、助滤剂或释放阻滞剂。
优选地，本发明所述药物的应用对象包括但不限于人类或动物。
优选的，本发明所提供的药物中，所述药物的剂型为注射液、片剂、粉剂、颗粒剂、胶囊剂、口服液、丸剂、或缓释剂。
优选的，本发明所述药物通过参与炎症环境下巨噬细胞的代谢重编程缓解RA的炎症反应。
更优选的，本发明所述药物通过修复炎症状态下的线粒体功能缓解RA的炎症反应。
本发明还提供犬尿喹啉酸在制备改善和/或缓解类风湿关节炎症状的食品、饮品、保健品、肠内营养剂、和/或膳食补充剂中的应用。
另一方面，本发明提供用于改善和/或缓解类风湿关节炎症状的食品、饮品、保健品、肠内营养剂、和/或膳食补充剂，其特征在于，包含犬尿喹啉酸。
本发明的有益效果在于：
本发明首次发现并证明了色氨酸代谢产物犬尿喹啉酸参与类风湿关节炎免疫调控的重要作用，其通过影响炎症环境下巨噬细胞的代谢重编程、修复炎症状态下的线粒体功能，实现了缓解炎症的效果。并且KynA可以缓解动物体内由胶原诱导的关节炎，减少炎症因子的产生。对类风湿关节炎患者的治疗、症状缓解、和生活质量提高具有重要意义，在制备缓解和/或治疗RA的产品中具有广阔的应用前景。
附图说明
图1显示了TRP代谢物在CIA小鼠体内的作用，其中：A. 灌胃TRP（n=6），KynA（n=6），XanA（n=6）和溶剂对照（n=7）的小鼠关节炎临床评分；B. 小鼠体重变化； C.小鼠血清抗II型胶原抗体滴度水平；D. 小鼠血清IL-6水平；*P≤0.05，**P≤0.01。
图2A显示了KynA对巨噬细胞的作用。
图2B显示了KynA对Th17细胞的作用。
图3显示了CIA小鼠关节的IHC染色，其中：黑色方框表示高倍镜放大区域，棕色表示F4/80阳性；**P≤0.01。
图4显示了TRP代谢物在原代骨髓来源巨噬细胞BMDM中的作用，其中：A. TRP代谢物处理后BMDM培养上清中TNF-α水平；B. TRP代谢物处理后BMDM培养上清中IL-6水平；C.TRP代谢物处理后BMDM培养上清中IL-1β水平；D. 不同浓度KynA处理后BMDM培养上清中TNF-α水平；E. 不同浓度KynA处理后BMDM培养上清中IL-6水平；F. 不同浓度KynA处理后BMDM培养上清中IL-1β水平；*P≤0.05，**P≤0.01，***P≤0.001，****P≤0.0001。
图5显示了KynA处理后BMDM转录组的结果。
图6显示了KynA防止功能障碍的线粒体的积累。
具体实施方式
本发明人通过在类风湿关节炎患者和健康对照人群中发现的色氨酸代谢物，特别是TRP、KynA和XanA的水平差异，进一步在胶原诱导关节炎（CIA）的小鼠模型中通过早期干预TRP、KynA和XanA这三种代谢物，发现了KynA组的小鼠关节炎临床评分较对照组、TRP及XanA组明显降低，KynA组小鼠的抗II型胶原抗体滴度明显降低，而TRP、XanA组的抗体滴度与对照组无明显差异；KynA组IL-6浓度明显低于对照组，表明KynA能够缓解CIA关节炎症。
为进一步了解KynA如何抑制CIA小鼠的关节炎症，通过流式细胞术分析对照组和KynA组中与RA发病相关的几种主要免疫细胞亚群的变化，发现KynA组小鼠DLNs的巨噬细胞较溶剂对照组减少，而在MLNs的巨噬细胞在两组间无明显差异。另外还发现KynA组小鼠DLNs及MLNs的CD4+IL-17+细胞比例（Th17）较溶剂对照组降低，表明KynA通过抑制Th17细胞而缓解关节炎症。
为了解KynA对关节局部巨噬细胞的作用，对小鼠关节切片做了F4/80免疫组化染色，研究发现对照组F4/80表达明显高于KynA组，表明KynA通过影响CIA滑膜巨噬细胞，从而减轻关节局部炎症。
在此基础上结合体外细胞实验，分别用TRP、KynA以及XanA处理原代骨髓来源巨噬细胞BMDM，用LPS激活巨噬细胞模拟炎症环境。经过KynA预处理后的巨噬细胞分泌的炎症因子TNF-α、IL-6、和IL-1β显著下降，且KynA对炎症因子的抑制能力随浓度升高而增强。
为进一步探索KynA如何调控巨噬细胞介导的炎症，通过转录组测序分析发现巨噬细胞KynA处理组的差异基因主要富集在细胞因子趋化因子信号通路、G蛋白受体内化通路以及氧化还原酶调节通路等。表明KynA可能介导炎症环境下巨噬细胞代谢的重编程。
下面结合实施例和附图对本发明的技术方案作出优选的详细说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例一
实验方法
1. 实验动物
健康7周龄DBA/1J雌鼠共25只，体重10-15 g，购自北京维通利华实验动物技术有限公司。小鼠于无特定病原体（SPF）级环境饲养，控制12 h光照/12 h无光照环境，环境温度控制在（25±1 ℃），相对湿度50-70%，自由进食及饮水。
2. 胶原诱导关节炎（collagen induced arthritis，CIA）小鼠造模及处理
（1）分组：用耳钉标记DBA/1J小鼠号数，并将其随机分为4组（TRP组6只，KynA组6只，XanA组6只，溶剂对照组vehicle 7只）。
（2）灌胃：在第一次免疫前1周起隔日灌胃TRP、KynA、XanA及溶剂，每只小鼠灌胃量为0.1 mL，化合物剂量为TRP 0.15 mg/只，KynA 0.24 mg/只，XanA 0.15 mg/只，一直持续至实验终点。
（3）第一次免疫：
1）配置0.05 M冰乙酸。
2）用电子天平称取II型胶原6 mg。
3）在冰上将称好的胶原溶解于3 mL配置好的0.05 M冰乙酸中，至胶原完全溶解，此时胶原乙酸溶液终浓度为2 mg/mL。
4）在冰上分别吸取3 mL完全弗氏佐剂（CFA，1mg/mL）与3 mL配置好的II型胶原乙酸溶液（2 mg/mL）以l：l等体积混合，用5 mL注射器反复吹打，直至完全乳化。
5）保持低温操作，将乳化后的液体转移至1 mL注射器，排空气泡。固定小鼠，尽量靠近尾根部向皮下注射乳液。每只小鼠注射量为0.1 mL，即100 μg II型胶原/只。每周对小鼠状态进行观察，对体重进行记录。
（4）第二次加强免疫：第一次免疫21天后给予小鼠第二针加强免疫。将等体积弗氏不完全佐剂IFA与II型胶原乙酸溶液（2 mg/mL）吹打乳化，具体方法同上述第一次免疫。待完全乳化后，将液体转移至1 mL注射器，排空气泡后，避开第一次免疫后造成的炎症组织，向尾部皮下注射乳液，每只小鼠注射量为0.1mL，即100 μg II型胶原/只。第二次免疫后1周左右小鼠陆续开始发病，主要表现为足趾、脚掌红肿，严重时可出现跛行。研究者隔日对小鼠每只足爪的关节炎症进行评分及记录，每周进行体重记录。
3. 实验取材
（1）取血及后续处理：
1）气体麻醉小鼠后，压迫小鼠颈部两侧，从内侧眼角处将毛细采血管斜向刺入2-3mm，使血液流入5 mL EDTA抗凝采血管。
2）采血结束后解除对小鼠颈部的压迫，待眼球复位后拔出毛细采血管，用干棉球止血。
3）将收集的小鼠外周血1500 rpm，10 min离心，吸取上清保存于1.5 mL EP管，于-80℃冰箱保存待测。
（2）肠系膜淋巴结分离及后续处理：
1）处死小鼠，固定四肢，用75%酒精消毒皮肤，沿腹中线剪开小鼠皮肤，钝性剥离腹膜打开腹腔，取延伸于肠系膜脂肪组织中的淋巴结，将其转入预冷的DPBS中。
2）去除肠系膜淋巴结周围脂肪、系膜等组织，用载玻片轻柔研磨，用DPBS冲洗，至研磨液清亮。
3）收集研磨液过70μm细胞筛至15 mL离心管，1500 rpm，离心5分钟。
4）弃上清，用DPBS重悬细胞，将单细胞悬液等分4份，置于4℃冰箱待测。
（3）关节引流区淋巴结分离及后续处理：
1）处死小鼠，固定四肢，75%酒精消毒皮肤，剪开小鼠上下肢皮肤，钝性剥离暴露小鼠腋窝及腘窝，分离浅表皮下脂肪组织中的腋窝淋巴结和腘窝淋巴结，转入预冷的DPBS中。
2）仔细去除DLN周围脂肪组织，用载玻片轻柔研磨，用DPBS冲洗，至研磨液清亮。
3）收集研磨液过70μm细胞筛至15 mL离心管，1500 rpm，离心5分钟。
4）弃上清，用DPBS重悬细胞，将单细胞悬液等分4份，置于4℃冰箱待测。
（4）关节固定及后续处理：从踝关节以上剪断小鼠后爪，剥离皮肤后将各关节压平置于包埋盒中，将其完全浸入通用型组织固定液中，4℃固定48 h，在10％乙二胺四乙酸中脱钙，并包埋在石蜡中待后续实验。
4. 流式检测
（1）Th1/Th17细胞染色：
1）制备单细胞悬液，计数细胞量，用1 mL完全培养基（RPMI 1640+10% FBS+1%二联抗生素）重悬，将细胞转入24孔细胞培养板。
2）37°C水浴中完全解冻细胞刺激混合物。
3）每孔加入2μL细胞刺激混合物（细胞浓度约1×106个细胞/mL），在37°C的CO2培养箱中培养细胞6 h。
5）收集刺激后的细胞于流式管，350 G离心5 min，弃上清。
6）死/活细胞染色：每管细胞用100μL DPBS重悬，加入1μL Zombie NIR dye，室温下避光孵育20 min。
7）每管细胞加入1 mL染色缓冲液，350 G，离心5 min，弃上清。
8）细胞表面标记物染色：每管细胞用100μL染色缓冲液重悬，加入1μL 鼠抗-CD4PerCP，4℃避光孵育30 min。
9）重复步骤7
10）固定破膜：每管细胞加入100μL 1×破膜固定液重悬，4℃避光固定60 min。
11）每管细胞加入1 mL 1×渗透缓冲液，500 G离心5 min，弃上清。
12）每管细胞加入100μL 1×渗透缓冲液，分别加入1μL 鼠抗-IFN-γ FITC、1μL鼠抗- IL-17A PE，室温下避光孵育60 min。
13）每管细胞加入1 mL 染色缓冲液，500 G离心5 min，弃上清。
14）每管细胞加入300μL染色缓冲液，用70μm细胞筛过滤后上机检测。
（2）Treg细胞染色：
1）细胞表面标记物染色：取1份上述“3. 实验取材”中制备的单细胞悬液，每管细胞用100μL染色缓冲液重悬，分别加入1μL 鼠抗-CD4 PerCP、1μL 鼠抗-CD25 PE，4℃避光孵育30 min。
2）每管细胞加入1 mL染色缓冲液，350 G，离心5 min，弃上清。
3）固定破膜：每管细胞加入100μL 1×破膜固定液重悬，4℃避光固定60 min。
4）每管细胞加入1 mL 1×渗透缓冲液，500 G离心5 min，弃上清。
5）每管细胞加入100μL 1×渗透缓冲液，加入1μL鼠抗- FOXP3 APC，室温下避光孵育60 min。
6）每管细胞加入1 mL染色缓冲液，500 G离心5 min，弃上清。
7）每管细胞加入300μL染色缓冲液，70μm细胞筛过滤后上机检测。
（3）巨噬细胞染色：
1）细胞表面标记物染色：取1份上述“3. 实验取材”中制备的单细胞悬液，每管细胞用100μL 染色缓冲液重悬，分别加入1μL 鼠抗-MHCII FITC、1μL鼠抗CD11b PE，4℃避光孵育30 min。
2）每管细胞加入1 mL染色缓冲液，350 G，离心5 min，弃上清。
3）每管细胞加入300μL染色缓冲液，70μm细胞筛过滤后上机检测。
（4）浆细胞染色：
1）细胞表面标记物染色：取1份上述“3. 实验取材”中制备的单细胞悬液，每管细胞用100μL 染色缓冲液重悬，分别加入1μL鼠抗-B220 FITC、1μL 鼠抗-CD138 PE，4℃避光孵育30 min。
2）每管细胞加入1 mL染色缓冲液，350 G，离心5 min，弃上清。
3）每管细胞加入300μL染色缓冲液，70μm细胞筛过滤后上机检测。
5. 血浆抗II型胶原抗体以及血浆IL-6炎症因子检测，按照使用说明书操作。
6. 关节H&E病理
（1）石蜡切片脱蜡：将切片按照下列顺序依次放入各溶液中：二甲苯20 min-二甲苯20 min-无水乙醇5 min-无水乙醇5 min-75%酒精5 min，后用去离子水清洗。
（2）苏木素染色：用苏木素染液染色3-5 min，去离子水清洗切片，加入分化液，再用去离子水清洗切片，加入返蓝液返蓝，再用去离子水冲洗。
（3）伊红染色：切片依次放入85%、95%的梯度酒精脱水各5 min，将切片放入伊红染液中染色5 min。
（4）脱水封片：将切片按照下列顺序依次放入各溶液中：无水乙醇5 min-无水乙醇5 min-无水乙醇5 min-二甲苯5 min-二甲苯5 min，用中性树胶封片。
（5）显微镜镜检，图像采集，用caseviewer软件读图，并如下表所示评分标准对关节病理评分。
7. 关节免疫组化
（1）石蜡切片脱蜡：将切片按照下列顺序依次放入各溶液中：环保型脱蜡液10min-环保型脱蜡液10 min-环保型脱蜡液10 min-无水乙醇5 min-无水乙醇5 min-无水乙醇5 min，再用去离子水清洗。
（2）热修复抗原，冷却后将玻片置于PBS（PH 7.4）中，脱色摇床上洗涤3次，每次5min。
（3）阻断内源性过氧化物酶：将切片放入3%双氧水溶液中，室温避光孵育25 min，将玻片置于PBS（PH 7.4）中，脱色摇床上洗涤3次，每次5 min。
（4）血清封闭：在组化圈内滴加3% BSA均匀覆盖组织，室温封闭30 min。
（5）加一抗：轻轻甩掉封闭液，在切片上滴加F4/80一抗，切片平放于湿盒内4℃孵育过夜。
（6）加二抗：将玻片置于PBS（PH 7.4）中，脱色摇床上洗涤3次，每次5 min。切片稍甩干后在圈内滴加HRP标记二抗覆盖组织，室温孵育50 min。
（7）DAB显色：将玻片置于PBS（PH 7.4）中，脱色摇床上洗涤3次，每次5 min。切片稍甩干后在圈内滴加DAB显色液。显微镜下可见阳性部分为棕黄色，用去离子水冲洗切片终止显色。
（8）复染细胞核：用苏木素染液复染3 min左右，去离子水冲洗，用苏木素分化液分化数秒，去离子水冲洗，再用苏木素返蓝液返蓝，去离子水冲洗。
（9）脱水封片：将切片依次放入75%酒精 5 min-85%酒精 5 min-无水乙醇 5 min-无水乙醇 5 min-正丁醇 5 min-二甲苯 5 min中脱水，晾干后用封片胶封片。
（10）显微镜镜检，同时进行图像采集，之后用caseviewer软件读图。苏木素染细胞核为蓝色，DAB显出的F4/80阳性表达的细胞为棕黄色，用ImageJ对具有代表性的图像进行平均光密度值（AOD）计算。
8. 统计分析
实验数据统计分析使用GraphPad Prism 9软件。对计量资料的数据，若符合正态分布，以平均值（标准差SD）形式表示，统计分析方法采用独立样本T检验；若不符合正态分布，则以中位数（四分位间距IQR）表示，统计学分析使用非参数检验方法。对计数资料的数据，统计学分析方法使用卡方检验。对多组数据样本分析时，正态分布数据用one wayanova分析，非正态分布数据用kruskal-wallis检验。由于动物模型的临床评分及体重的变化受到双因素交互作用的影响，统计方式采用two way anova分析。P值≤0.05认为差异具有显著性。
通过在CIA小鼠模型中早期干预TRP、KynA和XanA这三种代谢物，发现KynA组的小鼠关节炎临床评分较溶剂对照组（炎症对照组）、TRP及XanA组明显降低，KynA组小鼠的抗II型胶原抗体滴度明显降低，而TRP、XanA组的抗体滴度与溶剂对照组无明显差异；KynA组IL-6浓度明显低于溶剂对照组，结果如图1所示，表明KynA能够缓解CIA关节炎症。
通过流式细胞术分析溶剂对照组和KynA组中与RA发病相关的几种主要免疫细胞亚群的变化。我们首先对MHCII+CD11b+细胞群（巨噬细胞）进行分析，发现KynA组小鼠DLNs的巨噬细胞较溶剂对照组减少，而在MLNs的巨噬细胞在两组间无明显差异，结果如图6A所示。另外我们发现KynA组小鼠DLNs及MLNs的CD4+IL-17+细胞比例（Th17）较溶剂对照组降低，结果如图6B所示。上述结果表明KynA通过抑制Th17细胞而缓解关节炎症。
通过对小鼠关节切片做的F4/80免疫组化染色，研究发现溶剂对照组F4/80表达明显高于KynA组，结果如图3所示，表明KynA通过影响CIA滑膜巨噬细胞，从而减轻关节局部炎症。
因此，上述动物实验结果表明KynA可以缓解动物体内由胶原诱导的关节炎，减少炎症因子的产生。
实施例二
在实施例一的基础上，同时开展体外细胞实验，分别用TRP、KynA以及XanA处理原代骨髓来源的巨噬细胞BMDM，用脂多糖（LPS）激活巨噬细胞模拟炎症环境。经过KynA预处理后的巨噬细胞分泌的炎症因子TNF-α、IL-6、和IL-1β显著下降，且KynA对炎症因子的抑制能力随浓度升高而增强，结果如图4所示。
分别用TRP、KynA以及XanA处理巨噬细胞系Raw 264.7，用LPS激活巨噬细胞模拟炎症环境，然后通过酶联免疫吸附法（ELISA）检测细胞培养上清炎症因子分泌水平，转录组测序（RNA-seq）筛选和富集影响巨噬细胞炎症的基因及通路。
使用 Seahorse XF96 细胞外通量分析仪对巨噬细胞中的细胞外酸化率 (ECAR)和耗氧率 (OCR) 进行分析。在开始测定之前，洗涤细胞并在补充有10 mM 葡萄糖和1 mM丙酮酸钠的 Seahorse 测定培养基中，在37°C无CO2培养箱中孵育45分钟。在指定位置注射寡霉素（ATP 酶抑制剂，1 µM）、FCCP（1 µM）和鱼藤酮（0.5 µM），并实时测量 ECAR（mpH/min）和OCR（pMoles O2/min）。
对于巨噬细胞系Raw 264.7中上糖酵解、线粒体代谢谱的改变是否是由线粒体功能异常引起的研究，使用 MitoTracker Green（Δψm 独立的线粒体染色剂）和MitoTrackerDeep Red （Δψm 依赖的线粒体染色剂）来区分呼吸线粒体和功能障碍线粒体。
通过转录组测序分析发现巨噬细胞KynA处理组的差异基因主要富集在细胞因子趋化因子信号通路、G蛋白受体内化通路以及氧化还原酶调节通路等，结果如图5 中的A和B所示。其中KynA处理组的促炎细胞因子和趋化因子基因表达下调，抗氧化应激基因如Nqo1显著上调，而NOD样受体信号通路中基因Nod1、Nlrp3等显著下调。转录组分析进一步发现KynA可以上调IL-10受体β(IL10rb)，下调糖酵解通路关键基因，如葡萄糖转运基因Glut1(Slc2a1）、己糖激酶-2 (Hk2) 和乳酸脱氢酶 A (Ldha)、低氧诱导因子-1α (HIF-1α)等，以及下调线粒体代谢关键基因如沉默信息调节因子 3 (Sirt3) 和FoxO转录因子（Foxo1），结果如图5中的 B和C所示。以上结果表明KynA可通过HIF-1α/ Glut1通路下调糖酵解水平，通过Sirt3/ Foxo1通路下调线粒体呼吸，提示KynA介导了炎症环境下巨噬细胞的代谢重编程。
活化的巨噬细胞会发生代谢转换，包括糖酵解的快速上调、TCA 循环的重塑和线粒体呼吸的抑制。对于使用细胞能量代谢检测仪分析糖酵解和氧化磷酸化指标，通过Seahorse 分析 OCR，发现KynA通过改善耗氧量、调节巨噬细胞线粒体呼吸来恢复LPS刺激的巨噬细胞中的线粒体氧化功能，结果如图6中的A和B所示。使用 MitoTracker Green（Δψm 独立的线粒体染色剂）和MitoTracker Deep Red （Δψm 依赖的线粒体染色剂）染色LPS刺激的巨噬细胞系Raw 264.7，发现LPS使得功能障碍线粒体增加，而KynA可以降低功能障碍的线粒体比例，结果如图6中的C和D所示。上述结果表明KynA可以修复炎症状态下的线粒体功能。
因此，上述细胞实验表明KynA通过介导炎症环境下巨噬细胞的代谢重编程，进而缓解炎症。
最后需要说明的是，以上具体实施方式仅用以说明本发明的技术方案而非加以限制，尽管参照实施例对本发明进行了详细说明，所属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精神和范围，其均应涵盖在本发明的权利要求所请求保护的范围之内。
Claims (8)
1.犬尿喹啉酸在制备缓解和/或治疗类风湿关节炎的药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述药物以犬尿喹啉酸为活性成分，配以药学上可接受的辅料制备成制剂使用。
3.根据权利要求2 所述的应用，其特征在于：所述制剂选自注射液、片剂、粉剂、颗粒剂、胶囊剂、口服液、丸剂、缓释剂中的一种。
4.一种缓解和/或治疗类风湿关节炎的药物，其特征在于，所述药物中含有犬尿喹啉酸。
5.根据权利要求4所述的药物，其特征在于，所述药物还含有药学上可接受的辅料。
6.根据权利要求5所述的药物，其特征在于，所述药物的剂型为注射液、片剂、粉剂、颗粒剂、胶囊剂、口服液、丸剂、或缓释剂。
7.犬尿喹啉酸在制备改善和/或缓解类风湿关节炎症状的食品、饮品、保健品、肠内营养剂、和/或膳食补充剂中的应用。
8.用于改善和/或缓解类风湿关节炎症状的食品、饮品、保健品、肠内营养剂、和/或膳食补充剂，其特征在于，包含犬尿喹啉酸。
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