中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (35): 5700-5706.doi: 10.12307/2022.939

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

时辰艾灸干预类风湿性关节炎模型大鼠核心钟基因Clock、Bmal1表达与细胞焦亡的关系

余明芳1，2，陈白露1，何欣玲1，王嫒洋1，吴  晓1   

  1. 1西南医科大学中西医结合学院·附属中医医院，四川省泸州市  640000；2泸州市中医医院针灸科，四川省泸州市  646000
  • 收稿日期:2022-01-05 接受日期:2022-02-11 出版日期:2022-12-18 发布日期:2022-05-18
  • 通讯作者: 吴晓，博士，副主任医师，副教授，硕士生导师，西南医科大学中西医结合学院·附属中医医院，四川省泸州市 640000
  • 作者简介:余明芳，女，1988年生，四川省宜宾市人，汉族，西南医科大学在读硕士，主治中医师，主要从事针灸治疗骨代谢疾病的时间医学研究。
  • 基金资助:
    国家自然科学基金项目 (81804208)，项目负责人：吴晓，项目名称：不同时辰艾灸治疗RA的抗炎节律及核心钟基因的调控机制研究

Effects of Chrono-moxibustion on core clock genes Clock, Bmal1 and pyroptosis in rats with rheumatoid arthritis

Yu Mingfang1, 2, Chen Bailu1, He Xinling1, Wang Aiyang1, Wu Xiao1   

  1. 1College of Integrated Traditional Chinese and Western Medicine, Traditional Chinese Medicine Hospital Affiliated to Southwest Medical University, Luzhou 640000, Sichuan Province, China; 2Department of Acupuncture, Luzhou T.C.M. Hospital, Luzhou 646000, Sichuan Province, China
  • Received:2022-01-05 Accepted:2022-02-11 Online:2022-12-18 Published:2022-05-18
  • Contact: Wu Xiao, MD, Associate chief physician, Associate professor, Master’s supervisor, College of Integrated Traditional Chinese and Western Medicine, Traditional Chinese Medicine Hospital Affiliated to Southwest Medical University, Luzhou 640000, Sichuan Province, China
  • About author:Yu Mingfang, Master candidate, Attending physician, College of Integrated Traditional Chinese and Western Medicine, Traditional Chinese Medicine Hospital Affiliated to Southwest Medical University, Luzhou 640000, Sichuan Province, China; Department of Acupuncture, Luzhou T.C.M. Hospital, Luzhou 646000, Sichuan Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81804208 (to WX)

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摘要：

文题释义：
时辰艾灸：是基于中医时间针灸学，结合时间生物学的原理，根据机体生理和病理节律特点，在不同时辰针对不同疾病进行艾灸治疗，以达到最佳临床疗效。
核心钟基因：是在分子生物钟转录翻译反馈环中维持稳健的昼夜节律，调控其他生物钟基因之间协调表达的主要钟基因。其参与调节机体众多生理过程，以维持内环境稳态，目前主要的钟基因有Clock、Bmal1、Pers、Crys、Rev-erbα、Ror。

背景：类风湿关节炎是症状特点具有昼夜节律性的炎症性疾病，艾灸治疗类风湿关节炎疗效显著，但是其具体机制是否与钟基因调控及细胞焦亡的抑制有关尚不清楚。
目的：探究不同时辰艾灸治疗类风湿关节炎模型大鼠对核心钟基因和细胞焦亡的影响。
方法：随机将40只成年SD大鼠分为空白组、类风湿关节炎模型组(模型组)、辰时艾灸组(辰时组)、酉时艾灸组(酉时组)，雌雄各半，每组10只。空白组大鼠于右后足垫皮内注射无菌生理盐水，其余以弗氏完全佐剂制备实验性类风湿关节炎大鼠模型，辰时组(7：00-9：00)和酉时组(17：00-19：00)于造模后第7天在相应时辰取足三里、肾俞进行艾灸治疗，1次/d，每周治疗6 d后休息1 d，共治疗18 d。空白组及模型组行同法固定，不予艾灸干预。采用排水法分别测量大鼠模型制备前、制备后及每周艾灸干预后右足足容积；治疗结束后采集大鼠下丘脑和右膝关节滑膜，应用实时荧光定量PCR和Western blot观察下丘脑或膝关节滑膜组织中Clock、Bmal1基因、NOD样受体热蛋白结构域相关蛋白3(Nod-like receptor protein 3，NLRP3)、半胱氨酸蛋白酶1(Caspase-1)、消皮素D的表达情况。
结果与结论：①各组大鼠下丘脑中Clock、Bmal1 mRNA表达水平差异无显著性意义(P > 0.05)；在Clock、Bmal1 mRNA相对表达量中，下丘脑各组大鼠的表达丰度均比关节滑膜高(P < 0.01)；关节滑膜中，与空白组比较，模型组Clock、Bmal1 mRNA和蛋白表达均显著降低(P < 0.01)；与模型组相比，艾灸可显著升高滑膜Clock、Bmal1 mRNA和蛋白表达(P < 0.05，P < 0.01)；与辰时组比较，酉时组中Clock、Bmal1 mRNA和蛋白表达稍上调，但差异无显著性意义(P > 0.05)；②与空白组比较，模型组大鼠关节滑膜中NLRP3、Caspase-1、消皮素D mRNA表达量明显升高(P < 0.01)；与模型组比较，酉时艾灸可明显降低类风湿关节炎关节滑膜中NLRP3、Caspase-1、消皮素D mRNA表达量(P < 0.01)；与辰时组比较，酉时组中NLRP3、Caspase-1 mRNA表达量显著降低(P < 0.05，P < 0.01)；③结果说明艾灸可通过上调类风湿关节炎大鼠不同组织中Clock、Bmal1 mRNA和蛋白表达水平，下调细胞焦亡经典信号通路中效应分子NLRP3、Caspase-1、消皮素D mRNA表达，减轻滑膜炎症和细胞焦亡从而治疗类风湿关节炎；核心钟基因Clock、Bmal1在类风湿关节炎中发挥的抗炎机制可能与抑制细胞焦亡效应因子有关。
缩略语：NOD样受体热蛋白结构域相关蛋白3：Nod-like receptor family pyrin domain-containing 3，NLRP3；半胱氨酸蛋白酶1：Caspase-1；消皮素D：Gasdermin D，GSDMD

https://orcid.org/0000-0001-9604-4427 (余明芳) 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 类风湿关节炎, 艾灸, Clock基因, Bmal1基因, 细胞焦亡, 大鼠

Abstract: BACKGROUND: Rheumatoid arthritis is an inflammatory disease with a circadian variation of symptoms. Moxibustion has achieved remarkable efficacy in the treatment of rheumatoid arthritis, but it is unclear whether the specific mechanism is related to the regulation of the clock gene and the inhibition of pyroptosis.
OBJECTIVE: To explore the effects of moxibustion at different time points on core clock genes Clock, Bmal1 and pyroptosis in rats with rheumatoid arthritis. 
METHODS: Forty adult Sprague-Dawley rats of both sexes were randomized into a control group, a model group, a moxibustion at 7:00-9:00 A.M. group and a moxibustion at 5:00-7:00 P.M. group, with 10 rats in each group. To establish the rheumatoid arthritis model, the rats were injected with Freund’s Complete Adjuvant at the right foot. Rats in the control group were given the normal saline at the right foot. At the 7th after modeling, the two moxibustion groups began to be treated with moxibustion at Zusanli and Shenshu acupoints at 7:00-9:00 A.M. or 5:00-7:00 P.M., once a day, for total 18 treatments with a 1-day break every 6 days. We measured the right foot volume of rats using drainage method before model establishment, after model establishment and after treatment. The hypothalamus and the synovium of the right knee joint were collected from the rats after treatment. The expressions of Clock, Bmal1, Nod-like receptor family pyrin domain-containing 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD) in the hypothalamus or knee synovium were tested by real-time fluorescence quantitative PCR and western blot. 
RESULTS AND CONCLUSION: The mRNA expreession of Clock and BMAL1 in the hypothalamus had no obvious change among the four groups (P > 0.05). The relative mRNA expression of Clock and Bmal in the hypothalamus was higher than that in the synovium (P < 0.01). The expression of Clock and Bmal1 in the synovium was significantly down-regulated in the model group compared with the control group (P < 0.01). Moxibustion significantly increased the mRNA and protein expression of Clock and Bmal1 in the synoviums compared with the model group (P < 0.05). Compared with the moxibustion at 7:00-9:00 A.M. group, the mRNA and protein expressions of Clock and Bmal1 in the moxibustion at 5:00-7:00 P.M. group were slightly but insignificantly up-regulated (P > 0.05). The mRNA expressions of NLRP3, Caspase-1 and GSDMD in the synovium were significantly increased in the model group compared with the control group (P < 0.01). Compared with the model group, moxibustion at 5:00-7:00 P.M. could significantly reduce the relative mRNA expression of NLRP3, Caspase-1 and GSDMD in the synovium (P < 0.01). Compared with the moxibustion at 7:00-9:00 A.M. group, the relative mRNA expressions of NLRP3 and Caspase-1 were significantly lowered in the moxibustion at 5:00-7:00 P.M. group (P < 0.05, P < 0.01). All these findings indicate that moxibustion can treat rheumatoid arthritis rats by up-regulating the expression of Clock and Bmal1 mRNA and protein in different tissues, down-regulating the mRNA expression of NLRP3, Caspase-1 and GSDMD in the classical signal pathway of pyroptosis, and reducing synovitis and pyroptosis. The anti-inflammatory mechanism of core clock genes Clock and BMAL1 in rheumatoid arthritis may be related to the inhibition of pyrolytic effectors.

Key words: rheumatoid arthritis, moxibustion, Clock, Bmal1, pyroptosis, rat

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