中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (10): 1484-1489.doi: 10.12307/2024.261

• 组织工程骨材料 tissue-engineered bone • 上一篇    下一篇

褪黑素抑制NLRP3炎症小体激活缓解钴铬钼颗粒引起的骨溶解

张晨辉1，2，付婷婷2，吴阳林2，张  钦1，刘  昂1，杨惠林1，林  俊1，2   

  1. 1苏州大学附属第一医院骨科，江苏省苏州市  215006；2苏州独墅湖医院骨科，苏州大学附属独墅湖医院，苏州大学医学中心，江苏省苏州市 215001
  • 收稿日期:2023-02-11 接受日期:2023-04-20 出版日期:2024-04-08 发布日期:2023-08-17
  • 通讯作者: 林俊，博士，教授，硕士生导师，苏州大学附属第一医院骨科，江苏省苏州市 215006；苏州独墅湖医院骨科，苏州大学附属独墅湖医院，苏州大学医学中心，江苏省苏州市 215001 杨惠林，博士，教授，博士生导师，苏州大学附属第一医院骨科，江苏省苏州市 215006
  • 作者简介:张晨辉，男，1997年生，江苏省常州市人，汉族，在读硕士，主要从事巨噬细胞、炎症小体相关研究。 付婷婷，女，1996年生，安徽省亳州市人，汉族，在读硕士，主要从事自噬、炎症小体相关研究。
  • 基金资助:
    国家自然科学基金项目(81871789，82172387)，项目负责人：林俊

Melatonin alleviates CoCrMo particle-induced osteolysis by inhibiting NLRP3 inflammasome activation

Zhang Chenhui1, 2, Fu Tingting2, Wu Yanglin2, Zhang Qin1, Liu Ang1, Yang Huilin1, Lin Jun1, 2   

  1. 1Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China; 2Department of Orthopedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu Province, China
  • Received:2023-02-11 Accepted:2023-04-20 Online:2024-04-08 Published:2023-08-17
  • Contact: Lin Jun, PhD, Professor, Master’s supervisor, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China; Department of Orthopedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu Province, China Yang Huilin, PhD, Professor, Doctoral supervisor, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • About author:Zhang Chenhui, Master candidate, Department of Orthopedics, First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China; Department of Orthopedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu Province, China Fu Tingting, Master candidate, Department of Orthopedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 81871789, 82172387 (to LJ)

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摘要：


文题释义：

炎症小体：是一个大型的多聚蛋白复合物，它控制着Caspase-1的激活和促炎细胞因子白细胞介素1β的成熟，NLRP3炎症小体是最典型、功能最多的炎症小体之一。炎症小体在许多病理生理过程中发挥重要作用，有证据表明炎症小体的过度激活与骨溶解的发病息息相关。
骨溶解：作为终末期关节炎最有效的治疗手段，人工关节置换术有很多严重的并发症，而骨溶解是其中最常见的远期并发症之一。人工关节置换术后，假体周围的磨损颗粒诱发一系列炎症反应，导致骨溶解。由于临床上缺乏有效的治疗手段，骨溶解最终发展为无菌性松动，使得翻修手术成为唯一的治疗选择。


背景：假体周围骨溶解是人工关节置换术最常见的远期并发症，研究表明炎症小体可能在其发病过程中发挥重要作用。褪黑素是松果体分泌的节律调节激素，具有抗炎、抗氧化和抗肿瘤等多种功效，但其对骨溶解和炎症小体的影响还有待探索。

目的：探究褪黑素对磨损颗粒诱导的骨溶解与NLRP3炎症小体激活的作用。
方法：①体内实验：取15只C57BL/6小鼠，采用随机数字表法随机分为假手术组、骨溶解组、褪黑素组，每组5只。骨溶解组、褪黑素组通过向颅骨矢状缝内注射钴铬钼颗粒建立骨溶解模型，注射后，褪黑素组腹腔注射50 mg/(kg·d)的褪黑素，连续注射14 d。药物干预结束后取小鼠颅骨，进行Micro-CT分析，观察颅骨矢状缝周围微观结构变化。②体外实验：取小鼠骨髓源性巨噬细胞与THP-1细胞(已诱导分化为巨噬细胞)，均分为7组干预，分别为正常组、脂多糖组、脂多糖+钴铬钼组及褪黑素0.5，1，1.5，2 mmol/L组(褪黑素干预各组均加入脂多糖与钴铬钼)。药物干预6 h后，采用Western blot法检测细胞裂解液或细胞培养上清中炎症小体相关蛋白(NLRP3、Caspase-1、白细胞介素1β和焦孔素D、焦孔素D-N末端)的表达，通过乳酸脱氢酶释放与活死荧光染色观察细胞毒性和死亡情况。

结果与结论：①体内实验：micro-CT扫描3D重建图像显示，骨溶解组小鼠颅骨矢状缝周围骨量明显减少，骨组织结构被严重破坏；与骨溶解组相比，褪黑素组小鼠颅骨矢状缝周围骨量明显增加，组织结构破坏减少。②体外实验：对于小鼠骨髓源性巨噬细胞，脂多糖显著上调了细胞裂解液中NLRP3蛋白的表达，而褪黑素干预可呈剂量依赖性降低NLRP3蛋白的表达；钴铬钼颗粒显著上调了细胞裂解液中焦孔素D-N末端及细胞培养上清中Caspase-1、白细胞介素1β的蛋白表达，而褪黑素干预可呈剂量依赖性降低这些蛋白的表达。对于THP-1细胞，钴铬钼颗粒显著上调了细胞培养上清中Caspase-1、白细胞介素1β的蛋白表达，而褪黑素干预可呈剂量依赖性降低这些蛋白的表达。乳酸脱氢酶释放与活死荧光染色显示，钴铬钼颗粒显著增加了小鼠骨髓源性巨噬细胞培养上清中乳酸脱氢酶的释放及细胞的死亡，而褪黑素干预可减少乳酸脱氢酶的释放及细胞的死亡。③结果表明：褪黑素可以抑制磨损颗粒诱导的炎症小体激活和焦亡，抑制假体周围骨溶解。

https://orcid.org/0000-0002-3824-9253(张晨辉)

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料；口腔生物材料；纳米材料；缓释材料；材料相容性；组织工程

关键词: 钴铬钼颗粒, 骨溶解, 巨噬细胞, 炎症小体, 细胞焦亡, 褪黑素

Abstract: BACKGROUND: Periprosthetic osteolysis is the most common long-term complication of total joint arthroplasty. Many studies suggest that the inflammasome may play an important role during the osteolysis. Melatonin is a rhythm-regulated hormone secreted by the pineal gland with many functions including anti-inflammatory, anti-oxidation, and antitumor, but its effects on osteolysis and inflammasome have yet to be explored.
OBJECTIVE: To explore the effect of melatonin on the osteolysis induced by wear particles and the role of melatonin on the activation of NLRP3 inflammasome.
METHODS: (1) In vivo test: Fifteen C57BL/6 mice were randomly divided into sham operation group, osteolysis group and melatonin group by random number table method, with 5 mice in each group. The osteolysis model of the osteolysis group and the melatonin group was established by injecting cobalt-chromium-molybdenum (CoCrMo) particles into the sagittal suture of the skull. After injection, the melatonin group was intraperitoneally injected with 50 mg/(kg•d) of melatonin for 14 consecutive days. After drug intervention, the mouse calvarium was collected for micro-CT analysis to observe the micro-structural changes around the sagittal suture. (2) In vitro test: Mouse bone marrow-derived macrophages and THP-1 cells (which had been induced to differentiate into macrophages) were taken and divided into seven groups: normal group, lipopolysaccharide group, lipopolysaccharide+CoCrMo group and melatonin 0.5, 1, 1.5, 2 mmol/L groups (lipopolysaccharide and CoCrMo were added to the melatonin intervention groups). After the intervention for 6 hours, the expression of related proteins (NLRP3, Caspase-1, interleukin-1β, and gasdermin D, gasdermin D-N terminal) in the inflammasome of cell lysate or cell culture supernatant was detected by western blot assay. Cytotoxicity and cell death were observed through lactate dehydrogenase release and live-dead fluorescence staining. 
RESULTS AND CONCLUSION: (1) In vivo test: Micro-CT scanning 3D reconstruction images showed that the bone mass around the sagittal suture of the skull of mice in the osteolysis group was significantly reduced, and the bone tissue structure was severely damaged. Compared with the osteolysis group, the bone mass around the sagittal suture of the skull in the melatonin group was significantly increased, and the destruction of tissue structure was reduced. (2) In vitro test: For mouse bone marrow-derived macrophages, lipopolysaccharide significantly up-regulated NLRP3 protein expression in cell lysate, and melatonin intervention could reduce NLRP3 protein expression in a dose-dependent manner. CoCrMo particles significantly up-regulated the protein expressions of the gasdermin D-N terminal in cell lysate and Caspase-1 and interleukin-1β in the supernatant of cell culture, while melatonin intervention could reduce the expression of these proteins in a dose-dependent manner. For THP-1 cells, the protein expressions of Caspase-1 and interleukin-1β in the supernatant of cell culture were significantly up-regulated by CoCrMo particles, and the expression of these proteins was decreased dose-dependent by melatonin intervention. Lactate dehydrogenase release and live-dead fluorescence staining showed that CoCrMo particles significantly increased the release of lactate dehydrogenase and cell death in the supernatant of mouse bone marrow-derived macrophage culture, and melatonin intervention could reduce the release of lactate dehydrogenase and cell death. (3) The results show that melatonin can inhibit particle-induced inflammasome activation and pyroptosis to suppress periprosthetic osteolysis.

Key words: CoCrMo particle, osteolysis, macrophage, inflammasome, pyroptosis, melatonin

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