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  

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

作　　者：张晨辉 付婷婷[2] 吴阳林 张钦[1] 刘昂 杨惠林[1] 林俊[1,2] Zhang Chenhui;Fu Tingting;Wu Yanglin;Zhang Qin;Liu Ang;Yang Huilin;Lin Jun(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)

机构地区：[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

出　　处：《中国组织工程研究》2024年第10期1484-1489,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81871789,82172387);项目负责人:林俊。

摘　　要：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 antiinflammatory,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-chromiummolybdenum(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 su

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 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R684