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

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。

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 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-1βin 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-1βin 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.