消退素D1在甲状旁腺激素促进牵张成骨中的水平变化及其对巨噬细胞极化效应研究

Changes of Resolvin D1 and Its Polarization Effect on Macrophages in Mandibular Distraction Osteogenesis Promoted by Parathyroid Hormone Administration

目的:研究甲状旁腺激素(parathyroid hormone,PTH)促进牵张成骨过程中消退素D1(resolvin D1,RvD1)表达水平变化及其对巨噬细胞极化的影响。方法:48只兔建立下颌骨牵张成骨模型,随机分为对照组和实验组。对照组术后注射生理盐水,实验组间歇性注射PTH 20μg/kg。分别对各组兔牵张后第3、5、7天进行取样,通过苏木精-伊红(hematoxylin-eosin staining,HE)染色观察牵张区新骨组织;液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)检测新骨组织中RvD1含量;实时荧光定量PCR(quantitative real-time PCR,qPCR)检测新骨组织中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)及精氨酸酶1(Arginase 1,Arg1)的表达。体外构建由脂多糖(lipopolysaccharide,LPS)诱导的RAW264.7炎症模型,并添加100 ng/mL RvD1进行处理,qPCR和流式细胞术检测巨噬细胞极化标记物的表达。结果:PTH促进下颌骨牵张成骨兔牵张区新骨生成并明显提高新骨中RvD1含量(P<0.01)。同时,实验组牵张区新骨中巨噬细胞M2型极化标志物Arg1的表达升高,而iNOS的表达降低(P<0.01)。体外实验结果显示RvD1上调了LPS处理的RAW264.7细胞中M2型极化标志物Arg1、CD206和抑炎因子白细胞介素-10(interleukin-10,IL-10)的表达(P<0.05),并下调M1型标志物iNOS、CD86和促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达(P<0.01)。结论:PTH能提高兔下颌骨牵张新骨组织中RvD1含量,RvD1可进一步诱导巨噬细胞向M2型极化而发挥促进炎症消退和骨再生作用。

Objective:To study the changes of Resolvin D1(RvD1)and its effect on macrophage polarization during mandibular distraction osteogenesis promoted by parathyroid hormone(PTH).Methods:The mandibular distraction osteogenesis model was established in 48 rabbits and the rabbits were randomly divided into two groups.The experimental group was injected with PTH 20μg/kg intermittently after operation,while the control group was injected with normal saline.Samples were taken on the 3rd,5th,and 7th day of distraction,respectively,and the new bone tissue in the distraction area was observed by hematoxylin-eosin(HE)staining.LC-MS/MS was used to detect the content of RvD1 in the new bone tissue in the distraction zone.qPCR was used to detect the mRNA level of iNOS and Arg1 in new bone tissue.RAW264.7 cells were cultured and treated with LPS,RvD1,and LPS+RvD1,respectively.The expression level of macrophage polarization markers was detected by qPCR and flow cytometry.Results:HE staining and gross specimen observation showed that the new bone formation in the distraction zone of the experimental group was better than that of the control group.The content of RvD1 in the experimental group was significantly higher than that in the control group(P<0.01).qPCR results showed that PTH significantly increased the expression level of M2-type polarization marker Arg1 gene in macrophages,and decreased the expression level of M1-type polarization marker iNOS(P<0.01).In vitro,the expression level of Arg1 mRNA of M2-type polarization marker was up-regulated after RvD1 treatment,while the expression of iNOS mRNA of M1-type polarization marker was down-regulated(P<0.01).The results of flow cytometry showed that the LPS-induced increase in CD86 expression was significantly reduced and CD206 expression was significantly elevated after treatment of cells with RvD1.Conclusion:Intermittent injection of PTH can increase the content of RvD1 in rabbit mandibular distraction osteogenesis.RvD1 can polarize macrophages to M2,thus exerting anti-inflammatory effects.