微RNA-126对牙龈卟啉单胞菌脂多糖刺激下人巨噬细胞极化的调节作用  

Regulation of microRNA-126 on the polarization of human macrophages stimulated by Porphyromonas gingivalis lipopolysaccharide

作　　者：黎家君 刘玥 宋立婷 李长义[1] 蒋少云 Li Jiajun;Liu Yue;Song Liting;Li Changyi;Jiang Shaoyun(Department of Prosthodontics,Stomatological Hospital,Tianjin Medical University,Tianjin 300070,China;Stomatological Center,Peking University Shenzhen Hospital&Guangdong Provincial High-Level Clinical Key Specialty&Guangdong Province Engineering Research Center of Oral Disease Diagnosis and Treatment,Shenzhen 518036,China;Department of General Dentistry,Stomatological Hospital,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学口腔医院修复科,天津300070 [2]北京大学深圳医院口腔医学中心,广东省高水平临床重点专科,广东省口腔疾病诊疗技术工程技术研究中心,深圳518036 [3]天津医科大学口腔医院综合科,天津300070

出　　处：《中华口腔医学杂志》2022年第4期390-396,共7页Chinese Journal of Stomatology

基　　金：天津市自然科学基金(17JCYBJC26300);北京大学深圳医院科研项目(JCYJ2019004RC)。

摘　　要：目的研究微RNA-126(microRNA-126,miR-126)对牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)脂多糖(lipopolysaccharide,LPS)刺激下巨噬细胞极化的影响。方法用5 mg/L Pg-LPS刺激人单核细胞源性巨噬细胞48 h,以及转染miR-126模拟物或阴性对照物24 h后再行5 mg/L Pg-LPS刺激人单核细胞源性巨噬细胞48 h,实时定量PCR、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)、蛋白质印迹法分别检测miR-126、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-10(interleukin-10,IL-10)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、精氨酸酶-1(arginase-1,Arg-1)以及M1型极化相关通路:核因子-κB(nuclear factor-κB,NF-κB)和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的变化。结果Pg-LPS刺激巨噬细胞48 h后,与非Pg-LPS刺激组各因子mRNA水平(TNF-α:1.000±0.020,iNOS:1.125±0.064,miR-126:1.004±0.113,IL-10:1.003±0.053,Arg-1:1.130±0.061)相比,Pg-LPS刺激后TNF-α和iNOS的mRNA水平(3.105±0.278、4.296±0.003)均显著上升(t=6.53,P=0.003;t=42.63,P<0.001),miR-126、IL-10、Arg-1表达(0.451±0.038、0.545±0.004、0.253±0.017)均显著下降(t=7.95,P=0.001;t=7.36,P=0.002;t=11.94,P<0.001)。iNOS、TNF-α、Arg-1、IL-10蛋白表达量变化与mRNA变化趋势一致。同时,磷酸化NF-κB p65(phospho-NF-κB p65,p-p65)、磷酸化胞外信号调节激酶(phospho-extracellular signal-regulated kinase,p-ERK)、磷酸化p-38 MAPK(phospho-p38 MAPK,p-p38)相对蛋白表达均显著上升(t=2.78,P=0.013;t=18.70,P<0.001;t=5.65,P=0.005)。转染miR-126模拟物后,Pg-LPS刺激下与转染阴性对照物组各因子mRNA水平(TNF-α:1.141±0.197,iNOS:1.173±0.115,IL-10:1.032±0.138,Arg-1:0.933±0.044)相比,TNF-α、iNOS的mRNA水平(0.342±0.022、0.588±0.085)均显著下降(t=5.35,P=0.006;t=5.05,P=0.007),而IL-10、Arg-1的mRNA水平(1.786±0.221、2.152±0.229)均显著上升(t=3.71,P=0.021;t=6.21,P=0.003)。同时,iNOS、p-p65、p-ERK、p-p38相对�Objective To study the effect of microRNA-126(miR-126)on the polarization of human monocyte-derived macrophages stimulated by Porphyromonas gingivalis(Pg)lipopolysaccharide(LPS).Methods Macrophages derived from human myeloid leukemia mononuclear cells were stimulated by Pg-LPS(5 mg/L)and by Pg-LPS(5 mg/L)after 24 h-transfection of miR-126 mimic or negative control RNA for 48 h,respectively.Real-time quantitative-PCR(qRT-PCR),enzyme-linked immunosorbent assay(ELISA)and Western blotting were conducted to detect the changes in miR-126,pro-inflammatory factor tumor necrosis factor-α(TNF-α),anti-inflammatory factors interleukin-10(IL-10),inducible nitric oxide synthase(iNOS),arginase-1(Arg-1)and M1 polarization-related pathways such as nuclear factor kappa-B(NF-κB)and mitogen-activated protein kinase(MAPK)signaling pathways.Results Compared with non-LPS stimulation group(TNF-α:1.000±0.020,iNOS:1.125±0.064,miR-126:1.004±0.113,IL-10:1.003±0.053,Arg-1:1.130±0.061),the mRNA levels of TNF-α(3.105±0.278)and iNOS(4.296±0.003)increased significantly(t=6.53,P=0.003;t=42.63,P<0.001,respectively),while miR-126,IL-10 and Arg-1 expressions(0.451±0.038,0.545±0.004 and 0.253±0.017)decreased significantly(t=7.95,P=0.001;t=7.36,P=0.002;t=11.94,P<0.001,respectively)after Pg-LPS stimulated by human-derived macrophages for 48 h.The protein expression of iNOS,TNF-α,Arg-1 and IL-10 were consistent at mRNA levels.Meanwhile,the expressions of phospho-NF-κB p65(p-p65),phospho-extracellular signal-regulated kinase(p-ERK)and phospho-p38 MAPK(p-p38)increased significantly,while the expression of Arg-1 decreased significantly.Compared with the negative controls(scramble RNA)(TNF-α:1.141±0.197,iNOS:1.173±0.115,IL-10:1.032±0.138,Arg-1:0.933±0.044),the mRNA levels of TNF-α(0.342±0.022)and iNOS(0.588±0.085)expressions significantly decreased(t=5.35,P=0.006;t=5.05,P=0.007),while IL-10(1.786±0.221)and Arg-1 expressions(2.152±0.229)significantly increased(t=3.71,P=0.021;t=6.21,P=0.003)after Pg-LPS stimulation with miR-126 mimi

关 键 词：紫单胞菌 龈 微RNAs 微RNA-126 巨噬细胞 脂多糖类 细胞极化 

分 类 号：R781.42[医药卫生—口腔医学]