叉头框蛋白O对比格犬种植体周围炎症反应的抑制作用及机制研究

Inhibition effect of forkhead box O on peripheral inflammatory response of implant in Beagle dogs and its mechanism

目的研究叉头框蛋白O1(FOXO1)能否通过阻断丝裂原活化蛋白激酶(MAPK)信号通路抑制比格犬种植体周围的炎症反应。方法在局部用去骨+丝线结扎+高糖饮食联合法建立比格犬种植体周围炎骨缺损模型。按照体重将模型犬随机分为3组:模型组、转换模型组(感染pc DNA-3. 1空载体质粒)和质粒模型组(感染pc DNA-FOXO1质粒);另选正常犬作为正常组。分别将真核表达pc DNA-3. 1空载体和pc DNA-FOXO1载体(生物生工公司合成)瞬时转染至牙龈组织细胞。用酶联免疫吸附法检测龈沟液与细胞上清中的白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)和巨噬细胞炎症蛋白-1α(MIP-1α)的含量。用逆转录-聚合酶链反应法检测牙龈组织细胞中FOXO1及MAPK信号通路相关蛋白(p-ERK1/2、p-JNK和p-p38 MAPK) mRNA表达水平。结果正常组、模型组、转换模型组和质粒模型组的IL-8分别为(50±1),(151±18),(157±8)和(72±15) pg·m L-1;这4组的TNF-α分别为(80±20),(200±5),(181±38)和(115±12) pg·m L-1;这4组的MIP-1α分别为(35±5),(253±35),(269±55)和(83±9) pg·m L-1,模型组和转换模型组与正常组相比,上述炎症指标的差异均有统计学意义(P <0. 05,P <0. 01);质粒模型组与模型组比较或者质粒模型组与转换模型组比较,上述3种炎症因子的差异均有统计学意义(P <0. 05,P <0. 01)。这4组的p-ERK1/2 mRNA的表达水平分别为1. 18±0. 20,2. 38±0. 50,2. 11±0. 30和0. 84±0. 20;这4组的p-JNK mRNA的表达水平分别为:0. 86±0. 15,1. 75±0. 35,1. 96±0. 55和0. 83±0. 14;这4组的p-p38 MAPK mRNA的表达水平分别为0. 92±0. 10,1. 89±0. 35,2. 13±0. 60和0. 93±0. 30,模型组和转换模型组与正常组相比,上述3种基因表达水平的差异均有统计学意义(均P <0. 01);质粒模型组与模型组比较或者质粒模型组与转换模型组比较,上述3种基因的表达水平的差异均有统计学意义(P <0. 05,P <0. 01)。结论 FOXO1可能通过抑制MAPK通路的激活从而抑制比格犬种植体周围的炎症反应。

Objective To investigate whether forkhead box O1(FOXO 1)can inhibit the inflammatory response of around implant in Beagle dog by blocking the mitogen-activated protein kinase(MAPK)signaling pathway.Methods The combined method of local boning,silk thread ligation and high glucose diet was used to construct the peri-implantitis bone defect animal model.The model dogs were randomly divided into three groups according to their weight:model group,transfectionmodel group(infection of pcDNA-3.1 empty vector plasmid),transfection model+FOXO1 group(infection with pc DNA-FOXO1 plasmid);another normal dogs were selected as the normal group.Eukaryotic expression pcDNA-3.1 empty vector and pcDNA-FOXO1 vector(synthesized by Biotechnology Company)were transfected into gingival tissue cells instantaneously.The expression changes of interleukin-8(IL-8),tumor necrosis factor-α(TNF-α)and macrophage inflammatory protein-1α(MIP-1α)in gingival crevicular fluid and cells were determined by enzyme linked immunosorbent assay.The mRNA expression changes of FOXO1 and MAPK signal pathway related proteins[phosphorylated-extracellular regulated kinase 1/2(p-ERK1/2),phosphorylated-Jun N-terminal kinase(p-JNK),and phosphorylated-phosphorylated38 mitogen-activated protein kinase(p-p-P38 MAPK)]were detected by reverse transcription polymerase chain reaction.Results The levels of IL-8 in normal group,model group,transfection model group,transfection model+FOXO1 group were(50±1),(151±18),(157±8)and(72±15)pg·m L-1;the level of TNF-αin the 4 groups were(80±20),(200±5),(181±38)and(115±12)pg·m L-1;the level of MIP-1αin the 4 groups were(35±5),(253±35),(269±55)and(83±9)pg·m L-1;there were significant differences of the IL-8,TNF-αand MIP-1αbetween model group,transfection model group with normal group(P<0.05,P<0.01);there were significant differences of the factors between transfection model+FOXO1 group with model group or transfection model+FOXO1 group with transfection model group(P<0.05,P<0.01).The expression level of p-ERK1/2 mRNA in the 4 groups were 1.18±0.20,2.38±0.50,2.11±0.30,0.84±0.20,respectively;the expression level of p-JNK mRNA in the 4 groups were 0.86±0.15,1.75±0.35,1.96±0.55,and 0.83±0.14,respectively;the expression level of p-p38 MAPK mRNA in the 4 groups were 0.92±0.10,1.89±0.35,2.13±0.60,and0.93±0.30,respectively;there were significant differences of the p-ERK1/2,p-JNK,p-p38 MAPK between model group,transfection model group with normal group(P<0.05,P<0.01);there were significant differences of the factors between transfe ction model+FOXO1 group with model group or transfection model+FOXO1 group with transfection model group(P<0.05,P<0.01).Conclusion FOXO1 can inhibit inflammatory response in the peri-implantitis bone defect model in Beagles,which might be related to its inhibition on the activation of MAPK pathway.