基于中性粒细胞胞外诱捕网研究隔药灸减轻克罗恩病大鼠结肠炎症的效应机制

Effect mechanism investigation of herb-partitioned moxibustion on relieving colon inflammation in Crohn disease rats based on neutrophil extracellular traps

目的:从中性粒细胞胞外诱捕网(NETs)参与克罗恩病(CD)进展的角度,阐释隔药灸减轻CD大鼠肠道炎症的效应机制。方法:将大鼠随机分为正常组、模型组、隔药灸组和美沙拉嗪组。除正常组外,其余大鼠采用2,4,6-三硝基苯磺酸制备CD模型。正常组和模型组不治疗仅做与其他组相同的固定。隔药灸组予以隔药灸气海和双侧天枢穴。美沙拉嗪组接受美沙拉嗪肠溶片灌胃治疗。比较各组大鼠一般情况,苏木精-伊红染色观察结肠组织病理学变化并评分。采用ABC酶联免疫吸附测定法检测各组大鼠血清中性粒细胞胞外诱捕网DNA(NETs-DNA)、中性粒细胞弹性蛋白酶DNA(NE-DNA)、过氧化物酶DNA(MPO-DNA)浓度,采用免疫荧光和实时定量聚合酶链反应技术观察各组大鼠结肠组织瓜氨酸化组蛋白3(citH3)、MPO和NE蛋白和mRNA的表达。结果:与正常组比较,模型组结肠黏膜溃疡深达肌层,上皮不完整,杯状细胞明显减少,可见大量炎性细胞浸润,结肠黏膜损伤指数(CMDI)评分显著增加(P<0.01),模型组血清NETs-DNA、NE-DNA、MPO-DNA浓度均增加(P<0.05),模型组结肠组织NE、citH3和MPO蛋白和mRNA表达均上调(P<0.01或P<0.05);与模型组相比,隔药灸组和美沙拉嗪组结肠黏膜上皮修复,杯状细胞明显增多,可见少量炎性细胞浸润,CMDI评分明显降低(P<0.01),隔药灸组和美沙拉嗪组血清NETs-DNA、NE-DNA、MPO-DNA浓度均下降(P<0.05),隔药灸组和美沙拉嗪组NE、citH3和MPO蛋白和mRNA表达下调(P<0.01或P<0.05)。结论:隔药灸能降低血清中性粒细胞NETs复合物的浓度,抑制结肠组织中的NETs复合物的蛋白和mRNA表达,可能是隔药灸缓解CD结肠黏膜炎症的机制之一。

Objective:To explore the mechanism of herb-partitioned moxibustion in relieving rat intestinal inflammation by focusing on the neutrophil extracellular traps(NETs)in Crohn disease(CD)development.Methods:Rats were randomly divided into a normal group,a model group,a herb-partitioned moxibustion group,and a mesalazine group.The CD rat model was prepared with 2,4,6-trinitrobenzene sulfonic acid except for rats in the normal group.Rats in the normal group and model group did not receive any treatment but had the same fixation as the other groups.Rats in the herb-partitioned moxibustion group received herb-partitioned moxibustion at Qihai(CV6)and bilateral Tianshu(ST25).Rats in the mesalazine group received intragastric administration of mesalazine enteric-coated tablets.The general situation of rats in each group was recorded,and the histopathological changes in the colon were observed and scored by hematoxylin-eosin staining.The serum concentrations of NETs DNA(NETs-DNA),neutrophil elastase(NE)-DNA,and myeloperoxidase(MPO)-DNA were detected by ABC enzyme-linked immunosorbent assay,and the citrullinated histone 3(citH3),MPO,and NE protein and mRNA expression levels in rat colon tissue were observed by immunofluorescence and real-time quantitative polymerase chain reaction.Results:Compared with the normal group,the mucosal ulcer reached the muscularis,the epithelium was incomplete,the goblet cells decreased obviously with significant inflammatory cell infiltration in the colon;the colonic mucosa damage index(CMDI)score increased significantly(P<0.01);the serum NETs-DNA,NE-DNA,and MPO-DNA concentrations increased(P<0.05);the NE,citH3,and MPO protein and mRNA expression in the colonic tissue increased significantly in the model group(P<0.01 or P<0.05).Compared with the model group,the mucosal epithelium in the herb-partitioned moxibustion group and the mesalazine group was repaired and the goblet cells increased with a few infiltrating inflammatory cells in the colon;the CMDI score decreased(P<0.01);the serum NETs-DNA,NE-DNA,and MPO-DNA concentrations decreased(P<0.05);the NE,citH3,and MPO protein and mRNA expression in the colonic tissue was down-regulated(P<0.01 or P<0.05).Conclusion:Herb-partitioned moxibustion reduced the serum NETs complex and inhibited the protein and mRNA expression of NETs complex in the colon tissue,which may be one mechanism of herb-partitioned moxibustion in relieving colon mucosal inflammation in CD.