人β-防御素-3通过调控肠上皮细胞自噬对新生大鼠坏死性小肠结肠炎发挥保护作用的实验研究  

Protective effects of hBD3 on necrotizing enterocolitis via inhibiting excessive autophagy in neonatal rats

作　　者：陈丽萍 吕志宝[1] 葛贵杰 付东 路丽 余胜华 王雪莉[2] 盛庆丰[1] Chen Liping;Lyu Zhibao;Ge Guijie;Fu Dong;Lu Li;Yu Shenghua;Wang Xueli;Sheng Qingfeng(Department of General Surgery,Children's Hospital,Shanghai Jiao Tong University,Shanghai 200062,China;Department of Pathology,Children's Hospital,Shanghai Jiao Tong University,Shanghai 200062,China)

机构地区：[1]上海交通大学附属儿童医院,上海市儿童医院普外科,200062 [2]上海交通大学附属儿童医院,上海市儿童医院病理科,200062

出　　处：《中华小儿外科杂志》2021年第5期445-453,共9页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(81601322、81871194)。

摘　　要：目的研究人β-防御素-3(human beta-defensin-3,hBD3)对肠上皮细胞自噬的调节作用及其介导的自噬调控对肠上皮细胞迁移的影响。方法应用雷帕霉素(Rapamycin)诱导肠上皮细胞IEC-6构建细胞自噬模型。根据实验设计分为对照(Control)组、雷帕霉素(Rapamycin)组和人β-防御素-3(hBD3)+雷帕霉素组。蛋白质印迹法检测各组肠上皮细胞自噬相关蛋白表达水平,mRFP-GFP-LC3自噬双标腺病毒感染肠上皮细胞动态监测自噬流。应用CXCR4抑制剂和siRNA进一步验证hBD3调控自噬的潜在机制。划痕实验检测肠上皮细胞迁移能力,通过ATG7 siRNA转染的肠上皮细胞进一步佐证hBD3是通过调控自噬从而影响肠上皮细胞迁移。采用随机数字表法,将新生大鼠随机分为对照组+生理盐水(Control+NS,n=11),坏死性小肠结肠炎造模组+生理盐水(NEC+NS,n=12)和坏死性小肠结肠炎造模组+hBD3(NEC+hBD3,n=8)。通过配方奶喂养+缺氧冷刺激诱导构建坏死性小肠结肠炎(necrotizing enterocolitis,NEC)模型鼠,收集肠管组织检测自噬相关基因表达。结果蛋白免疫印迹法检测自噬相关蛋白结果显示,雷帕霉素组中应用200 nmol/L雷帕霉素处理IEC-6细胞24 h后,自噬相关蛋白p62、Beclin1和LC3Ⅱ/Ⅰ相较于对照组的表达量分别为0.44±0.05、1.17±0.05和1.32±0.05,均较对照组差异有统计学意义(P<0.001,P=0.026和0.0014),成功构建了肠上皮细胞自噬模型。hBD3+雷帕霉素组IEC-6细胞中p62、Beclin1和LC3Ⅱ/Ⅰ相较于对照组的表达量分别为0.86±0.04、0.75±0.04和0.95±0.03,与雷帕霉素组比较,差异亦有统计学意义(P=0.009、0.0027和0.003),提示hBD3预处理可以显著抑制肠上皮细胞自噬水平。雷帕霉素组mRFP+GFP+斑点和mRFP+GFP-斑点计数分别为(38.33±2.02)个和(72.33±2.61)个,较对照组(3.67±0.88)个和(4.67±1.20)个明显增加,组间比较,差异有统计学意义(P均<0.0001);hBD3+雷帕霉素组mRFP+GFP+斑点和mRFP+GFP-斑点数分别Objective To explore the regulation of human beta-defensin-3(hBD3)on autophagy in intestinal epithelial cells and to examine the effect of hBD3-mediated autophagy regulation on intestinal epithelial cell migration.Methods IEC-6 cells were induced by rapamycin and the effect of hBD3 on autophagy-related proteins in intestinal epithelial cell was detected by Western blot.Three groups of control,rapamycin and hBD3 plus rapamycin were designated.Autophagic flux of intestinal epithelial cells was measured by mRFP-GFP-LC3 adenovirus.And AMD3100(CXCR4 inhibitor)and CXCR4 small interfering RNA were utilized for confirming that CXCR4 signaling pathway was essential for the above process.Wound healing assay was employed for elucidating the relationship between hBD3-mediated autophagy regulation and intestinal epithelial cell migration.It was verified by Atg-7-deficient enterocyte after transfecting ATG7 siRNA.Neonatal rats were randomly divided into 3 groups of control+NS(n=11),NEC+NS(n=12)and NEC+hBD3(n=8).The model of cell autophagy was induced by hypertonic formula feeding and hypoxia-cold stress exposure.Intestinal tissues were collected for detecting the expressions of autophagy-related genes.Results Western blot indicated that the relative expressions of p62,Beclin1 and LC3Ⅱ/Ⅰwere(0.44±0.05),(1.17±0.05)and(1.32±0.05)in rapamycin group after 24h incubating with 200 nmol/L.There were significant differences from those in control group(P<0.001,P=0.026,P=0.0014).As compared with control group,the relative expression levels of p62,Beclin1 and LC3Ⅱ/I were(0.86±0.04),(0.75±0.04)and(0.95±0.03)in hBD3 plus rapamycin group.There were statistically significant differences as compared with rapamycin group(P=0.009,0.0027&0.003).It hinted that hBD3 pretreatment markedly suppressed the expressions of autophagy-related proteins.The numbers of mRFP+GFP+/mRFP+GFP-spots were(38.33±2.02)and(72.33±2.61)in rapamycin group.Both were significantly higher than those in control group(3.67±0.88&4.67±1.20).And the inter-group di

关 键 词：小肠结肠炎 坏死性 防御素 受体 自噬 

分 类 号：R722.1[医药卫生—儿科]