丁酸对新生儿坏死性小肠结肠炎新生小鼠模型的保护作用  

Protective effect of sodium butyrate on the neonatal mouse model of necrotizing enterocolitis

作　　者：李秋平 余加林[1] 胡坤 贺雨[1] 肖洒[1] 侯婷 艾青[1] LI Qiu-ping, YU Jia-lin, HU Kun, HE Yu, XIAO Sa, HOU Ting, AI Qing(Department of Neonatology, Children＇s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, China International Science and Technology Cooperation Base for Child Development and Critical Disorders, Chongqing 400014, Chin)

机构地区：[1]重庆医科大学附属儿童医院新生儿科、儿童发育疾病研究教育部重点实验室、儿科学重庆市重点实验室、儿童发育重大疾病国家国际科技合作基地,重庆400014

出　　处：《解放军医学杂志》2018年第3期201-205,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81571483); 儿童医院临床研究项目[(2014)254-lcyj2014-11]

摘　　要：目的探讨丁酸在新生儿坏死性小肠结肠炎(NEC)新生小鼠中的作用及其可能机制。方法将60只3日龄C57BL/6新生小鼠按随机数字表法分为磷酸盐缓冲液(PBS)组(n=30)及丁酸组(n=30),分别予以PBS或丁酸溶液灌胃,1次/d,连续干预7d后,采用缺氧+冷刺激+人工喂养的方法连续3d刺激建立NEC模型,隔夜空腹处死新生鼠。HE染色观察回盲部肠组织病理变化并行双盲病理评分。采用实时荧光定量PCR检测白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、转化生长因子-β_1(TGF-β_1)及肿瘤坏死因子(TNF-α)基因表达水平;ELISA检测肠组织匀浆上清液IL-10、TGF-β_1蛋白水平;采用流式细胞技术分析两组肠道固有层调节性T细胞(Treg)占CD4+T细胞比例。结果取标本时,丁酸组小鼠体重(4.50±0.42g)明显高于PBS组(4.16±0.60g,P<0.05);丁酸组生存率(76.34%)与PBS组生存率(67.95%)差异无统计学意义(P>0.05)。肠组织病理损伤评分结果显示,丁酸组肠组织损伤评分[1.33(1.33~1.67)]明显低于PBS组[2.00(1.67~2.25),P<0.05]。肠组织qPCR结果显示,丁酸组IL-6、TNF-αm RNA表达与PBS组比较明显降低(分别为0.85±0.30 vs.1.77±0.49,P<0.05;0.41±0.25 vs.0.96±0.56,P<0.05);而丁酸组IL-10、TGF-β_1 m RNA表达与PBS组比较明显升高(分别为1.91±0.82 vs.0.94±0.43,P<0.05;1.46±0.57 vs.0.88±0.29,P<0.05)。肠组织ELISA结果显示,丁酸组IL-10、TGF-β_1蛋白表达水平与PBS组比较明显升高(分别为68.60±15.06 vs.37.25±5.81,P<0.05;424.93±19.34v s.1 2 7.3 1±6 0.8 3,P<0.0 5)。流式细胞术分析结果显示,丁酸组肠道固有层Tr e g占C D 4+T细胞比例较P B S组高(12.68%±6.79%vs.3.57%±0.88%,P<0.05)。结论丁酸在NEC新生小鼠模型肠道损伤中起保护作用。其可能机制是丁酸下调炎症因子IL-6、TNF-α表达,上调细胞因子IL-10、TGF-β_1的表达,促进T细胞向Treg细胞分化,从而发挥抑制炎症反应的作用。Objective To investigate the protective effect of sodium butyrate on the neonatal mouse model of necrotizing enterocolitis and analyze its possible mechanism. Methods Sixty c57 BL/6 neonatal mice were randomly divided into two groups（n=30）： PBS group and butyric acid group. At the third day after birth, mice in both groups were respectively given PBS and sodium butyrate solution by gavage once a day for 7 days, and neonatal necrotizing enterocolitis（NEC） model was established by hypoxia, cold stimulation and artificial feeding. The newborn mice were sacrificed overnight after modeling. HE staining and double-blind pathological score were performed to observe the pathological changes of ileocecal intestinal tissue. The m RNA expressions of IL-6, IL-10, TGF-β_1 and TNF-a were tested by quantitative real-time PCR. The levels of IL-10 and TGF-β_1 in intestine tissues were evaluated using ELISA. Flow cytometry was used to analyze the ratio of regulatory T cells（Treg） on CD4＋ T cells in both groups. Results W hen mice were sacrificed overnight after NEC modeling, the body weight was significantly higher in butyric acid group（4.50±0.42 g） than in PBS group（4.16±0.60 g, P0.05）; No significant difference（P0.05） existed in sur vival rate of butyric acid group（76.34%） and PBS group（67.95%）. The pathological damage score of intestinal tissue showed that the median score of intestinal injury was significantly lower in butyric acid group [1.33（1.33-1.67）] than in PBS group [2.00（1.67-2.25）, P0.05]. q PCR demonstrated that the expressions of IL-6 and TNF-α m RNA were obviously lower in butyric acid group than in PBS group（0.85±0.30 vs. 1.77±0.49 and 0.41±0.25 vs. 0.96±0.56, respectively, P0.05）; and the expressions of IL-10 and TGF-β_1 m RNA were markedly higher in butyric acid group than in PBS group（1.91±0.82 vs. 0.94±0.43 and 1.46±0.57 vs. 0.88±0.29, respectively, P0.05）; Intestinal tissue ELISA results showed that the expressions of IL-10 and TGF-β_1 were hi

关 键 词：丁酸 新生儿坏死性小肠结肠炎 白细胞介素-10 转化生长因子-β_1 调节性T细胞 

分 类 号：R722.1[医药卫生—儿科]