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

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 higher in butyric acid group than in PBS group（68.60±15.06 vs. 37.25±5.81 and 424.93±19.34 vs. 127.31±60.83, respectively, P0.05）; Flow cytometry revealed that the proportion of regulatory T cells（Treg） of CD4＋ T cells was higher in butyric acid group than in PBS group（12.68%±6.79% vs. 3.57%±0.88%, P0.05）. Conclusions Butyric acid plays a protective effect in the intestinal injury of neonatal mouse model of necrotizing enterocolitis. The possible mechanism is that butyrate can down-regulate the expressions of cytokines IL-6 and TNF-α, up-regulate the expressions of cytokines IL-10 and TGF-β_1, and promote the differentiation of T cells into Treg cells.