叶酸缺乏对葡聚糖硫酸钠诱导的小鼠实验性结肠炎的影响

Effect of folate deficiency on experimental colitis in mice induced by dextran sodium sulfate

目的观察在葡聚糖硫酸钠（DSS）诱导的小鼠实验性结肠炎模型中，叶酸缺乏是否导致干扰素γ基因（IFNG）低甲基化，使IFN-γ高表达而促进UC的发生、发展。方法将24只雌性BALB／c小鼠分为4组，即叶酸缺乏并DSS造模（D／DSS^＋）组，标准饮食并DSS造模（C／DSS^＋）组，标准饮食未造模（C／DSS^-）组，叶酸缺乏未造模（D／DSS^-）组，每组6只。自饲养第6周起，造模组小鼠给予5％DSS溶液建立实验性结肠炎模型；于第6周末实验结束，计算结肠炎疾病活动指数（DAI）和进行组织学评价，应用ELISA检测小鼠外周静脉血清叶酸水平；免疫组织化学SP法检测小鼠结肠黏膜组织IFN-γ表达情况；甲基化特异性聚合酶链反应（MSP）检测IFNG启动子区CpG岛甲基化水平。计量资料比较行t检验。计数资料组间比较采用卡方检验，相关性采用Spearman相关分析。结果D／DSS^＋组和D／DSS^-组小鼠的外周静脉血叶酸水平[（2．70±0．19）、（2．80±0．25）μg／L]分别较C／DSS^＋组和C／DSS^-明显降低[（13．62±0．38）、（13．52±0．77）μg／L，t=-63．33、32．27，P均〈0．01）。D／DSS^＋组和C／DSS^＋组结肠黏膜组织IFN-γ表达水平明显高于D／DSS^-组和C／DSS^-组（χ^2=22．18，P〈0．05），且D／DSS^＋组较C／DSS^＋组也明显增高（χ^2=12．00，P〈0．05）。D／DSS^＋组和C／DSS^＋组的IFN-γ表达水平与DAI呈正相关（r=0．998、0．953，P均〈0．01）。D／DSS^＋组血清叶酸水平分别与IFN-γ表达水平、DAI呈负相关（r=-0．880、-0．926，P均〈0．05）；各组小鼠结肠黏膜组织中未检测到IFNG启动子区CpG岛甲基化异常。结论在DSS诱导的小鼠实验性结肠炎模型中，叶酸缺乏可导致炎性因子IFN-γ表达增高，加重结肠黏膜炎性活动度。

Objective To investigate whether folate deficiency cause high expression level of interferon gamma （IFN-γ） resulted from IFN-γ gene （IFNG） hypomethylation and then promote the pathogenesis and development of ulcerative colitis （UC） in a dextran sulfate sodium （DSS） induced experimental colitis model in mice. Methods A total of 24 female BALB/c mice were divided into four groups, six mice in each group, including folate deficient/DSS^＋ group, standard diet/DSS^＋ group, standard diet/DSS^- group and folate deficient/DSS^- group. At the beginning of the sixth week since fed, the mice of model groups were treated with 5% DSS to establish experimental colitis. By the end of the sixth week, disease activity index （DAI） of colitis and histological changes were evaluated. The folate level of peripheral blood serum of mice were detected by enzyme-linked immunosorbent assay （ELISA）. The expression of IFN-γ in colonic mueosa of mice was examined by immunohistochemistry. The methylation level of CpG island in the promoter region of IFNG was determined by methylation specific polymerase chain reaction （MSP）. The t test was used for measurement data. Chi square test was performed for comparison between groups of count data. Spearman correlation analysis was used for correlation analysis. Results The folate levels of peripheral blood serum of folate deficiency/DSS^＋ group and folate deficiency/DSS group （（2.70±0.19） and （2.80±0.25） tag/L） were significantly lower than those of standard diet/DSS^＋ group and standard diet/DSS^- group （（13. 62± 0. 38） and （13. 52±0. 77） μg/L, t= -63. 33.32. 27, both P〈 0. 05）, resepectively. The expression of IFN-γ in colonic mucosa of folate deficiency/DSS^＋ group and standard diet/DSS^＋ group were significantly higher than those of folate defieiency/DSS^- group and standard diet/DSS group （χ^2=22. 18, P 〈 0. 05）. And the expression of IFN-γ in colonic mucosa of folate deficiency/DSS^＋ group was also higher than that of standard diet/DSS^＋ group （χ^2= = 12. 00, P〈0. 05）. The expression level of IFN-γ of folate deficiency/ DSS^＋ group and standard diet/DSS^＋ group was positively correlated with （r=0. 998.0. 953, both P〈 0.01）. The folate levels of peripheral blood serum of folate deficiency/DSS^＋ group was negatively correlated with IFN-γ expression level and DAI （r=-0. 880 and -0. 926, both P〈0.05）. No abnormal methylation was detected in IFNG promoter CpG island in colonic mucosa tissues of mice of each group. Conclusion In the mice model of DSS induced acute experimental colitis, folate deficiency may increaee the expression of inflammatory factor IFN-γ and enhance the inflammation activity of colonic mucosa.