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  • Title: [Effects of vitamin D3 on intestinal mucosal barrier of mice with severe burns].
    Author: Liu XZ, You B, Zhang YL, Yang ZC, Chen P, Shi YL, Chen Y, Chen YJ, Chen J, Peng YZ.
    Journal: Zhonghua Shao Shang Za Zhi; 2019 Apr 20; 35(4):284-291. PubMed ID: 31060176.
    Abstract:
    Objective: To explore the effects of vitamin D3 on intestinal mucosal barrier of mice with severe burns. Methods: Forty-two C57BL/6C male mice aged eight to twelve weeks were divided into vitamin D3 vehicle+ sham injury group of seven mice, vitamin D3 vehicle+ burn injury group of fourteen mice, vitamin D3+ sham injury group of seven mice, and vitamin D3+ burn injury group of fourteen mice according to random number table. Mice in vitamin D3 vehicle+ sham injury group and vitamin D3 vehicle+ burn injury group were injected with vehicle of vitamin D3 at a dose of 0.1 mL intraperitoneally at 1, 24, and 48 h before burn experiment. Mice in vitamin D3+ sham injury group and vitamin D3+ burn injury group were injected with vitamin D3 at a dose of 100 ng/kg dissolved in 0.1 mL vehicle intraperitoneally at the same time points. Mice in vitamin D3 vehicle+ burn injury group and vitamin D3+ burn injury group were inflicted with 30% total body surface area full-thickness dermal scald (hereinafter referred to as burn) on the back by 98 ℃ hot water for 3 to 4 seconds. And mice in vitamin D3 vehicle+ sham injury group and vitamin D3+ sham injury were treated with 37 ℃ water on the back for 3 to 4 seconds to simulate injury. Seven mice in vitamin D3 vehicle+ sham injury group and seven mice in vitamin D3+ sham injury group at post injury hour (PIH) 24, and seven mice in vitamin D3 vehicle+ burn injury group and seven mice in vitamin D3+ burn injury group at PIH 6 and 24 were sacrificed respectively to collect mesentery lymph nodes, spleens, livers, and intestinal tissue. The mesentery lymph nodes, spleens, and livers of mice in each group were collected to observe growth of bacteria, and number of bacteria was counted. Intestinal tissue of mice in each group was collected to detect protein expressions of zonal occludin 1 (ZO-1) and occludin by immunohistochemistry staining method, distribution of ZO-1 by immunofluorescence staining method, and expression of occludin by Western blotting. Data were processed with Kruskal-Wallis H test, Nemenyi test, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) At PIH 6 and 24, bacterial counts of mesentery lymph nodes, livers, and spleens of mice in vitamin D3 vehicle+ burn injury group were significantly higher than those of mice in vitamin D3 vehicle+ sham injury group (P<0.05). At PIH 6, bacterial counts of livers and spleens of mice in vitamin D3+ burn injury group were significantly lower than those of mice in vitamin D3 vehicle+ burn injury group (P<0.05). At PIH 24, bacterial counts of mesentery lymph nodes and livers of mice in vitamin D3+ burn injury group were significantly lower than those of mice in vitamin D3 vehicle+ burn injury group (P<0.05). (2) At PIH 6 and 24, expressions of ZO-1 and occludin of intestinal tissue of mice in vitamin D3 vehicle+ burn injury group were significantly lower than those of mice in vitamin D3 vehicle+ sham injury group, and expressions of ZO-1 and occludin of intestinal tissue of mice in vitamin D3+ burn injury group were close to those of mice in vitamin D3+ sham injury group. At PIH 6 and 24, expressions of ZO-1 and occludin of intestinal tissue of mice in vitamin D3+ burn injury group were significantly higher than those of mice in vitamin D3 vehicle+ burn injury group. (3) At PIH 6 and 24, compared with that of mice in vitamin D3 vehicle+ sham injury group, distribution of ZO-1 of intestinal mucosal epithelium of mice in vitamin D3 vehicle+ burn injury group was discontinuous. Distribution of ZO-1 of intestinal mucosal epithelium of mice in vitamin D3+ sham injury group was normal, and the distribution of ZO-1 of intestinal mucosal epithelium of mice in vitamin D3+ burn injury group was with good continuity. (4) At PIH 6 and 24, expressions of occludin of intestinal tissue of mice in vitamin D3 vehicle+ burn injury group were 0.720±0.003, 0.638±0.052 respectively, significantly lower than 0.918±0.003 of mice in vitamin D3 vehicle+ sham injury group (t=57.33, 5.36, P<0.05). At PIH 6 and 24, expressions of occludin of intestinal tissue of mice in vitamin D3+ burn injury group were 0.994±0.058, 1.064±0.060, close to 0.938±0.023 of mice in vitamin D3+ sham injury group (t=0.91, 1.96, P>0.05). At PIH 6 and 24, expressions of occludin of intestinal tissue of mice in vitamin D3 vehicle+ burn injury group were significantly lower than those of mice in vitamin D3+ burn injury group (t=4.75, 5.35, P<0.05). Conclusions: Intestinal bacterial translocation can occur in the early stage of severe burn. And vitamin D3 plays a protective role in the intestinal mucosal barrier post severe burn to reduce the bacterial translocation by protecting tight junction proteins of intestinal epithelium. 目的：探讨维生素D3对严重烧伤小鼠肠黏膜屏障的作用。 方法：将42只C57BL/6C雄性8～12周龄小鼠按随机数字表法分为维生素D3溶剂＋假伤组7只、维生素D3溶剂＋烧伤组14只、维生素D3＋假伤组7只、维生素D3＋烧伤组14只。维生素D3溶剂＋假伤组及维生素D3溶剂＋烧伤组小鼠实验前1、24、48 h腹腔注射0.1 mL维生素D3溶剂，维生素D3＋假伤组和维生素D3＋烧伤组小鼠于相同时间点腹腔注射将按100 ng/kg计算的维生素D3溶于0.1 mL维生素D3溶剂中配制的维生素D3溶液。维生素D3溶剂＋烧伤组及维生素D3＋烧伤组小鼠背部浸于98 ℃热水中3～4 s致30%体表总面积Ⅲ度烫伤(以下称烧伤)。维生素D3溶剂＋假伤组及维生素D3＋假伤组小鼠背部浸于37 ℃温水中3～4 s致假伤。伤后24 h，取7只维生素D3溶剂＋假伤组、7只维生素D3＋假伤组小鼠断颈处死；伤后6、24 h，分别取7只维生素D3溶剂＋烧伤组、7只维生素D3＋烧伤组小鼠断颈处死，取肝脏、脾脏、肠系膜淋巴结及肠组织。取各组小鼠肝脏、脾脏、肠系膜淋巴结，观察细菌生长情况，并行细菌计数。取各组小鼠肠组织，采用免疫组织化学法检测带状闭合蛋白1(ZO－1)及咬合蛋白的表达，免疫荧光法检测ZO－1分布，蛋白质印迹法检测咬合蛋白的表达。对数据行Kruskal－Wallis H检验、Nemenyi检验、单因素方差分析、t检验及Bonferroni校正。 结果：(1)伤后6、24 h，维生素D3溶剂＋烧伤组小鼠肠系膜淋巴结、肝脏、脾脏细菌载量显著高于维生素D3溶剂＋假伤组(P<0.05)。伤后6 h，维生素D3＋烧伤组小鼠肝脏、脾脏的细菌载量明显低于维生素D3溶剂＋烧伤组(P<0.05)。伤后24 h，维生素D3＋烧伤组小鼠肠系膜淋巴结、肝脏细菌载量明显低于维生素D3溶剂＋烧伤组(P<0.05)。(2)伤后6、24 h，维生素D3溶剂＋烧伤组小鼠肠组织ZO－1及咬合蛋白表达明显低于维生素D3溶剂＋假伤组，维生素D3＋烧伤组小鼠肠组织ZO－1及咬合蛋白表达与维生素D3＋假伤组相近。伤后6、24 h，维生素D3＋烧伤组小鼠肠组织ZO－1及咬合蛋白表达明显高于维生素D3溶剂＋烧伤组。(3)伤后6、24 h，与维生素D3溶剂＋假伤组相比，维生素D3溶剂＋烧伤组小鼠肠黏膜上皮ZO－1分布不连续；维生素D3＋假伤组小鼠肠黏膜上皮ZO－1分布正常，维生素D3＋烧伤组小鼠肠黏膜上皮ZO－1分布连续性良好。(4)维生素D3溶剂＋烧伤组小鼠伤后6、24 h肠组织咬合蛋白表达分别为0.720±0.003、0.638±0.052，显著低于维生素D3溶剂＋假伤组的0.918±0.003(t＝57.33、5.36，P<0.05)；维生素D3 ＋烧伤组小鼠伤后6、24 h肠组织咬合蛋白表达为0.994±0.058、1.064±0.060，与维生素D3 ＋假伤组的0.938±0.023相近(t＝0.91、1.96，P>0.05)。维生素D3溶剂＋烧伤组小鼠伤后6、24 h肠组织咬合蛋白表达明显低于维生素D3 ＋烧伤组(t＝4.75、5.35，P<0.05)。 结论：严重烧伤早期小鼠即可发生肠道细菌移位，而维生素D3可通过保护肠上皮紧密连接蛋白，从而起到保护肠黏膜屏障，减少肠道细菌移位的作用。.
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