鼠李糖乳杆菌对斑马鱼脊髓损伤后肠道炎症的抑制作用及其机制

doi:10.13481/j.1671-587x.20200403

摘要/Abstract

摘要： 目的：探讨斑马鱼脊髓损伤（SCI）对肠道炎症发生发展的影响，阐明鼠李糖乳杆菌GG株（LGG）减轻肠道炎症的作用及机制。方法：14条正常斑马鱼随机分为正常饮食组（n=7）和LGG饮食组（LGG组，n=7），喂养21 d后取肠道组织，采用RT-qPCR法检测斑马鱼肠道组织中肠屏障相关分子β防御素样肽-1（DEFBL1）、紧密连接蛋白2a（TJP2a）、黏液素2.1（MUC2.1）和黏液素5.3（MUC5.3） mRNA表达水平。48条手术斑马鱼随机分为假手术+正常饮食组、假手术+LGG组、SCI+正常饮食组和SCI+LGG组，每组12条，术后喂养25 d（每组n=6）取肠道组织，采用RT-qPCR法检测肠道炎症相关分子肿瘤坏死因子（TNF-α）、Toll样受体2（TLR-2）和白细胞介素6（IL-6） mRNA表达水平，术后分别喂养7 d（每组n=3）和28d（每组n=3）取斑马鱼肠道组织，用肠道菌群高通量16S rRNA测序检测肠道菌群变化。结果：与正常饮食组比较，LGG组正常斑马鱼肠道组织中DEFBL1和TJP2a mRNA表达水平明显升高（t=-2.387，P<0.05；t=-12.482，P<0.01），MUC2.1和MUC5.3 mRNA表达水平明显降低（t=2.497，P<0.05；t=3.173，P<0.05）。与假手术+正常饮食组比较，SCI+正常饮食组斑马鱼肠道组织中TNF-α、TLR-2和IL-6 mRNA表达水平明显升高（P<0.05或P<0.01）；与SCI+正常饮食组比较，SCI+LGG组斑马鱼肠道组织中TNF-α、TLR-2和IL-6mRNA表达水平明显降低（P<0.05或P<0.01）。与正常饮食组（假手术+正常饮食组和SCI+正常饮食组）比较，假手术+LGG组和SCI+LGG组斑马鱼肠道内致病菌属（气单胞菌属、弧菌属）和条件致病菌属（脱硫弧菌属、短波单胞菌属）的丰度明显降低。结论：LGG能抑制斑马鱼SCI引起的肠道炎症，其机制可能与肠屏障相关分子表达水平增加和肠道致病菌丰度降低有关。

关键词: 鼠李糖乳杆菌, 脊髓损伤, 斑马鱼, 肠道炎症, 肠道菌群

Abstract: Objective: To study the influence of spinal cord injury (SCI) in intestinal inflammation in the zebrafishes, and to clarify the effect of alleviating the intestinal inflammation of Lactobacillus rhamnosus GG (LGG) and its mechanism. Methods: Fourteen normal zebrafishes were randomly divided into normal diet group (n=7) and LGG group (n=7).After 21 d of feeding, intestinal tissues were taken and the expression levels of intestinal barrier-related molecules defensin beta-like 1 (DEFBL1), tight junction protein 2a (TJP2a), mucin 2.1 (MUC2.1) and mucin 5.3 (MUC5.3) in intestinal tissue of the zebrafishes were detected by RT-qPCR method. Forty eight operated zebrafishes were randomly divided into sham operation + normal diet group (n=12), sham operation + LGG group (n=12), SCI + normal diet group (n=12) and SCI + LGG group (n=12).After 25 d of feeding after injury (n=6 in each group), intestinal tissues were taken and the expression levels of intestinal inflammatory cytokines tumor necrosis factor-α (TNF-α), Toll-like receptor 2 (TLR-2) and interleukin-6 (IL-6) were detected by RT-qPCR method. After 7 d (n=3 in each group) and 28 d (n=3 in each group) of feeding after injury, the intestinal tissues were taken for high-throughput 16S rRNA sequencing of intestinal flora to detect the changes in intestinal flora. Results: Compared with normal diet group,the expression levels of DEFBL1 and TJP2a in intestinal tissue of the zebrafishes in LGG group were significantly increased(t=-2.387, P<0.05;t=-12.482, P<0.001), but the expression levels of MUC2.1 and MUC5.3 were significantly decreased (t=2.497, P<0.05;t=3.173, P<0.05). Compared with sham operation +normal diet group, the expression levels of TNF-α, TLR-2 and IL-6 mRNA in the intestinal tissue of the zebrafishes in SCI + normal diet group were increased significantly(P<0.05 or P<0.01);compared with SCI + normal diet group, the expression levels of TNF-α, TLR-2, and IL-6 in intestinal tissue of the zebrafishes in SCI + LGG group were decreased(P<0.05 or P<0.01).Compared with normal diet groups (sham + normal diet group and SCI + normal diet group), the abundance of pathogenic bacteria (Aeromonas and Vibrio) and conditioned pathogenic bacteria (Desulphomonas and Brevundimonas) in intestinal tissue of the zebrafishes in sham + LGG group and SCI + LGG group were significantly decreased. Conclusion: LGG can inhibit the intestinal inflammation caused by SCI in the zebrafishes, and its mechanism may be related to increasing the expression levels of intestinal barrier-related molecules and decreasing the abundance of intestinal pathogenic bacteria.

Key words: Lactobacillus rhamnosus, spinal cord injury, zebrafish, intestinal inflammation, intestinal bacteria

中图分类号:

Q189

赵丽萍, 黄术兵, 张博枰, 周芝兰, 贾雪冰, 孙孟菲, 乔晨萌, 陈雪, 申延琴, 崔春. 鼠李糖乳杆菌对斑马鱼脊髓损伤后肠道炎症的抑制作用及其机制[J]. 吉林大学学报(医学版), 2020, 46(04): 680-686.

ZHAO Liping, HUANG Shubing, ZHANG Boping, ZHOU Zhilan, JIA Xuebing, SUN Mengfei, QIAO Chenmeng, CHEN Xue, SHEN Yanqin, CUI Chun. Inhibitory effect of Lactobacillus rhamnosus on intestinal inflammation after spinal cord injury in zebrafishes and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 680-686.

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