猪源乳杆菌Lactobacillus reuteri strain DSM和Lactobacillus taiwanensis strain BCRC对肠道炎症的影响

Effects of Lactobacillus reuteri strain DSM and Lactobacillus taiwanensis strain BCRC on intestinal inflammation

摘要:

[目的] 旨在以葡聚糖硫酸钠（DSS）诱导小鼠肠炎模型研究猪源乳杆菌Lactobacillus reuteri strain DSM（LR）和Lactobacillus taiwanensis strain BCRC（LT）对肠道炎症的影响。[方法] 选取24只8周龄体重相近的C57BL/6J雄性小鼠，随机分为4组（每组6个重复）：正常对照组、DSS组、LR菌处理组（DSS+LR）和LT菌处理组（DSS+LT）。适应期7 d，试验期16 d，随后处死小鼠并采样。[结果] 与正常对照组相比，DSS组小鼠体重、结肠长度显著降低，疾病活动指数（DAI）显著增加（P<0.05）；与DSS组相比，LR、LT菌处理组小鼠体重下降的程度并没有显著缓解，但是，其DAI评分显著降低；并且，LT菌处理组结肠长度显著增加（P<0.05）。正常对照组和LT菌处理组结肠组织病理学评分显著低于DSS组和LR菌处理组（P<0.05）。结肠髓过氧化物酶（MPO）活性在正常对照组和LT菌处理组也显著低于DSS组（P<0.05），在LR菌处理组与DSS组无显著差异（P>0.05）。与正常对照组相比，DSS组结肠和血浆的促炎因子TNF-α、IL-1β、IL-6水平显著升高，而抗炎因子IL-10水平显著降低；相比于DSS组，LT菌处理组显著降低了结肠和血浆的TNF-α、IL-1β、IL-6水平，升高了IL-10水平；LR菌处理组显著降低了结肠TNF-α水平、血浆IL-1β水平，升高IL-10水平（P<0.05）。免疫组化和ELISA结果显示，相比于正常对照组，DSS组降低了结肠紧密连接蛋白Claudin-1、Occludin、ZO-1和黏蛋白Muc-2表达水平，而LR或LT菌的处理则上调了DSS影响下的屏障相关蛋白表达。16S rDNA测序技术检测结肠菌群组成，PCoA分析得出，正常对照组和DSS组显著分离，LT处理组菌群组成更接近于正常对照组。门水平上，变形菌门丰度在正常对照组显著低于DSS组（P<0.05）；与DSS组相比，LT菌处理组也能降低变形菌门丰度，但未达显著水平（P>0.05）。属水平上，Muribaculaceae_norank属在正常对照组显著高于其他3组，Bacteroides在正常对照组显著低于其他3组（P<0.05）；与DSS组相比，LR或LT菌的处理增加了Muribaculaceae_norank丰度，降低了Bacteroides菌丰度，但差异不显著（P>0.05）。[结论] 乳杆菌Lactobacillus reuteri和Lactobacillus taiwanensis能增强肠上皮屏障，对DSS引起的肠道炎症起到了一定的缓解作用；其中，Lactobacillus taiwanensis对肠上皮屏障的保护效果更为明显。

Abstract:

[Objective] This study aims to investigate the effects of Lactobacillus reuteri strain DSM (LR) and Lactobacillus taiwanensis strain BCRC (LT) on intestinal inflammation using a mouse model of dextran sodium sulfate (DSS) induced colitis. [Methods] Twenty-four 8-week-old C57BL/6J male mice with similar body weight were selected and randomly divided into 4 groups (6 replicates in each group):normal group, DSS group, LR bacteria treatment group and LT bacteria treatment group. After 7 days adaptation period and 16 days experiment period, the mice were sacrificed and sampled. [Results] Compared with the normal group, the body weight and colon length of the DSS group were significantly reduced, and the disease activity index (DAI) scores as significantly increased (P<0.05). There was no significant difference in body weight between the LR and LT bacteria treatment groups and the DSS group (P>0.05), whereas the LR and LT bacteria treatment significantly reduced the DAI scores and the LT bacteria treatment treatment increased the colon length. The colonic histopathological scores of the normal group and the LT bacteria treatment group were significantly lower than those of the DSS and LR bacteria treatment groups (P<0.05). Colonic myeloperoxidase (MPO) activity was also significantly lower in the normal group and the LT bacteria treatment group than the DSS group, and there was no significant difference between the LR bacteria treatment group and the DSS group (P>0.05). Compared with the normal group, the levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colon and plasma of the DSS group were significantly increased, while the levels of the anti-inflammatory factor IL-10 were significantly decreased. Compared with the DSS group, LT bacteria treatment group significantly reduced the levels of TNF-α, IL-1β, and IL-6 in the colon and plasma, and increased the level of IL-10; the LR bacteria treatment group significantly reduced the levels of colonic TNF-α and plasma IL-1β, and increase the level of IL-10 (P<0.05). The results of immunohistochemistry and ELISA showed that compared with the normal group, the DSS group reduced the expression levels of colonic tight junction proteins including Claudin-1, Occludin, ZO-1, and mucin Muc-2, while the treatment of LR and LT bacteria up-regulated the expression of those barrier-related proteins under the influence of DSS. The composition of colonic flora was detected by using 16S rDNA sequencing technology. PCoA analysis showed that the normal group and the DSS group are significantly separated, and the flora composition of the LT treatment group was closer to the normal group. At the phylum level, the abundance of Proteobacteria in the normal group was significantly lower than that of the DSS group (P<0.05). Compared with the DSS group, the LT bacteria treatment group could also reduce the abundance of Proteobacteria, but did not reach a significant level (P>0.05). At the genus level, Muribaculaceae_norank in the normal group was significantly higher than the other three groups, and Bacteroides in the normal control group was significantly lower than the other three groups (P<0.05). Compared with the DSS group, the treatment of LR and LT bacteria increased Muribaculaceae_norank abundance, and reduced Bacteroides abundance, but the difference was not significant (P>0.05). [Conclusion] Lactobacillus reuteri and Lactobacillus taiwanensis can strengthen the intestinal epithelial barrier and relieve the intestinal inflammation caused by DSS to a certain extent. Among them, Lactobacillus taiwanensis has a more significant protective effect on the intestinal epithelial barrier.