丁酸梭菌影响肠炎沙门氏菌对SPF小鼠致病性的作用

Effect of Clostridium butyricum on the pathogenicity of Salmonella enteritidis in SPF mice

摘要:

[目的] 旨在探究丁酸梭菌影响肠炎沙门氏菌（CVCC3377）对SPF小鼠的致病性及对小鼠的保护作用。[方法] 选用72只6周龄SPF级雌性C57BL/6小鼠，按照完全随机区组设计分为阴性对照（CON）、饲喂丁酸梭菌组（CB）、沙门氏菌感染组（SE）和丁酸梭菌保护组（CB+SE）4组，每组18只。CON组和SE组饲喂基础日粮和饮水，CB组和CB+SE组饲喂基础日粮和每天更换添加丁酸梭菌（1×10⁷ CFU/mL）的饮水，连续饲喂一周；第8天进行肠炎沙门氏菌感染实验，分别对SE组和CB+SE组灌服0.2 mL 1.1×10⁴ CFU/只的肠炎沙门氏菌，同时对阴性对照组和丁酸梭菌组灌服0.2 mL生理盐水。在感染后第6天处死小鼠并采集肝脏、脾脏和盲肠及内容物样品。采用H.E染色检测肝脏和脾脏组织的病理学变化，免疫组化检测脾脏组织中TLR4、MyD88蛋白的表达水平，荧光定量PCR检测盲肠组织中TNF-α和IL-6的表达水平，并通过高通量测序方法分析其对肠道菌群的影响。[结果] 结果显示，丁酸梭菌能够缓解肠炎沙门氏菌感染引起的小鼠体重下降；H.E染色结果显示，丁酸梭菌可以缓解肠炎沙门氏菌感染造成的组织损伤；免疫组化结果显示，丁酸梭菌可降低脾脏组织细胞中TLR4、MyD88蛋白的表达水平，荧光定量PCR结果显示肠炎沙门氏菌能够显著升高盲肠组织中TNF-α（P<0.05）和IL-6的表达水平，在此基础上饲喂丁酸梭菌能够降低TNF-α和IL-6的表达水平。高通量测序结果显示，丁酸梭菌组Chao1指数、ACE指数和Shannon指数均高于对照组和沙门氏菌感染组（P>0.05），丁酸梭菌可显著降低放线菌门（Actinobacteria）和疣微菌门（Verrucomicrobia）在肠道微生物中的比例（P<0.05），另外，丁酸梭菌可以增加乳酸菌属在肠道微生物群落的相对丰度（P>0.05）。[结论] 结果表明，丁酸梭菌可以提高小鼠免疫力，缓解由于沙门氏菌感染引起的炎症反应，增加肠道微生物多样性，调节肠道菌群结构。

Abstract:

[Objective] To explore the effect of Clostridium butyricum on pathogenicity and protection mechanism of Salmonella enteritidis (CVCC3377) in SPF mice. [Methods] Mice were sacrificed on the 6th day post infection and samples of liver, spleen and cecum samples were collected. The pathological changes of liver and spleen tissue were detected by H.E staining, the expression levels of TLR4 and MyD88 protein in spleen tissue were detected by immunohistochemistry, and the expression levels of TNF-α and IL-6 in cecum tissue were detected by fluorescence quantitative PCR. The effect on intestinal flora was analyzed by high-throughput sequencing. [Results] C. butyricum could alleviate the weight loss of mice caused by Salmonella enteritidis infection. The results of HE staining show that Clostridium butyricum could alleviate the tissue damaged by Salmonella enteritidis infection. The results of immunohistochemistry show that Clostridium butyricum could decrease the expression levels of TLR4 and MyD88 protein in liver and spleen tissue cells. Fluorescence quantitative PCR shows that Salmonella enteritidis could significantly increase TNF-α in cecal tissue (P<0.05). The expression of TNF-alpha and IL-6 decreased by feeding Clostridium butyricum. The results of high throughput sequencing show that the Chao 1 index, ACE index and Shannon index of Clostridium butyricum treatment group were higher than those of the control group and Salmonella infection group (P>0.05). Clostridium butyricum could significantly reduce the proportion of Actinobacteria and Verrucomicrobia in intestinal microorganisms (P<0.05). In addition, the relative abundance of Lactobacillus in intestinal microbial community increased by bacteria (P>0.05). [Conclusion] Clostridium butyricum could improve the immunity of mice, alleviate the inflammatory reaction caused by Salmonella infection, increase intestinal microflora diversity and regulate intestinal flora structure.