移植菌液pH值对粪便菌群移植治疗小鼠溃疡性结肠炎肠道黏膜免疫影响及机制研究

Study on the effect and mechanism of pH value of transplantation bacteria liquid on the intestinal mucosal immunity of mice ulcerative colitis with treated with fecal flora transplantation

目的研究移植菌液pH值对粪便菌群移植治疗溃疡性结肠炎(UC)小鼠肠道黏膜免疫的影响及芳香烃受体(AHR)信号通路在其中的可能作用机制。方法①将50只C57BL/6小鼠随机分为对照组、模型组、粪便菌群低pH值组、粪便菌群中pH值组和粪便菌群高pH值组,每组10只。除对照组外,其余组采用2%葡聚糖硫酸钠连续饮用7 d建立UC模型。造模后,粪便菌群低pH值组、中pH值组和高pH值组分别给予酸性、中性、碱性粪便滤液灌肠,对照组和模型组给予等量生理盐水灌肠,均2次/d,连续7 d。处死小鼠,ELISA法检测血浆中白细胞介素-10(IL-10)、转化生长因子β_(1)(TGF-β_(1))、白细胞介素-17A(IL-17A)含量,PCR法检测结肠组织中IL-10、TGF-β_(1)、IL-17A、AHR、CYP1A1 mRNA表达量,免疫组化法检测结肠组织中IL-10、TGF-β_(1)、IL-17A蛋白表达情况。②无菌分离C57BL/6小鼠脾脏,制备脾细胞悬液,体外诱导分化为调节性T细胞(Tregs)和辅助性T细胞17(Th17),培养5 d后分为2组,对照组常规培养,AHR激动剂组加入吲哚甲醇培养,培养5 d后,ELISA法检测Tregs细胞上清液中IL-10、TGF-β_(1)和Th17细胞上清液中IL-17A含量,PCR法检测Tregs细胞上清液中IL-10、TGF-β_(1)、CYP1A1 mRNA和Th17细胞上清液中IL-17A、CYP1A1 mRNA表达量。结果①与对照组比较,模型组血浆中IL-10、TGF-β_(1)含量和结肠组织中IL-10、TGF-β_(1)、AHR、CYP1A1 mRNA表达量均明显降低(P均<0.05),血浆中IL-17A含量和结肠组织中IL-17A mRNA表达量均明显升高(P均<0.05),结肠原位蛋白标记IL-10、TGF-β_(1)、IL-17A结果与mRNA表达一致;粪便菌群各组血浆中IL-10、TGF-β_(1)含量和结肠组织中IL-10、TGF-β_(1)、AHR、CYP1A1 mRNA表达量均明显高于模型组(P均<0.05),血浆中IL-17A含量和结肠组织中IL-17A mRNA表达量均明显低于模型组(P均<0.05),粪便菌群中pH值组各指标改善最显著。②AHR激动剂组Tregs细胞上清液中IL-10、TGF-β_(1)含量和IL-10、TGF-β_(1)、CYP1A1 mRNA表达量均明显高于对照组(P均<0.05);Th17细胞上清液中IL-17A含量和IL-17A mRNA表达量均明显低于对照组(P均<0.05),CYP1A1 mRNA表达量明显高于对照组(P<0.05)。结论粪便菌群移植能通过激活AHR信号通路调节结肠中Tregs和Th17的表达,改善肠道黏膜免疫功能,且其作用与移植菌液的pH值相关,中性pH值移植菌液作用更明显。

Objective It is to study the effect of the pH value of the transplanted bacterial solution on the intestinal mucosal immunity of mice with ulcerative colitis(UC)treated with fecal flora transplantation and the possible mechanism of the aromatic hydrocarbon receptor(AHR)signaling pathway in it.Methods①Fifty C57BL/6 mice were randomly divided into control group,model group,fecal flora low pH group,fecal flora medium pH group,and fecal flora high pH group,with 10 mice in each group.Except for the control group,the mice in the other groups were treated with 2%dextran sodium sulfate solution continuously for 7 days to establish UC models.After modeling,the fecal flora low pH group,medium pH group,and high pH group were respectively given acidic,neutral,and alkaline fecal filtrate by enema,and the control group and model group were given the same amount of normal saline by enema,all twice/d,continuously treated for 7 days.The mice were sacrificed,the plasma levels of interleukin-10(IL-10),transforming growth factorβ_(1)(TGF-β_(1)),and interleukin-17A(IL-17A)were detected by ELISA,the expression of IL-10,TGF-β_(1),IL-17A,AHR,and CYP1A1 mRNA in the colon tissue were detected by PCR method,and the expression of IL-10,TGF-β_(1),and IL-17A protein in the colon tissue were detected by immunohistochemical method.②The spleen of C57BL/6 mice were aseptically isolated,a spleen cell suspension was prepared,and the differentiation of regulatory T cells(Tregs)and helper T cells 17(Th17)was induced in vitro.After culturing for 5 days,they were divided into two groups,the control group was cultured routinely,the AHR agonist group was cultured with indole methanol.After 5 days of culture,the contents of IL-10,TGF-β_(1) in Tregs cell supernatant and the content of IL-17A in Th17 cell supernatant were detected by ELISA,the expression of IL-10,TGF-β_(1),CYP1A1mRNA in the Tregs cell supernatant and the expression of IL-17A,CYP1A1mRNA in the Th17 cells supernatant was detected by PCR method.Results①Compared with the control group,the plasma levels of IL-10 and TGF-β_(1) and the expression levels of IL-10,TGF-β_(1),AHR,and CYP1A1 mRNA in the colon tissue in the model group were significantly reduced(all P<0.05),and the plasma level of IL-17A and the IL-17A mRNA expression in the colon tissue were significantly increased(all P<0.05).The results of the colon in situ protein markers of IL-10,TGF-β_(1),and IL-17A were consistent with the mRNA expression;the levels of IL-10 and TGF-β_(1) in plasma and the expression of IL-10,TGF-β_(1),AHR,and CYP1A1 mRNA in colon tissue in each group of fecal flora were significantly higher than those in the model group(all P<0.05),the levels of IL-17A in plasma and the expression of IL-17A mRNA in colon tissue were significantly lower than that of the model group(all P<0.05),and the improvement of each index was the most significant in neutral pH value group.②The contents of IL-10,TGF-β_(1) and the expression of IL-10,TGF-β_(1),CYP1A1 mRNA in the Tregs cell supernatant in the AHR agonist group were significantly higher than those in the control group(all P<0.05);the contents of IL-17A and the expression of IL-17A mRNA in the Th17 cell supernatant were significantly lower and the expression of CYP1A1mRNA was significantly higher than those in the control group(all P<0.05).Conclusion Fecal microbiota transplantation can regulate the expression of Tregs and Th17 in colon and improve the immune function of the intestinal mucosa by activating the AHR signaling pathway,and its effect is related to the pH of the transplanted bacterial solution.The neutral pH value of the transplanted bacterial solution is more obvious.