1型糖尿病小鼠肠道菌群与免疫状态演变的实验研究

Evolution of intestinal flora and immune status in type 1 diabetic mice

目的探究链脲佐菌素(STZ)诱导的1型糖尿病(T1DM)小鼠在不同时期的肠道功能、血清相关抗体、脾脏细胞因子谱表达与肠道菌群的演变。方法取3~4周龄雄性C57BL/6小鼠28只,采用随机抽样法分为对照组(NC组,13只)和STZ组(15只)。STZ组连续5 d空腹腹腔注射STZ 50 mg·kg-1·d-1,NC组注射等体积溶剂。实验第3周测定随机血糖>16.7 mmol/L为建模成功,定义为基线水平,两组各取6只小鼠处死,剩余小鼠观察4周,即实验第7周(实验终点)处死剩余小鼠。每周测量体重、随机血糖和每日饮水量。采用酶联免疫吸附测定(ELISA)法检测脾脏细胞因子谱[肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素(IL)-6、IL-17、IL-4、IL-10、转化生长因子-β(TGF-β)]、血清相关抗体[胰岛细胞抗体(ICA)、胰岛素自身抗体(IAA)与谷氨酸脱羧酶抗体(GADA)]和结肠紧密连接蛋白1(ZO-1)的表达;采用流式细胞术检测脾脏CD4+FoxP3+调节性T细胞(Treg)水平;对粪便进行16SrRNA菌群测序分析。组间比较采用独立样本t检验。采用Spearman相关分析法分析CD4+FoxP3+Treg细胞、肠道菌群、各免疫生化指标之间的相关性。结果流式细胞术与ELISA结果显示,在基线水平,与NC组相比,STZ组小鼠脾脏CD4+FoxP3+Treg、TGF-β、TNF-α、IFN-γ、IL-6、IL-17与血清ICA、IAA表达水平增加,脾脏IL-4、IL-10表达水平下降(均P<0.05);在实验终点,与NC组相比,STZ组小鼠脾脏TNF-α、IFN-γ、IL-6,血清GADA、ICA与结肠ZO-1表达水平增加(均P<0.05)。16SrRNA测序结果显示,与NC组相比,STZ组乳杆菌属丰度明显下降(P<0.05),拟杆菌属、阿克曼菌属、普雷沃菌属和颤螺菌属丰度明显上升(均P<0.05)。Spearman相关性分析结果表明,乳杆菌属丰度与IL-10水平呈正相关(r=0.65,P<0.05),与TNF-α、IFN-γ、IL-6、IL-17和GADA水平呈负相关(r值-0.61~-0.58,均P<0.05);颤螺菌属丰度与TGF-β、TNF-α、IL-6和IAA水平呈正相关(r值0.55~0.72,均P<0.05),与IL-10水平呈负相关(r=-0.59,P<0.05)。结论多次低剂量STZ诱导的T1DM小鼠可能存在CD4+Foxp3+Treg及TGF-β介导的负性免疫调节和以增加结肠ZO-1表达的肠道调节,其中颤螺菌属与乳杆菌属丰度改变可能参与免疫调节。

Objective To explore the intestinal function,serum-related antibodies,expression of the spleen cytokine profile and intestinal flora evolution in streptozotocin(STZ)-induced type 1 diabetes mellitus(T1DM)mice at different stages.Methods Twenty-eight male C57BL/6 mice aged 3-4 weeks were randomly divided into control group(NC group,13 mice)and streptozotocin group(STZ group,15 mice).The STZ group was injected intraperitoneally with STZ 50 mg·kg-1·d-1 for 5 consecutive days on a fasting state,and the NC group was injected with an equal volume of solvent.In the third week of the experiment,the random blood glucose>16.7 mmol/L was determined as the successful modeling,which was defined as the baseline level.Six mice in each group were sacrificed,and the remaining mice were observed for 4 weeks,i.e.the remaining mice were sacrificed at the seventh week of the experiment(the end of the experiment).Body weight,random blood glucose and daily water intake were measured weekly.The expression of cytokines in spleen[tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin(IL)-6,IL-17,IL-4,IL-10,transforming growth factor-β(TGF-β)],serum-related antibodies[islet cell antibody(ICA),insulin autoantibody(IAA)and glutamic acid decarboxylase antibody(GADA)]and colonic tight junction protein 1(ZO-1)were detected by enzyme-linked immunosorbent assay(ELISA).The level of CD4+FoxP3+Treg in spleen was determined by flow cytometry.Faeces were subjected to 16SrRNA flora sequencing analysis.Differences in expression between groups were compared using a two-sided unpaired t test.Spearman correlation analysis was used to compare the correlation between CD4+FoxP3+Treg cells and various immune biochemistry of intestinal flora.Results The results of flow cytometry and ELISA showed that at the baseline level,compared with the NC group,the expression of CD4+FoxP3+Treg,TGF-β,TNF-α,IFN-γ,IL-6,IL-17 in spleen and serum ICA and IAA increased(all P<0.05),and the expression of IL-4 and IL-10 in spleen decreased(all P<0.05).At the end of the experiment,the expression of TNF-α,IFN-γ,IL-6 in spleen,GADA,ICA in serum and ZO-1 in colon increased in STZ group(all P<0.05).The results of 16 SrRNA sequencing showed that compared with the NC group,the abundance of Lactobacillus in the STZ group was significantly decreased(P<0.05),and the abundance of Bacteroides,Akermansia,Prevotella and Oscillatoria was significantly increased(all P<0.05).Spearman correlation results showed that the abundance of Lactobacillus was positively correlated with IL-10 level(r=0.65,P<0.05),and negatively correlated with TNF-α,IFN-γ,IL-6,IL-17 and GADA levels(r=-0.61--0.58,all P<0.05).The abundance of Oscillatoria was positively correlated with the levels of TGF-β,TNF-α,IL-6 and IAA(r=0.55-0.72,all P<0.05),and negatively correlated with the level of IL-10(r=-0.59,P<0.05).Conclusions Multiple low-dose STZ-induced T1DM mice may have negative immune regulation mediated by CD4+Foxp3+Treg and TGF-βand intestinal regulation to increase the expression of ZO-1 in the colon.Among these,the changes in the abundance of Oscillatoria and Lactobacillus may be involved in immune regulation.