二甲双胍促进放射性肠炎黏膜屏障修复的作用及机制

Effects of metformin on intestinal barrier repair in radiation proctitis and its mechanism

目的:探讨二甲双胍对放射性肠炎(RP)黏膜屏障功能的影响及机制。方法:收集接受盆腔外照射后出现RP的宫颈癌患者61例血液及尿液样本。ELISA法、鲎试剂法检测血清TNF-α、IL-1β和内毒素的水平;高压液相色谱法检测尿液乳果糖和甘露醇(L/M)水平。构建小鼠RP模型,HE、IHC染色、透射电镜观察及荧光素示踪法评价肠黏膜组织病理学及紧密连接的变化。Western blot检测ZO-1、MUC2、AMPK和mTOR磷酸化蛋白表达。ELISA法检测TNF-α和IL-1β的水平。结果:二甲双胍组患者血清TNF-α、IL-1β和内毒素水平均明显低于空白对照组患者[TNF-α:(37.60±11.93)μg/L比(89.40±7.52)μg/L,IL-1β:(0.29±0.03)pg/L比(0.84±0.01)pg/L,P均<0.001],尿液L/M比值明显高于(0.07±0.02比0.04±0.03,P=0.020)。RP组小鼠DAI指数随时间延长逐渐升高,组织病理学检查见肠黏膜出现损伤。RP+Met组DAI指数明显减低,肠黏膜损伤修复。RP+Met组血浆FD4、TNF-α和IL-1β均低于RP组[FD4:(26.60±3.84)mg/L比(54.26±5.28)mg/L,TNF-α:(0.35±0.26)pg/L比(0.71±0.20)pg/L,IL-1β:(0.24±0.16)pg/L比(0.45±0.23)pg/L,P<0.001];ZO-1、MUC2、p-AMPK蛋白表达高于RP组,而p-mTOR表达低于RP组。结论:二甲双胍能够改善肠道炎症反应,促进RP肠黏膜屏障的修复,其机制可能与二甲双胍激活AMPK通路,抑制mTOR通路,维持肠紧密连接完整性,促进MUC2的合成有关。

Objective To explore the effects and mechanism of metformin on intestinal barrier repair in radiation proctitis(RP).Methods Serum and urine samples were collected from 61 cervical cancer patients with radiation proctitis during radiotherapy.ELISA and tachypleus amebocyte lysate was used to detect levels of TNF-α,IL-1βand endotoxin.High pressure liquid chromatography was used to detect lactulose/mannital(L/M)ratio.A radiation-induced mouse proctitis model was established,and HE staining,immunohistochemical(IHC)staining as well as electron microscope observation and fluorescein tracer were applied to analyze the changes of intestinal tissue pathology and tight junction.Western blot was applied to detect protein expressions of ZO-1,MUC2,p-AMPK and p-mTOR.ELISA was used to detect the levels of TNF-αand IL-1β.Results In RP patients,compared to control group(without metformin),serum levels of TNF-α[(37.60±11.93)μg/L vs.(89.40±7.52)μg/L,P<0.001]and IL-1β[(0.29±0.03)pg/L vs.(0.84±0.01)pg/L,P<0.001]were decreased,and urine L/M ratio(0.07±0.02 vs.0.04±0.03,P=0.020)was increased in metformin group.In RP mice model,DAI index increased gradually.Histopathological examination revealed incomplete intestinal mucosa.After metformin treatment,the above changes were alleviated.Meanwhile,compared to mice in RP group,plasma FD4[(26.60±3.84)mg/L vs.(54.26±5.28)mg/L,P<0.001],TNF-α[(0.35±0.26)μg/L vs.(0.71±0.20)μg/L,P<0.001]and IL-1β[(0.24±0.16)pg/L vs.(0.45±0.23)pg/L,P<0.001]were all decreased,protein expression of ZO-1,MUC2 and p-AMPK were up-regulated,while p-mTOR was down-regulated in RP+Metformin group.Conclusions Metformin can improve intestinal inflammation and promote the repair of intestinal barrier in radiation proctitis.The mechanism may be related to the role of metformin′s activating the AMPK pathway,inhibiting the mTOR pathway,maintaining the intestinal tight junction and promoting the synthesis of MUC2.