丁酸钠对Caco-2细胞肠屏障的作用及NF-κB途径的影响  

Effects of sodium butyrate and NF-κB pathway on intestinal barrier function in a Caco-2 cell monolayer model of intestinal barrier

作　　者：黄晓忠[1] 郭世坤 胡笑笑 林孝坤 包小周[1] 夏肇波 朱利斌[1] 李仲荣[1] Huang Xiaozhong;Guo Shikun;Hu Xiaoxiao;Lin Xiaokun;Bao Xiaozhou;Xia Zhaobo;Zhu Libin;Li Zhongrong(Department of Pediatric Surgery,Second Affiliated Hospital&Yuying Children's Hospital,Wenzhou Medical University,Wenzhou 325027,China)

机构地区：[1]温州医科大学附属第二医院育英儿童医院小儿外科,325027

出　　处：《中华小儿外科杂志》2020年第10期948-954,共7页Chinese Journal of Pediatric Surgery

基　　金：国家自然科学基金(81903235);浙江省自然科学基金(LQ18H020004,LY17H040010);浙江省医药卫生科技计划项目(2017ZD025)。

摘　　要：目的通过观察高浓度丁酸钠(sodium butyrate,NaB)(5 mmol/L,8 mmol/L)对单层Caco-2细胞肠屏障模型的作用,及该过程中对NF-κB通路的影响,旨在探讨高浓度NaB破坏单层Caco-2细胞肠屏障模型的可能分子机制。方法使用不同浓度的NaB及雷公藤甲素(triptolide,TP)(特异性抑制NF-κB)50 nmol/L作用体外培养的Caco-2细胞及单层Caco-2细胞肠屏障模型,按照作用的试剂和浓度分为0 mmol/L NaB(对照组)、50 nmol/L TP组(TP组)、2 mmol/L NaB组(NaBⅠ组)、5 mmol/L NaB(NaBⅡ组)、8 mmol/L NaB(NaBⅢ组)、5 mmol/L NaB+50 nmol/L TP组(NaB+TP组)。测定单层Caco-2细胞肠屏障模型的跨上皮电阻(transepithelial electrical resistance,TER)及菊粉-FITC(inulin-FITC)的通透性;用实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR)及蛋白质印迹法检测各组IL-1β和TNF-α的mRNA及蛋白水平;用核质分离试验检测p65蛋白入核水平的变化。结果①高浓度NaB(5 mmol/L、8 mmol/L)作用于单层Caco-2细胞肠模型72 h后,与对照组相比,TER显著下降,inulin-FITC通透性显著升高;如5 mmol/L NaB作用于单层Caco-2细胞肠屏障72 h,TER降低至(106.33±4.04)(Ω×cm^2),inulin-FITC的通透性升高至(12.52±1.82)%,与对照组相比,差异均具有统计学意义(P<0.05);而使用50 nmol/L TP特异性抑制NF-κB后,其TER升高至(398.33±3.06)(Ω×cm^2),inulin-FITC的通透性降低至(7.24±0.25)%,差异具有统计学意义(P<0.05),提示5 mmol/L NaB对肠屏障的破坏作用被显著抑制;②高浓度NaB(5 mmol/L、8 mmol/L)作用Caco-2细胞72 h,IL-1β和TNF-αmRNA及蛋白的表达水平较对照组显著提高,且p65蛋白入核水平也显著提高,差异具有统计学意义(P<0.05);③在5 mmol/L NaB的基础上,使用TP抑制NF-κB后,可显著抑制高浓度NaB诱导IκB-α的磷酸化及p65蛋白入核,并显著降低IL-1β与TNF-α的表达水平。结论高浓度NaB可破坏肠屏障功能,其机制可能与活化NF-κB通路,促进炎症因子Objective Researches have demonstrated that physiological concentrated of sodium butyrate plays an important role in promoting intestinal barrier function while an excessive level of sodium butyrate is correlated with the occurrence of neonatal necrotizing enterocolitis (NEC). However, the underlying mechanism has remained elusive. Here the authors explored the effect of high-concentration sodium butyrate on monolayer Caco-2 cell model of intestinal barrier and examine the effect of nuclear factor-kappa B (NF-κB) pathway on this process for elucidating the possible molecular mechanisms of high-concentration sodium butyrate impairing intestinal epithelial barriers.Methods Different concentrations of sodium butyrate and triptolide (NF-κB specific inhibitor, 50 nmol/L) were employed for Caco-2 cell monolayer model of intestinal barrier and Caco-2 cell in vitro. The transepithelial electrical resistance (TER) and the permeability of inulin-FITC were measured. The mRNA and protein levels of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α ) were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Nuclear and cytosolic extracts of Caco-2 cells were isolated for analyzing the nuclear translocation of p65 by immunoblot.Results After 72h incubation of high-concentration sodium butyrate (5/8 mmol/L), TERs declined while inulin-FITC permeability increased significantly as compared with control group. TER of 5 mmol/L sodium butyrate declined to (106.33±4.04 Ω×cm^2) and the permeability of inulin-FITC spiked to (12.52%±1.82%). Both were significantly different with control group. After inhibiting NF-κB with 50 nmol/L triptolide, TER increased to (398.33±3.06 Ω×cm^2) and the permeability of inulin-FITC declined to (7.24%±0.25%). And both were significantly different from high-concentration sodium butyrate (5 mmol/L) group. As compared with control group, after treating with high-concentration sodium butyrate (5/8 mmol/L), mRNA and protein levels of IL-

关 键 词：肠黏膜 丁酸钠 NF-κB炎症信号通路 

分 类 号：R965[医药卫生—药理学]