缺氧调控大鼠小肠隐窝上皮细胞自噬的变化规律及分子机制  

Changes pattern of autophagy in small intestinal crypt epithelial cells regulated by hypoxia and its molecular mechanism

作　　者：王彬彬 张娅 罗自珍 陈红纲[1] 张红 Wang Binbin;Zhang Ya;Luo Zizhen;Chen Honggang;Zhang Hong(Department of Emergency,Gansu Provincial Hospital,Lanzhou 730000,China)

机构地区：[1]甘肃省人民医院急诊科,兰州730000

出　　处：《临床内科杂志》2025年第2期155-159,共5页Journal of Clinical Internal Medicine

基　　金：甘肃省人民医院院内科研基金项目(24GSSYE-8)。

摘　　要：目的 探讨缺氧调控大鼠小肠隐窝上皮细胞自噬的变化规律及分子机制。方法分别在常氧及缺氧条件下处理大鼠小肠隐窝上皮细胞(IEC-6),将所有细胞分为对照组(常氧状态)、缺氧组(缺氧状态)及3-甲基腺嘌呤(MA)+缺氧组(缺氧状态下加入5 mmol/L的3-MA)。采用甲基噻唑基四唑(MTT)法检测细胞存活率、单丹磺酰尸胺(MDC)荧光染色观察细胞内自噬荧光颗粒、透射电子显微镜观察细胞内双膜结构自噬溶酶体形成、Western blot检测相关蛋白表达水平。结果 缺氧组12 h、24 h及48 h肠上皮细胞存活率均低于同期对照组(P<0.05)。MDC荧光染色及透射电子显微镜观察显示,缺氧组肠上皮细胞内自噬荧光颗粒及双层膜结构的自噬溶酶体在6~24 h均明显增加,而在48 h时开始下降。与对照组比较,缺氧组6 h、12 h、24 h微管相关蛋白轻链3(LC3)-Ⅱ蛋白表达水平及LC3-Ⅱ/Ⅰ比值均明显上升,12 h、2 4 h Beclin-1蛋白表达水平均明显上升,且上述指标水平均于缺氧24 h达峰值后回落,48 h下降后的水平仍显著高于对照组(P<0.05)。对照组、3-MA+缺氧组及缺氧24 h组细胞LC3-Ⅱ、Beclin-1及磷酸化蛋白激酶B(p-Akt)蛋白表达水平、LC3-Ⅱ/Ⅰ比值均依次升高(P<0.05)。结论 缺氧可诱导IEC-6大鼠小肠隐窝上皮细胞损伤时激活自噬,并上调自噬相关蛋白LC3-Ⅱ、LC3-Ⅱ/Ⅰ比值、Beclin-1和p-Akt水平。Objective To explore the changing pattern and molecular mechanism of hypoxia regulating autophagy in small intestinal crypt epithelial cells in rats.Methods Rat small intestinal crypt epithelial cells(IEC⁃6)were treated under normal oxygen state and hypoxia state,and all cells were divided into control group(normal oxygen state),hypoxia group(hypoxia state)and 3⁃methyladenine(MA)+hypoxia group(adding 5 mmol/L 3⁃MA under hypoxia state).Cell survival rate was detected by Methyc Thiazolyl Tetrazolium(MTT)assay,autophagy fluorescent particles were observed by MDC fluorescence staining,the formation of autophagy lysosomes with double membrane structure was observed by transmission electron microscopy,the protein expression levels were detected by Westernt blot.Results The survival rate of intestinal epithelial cells at 12 h,24 h and 48 h in hypoxia group were lower than that in control group at the same period(P<0.05).The results of MDC fluorescence staining and transmission electron microscopy showed that the autophagy fluorescence particles and the autophagy lysosomes in the intestinal epithelial cells of hypoxia group increased significantly from 6 h to 24 h,but began to decrease at 48 h.Compared with control group,the microtubule⁃associated protein light chain 3(LC3)⁃Ⅱprotein expression level and LC3⁃Ⅱ/Ⅰratio in hypoxia group were significantly increased at 6 h,12 h and 24 h,and the Beclin⁃1 protein expression level was significantly increased at 12 h and 24 h,and the above index levels fell after reaching the peak at 24 h,and began to decline at 48 h,but still significantly higher than control group at the same period(P<0.05).LC3⁃Ⅱ,Beclin⁃1,phosphorylated protein kinase B(p⁃Akt)protein expression levels and LC3⁃Ⅱ/Ⅰratio of cells in control group,3⁃MA+hypoxia group and 24 h hypoxia group were increased successively(P<0.05).Conclusion Hypoxia induced enhanced autophagy and up⁃regulated the levels of autophagy⁃related proteins LC3⁃Ⅱ,LC3⁃Ⅱ/Ⅰratio,Beclin⁃1 and p⁃Ak

关 键 词：缺氧 肠上皮细胞 自噬 

分 类 号：R574.5[医药卫生—消化系统]