内质网应激诱导的细胞自噬机制在坏死性小肠结肠炎大鼠中的研究

Endoplasmic reticulum stress induced cell autophagy in the pathogenesis of necrotizing enterocolitis rats

目的探讨内质网应激(endoplasmic reticulum stress,ERS)与细胞自噬机制在坏死性小肠结肠炎(necrotizing enterocolitis,NEC)大鼠中的作用。方法新生SD大鼠39只,随机分为3组,建立NEC组(人工喂养+缺氧刺激+胃内注射LPS)、ERS拮抗组(人工喂养+缺氧刺激+胃内注射脂多糖+腹腔注射4-苯基丁酸)、ERS诱发组(人工喂养+缺氧刺激+胃内注射脂多糖+腹腔注射衣霉素)大鼠模型。造模成功后处死大鼠,留取肠道组织,电镜下观察大鼠肠道病理,酶联免疫吸附法检测肠型脂肪酸结合蛋白(intestinal fatty acid binding protein,I-FABP)水平,实时定量聚合酶链反应法测定葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)mRNA、氧调节蛋白150(oxygen-regulated protein 150,ORP150)mRNA表达水平,Western-blot检测p62和自噬相关蛋白微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)Ⅱ、LC3Ⅰ的表达水平,计算LC3Ⅱ/LC3Ⅰ比值。结果(1)造模结束后,大鼠肠道病理评分均≥2分。(2)ERS诱发组临床表现评分、病理评分、I-FABP值、GRP78 mRNA值、ORP150 mRNA值、LC3Ⅱ/LC3Ⅰ高于NEC组和ERS拮抗组,NEC组高于ERS拮抗组,差异均有统计学意义(P<0.05);ERS诱发组p62表达量低于NEC组和ERS拮抗组,NEC组低于ERS拮抗组,差异均有统计学意义(P<0.05)。(3)LC3Ⅱ/LC3Ⅰ比值和临床表现评分、病理评分、I-FABP均呈正相关(P<0.05);p62表达量和临床表现评分、病理评分、I-FABP均呈负相关(P<0.05);GRP78、ORP150 mRNA和临床表现评分、病理评分、I-FABP均呈正相关(P<0.05);LC3Ⅱ/LC3Ⅰ比例和GRP78、ORP150 mRNA值呈正相关(P<0.05);p62表达量和GRP78、ORP150 mRNA值呈负相关(P<0.05)。结论内质网应激和NEC发病有关,抑制内质网应激可抑制自噬反应,改善NEC肠道屏障功能及临床症状。

Objective To study the effects of endoplasmic reticulum stress(ERS)and autophagy mechanisms in the pathogenesis of necrotizing enterocolitis(NEC).Methods A total of 39 newborn SD rats were randomly assigned into 3 groups:the NEC group(NEC model:artificial feeding+hypoxic stimulation+intragastric injection of lipopolysaccharides),the ERS antagonist group(NEC model+intraperitoneal injection of 4-phenylbutyric acid)and the ERS inducer group(NEC model+intraperitoneal injection of tunicamycin).After successful modeling,the rats were sacrificed and intestinal tissues were obtained.The intestinal pathology was observed using electronic microscope.Intestinal fatty acid binding protein(I-FABP)was detected using ELISA.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to examine mRNA levels of glucose-regulated protein 78(GRP78)and oxygen-regulated protein 150(ORP150).Western-blot was used to detect p62 and autophagy-related proteins(microtubule-associated protein 1 light chain 3(LC3)Ⅱand LC3Ⅰ)and LC3Ⅱ/LC3Ⅰratio was calculated.Results(1)For all 3 groups,the pathological scores of rat intestines were≥2.(2)The ERS inducer group showed significantly higher clinical score,pathological score,I-FABP level,GRP78 and ORP150 mRNA levels and LC3Ⅱ/LC3Ⅰratio than the NEC group and ERS antagonist group,and the NEC group higher than the ERS antagonist group(P<0.05).The p62 level in the ERS inducer group was significantly lower than the NEC group and the ERS antagonist group,and the NEC group lower than the ERS antagonist group(P<0.05).(3)The LC3Ⅱ/LC3Ⅰratio was positively correlated with clinical score,pathological score and I-FABP level(P<0.05).The p62 level was negatively correlated with clinical score,pathological score and I-FABP level(P<0.05).The mRNA levels of GRP78 and ORP150 were positively correlated with clinical score,pathological score and I-FABP level(P<0.05).LC3Ⅱ/LC3Ⅰratio was positively correlated with the mRNA levels of GRP78 and ORP150(P<0.05).The p62 level was negatively correlated with the mRNA levels of GRP78 and ORP150(P<0.05).Conclusions ERS is associated with the pathogenesis of NEC.Inhibition of ERS can reduce autophagy and improve intestinal barrier function and clinical symptoms of NEC.