目的 探究褪黑素(Mel)是否能够通过调控蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)通路对坏死性小肠结肠炎(NEC)新生大鼠肠道屏障功能发挥保护作用。 方法 90只新生大鼠随机分为正常组、模型组、Mel低(15 mg/kg)、高剂量组(30 mg/kg)与Mel(30 mg/kg)+Akti(5 μmol/L)组,除正常组外,其余4组建立NEC模型。实验结束后,取眼静脉血分离血清,检测D-乳酸、二胺氧化酶(DAO)和内毒素含量;取回盲部肠组织,用于HE染色及检测IL-6、IL-1β、TNF-α含量与自噬基因(Beclin-1)、LC3B、p-Akt/Akt、p-mTOR/mTOR表达水平。结果 与正常组比较,模型组大鼠回盲部肠组织细胞出现变性坏死,黏膜肌层水肿,绒毛脱落、缺失等现象;血清中D-乳酸、DAO、内毒素含量与肠组织中IL-6、IL-1β、TNF-α含量和Beclin-1、LC3B蛋白阳性率均明显升高(P＜0.05),p-Akt/Akt、p-mTOR/mTOR明显降低(P＜0.05);与模型组比较,Mel低、高剂量组大鼠回盲部肠组织病变明显改善;D-乳酸、DAO、内毒素含量与IL-6、IL-1β、TNF-α含量和Beclin-1、LC3B蛋白阳性率均依次降低(P＜0.05),p-Akt/Akt、p-mTOR/mTOR升高(P＜0.05);与Mel高剂量组比较,Mel+Akti组大鼠回盲部肠组织病变加重;D-乳酸、DAO、内毒素含量与肠组织中IL-6、IL-1β、TNF-α含量和Beclin-1、LC3B蛋白阳性率均明显升高(P＜0.05),p-Akt/Akt、p-mTOR/mTOR明显降低(P＜0.05)。 结论 Mel能够通过上调Akt/mTOR信号通路蛋白表达,抑制自噬,对NEC新生大鼠肠道屏障功能发挥保护作用。
Abstract
Objective To explore whether melatonin(Mel) can protect the intestinal barrier function of neonatal rats with necrotizing enterocolitis(NEC) by regulating the Akt/mTOR pathway.Methods Ninety newborn rats were randomly divided into normal group, model group, low(15 mg/kg) and high(30 mg/kg) dose Mel groups, and Mel(30 mg/kg) + Akti(Akt inhibitor, 5 μmol/L) group. Except for the normal group, NEC models were established in rats of the other 4 groups. After the experiment, the ocular venous blood was collected to detect the contents of serum D-lactic acid, diamine oxidase(DAO) and endotoxin. The ileocecum was retrieved for HE staining and detection of IL-6, IL-1β, TNF-α contents and autophagy gene(Beclin-1), LC3B, p-Akt/Akt, p-mTOR/mTOR expression levels.Results Compared with the normal group, the cells in ileocecum of the model group obviously showed degeneration and necrosis, mucosal muscular layer edema and loss of villi. Serum D-lactic acid, DAO, endotoxin content and intestinal tissue IL-6, IL-1β, TNF-α contents and Beclin-1, LC3B protein positive rates significantly increased(P<0.05), p-Akt/Akt and p-mTOR/mTOR levels were significantly reduced(P<0.05). Compared with the model group, the pathological changes of the ileocecal intestine tissue of the rats in the low and high dose Mel groups were significantly improved. Serum D-lactic acid, DAO, endotoxin content and IL-6, IL-1β, TNF-α contents and the positive rates of Beclin-1 and LC3B protein were reduced in sequence(P<0.05), p-Akt/Akt and p-mTOR/mTOR were increased(P<0.05). Compared with the Mel high-dose group, the ileocecal intestinal tissue lesions in the Mel+Akti group were worse. Serum D-lactic acid, DAO, endotoxin content and IL-6, IL-1β, TNF-α contents in intestinal tissue and the positive rates of Beclin-1, LC3B protein were significantly increased(P<0.05), p-Akt/Akt and p-mTOR/mTOR were significantly reduced(P<0.05).Conclusions Mel can up-regulate the expression of Akt/mTOR signaling pathway protein to inhibit autophagy and protect the intestinal barrier function of NEC neonatal rats.
关键词
褪黑激素 /
蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路 /
坏死性小肠结肠炎 /
肠道屏障
Key words
melatonin /
protein kinase B/mammalian target of rapamycin signaling pathway /
necrotizing enterocolitis /
intestinal barrier
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基金
2016年度海南省卫生计生行业科研项目(15A200076)
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