褪黑激素调节Akt/mTOR信号通路对坏死性小肠结肠炎新生大鼠肠道屏障功能的影响

doi:10.11852/zgetbjzz2021-1918

摘要/Abstract

摘要： 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。

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

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.

Key words: melatonin, protein kinase B/mammalian target of rapamycin signaling pathway, necrotizing enterocolitis, intestinal barrier

中图分类号:

R722.1

周丽霞, 蔡冬, 陈有平. 褪黑激素调节Akt/mTOR信号通路对坏死性小肠结肠炎新生大鼠肠道屏障功能的影响[J]. 中国儿童保健杂志, 2022, 30(6): 622-626.

ZHOU Li-xia, CAI Dong, CHEN You-ping. Effect of melatonin regulating Akt/mTOR signal pathway on intestinal barrier function in neonatal rats with necrotizing enterocolitis[J]. Chinese Journal of Child Health Care, 2022, 30(6): 622-626.

参考文献

[1] 中国医师协会新生儿科医师分会循证专业委员会. 新生儿坏死性小肠结肠炎临床诊治指南(2020)[J]. 中国当代儿科杂志, 2021, 23(1):1-11.
[2] 张卉, 冯永文, 姚咏明. 自噬在脓毒症免疫反应中的潜在作用与意义[J]. 中华急诊医学杂志, 2019, 28(2):131-134.
[3] Sampath V, Bhandari V, Berger J, et al. A functional ATG16L1(T300A) variant is associated with necrotizing enterocolitis in premature infants[J]. Pediatr Res, 2017, 81(4):582-588.
[4] 康富贵, 聂静云, 杨泽, 等. 硫化氢介导自噬相关基因在脓毒症肠功能损伤中的作用[J]. 中华危重病急救医学, 2020, 32(1):118-120.
[5] Li GM, Liu SS, Wang HL, et al.Ligustrazine amelio-rates lipopolysaccharide-induced neurocognitive impairment by activating autophagy via the PI3K/AKT/mTOR pathway[J]. Int J Mol Med, 2020, 45(6):1711-1720.
[6] 陈丽萍, 吕志宝, 葛贵杰, 等. 人β-防御素-3通过调控肠上皮细胞自噬对新生大鼠坏死性小肠结肠炎发挥保护作用的实验研究[J]. 中华小儿外科杂志, 2021, 42(5):445-453.
[7] 孟宪红, 李立, 曹巍, 等. 褪黑素对非酒精性脂肪性肝病中NLRP3炎症小体和肝脏纤维化的影响及机制研究[J]. 哈尔滨医科大学学报, 2021, 55(5):451-456.
[8] Wang X,Bian Y, Zhang R, et al. Melatonin alleviates cigarette smoke-induced endothelial cell pyroptosis through inhibiting ROS/NLRP3 axis[J]. Biochem Biophys Res Commun, 2019, 519(2):402-408.
[9] 席世兵, 杨敏, 谢集建, 等. 新生鼠坏死性小肠结肠炎模型建立方法的改进及评价[J]. 实验动物科学, 2019, 36(1):36-40.
[10] 赵铭, 姜红, 秦苗, 等. 褪黑素对坏死性小肠结肠炎新生大鼠NLRP3及相关炎性因子影响的研究[J]. 中华新生儿科杂志, 2021, 36(2):53-58.
[11] 徐臣年, 尹文超, 王建浩, 等. 褪黑素通过Akt/mTOR信号通路对自噬和H9C2心肌细胞缺血再灌注损伤的影响[J]. 解放军医药杂志, 2019, 31(3):8-14.
[12] Müller MJ, Paul T,Seelinger S. Necrotizing enterocolitis in premature inflants and new borns[J]. J Neonatal Perinatal Med, 2016, 9(3):233-242.
[13] 何颖, 许景林, 陈冬梅, 等. 肠型脂肪酸结合蛋白在新生大鼠坏死性小肠结肠炎肠道组织中的表达及意义[J]. 中华新生儿科杂志, 2019, 34(5):385-389.
[14] 马静, 乔彦霞, 曹琴英, 等. 新生儿坏死性小肠炎41例相关因素分析[J]. 中华新生儿科杂志, 2019, 34(4):291-294.
[15] Gopalakrishna KP, Macadangdang BR, Rogers MB, et al. Maternal IgA protects against the development of necrotizing enterocolitis in preterm infants[J]. Nat Med, 2019, 25(7):1110-1115.
[16] 张岚, 白铂亮, 王莉, 等. SIRT1信号通路在新生儿坏死性小肠结肠炎肠组织中的表达研究[J]. 海南医学院学报, 2019, 25(8):578-582.
[17] Lim JA, Li L,Shirihai OS, et al. Modulation of mTOR signaling as a strategy for the treatment of Pompe disease[J]. EMBO Mol Med, 2017, 9(3):353-370.
[18] 张一然, 刘风玲. 褪黑素减少炎症性肠病及结直肠癌肠道炎症发生的研究进展[J]. 消化肿瘤杂志(电子版), 2020, 12(3):181-188.
[19] Wang X,Bian Y, Zhang R, et al. Melatonin alleviates cigarette smoke-induced endothelial cell pyroptosis through inhibiting ROS/NLRP3 axis[J]. Biochem Biophys Res Commun, 2019, 519(2):402-408.
[20] 胡璐璐, 张方信. PI3K/Akt/mTOR信号通路在高原缺氧所致肠黏膜损伤的自噬调节中的作用[J]. 国际消化病杂志, 2018, 38(2):94-97,110.
[21] Wang N,Wang MY.Dexmedetomidine suppresses sevo-flurane anesthesia-induced neuroinflammation through activation of the PI3K/Akt/mTOR pathway[J]. BMC Anesthesiol, 2019, 19(1):134-147.
[22] Nighot PK, Hu CA, Ma TY. Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation[J]. J Biol Chem, 2015, 290(11):7234-7246.