miR-146a-5p表达与新生儿坏死性小肠结肠炎肠道损伤严重程度的相关研究

doi:10.3969/j.issn.1674-8115.2021.09.003

摘要/Abstract

摘要：

目的·检测新生儿坏死性小肠结肠炎（necrotizing enterocolitis，NEC）肠道miR-146a-5p表达水平，并分析其与NEC患儿肠道损伤严重程度的相关性。方法·收集2014年1月—2018年12月于上海交通大学附属儿童医院普外科就诊的NEC和肠闭锁（intestinal atresia，IA）患儿的临床资料。根据肠道组织标本的取材部位，将NEC患儿分为NEC炎症组与NEC未受影响组，以IA患儿的肠道组织标本为对照组（即IA组）。选取5~7日龄C57BL/6J新生小鼠，根据随机数字表将其分为NEC造模组与对照组；前者行造模处理，后者不做处理。处死后，取小鼠的末端回肠组织标本。分别对各组新生小鼠和患儿的组织标本行苏木精-伊红染色，并对小鼠标本行组织病理评级。采用原位杂交检测各组新生小鼠与患儿肠道中miR-146a-5p的表达水平，并分别比较2组新生小鼠、2组患儿的表达差异。对NEC患儿肠道中miR-146a-5p表达水平与组织病理评级行相关性分析，并比较miR-146a-5p高表达（阳性细胞个数≥100个）与低表达（阳性细胞个数<100个）患儿在不同临床资料中的分布情况。结果·①与对照组相比，NEC造模组小鼠的末端回肠组织的病理评级较高，且miR-146a-5p表达水平亦较高（P=0.000）。②与IA组和NEC未受影响组相比，NEC炎症组患儿肠道组织中miR-146a-5p表达水平较高（均P=0.000）；NEC患儿miR-146a-5p表达水平与肠道组织病理评级呈正相关（P=0.015，r=0.578）。③与miR-146a-5p低表达相比，miR-146a-5p高表达患儿中肠道切除长度≥5 cm者较多（P=0.005）。结论·miR-146a-5p高表达与NEC肠道损伤严重程度相关，提示miR-146a-5p参与了NEC的发生与发展过程。

关键词: miR-146a-5p, 坏死性小肠结肠炎, 肠道损伤

Abstract:

Objective·To detect the expression level of miR-146a-5p in the intestinal tract of neonatal necrotizing enterocolitis (NEC), and analyze its correlation with the severity of intestinal injury in children with NEC.

Methods·The clinical data of children with NEC and intestinal atresia (IA) admitted to the Department of General Surgery of Shanghai Children's Hospital, Shanghai Jiao Tong University from January 2014 to December 2018 were collected, respectively. According to the location of intestinal tissue samples, children with NEC were divided into NEC-inflamed group and NEC-unaffected group. The intestinal tissue samples of children with IA were used as control group (i.e. IA group). Newborn C57BL/6J mice aged 5-7 days were randomly divided into NEC model group and control group. The former group was treated by modeling, while the latter was not. After the mice were killed, the terminal ileum tissue samples were taken. Hematoxylin-eosin staining was performed on the tissue samples of newborn mice and children in each group, and histopathological grade of mice was performed. The expression level of miR-146a-5p in the intestine of the newborn mice and the children in each group were detected by in situ hybridization, and the differences of expression between the two groups were compared, respectively. The correlation between the expression level of miR-146a-5p and histopathological grade in intestinal tract of children with NEC was analyzed. The distribution of children with miR-146a-5p high expression (positive cells≥100) and low expression (positive cells <100) in different clinical data was compared.

Results·① Compared with the control group, the pathological grade and the expression level of miR-146a-5p (P=0.000) in the terminal ileum of NEC model mice were higher. ② Compared with IA group and NEC-unaffected group, the expression level of miR-146a-5p was higher in NEC-inflamed group (both P=0.000). And the expression level of miR-146a-5p was positively correlated with the pathological grade of intestinal tissues in children with NEC (P=0.015, r=0.578). ③ Compared with miR-146a-5p low expression, the number of children with intestinal resection length ≥5 cm in miR-146a-5p high expression was more (P=0.005).

Conclusion·The high expression of miR-146a-5p is related to the severity of intestinal injury in NEC, suggesting that miR-146a-5p is involved in the occurrence and development of NEC.

Key words: miR-146a-5p, necrotizing enterocolitis (NEC), intestinal injury

中图分类号:

R722.19

陈江龙, 陈砼, 吕志宝, 王雪莉, 盛庆丰. miR-146a-5p表达与新生儿坏死性小肠结肠炎肠道损伤严重程度的相关研究[J]. 上海交通大学学报(医学版), 2021, 41(9): 1147-1153.

Jiang-long CHEN, Tong CHEN, Zhi-bao LÜ, Xue-li WANG, Qing-feng SHENG. Study on the correlation between the expression of miR-146a-5p and the severity of intestinal injury in neonatal necrotizing enterocolitis[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(9): 1147-1153.

图/表 6

图1 2组小鼠末端回肠组织标本的H-E染色（×100）及组织病理评级

Fig 1 H-E staining (×100) and histopathological grade of terminal ileum tissue samples of mice in the two groups

图 2 原位杂交检测2组小鼠末端回肠组织标本中miR-146a-5p的表达Note：A. In situ hybridization results of miR-146a-5p (×400). Green fluorescence indicates miR-146a-5p stained by in situ hybridization, blue fluorescence indicates nucleus stained by DAPI, white arrow indicates miR-146a-5p and DAPI co-expression-positive cell. B. Statistical chart of miR-146a-5p-positive cells.

Fig 2 Expression of miR-146a-5p in terminal ileum tissue samples of mice in the two groups by in situ hybridization

图 3 3组患儿肠道组织标本的H-E染色（×100）

Fig 3 H-E staining of intestinal tissue samples in the three groups (×100)

图 4 原位杂交检测3组患儿肠道组织标本中miR-146a-5p的表达Note： A. In situ hybridization results of miR-146a-5p (×400). Green fluorescence indicates miR-146a-5p stained by in situ hybridization, blue fluorescence indicates nucleus stained by DAPI, white arrow indicates miR-146a-5p and DAPI co-expression-positive cell. B. Statistical chart of miR-146a-5p-positive cells.

Fig 4 Expression of miR-146a-5p in intestinal tissue samples of children in the three groups by in situ hybridization

图5 NEC患儿肠道组织标本中miR-146a-5p表达水平与组织病理评级的相关性分析

Fig 5 Correlation analysis between expression of miR-146a-5p and histopathological grade in intestinal tissues samples of children with NEC

表 1 NEC患儿肠道组织标本中miR-146a-5p高表达与低表达在不同临床资料中的分布比较

Tab 1 Comparison of high and low expression of miR-146a-5p in intestinal tissues samples of NEC children in different clinical data

Item	miR-146a-5p positive cells		P value
Item	<100	≥100	P value
Gender/n			0.395
Male	5	7
Female	1	4
Birth weight/n			0.339
<1 500 g	3	3
≥1 500 g	3	8
Gestational week/n			0.728
<32 weeks	5	9
≥32 weeks	1	2
Feeding method/n			0.353
Simple breast milk	1	0
Artificial or mixed feeding	5	11
Intestinal resection length/n			0.005
<5 cm	5	1
≥5 cm	1	10
NLR/n			0.555
<2	5	8
≥2	1	3
CRP/n			0.339
<10 mg·L^-1	3	3
≥10 mg·L^-1	3	8
Hospitalized time/n			0.373
<80 d	4	5
≥80 d	2	6
Prognosis/n			0.596
Death	1	1
Recurred or get better	5	10

参考文献 26

1	Stoll BJ, Hansen NI, Bell EF, et al. Trends in care practices, morbidity, and mortality of extremely preterm neonates, 1993-2012[J]. JAMA, 2015, 314(10): 1039-1051.
2	Fitzgibbons SC, Ching Y, Yu D, et al. Mortality of necrotizing enterocolitis expressed by birth weight categories[J]. J Pediatr Surg, 2009, 44(6): 1072-1075.
3	Moschino L, Duci M, Fascetti Leon F, et al. Optimizing nutritional strategies to prevent necrotizing enterocolitis and growth failure after bowel resection[J]. Nutrients, 2021, 13(2): 340.
4	Agnoni A, Lazaros Amendola C. Necrotizing enterocolitis: current concepts in practice[J]. JAAPA, 2017, 30(8): 16-21.
5	Ng PC, Chan KY, Leung KT, et al. Comparative miRNA expressional profiles and molecular networks in human small bowel tissues of necrotizing enterocolitis and spontaneous intestinal perforation[J]. PLoS One, 2015, 10(8): e0135737.
6	Wu YZ, Chan KYY, Leung KT, et al. Dysregulation of miR-431 and target gene FOXA1 in intestinal tissues of infants with necrotizing enterocolitis[J]. FASEB J, 2019, 33(4): 5143-5152.
7	Chen H, Zeng LC, Zheng W, et al. Increased expression of microRNA-141-3p improves necrotizing enterocolitis of neonates through targeting MNX1[J]. Front Pediatr, 2020, 8: 385.
8	Chassin C, Kocur M, Pott J, et al. miR-146a mediates protective innate immune tolerance in the neonate intestine[J]. Cell Host Microbe, 2010, 8(4): 358-368.
9	Saba R, Sorensen DL, Booth SA. MicroRNA-146a: a dominant, negative regulator of the innate immune response[J]. Front Immunol, 2014, 5: 578.
10	Chassin C, Hempel C, Stockinger S, et al. MicroRNA-146a-mediated downregulation of IRAK1 protects mouse and human small intestine against ischemia/reperfusion injury[J]. EMBO Mol Med, 2012, 4(12): 1308-1319.
11	Teodori L, Petrignani I, Giuliani A, et al. Inflamm-aging microRNAs may integrate signals from food and gut microbiota by modulating common signalling pathways[J]. Mech Ageing Dev, 2019, 182: 111127.
12	Caplan MS, Hedlund E, Adler L, et al. Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis[J]. Pediatr Pathol, 1994, 14(6): 1017-1028.
13	Barlow B, Santulli TV. Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model[J]. Surgery, 1975, 77(5): 687-690.
14	Nadler EP, Dickinson E, Knisely A, et al. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis[J]. J Surg Res, 2000, 92(1): 71-77.
15	Dvorak B, Halpern MD, Holubec H, et al. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model[J]. Am J Physiol Gastrointest Liver Physiol, 2002, 282(1): G156-G164.
16	Pisano C, Galley J, Elbahrawy M, et al. Human breast milk-derived extracellular vesicles in the protection against experimental necrotizing enterocolitis[J]. J Pediatr Surg, 2020, 55(1): 54-58.
17	Niemarkt HJ, de Meij TG, van de Velde ME, et al. Necrotizing enterocolitis: a clinical review on diagnostic biomarkers and the role of the intestinal microbiota[J]. Inflamm Bowel Dis, 2015, 21(2): 436-444.
18	Mohamed A, Shah PS. Transfusion associated necrotizing enterocolitis: a meta-analysis of observational data[J]. Pediatrics, 2012, 129(3): 529-540.
19	Ip S, Chung M, Raman G, et al. Breastfeeding and maternal and infant health outcomes in developed countries[J]. Evid Rep Technol Assess (Full Rep), 2007(153): 1-186.
20	Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis[J]. Lancet, 1990, 336(8730): 1519-1523.
21	Bazacliu C, Neu J. Necrotizing enterocolitis: long term complications[J]. Curr Pediatr Rev, 2019, 15(2): 115-124.
22	Papillon S, Castle SL, Gayer CP, et al. Necrotizing enterocolitis: contemporary management and outcomes[J]. Adv Pediatr, 2013, 60(1): 263-279.
23	Ghorpade DS, Sinha AY, Holla S, et al. NOD2-nitric oxide-responsive microRNA-146a activates sonic hedgehog signaling to orchestrate inflammatory responses in murine model of inflammatory bowel disease[J]. J Biol Chem, 2013, 288(46): 33037-33048.
24	Chen JL, Chen T, Zhou J, et al. miR-146a-5p mimic inhibits NLRP3 inflammasome downstream inflammatory factors and CLIC₄ in neonatal necrotizing enterocolitis[J]. Front Cell Dev Biol, 2020, 8: 594143.
25	He XM, Zheng YQ, Liu SZ, et al. miR-146a protects small intestine against ischemia/reperfusion injury by down-regulating TLR4/TRAF6/NF‑κB pathway[J]. J Cell Physiol, 2018, 233(3): 2476-2488.
26	Chen YH, Wu ZF, Yuan BY, et al. MicroRNA-146a-5p attenuates irradiation-induced and LPS-induced hepatic stellate cell activation and hepatocyte apoptosis through inhibition of TLR4 pathway[J]. Cell Death Dis, 2018, 9(2): 22.