沉默信息调节因子1在新生儿坏死性小肠结肠炎肠组织中的表达特点

doi:10.3969/j.issn.1674-8115.2021.09.004

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (9): 1154-1161.doi: 10.3969/j.issn.1674-8115.2021.09.004

• 新生儿外科专题 • 上一篇

沉默信息调节因子1在新生儿坏死性小肠结肠炎肠组织中的表达特点

陈锐¹^,²(), 赵云²^,³, 赵晓霞¹^,², 马东¹^,², 韩一江¹^,², 赖登明¹^,², 顾伟忠²^,³, 钭金法¹^,²()

^1.浙江大学医学院附属儿童医院新生儿外科，杭州 310052
^2.国家儿童健康与疾病临床医学研究中心，杭州 310052
^3.浙江大学医学院附属儿童医院病理科，杭州 310052

收稿日期:2021-03-19 出版日期:2021-08-24 发布日期:2021-08-24
通讯作者: 钭金法 E-mail:chenruiwz@zju.edu.cn;toujinfa@zju.edu.cn
作者简介:陈锐（1987—），男，主治医师，硕士；电子信箱：chenruiwz@zju.edu.cn。
基金资助:
浙江省自然科学基金青年项目(LQ19H030012)

Expression characteristics of silent information regulator transcript 1 in intestinal tissues of neonatal necrotizing enterocolitis

Rui CHEN¹^,²(), Yun ZHAO²^,³, Xiao-xia ZHAO¹^,², Dong MA¹^,², Yi-jiang HAN¹^,², Deng-ming LAI¹^,², Wei-zhong GU²^,³, Jin-fa TOU¹^,²()

^1.Department of Neonatal Surgery, The Children's Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China
^2.National Clinical Research Center for Child Health, Hangzhou 310052, China
^3.Department of Pathology, The Children's Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China

Received:2021-03-19 Online:2021-08-24 Published:2021-08-24
Contact: Jin-fa TOU E-mail:chenruiwz@zju.edu.cn;toujinfa@zju.edu.cn
Supported by:
Youth Program of Natural Science Foundation of Zhejiang Province(LQ19H030012)

摘要/Abstract

摘要：

目的·探究沉默信息调节因子1（silent information regulator transcript 1，SIRT1）在坏死性小肠结肠炎（necrotizing enterocolitis，NEC）肠管组织中的表达特点，初步探讨SIRT1对NEC的影响及可能机制。方法·纳入2018年6月—2020年10月经浙江大学医学院附属儿童医院新生儿外科手术治疗的NEC患儿80例，分为观察组和对照组。观察组组织样本为炎症坏死肠管；对照组为经保守治疗后发生肠狭窄，再次选择外科手术治疗的NEC患儿，组织样本为切缘肠管。收集2组患儿术前24 h内的血清中降钙素原（procalcitonin，PCT）、超敏C反应蛋白（hypersensitive C reactive protein，hs-CRP）、细胞因子［白细胞介素-2（interleukin-2，IL-2）、IL-4、IL-6、IL-10、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、γ干扰素（interferon-γ，IFN-γ）］以及性别、孕周等临床资料。采用免疫组织化学法检测2组患儿组织样本中的SIRT1、核因子κB（nuclear factor-κB，NF-κB）、转化生长因子β1（transforming growth factor β1，TGF-β1）和Smad3蛋白的表达水平。体外培养SD雌性大鼠小肠上皮IEC-6细胞，采用干扰小RNA（small interfering RNA，siRNA）技术抑制IEC-6细胞中的SIRT1表达后，使用蛋白质印迹检查NF-κB表达变化，通过Cell Counting Kit-8（CCK8）和Transwell迁移实验检测SIRT1对IEC-6增殖和迁移能力的影响。结果·2组患儿的性别、孕周、出生体质量和分娩方式等一般资料差异无统计学意义。与对照组相比，观察组术前24 h内血清hs-CRP、PCT、IL-6、IL-10升高（均P=0.000）。与对照组NEC肠狭窄切缘肠管组织相比，观察组NEC坏死肠管组织中的SIRT1呈低表达，NF-κB呈过表达（均P=0.000）。Smad3蛋白和TGF-β1蛋白表达2组差异无统计学意义。NEC坏死肠管组织中SIRT1与NF-κB表达呈负相关（r=-0.592，P=0.000）。抑制SIRT1的mRNA和蛋白表达后，IEC-6细胞中的NF-κB相对表达量升高，细胞的增殖能力和迁移能力明显降低。结论·SIRT1可能通过抑制NF-κB的表达来降低NEC中炎症因子的表达，从而减轻NEC的进展。另一个可能的机制是SIRT1通过保护肠上皮细胞，减少其凋亡和迁移，从而减轻NEC的进展。

关键词: 沉默信息调节因子1, 核因子κB, 坏死性小肠结肠炎, 炎症细胞因子, 新生儿

Abstract:

Objective·To explore the expression characteristics of silent information regulator transcript 1 (SIRT1) in intestinal tissues of necrotizing enterocolitis (NEC), and preliminarily discuss the effect and possible mechanisms of SIRT1 in NEC.

Methods·From June 2018 to October 2020, 80 children with NEC who were treated by neonatal surgery in the Children's Hospital, Zhejiang University School of Medicine were divided into observation group and control group. The tissue samples of the observation group were inflammatory necrotic intestinal tubes, while those of the control group were incised intestinal tubes. The NEC children in the control group were with intestinal strictures after conservative treatment, and then were treated by surgery again. The clinical data of the two groups were collected 24 h before surgery, including procalcitonin (PCT), hypersensitive C reactive protein (hs-CRP), cytokines [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)] in the serum, gender, gestational age and so on. The expression characteristics of SIRT1, nuclear factor-κB (NF-κB), transforming growth factor β1 (TGF-β1) and Smad3 proteins of the tissues in the two groups were detected by immunohistochemistry. IEC-6 cells of small intestinal epithelium of SD female rats were cultured in vitro. After SIRT1 expression in IEC-6 cells was inhibited by small interfering RNA (siRNA) technology, the protein expression of NF-κB was detected by Western blotting and the effects of SIRT1 on IEC-6 cells were detected by Cell Counting Kit-8 (CCK8) and Transwell migration assay.

Results·There was no significant difference in gender, gestational age, birth weight and delivery mode between the two groups. Compared with the control group, the levels of hs-CRP, PCT, IL-6 and IL-10 in the serum in the observation group increased within 24 h before surgery (all P=0.000).Compared with the margin tissues of narrow intestines in the control group, the positive expression of SIRT1 in NEC necrotic intestinal tissues in the observation group showed low expression, and the positive expression of NF-κB was overexpressed (both P=0.000). There was no significant difference in the expression of Smad3 and TGF-β1 between the two groups. The expression of SIRT1 in NEC necrotic intestinal tissue was negatively correlated with the expression of NF-κB (r=-0.592, P=0.000). After inhibiting the mRNA and protein expression of SIRT1, the relative expression of NF-κB in IEC-6 cells was increased, and the proliferation and migration ability of the cells was significantly decreased.

Conclusion·The mechanism for the SIRT1 reducing progression of NEC may be that SIRT1 can inhibit the expression of NF-κB to reduce the expression of inflammatory factors in NEC, and another possible mechanism may be that SIRT1 can protect intestinal epithelial cells by promoting cell proliferation and migration.

Key words: silencing information regulator factor 1 (SIRT1), nuclear factor-κB (NF-κB), necrotizing enterocolitis (NEC), inflammatory cytokine, neonate

中图分类号:

R722.1

陈锐, 赵云, 赵晓霞, 马东, 韩一江, 赖登明, 顾伟忠, 钭金法. 沉默信息调节因子1在新生儿坏死性小肠结肠炎肠组织中的表达特点[J]. 上海交通大学学报(医学版), 2021, 41(9): 1154-1161.

Rui CHEN, Yun ZHAO, Xiao-xia ZHAO, Dong MA, Yi-jiang HAN, Deng-ming LAI, Wei-zhong GU, Jin-fa TOU. Expression characteristics of silent information regulator transcript 1 in intestinal tissues of neonatal necrotizing enterocolitis[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(9): 1154-1161.

图/表 7

表1 2组患儿的一般资料比较

Tab 1 Comparison of the general data between the two groups

Item	Control group （n=40）	Obeservation group （n=40）	χ²/t value	P value
Gender/n			0.202	0.653
Male	23	21
Female	17	19
Gestational age/n			0.139	0.709
Premature infant（<37 week）	37	35
Term infant（≥37 week）	3	5
Birth weight/g
Premature infant	1 614.1±477.1	1 689.0±493.6	0.109	0.913
Term infant	2 985.0±551.2	3 181.0±583.7	0.224	0.830
Delivery mode/n			0.058	0.809
Vaginal delivery	12	13
Cesarean section	28	27

图1 2组患儿肠管组织的术中照片和H-E染色（×50）Note： A. Intraoperative photograph of inflammatory and necrotic bowel in the observation group. B. Intraoperative photograph of intestinal stenosis after conservatively treatment in the control group. C. H-E staining of inflammatory and necrotic bowel in the observation group. D. H-E staining of margin intestinal tissue after conservatively treatment in the control group.

Fig 1 Intraoperative photographs and H-E staining images of intestinal tissues in the two groups (×50)

表2 2组患儿血清中炎症因子表达水平比较

Tab 2 Comparison of expression levels of inflammatory factors in serum of the two groups

Inflammatory factor	Control group （n=40）	Obeservation group （n=40）	U value	P value
hs-CRP/（mg·L^-1）	1.00 （0.50， 2.00）	58.91 （32.74， 107.40）	89.50	0.000
PCT/（ng·mL^-1）	0.25 （0.22， 0.46）	10.25 （6.51， 17.93）	0.00	0.000
IL-2/（pg·mL^-1）	4.70 （2.98， 6.80）	4.55 （3.43， 6.53）	796.00	0.971
IL-4/（pg·mL^-1）	2.15 （1.59， 2.66）	2.29 （1.60， 2.75）	730.00	0.504
IL-6/（pg·mL^-1）	11.85 （9.75， 25.03）	719.30 （156.30， 1 555.00）	20.00	0.000
IL-10/（pg·mL^-1）	3.20 （2.20， 4.95）	23.60 （10.15， 51.08）	0.00	0.000
TNF-α/（pg·mL^－1）	3.09 （2.44， 3.98）	3.27 （2.44， 3.94）	786.00	0.895
IFN-γ/（pg·mL^-1）	3.50 （2.45， 6.93）	5.70 （2.80， 10.27）	614.00	0.074

图2 光学显微镜下观察SIRT1、NF-κB、TGF-β1和Smad3在2组间的表达（×200）Note： The arrows in the image show the positive expression of SIRT1, NF-κB, Samd3 and TGF-β1 in the intestine tissus, respectively.

Fig 2 Expression of SIRT1, NF-κB, TGF-β1 and Smad3 in the two groups by optical microscope (×200)

表3 SIRT1、NF-κB、TGF-β1和Smad3在2组间的表达差异

Tab 3 Differential expression of SIRT1， NF-κB， TGF-β1 and Smad3 in the two groups

Protein	Control group （n=40）	Observation group （n=40）	χ² value	P value
SIRT1/n			33.885	0.000
+	34	8
-	6	32
NF-κB/n			24.444	0.000
+	11	33
-	29	7
Smad3/n			0.238	0.626
+	27	29
-	13	11
TGF-β1/n			1.067	0.302
+	8	12
-	32	28

图3 IEC-6细胞中SIRT1蛋白与NF-κB蛋白的表达关系Note：A. siRNA-Sirt1 sequence screening. B. Expression of SIRT1 and NF-κB in IEC-6 cells. C. Statistical analysis of relative expression of SIRT1 and NF-κB in IEC-6 cells. ①P=0.000, compared with the NC group; ②P=0.001, ③P=0.002, compared with the NC group.

Fig 3 Relationship between the expression of SIRT1 protein and NF-κB protein in IEC-6 cells

图4 SIRT1对大肠上皮细胞的增殖和迁移能力的影响Note： A. Inhibition of the expression of SIRT1 can reduce the proliferation of IEC-6 cells. B. Statistical analysis of the number of IEC-6 cells migration after inhibiting SIRT1 expression. C. IEC-6 cells migration after inhibiting SIRT1 expression under fluorescence microscope (×200). ①P=0.000, compared with the NC group.

Fig 4 Effects of SIRT1 on the proliferation and migration of intestinal epithelial cells

参考文献 23

1	Niño DF, Sodhi CP, Hackam DJ. Necrotizing enterocolitis: new insights into pathogenesis and mechanisms[J]. Nat Rev Gastroenterol Hepatol, 2016, 13(10): 590-600.
2	Cho SX, Berger PJ, Nold-Petry CA, et al. The immunological landscape in necrotising enterocolitis[J]. Expert Rev Mol Med, 2016, 18: e12.
3	Cho SX, Rudloff I, Lao JC, et al. Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities[J]. Nat Commun, 2020, 11(1): 5794.
4	Agakidou E, Agakidis C, Gika H, et al. Emerging biomarkers for prediction and early diagnosis of necrotizing enterocolitis in the era of metabolomics and proteomics[J]. Front Pediatr, 2020, 8: 602255.
5	Chadha S, Wang LQ, Hancock WW, et al. Sirtuin-1 in immunotherapy: a Janus-headed target[J]. J Leukoc Biol, 2019, 106(2): 337-343.
6	施诚仁, 金先庆, 李仲智. 小儿外科学[M]. 4版. 北京: 人民卫生出版社, 2009: 309-312.
7	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.
8	窦敏, 郑英霞, 韩丽, 等. PRMT4在胃癌发生和发展中的作用和机制研究[J]. 上海交通大学学报(医学版), 2020, 40(5): 609-618.
9	Denning NL, Prince JM. Neonatal intestinal dysbiosis in necrotizing enterocolitis[J]. Mol Med, 2018, 24: 4.
10	Hackam DJ, Sodhi CP. Toll-like receptor-mediated intestinal inflammatory imbalance in the pathogenesis of necrotizing enterocolitis[J]. Cell Mol Gastroenterol Hepatol, 2018, 6(2): 229-238.e1.
11	Mihi B, Good M. Impact of Toll-like receptor 4 signaling in necrotizing enterocolitis: the state of the science[J]. Clin Perinatol, 2019, 46(1): 145-157.
12	Lan KC, Chao SC, Wu HY, et al. Salidroside ameliorates sepsis-induced acute lung injury and mortality via downregulating NF‑κB and HMGB1 pathways through the upregulation of SIRT1[J]. Sci Rep, 2017, 7(1): 12026.
13	Li GQ, Xia ZB, Liu Y, et al. SIRT1 inhibits rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and inflammatory response via suppressing NF-κB pathway[J]. Biosci Rep, 2018, 38(3): BSR20180541.
14	Wang L, Wang MM, Dou HJ, et al. Sirtuin 1 inhibits lipopolysaccharide-induced inflammation in chronic myelogenous leukemia k562 cells through interacting with the Toll-like receptor 4-nuclear factor κB-reactive oxygen species signaling axis[J]. Cancer Cell Int, 2020, 20: 73.
15	Mishra M, Duraisamy AJ, Kowluru RA. Sirt1: a guardian of the development of diabetic retinopathy[J]. Diabetes, 2018, 67(4): 745-754.
16	郑文香, 丛立春, 韩斌. 沉默信息调节因子1在具核梭杆菌诱导肠上皮细胞炎症和凋亡中的作用[J]. 中国病原生物学杂志, 2019, 14(6): 643-649, 655.
17	尹海朦, 何鑫, 单颖, 等. 沉默信息调节因子1促进鼻咽癌增殖、迁移和脂质代谢机制的研究[J]. 中华耳鼻咽喉头颈外科杂志, 2020, 55(10): 934-943.
18	Wellman AS, Metukuri MR, Kazgan N, et al. Intestinal epithelial Sirtuin 1 regulates intestinal inflammation during aging in mice by altering the intestinal microbiota[J]. Gastroenterology, 2017, 153(3): 772-786.
19	Markel TA, Martin CA, Chaaban H, et al. New directions in necrotizing enterocolitis with early-stage investigators[J]. Pediatr Res, 2020, 88(): 35-40.
20	Zhang WJ, Zhang YY, Guo XH, et al. Sirt1 protects endothelial cells against LPS-induced barrier dysfunction[J]. Oxid Med Cell Longev, 2017, 2017: 4082102.
21	Hodzic Z, Bolock AM, Good M. The role of mucosal immunity in the pathogenesis of necrotizing enterocolitis[J]. Front Pediatr, 2017, 5: 40.
22	Burge K, Bergner E, Gunasekaran A, et al. The Role of glycosaminoglycans in protection from neonatal necrotizing enterocolitis: a narrative review[J]. Nutrients, 2020, 12(2): 546.
23	Hansen LW, Khader A, Yang WL, et al. Sirtuin 1 activator SRT1720 protects against organ injury induced by intestinal ischemia-reperfusion[J]. Shock, 2016, 45(4): 359-366.

[1]	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.
[2]	谢雨婕, 谢利娟, 朱天闻, 王依闻. 白介素-10受体A基因突变致新生儿极早发炎性肠病2例[J]. 上海交通大学学报(医学版), 2021, 41(3): 409-412.
[3]	和斌，李祺越，洪岭，伍园园，滕晓明，唐传玲. SIRT1对H2O2诱导的人卵巢颗粒细胞氧化应激损伤的影响[J]. 上海交通大学学报(医学版), 2020, 40(12): 1591-1597.
[4]	童兴瑜，尤纱纱，曹慧敏，何斌. 湿化高流量鼻导管通气治疗儿科患者的临床研究进展[J]. 上海交通大学学报（医学版）, 2018, 38(5): 565-.
[5]	周卓超，陈颖，朱书仪，沈雯琦，周爱武. Z 突变 α1- 抗胰蛋白酶缺乏症的研究进展#br#[J]. 上海交通大学学报（医学版）, 2017, 37(8): 1179-.
[6]	贾思，易正辉 . 改良电休克治疗在妊娠期精神疾病中应用的研究进展[J]. 上海交通大学学报（医学版）, 2017, 37(7): 1038-.
[7]	盛王涛，方旭华，李菁，孙建华，贝斐，张国庆 . 新生儿重度高胆红素血症与听力损伤的相关性研究#br#[J]. 上海交通大学学报（医学版）, 2017, 37(10): 1383-.
[8]	雷一慧，沈海青，何雪梅，等. 乳凝集素对新生大鼠坏死性小肠结肠炎肠道损伤修复和TLR4表达的影响[J]. 上海交通大学学报（医学版）, 2015, 35(7): 967-.
[9]	郭静，许洪平，姚静，等. 上海市新生儿葡萄糖-6-磷酸脱氢酶缺乏症筛查及临界值确立[J]. 上海交通大学学报（医学版）, 2015, 35(4): 618-.
[10]	朱泓，杨祖菁，惠宁，等. 孕期补锌对母胎汞毒性的拮抗作用及其抗氧化损伤机制研究[J]. 上海交通大学学报（医学版）, 2014, 34(2): 193-.
[11]	黄海源，潘兴寿，黄显南，等. 阿托伐他汀对高血压合并血脂异常患者的疗效及对机体炎症反应的影响[J]. 上海交通大学学报（医学版）, 2014, 34(11): 1642-.
[12]	阮莉莉，杜俊君，林艳，等. CD64指数在新生儿脓毒症早期诊断中的意义[J]. 上海交通大学学报（医学版）, 2014, 34(10): 1503-.
[13]	朱天闻，张永红，陈妍，等. 晚期早产儿与足月新生儿呼吸衰竭发生及预后的影响因素[J]. 上海交通大学学报（医学版）, 2013, 33(7): 920-.
[14]	李华君，朱建幸, 谢利娟. 胸膜腔内注入红霉素治疗新生儿乳糜胸的疗效分析[J]. 上海交通大学学报（医学版）, 2013, 33(7): 936-.
[15]	颜崇兵，裘刚，龚小慧，等. 新生儿科鲍曼不动杆菌感染现状及舒巴坦治疗的评价[J]. 上海交通大学学报（医学版）, 2013, 33(7): 940-.

沉默信息调节因子1在新生儿坏死性小肠结肠炎肠组织中的表达特点

Expression characteristics of silent information regulator transcript 1 in intestinal tissues of neonatal necrotizing enterocolitis

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 23

相关文章 15

编辑推荐

Metrics