褪黑素缓解空肠黏膜上皮细胞氧化损伤的效果研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0190

生物技术通报 ›› 2023, Vol. 39 ›› Issue (9): 291-299.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0190

褪黑素缓解空肠黏膜上皮细胞氧化损伤的效果研究

康凌云¹^,²(), 韩露露², 韩德平³, 陈建胜⁴, 甘瀚凌⁴, 邢凯⁴, 马友记¹(), 崔凯²()

1.甘肃农业大学动物科学技术学院，兰州 730070
2.中国农业科学院饲料研究所，农业农村部饲料生物技术重点实验室，北京 100081
3.中国农业大学动物医学院，北京 100193
4.北京农学院动物科学技术学院，北京 102206

收稿日期:2023-03-03 出版日期:2023-09-26 发布日期:2023-10-24
通讯作者: 马友记，男，博士，教授，研究方向：绵羊分子育种与生产；E-mail: yjma@gsau.edu.cn；
崔凯，男，博士，副研究员，研究方向：反刍动物营养与饲料科学；E-mail: cuikai@caas.com
作者简介:康凌云，男，硕士研究生，研究方向：E-mail: 18821622269@163.com
基金资助:
国家自然科学基金面上项目(32172764);北京市2020年高水平人才交叉培养“实培计划”项目

Effect of Melatonin on Protecting the Jejunum Mucosal Epithelial Cells from Oxidative Stress Damage

KANG Ling-yun¹^,²(), HAN Lu-lu², HAN De-ping³, CHEN Jian-sheng⁴, GAN Han-ling⁴, XING Kai⁴, MA You-ji¹(), CUI Kai²()

1. College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070
2. Key Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, China, Institute of Feed Research of Chinese Academy of Agricultural Sciences, Beijing 100081
3. College of Veterinary Medicine, China Agricultural University, Beijing 100193
4. College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206

Received:2023-03-03 Published:2023-09-26 Online:2023-10-24

摘要/Abstract

摘要：

肠道黏膜氧化损伤与畜禽的生长发育和腹泻等疾病的发生密切相关，而褪黑素作为一种吲哚胺类化合物，具有明显抗氧化保护作用。为探究褪黑素对肠黏膜上皮细胞氧化损伤时的保护作用，本研究选取原代小鼠空肠黏膜上皮细胞，利用硫酸亚铁和过氧化氢处理建立氧化损伤模型，并通过添加不同浓度的褪黑素预处理上皮细胞，检测细胞损伤、氧化产物、抗氧化酶和炎性相关细胞因子的表达变化。结果发现，硫酸亚铁和过氧化氢处理后，黏膜上皮细胞明显受损，丙二醛含量显著增加、抗氧化酶表达水平降低。而褪黑素能明显减轻细胞氧化损伤程度、抑制丙二醛的产生并增加抗氧化酶的表达。同时，褪黑素还能显著减少炎性因子白介素-6的表达，并增加趋化因子白介素-8的表达。因此，褪黑素可以缓解自由基增加诱发的空肠黏膜上皮细胞氧化损伤，并能够减轻细胞损伤引起的炎症反应。

关键词: 褪黑素, 氧化应激, 抗氧化, 黏膜免疫

Abstract:

The oxidative injury of intestinal mucosa is strongly associated with the development and intestinal diseases such as diarrhea for animals. Melatonin（N-acetyl-5-methoxy-tryptamine）, as an indoleamine neurohormone, has important role in the processes of antioxidant protection. To investigate the protective effect of melatonin on the oxidative injury to the mucosal epithelial cells, the primary mouse jejunum mucosal epithelial cells were isolated and exposed to the FeSO₄and H₂O₂ to establish the oxidative damage model. After adding different doses of melatonin to the epithelial cells, we observed the cells’ pathological changes and then detected the changes of oxidative products, antioxidant enzymes, and inflammatory-related cytokines. It is found that after treatment with FeSO₄and H₂O₂, the mucosal epithelial cells programmed severe injury with significant increase of malonaldehyde and down-regulation of antioxidant enzymes. When adding melatonin to epithelial cells, the significantly-decreased malonaldehyde and highly-expressed antioxidant enzymes were detected. Meanwhile, melatonin also significantly reduced the expression of pro-inflammatory factor interleukin-6, and increased the expression of chemotactic factor interleukin-8. Therefore, melatonin may alleviate the oxidative damage of mucosal epithelial cells of jejunum caused by the increase of free radicals, and reduce the inflammatory response induced by oxidative damage.

Key words: melatonin, oxidative stress, antioxidant, mucosal immunity

康凌云, 韩露露, 韩德平, 陈建胜, 甘瀚凌, 邢凯, 马友记, 崔凯. 褪黑素缓解空肠黏膜上皮细胞氧化损伤的效果研究[J]. 生物技术通报, 2023, 39(9): 291-299.

KANG Ling-yun, HAN Lu-lu, HAN De-ping, CHEN Jian-sheng, GAN Han-ling, XING Kai, MA You-ji, CUI Kai. Effect of Melatonin on Protecting the Jejunum Mucosal Epithelial Cells from Oxidative Stress Damage[J]. Biotechnology Bulletin, 2023, 39(9): 291-299.

图/表 7

表1 引物序列信息

Table 1 Primer sequence information

基因Gene	登录号GenBank ID	引物序列Primer sequence（5'-3'）	产物长度Product length/bp
TNF-α	NM_001024860.1	F: GAGAAGATTCGTGGGCTGAT	169
TNF-α	NM_001024860.1	R: CCTTGTTCTTGTCCTGTGGG	169
IL-1β	NM_001009465.2	F: TGATGGCTTATTACAGTGGCAA	183
IL-1β	NM_001009465.2	R: CCATGGCCACAACAACTGAC	183
IL6	NM_001009392.1	F: CCTTCGGTCCAGTTGCCTTCT	234
IL6	NM_001009392.1	R: CCAGTGCCTCTTTGCTGCTTTC	234
β-actin	NM_0010009784.3	F: GATTCCTATGTGGGCGACGA	229
β-actin	NM_0010009784.3	R: AGGTCTCAAACATGATCTGGGT	229

图1 空肠黏膜上皮细胞形态及CK-18免疫荧光染色

Fig.1 Jejunum epithelial cell morphology and CK-18 immunofluorescence staining

图2 FeSO4/H2O2处理可明显引起黏膜上皮细胞氧化损伤不同字母代表差异显著（P<0.05），下同

Fig. 2 FeSO4/H2O2 treatment significantly causing the oxidative damage to mucosal epithelial cells pifevent letters indicate significant dlifference at（P<0.05）level, the same below

图3 褪黑素处理可明显缓解黏膜上皮细胞的氧化损伤

Fig. 3 Melatonin treatment significantly ameliorating the oxidative damage to mucosal epithelial cells

表2 不同浓度褪黑素预处理对黏膜上皮细胞抗氧化因子的表达影响

Table 2 Effects of pretreatment with different concentrations of melatonin on the expressions of antioxidant factors in mucosal epithelial cells

抗氧化因子 Antioxidant factor	不同处理组 Treatment groups							SEM	P
抗氧化因子 Antioxidant factor	CON	FeSO₄/H₂O₂	MT0.01	MT0.1	MT1.0	MT5.0	MT10	SEM	P
MDA	4.31^bc	5.21^a	4.37^bc	4.17^c	4.37^bc	4.57^b	4.57^b	0.099	<0.0001
LDH	3.74^a	3.88^a	3.14^b	3.44^ab	3.08^b	3.85^a	3.63^a	0.132	0.0011
SOD	1.94^ab	1.90^b	1.90^b	1.97^ab	1.94^ab	1.97^ab	2.00^a	0.022	0.068
GSH-Px	3.76^bc	3.60^c	3.96^ab	4.09^ab	4.25^a	4.12^ab	4.34^a	0.104	0.002
CAT	216.58^a	167.22^b	165.83^b	174.87^b	163.45^b	179.04^b	169.65^b	6.008	<0.01

图4 褪黑素调节黏膜上皮细胞氧化损伤时IL-6和IL-8的表达

Fig. 4 Melatonin regulating the expressions of IL-6 and IL-8 during oxidative injury of mucosal epithelial cells

图5 褪黑素调节粘膜上皮细胞氧化损伤时TNF-α、IL-1β及IL-6的mRNA表达水平

Fig. 5 Melatonin regulateing the mRNA expression of TNF-α, IL-1β and IL-6 during oxidative injury of mucosal epithelial cells

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褪黑素缓解空肠黏膜上皮细胞氧化损伤的效果研究

Effect of Melatonin on Protecting the Jejunum Mucosal Epithelial Cells from Oxidative Stress Damage

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