维生素A对大豆7S球蛋白致仔猪肠上皮细胞屏障功能损伤的影响

doi:10.3969/j.issn.1004-1524.2021.11.04

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (11): 2026-2033.DOI: 10.3969/j.issn.1004-1524.2021.11.04

维生素A对大豆7S球蛋白致仔猪肠上皮细胞屏障功能损伤的影响

杨菊(), 邓俊良^*(), 夏江英, 宋天浩, 庞莲凤, 任志华

四川农业大学动物医学院/动物疫病与人类健康四川省重点实验室/环境公害与动物疾病四川省高校重点实验室,四川成都 611130

收稿日期:2021-01-08 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 邓俊良
作者简介:*邓俊良,E-mail: dengjl213@126.com
杨菊(1996—),女,四川梓潼人,硕士研究生,研究方向为动物营养代谢病。E-mail: 1481820037@qq.com
基金资助:
国家“十三五”重点研发计划(2016YFD0501204)

Effect of vitamin A on barrier function damage of piglet intestinal epithelial cells induced by soybean 7S globulin

YANG Ju(), DENG Junliang^*(), XIA Jiangying, SONG Tianhao, PANG Lianfeng, REN Zhihua

College of Veterinary Medicine/Key Laboratory of Animal Disease and Human Health of Sichuan Province/Key Laboratory of Environmental Hazards and Animal Diseases of Sichuan Province Colleges and Universities, Sichuan Agriculture University, Chengdu 611130, China

Received:2021-01-08 Online:2021-11-25 Published:2021-11-26
Contact: DENG Junliang

摘要/Abstract

摘要：

以仔猪肠上皮细胞(IPEC-J2)为研究对象,用大豆7S球蛋白作为诱导因素,通过检测细胞活力、细胞跨膜电阻(TEER)、荧光素钠渗透率、细胞膜完整性相关指标与紧密连接蛋白的mRNA表达情况,探究维生素A(V_A)对大豆7S球蛋白致IPEC-J2细胞屏障功能损伤的影响。结果显示:5.0 mg·mL^-1大豆7S球蛋白导致IPEC-J2活力、TEER与ZO-1、Claudin-1、Occludin的mRNA表达量极显著(P<0.01)降低,而荧光素钠渗透率和细胞培养液中碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)、二胺氧化酶(DAO)、肠型脂肪酸结合蛋白1(IFABP1)的含量极显著(P<0.01)升高;添加0.1和1 μmol·L^-1 V_A极显著提高了细胞活力(P<0.01),0.1~10 μmol·L^-1 V_A显著(P<0.05)缓解了大豆7S球蛋白导致的TEER、荧光素钠渗透率、细胞膜完整性相关指标与紧密连接蛋白mRNA表达的变化;10 000 μmol·L^-1 V_A反而加剧了这些影响。总体上,0.1~10 μmol·L^-1 V_A能通过保护细胞活力和细胞完整性、影响紧密连接蛋白的mRNA表达对大豆7S球蛋白导致的IPEC-J2细胞屏障功能损伤发挥保护作用,而10 000 μmol·L^-1 V_A反而加重了这种损伤。

关键词: 大豆7S球蛋白, 维生素A, 仔猪空肠上皮细胞, 通透性, 紧密连接蛋白

Abstract:

To explore effect of vitamin A (V_A) on barrier function damage of piglet intestinal epithelial cells (IPEC-J2) induced by soybean 7S globulin, took IPEC-J2 as the research object and soybean 7S globulin as the inducing factor, cell viability, trans-epithelial electrical resistance (TEER), permeability of sodium fluorescein, cell membrane integrity related indicators and tight junction protein mRNA expression were assessed. The results showed that 5 mg·mL^-1 soybean 7S globulin caused IPEC-J2 viability, TEER, ZO-1, Claudin-1 and Occludin mRNA expression significantly reduced (P<0.01), and permeability of fluorescein sodium, content of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), diamine oxidase (DAO) and intestinal fatty acid binding protein 1 (IFABP1) in the cell culture fluid significantly increased (P<0.01). 0.1 and 1 μmol·L ^-1 V_A could significantly protect cell viability (P<0.01), 0.1-10 μmol·L^-1 V_A could significantly (P<0.05) alleviate the changes of TEER, fluorescein sodium permeability, cell membrane integrity related indicators and tight junction protein mRNA expression caused by 7S globulin; 10 000 μmol·L^-1 V_A exacerbated these effects. The results indicated that 0.1-10 μmol·L^-1 V_A could protect the cell barrier function of IPEC-J2 caused by soybean 7S globulin through protecting cell viability and cell integrity, and affecting expression of tight junction protein mRNA. However, 10 000 μmol·L^-1 V_A aggravated this damage.

Key words: soybean 7S globulin, vitamin A, piglet intestinal epithelial cell, permeability, tight junction protein

中图分类号:

S852.4

杨菊, 邓俊良, 夏江英, 宋天浩, 庞莲凤, 任志华. 维生素A对大豆7S球蛋白致仔猪肠上皮细胞屏障功能损伤的影响[J]. 浙江农业学报, 2021, 33(11): 2026-2033.

YANG Ju, DENG Junliang, XIA Jiangying, SONG Tianhao, PANG Lianfeng, REN Zhihua. Effect of vitamin A on barrier function damage of piglet intestinal epithelial cells induced by soybean 7S globulin[J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2026-2033.

图/表 7

表1 荧光定量PCR引物

Table 1 Primers for amplification of genes by qRT-PCR

基因名称 Gene name	基因ID Gene ID	上游引物序列(5'-3') Forward primer sequence (5'-3')	下游引物序列(5'-3') Reverse primer sequence (5'-3')	产物大小 Product size/bp
ZO-1	396567	CCAGGGAGAGAAGTGCCAGTAGG	TTTGGTGGGTTTGGTGGGTTGAC	92
Claudin-1	100625166	CCATCGTCAGCACCGCACTG	CGACACGCAGGACATCCACAG	107
Occludin	397236	TGGCTGCCTTCTGCTTCATTGC	GAACACCATCACACCCAGGATAGC	130
β-actin	414396	GGCACCACACCTTCTACAACGAG	TCATCTTCTCACGGTTGGCTTTGG	102

图1 大豆7S球蛋白诱导下VA对IPEC-J2活力的影响柱形图中无相同大写字母表示差异极显著(P<0.01),无相同小写字母表示差异显著(P<0.05)。下同。

Fig.1 Effect of VA on viability of IPEC-J2 injured by soybean 7S globulin Data on the bars marked without the same uppercase letter indicated significant differences at P<0.01, data on the bars marked without the same lowercase letter indicated significant differences at P<0.05. The same as below.

图2 IPEC-J2的细胞跨膜电阻

Fig.2 Trans-epithelial electrical resistance of IPEC-J2

图3 大豆7S球蛋白诱导下VA对IPEC-J2单层细胞细胞跨膜电阻的作用

Fig.3 Effect of VA on trans-epithelial electrical resistance of IPEC-J2 monolayer cells induced by soybean 7S globulin

图4 大豆7S球蛋白诱导下VA对IPEC-J2单层细胞荧光素钠渗透率的影响

Fig.4 Effect of VA on permeability of sodium fluorescein of IPEC-J2 monolayer cells induced by soybean 7S globulin

图5 大豆7S球蛋白诱导下VA对IPEC-J2细胞膜完整性的影响

Fig.5 Effect of VA on destruction of IPEC-J2 integrity by soybean 7S globulin

图6 大豆7S球蛋白诱导下VA对IPEC-J2紧密连接蛋白基因表达的影响

Fig.6 Effect of VA on expression of tight junction protein genes in IPEC-J2 induced by soybean 7S globulin

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维生素A对大豆7S球蛋白致仔猪肠上皮细胞屏障功能损伤的影响

Effect of vitamin A on barrier function damage of piglet intestinal epithelial cells induced by soybean 7S globulin

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