维生素C对β-伴大豆球蛋白诱导的仔猪肠上皮细胞炎性损伤的保护作用

doi:10.3969/j.issn.1004-1524.2021.11.03

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (11): 2017-2025.DOI: 10.3969/j.issn.1004-1524.2021.11.03

维生素C对β-伴大豆球蛋白诱导的仔猪肠上皮细胞炎性损伤的保护作用

夏江英(), 杨菊, 宋天浩, 庞莲凤, 叶婷, 任志华, 邓俊良^*()

四川农业大学动物医学院,四川成都 611130

收稿日期:2020-12-29 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
夏江英(1995—),女,四川南充人,硕士研究生,研究方向为动物营养代谢病。E-mail: 962268356@qq.com
基金资助:
国家“十三五”重点研发计划(2016YFD0501204)

Protective effect of vitamin C in piglet intestinal epithelial cells inflammatory injury induced by β-conglycinin

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

College of Veterinary Medicine, Sichuan Agricultural University,Chengdu 611130, China

Received:2020-12-29 Online:2021-11-25 Published:2021-11-26
Contact: DENG Junliang

摘要/Abstract

摘要：

为分析不同浓度维生素C(vitamin C,V_C)对β-伴大豆球蛋白(7S)诱导的仔猪肠上皮细胞(IPEC-J2)炎性损伤的保护作用,取对数生长期的IPEC-J2用于试验,随机分为对照组、7S模型组(5 mg·mL^-1 7S)和V_C(25、50、100、200、400、600、800、1 000 μmol·L^-1)保护组。细胞培养24 h后采用CCK-8法检测细胞存活率,倒置显微镜观察细胞形态学变化,ELISA法检测细胞上清液中LDH、ALP、DAO、IFABP1含量和IL-1β、IL-6、TNF-α、IL-4、IL-10的分泌水平,用qRT-PCR法检测IL-1β、IL-6、TNF-α、IL-4和IL-10 mRNA相对表达量。结果显示:7S可显著(P<0.01)降低IPEC-J2活力、破坏细胞膜完整性,上调细胞促炎性因子和下调抗炎因子的产生;与7S模型组相比,同时添加7S和V_C的试验组细胞活力增加,细胞上清液中LDH、ALP、DAO、IFABP1含量显著(P<0.01)降低,促炎因子IL-1β、IL-6、TNF-α分泌水平降低,抗炎因子IL-4和IL-10的分泌水平升高。因此,不同浓度V_C均可保护由7S诱导引起的仔猪肠上皮细胞损伤,100 μmol·L^-1 V_C的修复和保护作用最佳。

关键词: 维生素C, β-伴大豆球蛋白, 仔猪肠上皮细胞, 膜完整性, 细胞因子

Abstract:

To investigate protective effects of different concentrations of vitamin C (V_C) on inflammatory injury of piglet intestinal epithelial cells (IPEC-J2) induced by β-conglycinin (7S). IPEC-J2 in logarithmic growth phase were used for test. The cells were randomly divided into control group, 7S model group (5 mg·mL^-1 7S), and V_C(25, 50, 100, 200, 400, 600, 800, 1 000 μmol·L^-1)protected groups.After cells were cultured for 24 h, cell viability was assessed by CCK-8 assay and morphological changes of cells were observed by an inverted microscope. Contents of LDH, ALP, DAO, IFABP1, IL-1β, IL-6, TNF-α, IL-4 and IL-10 in the cell supernatant were detected by ELISA assay. The relative expression of IL-1β, IL-6, TNF-α,IL-4 and IL-10 mRNA were detected by qRT-PCR. The results showed that 7S significantly (P<0.01) reduced the viability of IPEC-J2 and destroyed the integrity of cell membranes. 7S also elevated pro-inflammatory and decreased anti-inflammatory factors in cells.Compared with 7S model group, cell viability in the test group added with 7S and V_C at the same time was significantly increased, and contents of LDH, ALP, DAO, IFABP1 in cell supernatant were significantly (P<0.01) reduced. Compared with 7S model group, V_C protection groups reduced the secretion levels of cell pro-inflammatory factors IL-1β, IL-6 and TNF-α, and increased the secretion levels of anti-inflammatory factors IL-4 and IL-10. Therefore, various doses of V_C protected against intestinal epithelial cell injury in piglets induced by 7S, and 100 μmol·L^-1 V_C had the best repair and protection effect on 7S injured IPEC-J2.

Key words: vitamin C, β-conglycinin, piglet intestinal epithelial cells, membrane integrity, cytokines

中图分类号:

S852.4

夏江英, 杨菊, 宋天浩, 庞莲凤, 叶婷, 任志华, 邓俊良. 维生素C对β-伴大豆球蛋白诱导的仔猪肠上皮细胞炎性损伤的保护作用[J]. 浙江农业学报, 2021, 33(11): 2017-2025.

XIA Jiangying, YANG Ju, SONG Tianhao, PANG Lianfeng, YE Ting, REN Zhihua, DENG Junliang. Protective effect of vitamin C in piglet intestinal epithelial cells inflammatory injury induced by β-conglycinin[J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2017-2025.

图/表 8

表1 各基因real-time PCR引物序列

Table 1 Primer sequence of genes for real-time PCR

基因 Gene	登录号 Accession number	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
IL-1β	NM_001302388.2	GAAGGCTCTCCACCTCCTCACAG	TTGTCATCGCTGTCATCTCCTTGC	87
IL-6	NM_214399.1	TGCAGTCACAGAACGAGTGG	CAGGTGCCCCAGCTACATTAT	116
TNF-α	NM_214022.1	GCACTGAGAGCATGATCCGAGAC	CGACCAGGAGGAAGGAGAAGAGG	120
IL-4	NM_214123.1	TCGGCACATCTACAGACACCAC	TCTCCTTCATAATCGTCTTTAGCCT	158
IL-10	NM_214041.1	TGCTCTATTGCCTGATCTTCCTG	AGTCGCCCATCTGGTCCTTC	162
β-actin	NM_001170517.2	GGCACCACACCTTCTACAACGAG	TCATCTTCTCACGGTTGGCTTTGG	102

图1 VC和7S对IPEC-J2存活率的影响 a,VC对IPEC-J2存活率的影响。b,VC对7S诱导下IPEC-J2存活率的影响。A,对照组;B,7S模型组;C,7S+25 μmol·L-1 VC组;D,7S+50 μmol·L-1 VC组;E,7S+100 μmol·L-1 VC组;F,7S+200 μmol·L-1 VC组;G,7S+400 μmol·L-1 VC组;H,7S+600 μmol·L-1 VC组;I,7S+800 μmol·L-1 VC组;J,7S+1 000 μmol·L-1 VC组。*表示与对照组相比差异显著(P<0.05),**表示与对照组相比差异极显著(P<0.01),#表示与7S模型组相比差异显著(P<0.05),##表示与7S模型组相比差异极显著(P<0.01)。下同。

Fig.1 Effect of VC and 7S on survival rate of IPEC-J2 a, Effect of VC on survival rate of IPEC-J2.b, Effect of VC on survival rate of IPEC-J2 induced by 7S.A, Control group; B, 7S model group; C, 7S+25 μmol·L-1 VC group; D, 7S+50 μmol·L-1 VC group; E, 7S+100 μmol·L-1 VC group; F, 7S+200 μmol·L-1 VC group; G, 7S+400 μmol·L-1 VC group; H, 7S+600 μmol·L-1 VC group; I, 7S+800 μmol·L-1 VC group; J, 7S+1 000 μmol·L-1 VC group. * and **showed significant difference compared with the control group at P<0.05 and P<0.01, # and ## showed significant difference compared with 7S model group at P<0.05 and P<0.01. The same as below.

图2 VC对7S刺激IPEC-J2后细胞形态的影响(×100)

Fig.2 Effect of VC on morphology of IPEC-J2 stimulated by 7S (×100)

图3 VC对7S诱导下IPEC-J2细胞膜完整性的影响

Fig.3 Effect of VC on membrane integrity of IPEC-J2 induced by 7S

图4 VC对7S诱导下IPEC-J2上清液中IL-1β、IL-6、TNF-α分泌水平的影响

Fig.4 Effects of VC on secretion levels of IL-1β, IL-6 and TNF-α in supernatant ofIPEC-J2 induced by 7S

图5 VC对7S诱导下IPEC-J2上清液中IL-4和IL-10分泌水平的影响

Fig.5 Effects of VC on IL-4 and IL-10 secretion levels in supernatants ofIPEC-J2 induced by 7S

图6 VC对7S诱导下IPEC-J2促炎因子mRNA表达的影响

Fig.6 Effect of VC on mRNA expression of proinflammatory factors in IPEC-J2 induced by 7S

图7 VC对7S诱导下IPEC-J2中抗炎因子mRNA表达的影响

Fig.7 Effect of VC on mRNA expression of anti-inflammatory factor in IPEC-J2 induced by 7S

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维生素C对β-伴大豆球蛋白诱导的仔猪肠上皮细胞炎性损伤的保护作用

Protective effect of vitamin C in piglet intestinal epithelial cells inflammatory injury induced by β-conglycinin

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