中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (12): 4702-4709.doi: 10.16431/j.cnki.1671-7236.2021.12.038

• 基础兽医 • 上一篇    下一篇

富硒乳酸菌对黄曲霉毒素B1诱导鸡小肠上皮细胞凋亡的影响

李元辉, 傅春妮, 李鹏程, 彭玉凯, 李留安, 张建斌, 秦顺义   

  1. 天津农学院动物科学与动物医学学院, 天津市农业动物繁育与健康养殖重点实验室, 天津 300384
  • 收稿日期:2021-04-14 出版日期:2021-12-20 发布日期:2021-12-02
  • 通讯作者: 秦顺义 E-mail:qinshunyi@163.com
  • 作者简介:李元辉(1994-),女,河北保定人,硕士,研究方向:动物生长调控与生理学,E-mail:lyh_8590@163.com
  • 基金资助:
    天津市自然科学基金重点项目(20JCZDJC00170);天津市"131"创新型人才团队建设项目(20180318);天津农学院研究生科研创新项目(2019XY022);天津市科技帮扶提升重大工程项目(20ZYCGSN00260);天津市高校"中青年骨干创新人才培养计划"人选资助项目

Effect of Selenium-Enriched Lactic Acid Bacteria on Apoptosis of Chicken Small Intestinal Epithelial Cells Induced by Aflatoxin B1

LI Yuanhui, FU Chunni, LI Pengcheng, PENG Yukai, LI Liuan, ZHANG Jianbin, QIN Shunyi   

  1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China
  • Received:2021-04-14 Online:2021-12-20 Published:2021-12-02

摘要: 试验旨在探究富硒乳酸菌对黄曲霉毒素B1(AFB1)对鸡小肠上皮细胞(CSIEC)凋亡的影响。将体外培养的CSIEC细胞分为对照组(C)、AFB1(300 μmol/L AFB1)、AFB1+0.01 Se(300 μmol/L AFB1+0.01 μmol/L富硒乳酸菌,以Se计)、AFB1+0.05 Se和AFB1+0.1 Se组,待细胞长至60%~70%汇合时,AFB1+0.01 Se、AFB1+0.05 Se和AFB1+0.1 Se组培养液更换为含对应Se浓度的培养液,对照组和AFB1组更换为正常培养液,8 h后向含AFB1组加入300 μmol/L AFB1,继续培养24 h。用CCK-8法检测细胞存活率,ELISA法检测培养液中乳酸脱氢酶(LDH)的活性,TUNEL法检测细胞凋亡率,Western blotting检测线粒体通路和内质网通路相关蛋白Bcl-2、Bax、PERK、ATF4、CHOP、GRP78、p53和p21的表达。细胞存活率检测结果表明,与对照组相比,其余各组CSIEC存活率均极显著降低(P<0.01);与AFB1组相比,加硒各组存活率均极显著升高(P<0.01)。LDH活性检测结果表明,与对照组相比,其余各组LDH活性均极显著升高(P<0.01);与AFB1组相比,加硒各组LDH活性均极显著降低(P<0.01)。细胞凋亡率检测结果表明,与对照组相比,AFB1组TUNEL阳性细胞数极显著升高(P<0.01),且与加硒各组差异均不显著(P>0.05)。Bcl-2/Bax表达量检测结果表明,与对照组相比,AFB1组Bcl-2/Bax极显著降低(P<0.01),AFB1+0.05 Se组显著升高(P<0.05)。内质网应激通路相关蛋白表达量检测结果表明,与对照组相比,AFB1组PERK、CHOP、GRP78和p53的表达量均极显著升高(P<0.01);与AFB1组相比,加硒各组PERK、GRP78表达量均极显著降低(P<0.01),AFB1+0.05 Se和AFB1+0.1 Se组CHOP和p53的表达量均极显著或显著降低(P<0.01;P<0.05)。因此,富硒乳酸菌能够缓解AFB1诱导的CSIEC凋亡。

关键词: 富硒乳酸菌; 黄曲霉毒素; 鸡小肠上皮细胞; 细胞凋亡

Abstract: In order to investigate the effect of selenium-enriched lactic acid bacteria on the apoptosis of chicken small intestinal epithelial cells (CSIEC) induced by aflatoxin B1 (AFB1), CSIEC cultured in vitro were divided into 5 groups:control group (C), AFB1 group (300 μmol/L AFB1), AFB1+0.01 Se group (300 μmol/L AFB1+0.01 μmol/L selenium-enriched lactic acid bacteria, in terms of Se), AFB1+0.05 Se group and AFB1+0.1 Se group.When the cells grew to 60%-70% confluence, the culture medium of AFB1+0.01 Se, AFB1+0.05 Se and AFB1+0.1 Se groups was replaced with the culture medium containing the corresponding Se concentration, and C and AFB1 groups were replaced with normal culture medium. After 8 h, 300 μmol/L AFB1 was added to the AFB1-containing groups, and the culture was continued for 24 h.CCK-8 was used to detect the antagonistic effect of selenium-enriched lactic acid bacteria on the cytoactive of CSIEC induced by AFB1.ELISA was used to detect the change of lactate dehydrogenase (LDH) activity in the culture medium.TUNEL was used to observe the effect of selenium-enriched lactic acid bacteria against AFB1 on cell apoptosis rate.The protein levels of Bcl-2/Bax, PERK, ATF4, CHOP, GRP78, p53 and p21 associated with mitochondrial and endoplasmic reticulum stress pathways were detected by Western blotting.The results showed that the cytoactive of CSIEC in the other groups was extremely significantly lower than that in control group (P<0.01), and those in selenium-added groups were extremely significantly higher than those in AFB1 group (P<0.01).The LDH activities in the other groups were extremely significantly higher than those in control group (P<0.01), and LDH activities in selenium-added groups were extremely significantly lower than those in AFB1 group (P<0.01).Compared with control group, the number of TUNEL positive cells in AFB1 group was extremely significantly increased (P<0.01);and no significant difference were found between selenium-added groups and AFB1 group (P>0.05).Compared with control group, the protein expression levels of Bcl-2/Bax in AFB1 group were extremely significantly decreased (P<0.01), and that in AFB1+0.05 Se group was increased (P<0.05).Endoplasmic reticulum stress pathway protein levels showed that the protein levels of PERK, CHOP, GRP78 and p53 in AFB1 group were extremely significantly higher than those in control group (P<0.01), protein levels of PERK and GRP78 in selenium-added groups were significantly lower than those in AFB1 group (P<0.01).Protein levels of CHOP and p53 in AFB1+0.05 Se and AFB1+0.1 Se groups were extremely significantly lower or significantly lower than those in AFB1 group (P<0.01;P<0.05).Therefore, selenium-enriched lactic acid bacteria could alleviate the apoptosis of CSIEC induced by AFB1.

Key words: selenium-enriched lactic acid bacteria; aflatoxin; chicken small intestinal epithelial cells; cell apoptosis

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