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

doi:10.16431/j.cnki.1671-7236.2021.12.038

摘要/Abstract

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

中图分类号:

S831.7

李元辉, 傅春妮, 李鹏程, 彭玉凯, 李留安, 张建斌, 秦顺义. 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[J]. 中国畜牧兽医, 2021, 48(12): 4702-4709.

LI Yuanhui, FU Chunni, LI Pengcheng, PENG Yukai, LI Liuan, ZHANG Jianbin, QIN Shunyi. Effect of Selenium-Enriched Lactic Acid Bacteria on Apoptosis of Chicken Small Intestinal Epithelial Cells Induced by Aflatoxin B₁[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4702-4709.

参考文献

[1] 冯建蕾.黄曲霉毒素的危害和防治[J].中国畜牧兽医,2005,32(12):5-7. FENG J L.Harm and prevention of aflatoxin[J].China Animal Husbandry & Veterinary Medicine,2005,32(12):5-7.(in Chinese)
[2] HUANG W,LIU M,XIAO B,et al.Aflatoxin B₁ disrupts blood-testis barrier integrity by reducing junction protein and promoting apoptosis in mice testes[J].Food and Chemical Toxicology,2021,148:111972.
[3] 庞惠萍,丁泽,苏娜,等.黄曲霉毒素B₁致肝脏损伤的机制[J].动物医学进展,2019,40(12):110-113. PANG H P,DING Z,SU N,et al.Mechanism of liver injury caused by aflatoxin B₁[J].Progress in Veterinary Medicine,2019,40(12):110-113.(in Chinese)
[4] PENG X,YU Z,LIANG N,et al.The mitochondrial and death receptor pathways involved in the thymocytes apoptosis induced by aflatoxin B₁[J].Oncotarget,2016,7(11):12222-12234.
[5] LIU Y,WANG W.Aflatoxin B₁ impairs mitochondrial functions,activates ROS generation,induces apoptosis and involves Nrf2 signal pathway in primary broiler hepatocytes[J].Animal Science Journal,2016(12):1490-1500.
[6] HETZ C,PAPA FR.The unfolded protein response and cell fate control[J].Molecular Cell,2018,69:169-181.
[7] LIAO S,SHI D,CLEMONS-CHEVIS C L,et al.Protective role of selenium on aflatoxin B₁-induced hepatic dysfunction and apoptosis of liver in ducklings[J].Biological Trace Element Research,2014,162:296-301.
[8] YUAN S,WU B,YU Z,et al.The mitochondrial and endoplasmic reticulum pathways involved in the apoptosis of bursa of Fabricius cells in broilers expose to dietary aflatoxin B₁[J].Oncotarget,2016,7:65295-65306.
[9] 朱盼盼.黄曲霉毒素B₁对雏鸡脾脏细胞凋亡信号通路的影响及硒的拮抗作用研究[D].成都:四川农业大学,2018. ZHU P P.The protective effect of Se on the apoptosis pathways of chicken’s spleen caused by aflatoxin B₁[D].Chengdu:Sichuan Agricultural University,2018.(in Chinese)
[10] 姚婉,赵方红,刘丽娜,等.不同浓度硒含量的富硒乳酸菌抑制黄曲霉菌生长以及产毒效果的研究[J].中国饲料,2016,(12):19-21. YAO W,ZHAO F H,LIU L N,et al.Study on Se-enriched Lactobacillus with different selenium concentration inhibiting the growth and toxin-producing effects of aspergillus flavus[J].China Feed,2016,(12):19-21.(in Chinese)
[11] PENG X,ZHANG S,FANG J,et al.Protective roles of sodium selenite against aflatoxin B₁-induced apoptosis of jejunum in broilers[J].International Journal of Environmental Research and Public Health,2014,11(12):13130-13143.
[12] CHAN F K M,MORIWAKI K,DE ROSA M J.Detection of necrosis by release of lactate dehydrogenase activity[J].Methods in Molecular Biology,2013,979:65-70.
[13] 谢佳雨,张雯,杨靖亚,等.壳寡糖对黄曲霉毒素B₁诱导大鼠肝细胞毒性损伤的干预作用[J].上海海洋大学学报,2021,2:1-14. XIE J Y,ZHANG W,YANG J Y,et al.Intervention effect of chitooligosaccharides on aflatoxin B₁ induced toxic damage of rat liver cells[J].Journal of Shanghai Ocean University,2021,2:1-14.(in Chinese)
[14] ZHENG N,ZHANG H,LI S L,et al.Lactoferrin inhibits aflatoxin B₁ and aflatoxin M₁-induced cytotoxicity and DNA damage in Caco-2,HEK,Hep-G2,and SK-N-SH cells[J].Toxicon,2018,150:77-85.
[15] 贾红豆.AFB₁通过ROS-Caspase通路诱导的奶牛肝细胞凋亡[J].甘肃畜牧兽医,2021,51(1):49-52. JIA H D.Apoptosis of bovine hepatocytes induced by AFB₁ via ROS-Caspase pathway[J].Gansu Animal Husbandry and Veterinary,2021,51(1):49-52.(in Chinese)
[16] ROZPEDEK W,PYTEL D,DIEHL JA,et al.Small molecule inhibitors of PERK-dependent signaling pathway as a novel,therapeutic molecular strategy in Alzheimer’s disease treatment[J].Organ Polskiego Towarzystwa Lekarskiego,2016,41(241):5-10.
[17] LANGER R,FEITH M,SIEWERT J R,et al.Expression an clinical significance of glucose regulated proteins GRP78 (BiP) and GRP94 (GP96) in human adenocarcinomas of the esophagus[J].BMC Cancer,2008,8:70.
[18] RAJESH K,KRISHNAMOORTHY J,GUPTA J,et al.The eIF2α serine 51 phosphorylation-ATF4 arm promotes HIPPO signaling and cell death under oxidative stress[J].Oncotarget,2016,7(32):51044-51058.
[19] LIU D,ZHANG M,YIN H.Signaling pathways involved in endoplasmic reticulum stress-induced neuronal apoptosis[J].International Journal of Neuroscience,2013,123(3):155-162.
[20] WU Y,YUE Y,FU S,et al.Icariside Ⅱ prevents hypertensive heart disease by alleviating endoplasmic reticulum stress via the PERK/ATF-4/CHOP signalling pathway in spontaneously hypertensive rats[J].Journal of Pharmacy and Pharmacology,2019,71(3):400-407.
[21] ZHENG Z,ZUO Z,ZHU P,et al.2017.A study on the expression of apoptotic molecules related to death receptor and endoplasmic reticulum pathways in the jejunum of AFB₁-intoxicated chickens[J].Oncotarget,2017,8:89655-89664.
[22] ZHU P,ZUO Z,ZHENG Z,et al.Aflatoxin B₁ affects apoptosis and expression of death receptor and endoplasmic reticulum molecules in chicken spleen[J].Oncotarget,2017,8:99531-99540.
[23] LIMAYE A,YU R C,CHOU C C,et al.Protective and detoxifying effects conferred by dietary selenium and curcumin against AFB₁-mediated toxicity in livestock:A review[J].Toxins,2018,10(1):25.
[24] 尹恒.黄曲霉毒素B₁致雏鸡空肠黏膜细胞周期G2/M期阻滞及硒对其保护作用的研究[D].成都:四川农业大学,2017. YIN H.Studies of G2/M cell cycle arrest caused by aflatoxin B₁ and protective effect of Se in jejunum of broilers[D].Chengdu:Sichuan Agricultural University,2017.(in Chinese)

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

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	钟粤韵, 杨舒展, 陈非玥, 陆智儿, 李冰心, 李婉雁, 田允波, 黄运茂, 许丹宁, 曹楠. LPS通过ROS/p38/MAPK信号通路对鹅胚肝细胞凋亡的影响[J]. 中国畜牧兽医, 2023, 50(6): 2224-2232.
[2]	郭红伟, 蔡永超, 樊春光, 郝凯旋, 常娟, 王平, 刘超齐, 尹清强. 复合生物解毒剂在人工胃肠液中对黄曲霉毒素B₁降解率的影响[J]. 中国畜牧兽医, 2023, 50(4): 1395-1403.
[3]	张振宇, 王勇. 乳与乳制品中黄曲霉毒素检测研究进展[J]. 中国畜牧兽医, 2022, 49(7): 2820-2830.
[4]	顾先哲, 侯宁, 吕晓萍, 高雪丽, 刘超男, 吴文杰, 郑世民. 禽网状内皮组织增生病病毒感染对SPF雏鸡法氏囊细胞Bcl-2和C-myc基因表达及凋亡的影响[J]. 中国畜牧兽医, 2022, 49(4): 1488-1496.
[5]	吴艳, 皮劲松, 张昊, 梁振华, 杜金平, 潘爱銮, 申杰, 蒲跃进. miR-145-4调控蛋鸭卵泡发育机制的研究[J]. 中国畜牧兽医, 2022, 49(3): 809-816.
[6]	乔春雨, 刘家和, 张博熙, 何雨曦, 郑雨微, 吕红明. 黄曲霉毒素B₁致病机制及防治研究进展[J]. 中国畜牧兽医, 2022, 49(2): 765-775.
[7]	孙向丽, 刘宇翾, 时子瑶, 温长印, 田亚东, 康相涛, 王彦彬. 降解AFB₁的枯草芽孢杆菌S₁分离与鉴定[J]. 中国畜牧兽医, 2022, 49(12): 4654-4664.
[8]	张颖, 李先强, 李亚娜, 王家乡, 吴艳, 皮劲松, 陈林. 颗粒细胞凋亡影响家禽卵泡闭锁的研究进展[J]. 中国畜牧兽医, 2022, 49(12): 4725-4733.
[9]	姚家威, 黄钰洁, 黄丽贞, 阮慧敏, 陈志胜, 王丙云, 陈胜锋, 张晖, 白银山, 刘璨颖, 计慧琴. α-酮戊二酸二甲酯预处理对犬脂肪间充质干细胞氧化应激损伤的保护作用[J]. 中国畜牧兽医, 2021, 48(12): 4735-4743.
[10]	王黎霞, 宋丽聪, 张建军, 安健. 柔嫩艾美耳球虫感染对鸡血清凋亡蛋白的影响[J]. 中国畜牧兽医, 2020, 47(4): 1274-1281.
[11]	杨浩, 金三俊, 庞倩, 冯兴军. 白藜芦醇对畜禽常见肝损伤保护机制及应用的研究进展[J]. 中国畜牧兽医, 2020, 47(11): 3528-3535.
[12]	张生英, 武小椿, 邢小勇, 刘佳, 岳亚辉, 张阳阳, 贺健, 温峰琴, 包世俊. 牛支原体P48蛋白诱导EBL细胞增殖和凋亡的研究[J]. 中国畜牧兽医, 2020, 47(10): 3149-3157.
[13]	张牧臣, 郑楠, 赵圣国, 张养东, 李松励, 王加启. 黄曲霉毒素B₁与M₁对小鼠肠道细菌多样性的影响[J]. 《中国畜牧兽医》, 2019, 46(2): 433-441.
[14]	马兴树, 宋金祥. 非洲猪瘟病毒免疫及基因工程疫苗研究进展[J]. 中国畜牧兽医, 2019, 46(11): 3404-3413.
[15]	张美美, 蒋梦宇, 孙悠然, 魏川子, 谢小来. 不同氨化处理黄曲霉毒素B₁脱毒效果及其对奶牛瘤胃体外发酵的影响[J]. 《中国畜牧兽医》, 2019, 46(1): 130-139.