硒缺乏通过TLR4/MyD88/NF-κB信号通路介导鸡胸腺细胞凋亡

doi:10.11843/j.issn.0366-6964.2022.11.029

摘要/Abstract

摘要： 本试验旨在探讨硒缺乏是否通过激活Toll样受体信号通路诱导鸡胸腺细胞凋亡。复制硒缺乏模型，将200只1日龄健康的肉鸡随机分为对照组（C组）和缺硒组（L组），对照组饲喂硒含量0.2 mg·kg^-1的正常日粮，缺硒组饲喂硒含量0.004 mg·kg^-1的缺硒日粮。在15、25、35、45、55日龄时，每组分别选取15只鸡，观察胸腺组织病理形态变化和超微结构变化；检测胸腺组织中TLR4信号转导通路与细胞凋亡相关因子的表达。又建立了MDCC-MSB1（MSB1）细胞硒缺乏模型，并在此基础上设立6个分组：对照（C group）组、缺硒（L group）组、缺硒+转染空质粒（L+pCMV-HA-N）组、缺硒+siRNA阴性对照（L+NCsiRNA）组和缺硒+过表达TLR4（L+pCMV-HA-TLR4）组、缺硒+干扰TLR4（L+siChTLR4）组，低硒处理5 d后，检测细胞活力、细胞凋亡情况、TLR4信号转导通路与细胞凋亡相关因子的表达。结果显示：1）与C组相比，L组皮质髓质的淋巴细胞数量减少、细胞排列紊乱、皮质髓质充血、核碎裂，广泛的局灶性坏死。L组鸡胸腺组织淋巴细胞间出现裂隙，体积缩小，细胞碎裂，核染色质边集，线粒体肿胀，嵴断裂。2）与15日龄C组相比，L组鸡胸腺中TLR4、MyD88、NF-κB、Caspase-3、Caspase-9、Bax mRNA和蛋白表达水平在15~55日龄中均显著上调，Bcl-2 mRNA和蛋白表达水平呈相反趋势。3）与C组相比，L组MSB1细胞活力显著下降、细胞凋亡数量明显增加、TLR4、MyD88、NF-κB、Caspase-3、Caspase-9、Bax mRNA和蛋白的表达均显著增加、Bcl-2 mRNA和蛋白表达水平极显著降低；与L组相比，L+siChTLR4组细胞活力明显增强、细胞凋亡数量明显减少、相关因子mRNA和蛋白表达显著下降，而L+pCMV-HA-TLR4组细胞活力明显降低、细胞凋亡数量明显增加、相关因子mRNA和蛋白表达均明显增加。综上所述，硒缺乏通过TLR4/MyD88/NF-κB信号通路介导鸡胸腺细胞凋亡，并进一步诱导鸡胸腺损伤。

关键词: 硒缺乏, TLR4, MyD88, NF-κB, 胸腺, 凋亡

Abstract: This study aimed to investigate whether selenium deficiency induced apoptosis of chicken thymocytes by activating toll-like receptor signaling pathway. In vivo experiment, 200 healthy one-day-old broilers were randomly divided into control group (C group) and selenium deficiency group (L group) by replicating selenium deficiency model, the control group was fed a normal diet with 0.2 mg·kg^-1 selenium, and the se-deficient group was fed a se-deficient diet with 0.004 mg·kg^-1 selenium. At 15, 25, 35, 45, and 55 days of age, 15 chicks were selected from each group, the morphological and ultrastructural changes of thymus tissue were observed. TLR4 signaling pathway and apoptosis-related factors were detected in thymus tissue. The selenium deficiency model of MDCC-MSB1(MSB1) cells in vitro was established, and six groups were established on this basis:Control (C) group, Selenium deficiency (L) group, selenium deficiency+transfection empty plasmid (L+pCMV-HA-N) group, selenium deficiency+siRNA negative control (L+ NCsiRNA) group, selenium deficiency + overexpression of TLR4 (L+ pCMV-HA-TLR4) group, selenium deficiency +siChTLR4 (L+ siChTLR4) group. After 5 days of low selenium treatment, cell viability, apoptosis, TLR4 signal transduction pathway and expression of apoptosis-related factors were detected. Results were as follows:1) Compared with the C group, the number of lymphocytes in the medulla of the cortex in the L group was reduced, the cells were disordered, the medulla of the cortex was hyperemic, the nucleus was fragmented, and extensive focal necrosis was observed. The thymus tissue of chickens in L group showed fissure, reduced volume, cell fragmentation, and nuclear chromatin edge aggregation, mitochondrial swelling, cristae fracture; 2) Compared with C group at 15 days of age, mRNA and protein expressions of TLR4, MyD88, NF-κB, Caspase-3, Caspase-9 and Bax in the thymus of L group were up-regulated at 15 to 55 days of age, the expression levels of Bcl-2 mRNA and protein showed opposite trends; 3) Compared with C group, the viability of MSB1 cells in L group was significantly decreased, the number of apoptosis was significantly increased, the mRNA and protein expressions of TLR4, MyD88, NF-κB, Caspase-3, Caspase-9 and Bax were significantly increased, and the mRNA and protein expression levels of Bcl-2 were significantly decreased. Compared with the L group, the cell viability was significantly enhanced, the number of apoptosis was significantly decreased, and the mRNA and protein expression of related factors were significantly decreased in the L+siChTLR4 group, while the cell viability was significantly decreased, the number of apoptosis was significantly increased, and the mRNA and protein expression of related factors were significantly increased in the L+ pCMV-HA-TLR4 group. In conclusion, selenium deficiency mediates the apoptosis of chicken thymocyte through the TLR4/MyD88/NF-κB signaling pathway, and further induces thymus injury in chickens.

Key words: selenium deficiency, TLR4, MyD88, NF-κB, thymus, apoptosis

中图分类号:

S852.3

张瑞莉, 张迪, 郭荣, 陈阳, 李广兴, 黄小丹. 硒缺乏通过TLR4/MyD88/NF-κB信号通路介导鸡胸腺细胞凋亡[J]. 畜牧兽医学报, 2022, 53(11): 4035-4047.

ZHANG Ruili, ZHANG Di, GUO Rong, CHEN Yang, LI Guangxing, HUANG Xiaodan. Selenium Deficiency Induced Thymocytes Apoptosis of Broilers via the TLR4/MyD88/NF-κB Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4035-4047.

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