TBHQ对鸡舍PM2.5诱导鸡胚肺组织细胞焦亡、坏死和炎症损伤的影响

doi:10.3864/j.issn.0578-1752.2023.04.015

摘要/Abstract

摘要：

【目的】探讨特丁基对苯二酚（tert-Butylhydroquinone，TBHQ）对鸡舍细颗粒物（fine particulate matter，PM_2.5）诱导鸡胚肺损伤的缓解作用及机制，为预防和缓解鸡舍PM_2.5污染引起的鸡呼吸道健康问题提供理论依据。【方法】选用14日龄鸡胚作为研究对象，首先建立鸡舍PM_2.5诱导鸡胚肺组织损伤模型，再利用鸡胚肺损伤模型研究TBHQ的缓解作用。在建立鸡舍PM_2.5诱导鸡胚肺组织损伤模型试验中，选取不同浓度的PM_2.5（0、0.25、0.5、1 mg·mL^-1）在14日龄鸡胚卵白处注射，5 d后，观察鸡胚存活率以及鸡胚肺组织形态，选取合适的PM_2.5处理浓度（0.25 mg·mL^-1），建立鸡胚肺损伤模型。在TBHQ对PM_2.5诱导鸡胚肺损伤的影响试验中，将14日龄鸡胚分为对照组（生理盐水）、PM_2.5组（0.25 mg·mL^-1 PM_2.5）、TBHQ组（0.1 μg·mL^-1 TBHQ）、PM_2.5+TBHQ组（0.25 mg·mL^-1 PM_2.5+ 0.1 μg·mL^-1 TBHQ），分别注入鸡胚卵白处，试验持续5 d，采集肺组织，记录种蛋重量、胚胎重量以及肺组织重量；对鸡胚肺脏进行组织学观察；检测肺组织丙二醛（malondialdehyde，MDA）、总超氧化物歧化酶（total superoxide dismutase，T-SOD）、总抗氧化能力（total antioxidant capacity，T-AOC）和细胞焦亡（NLRP3、Caspase-1、IL-18、IL-1β）以及细胞程序性坏死（RIPK1、RIPK3、MLKL）相关基因的表达。【结果】在建立鸡舍PM_2.5诱导鸡胚肺组织损伤模型试验中，不同浓度的PM_2.5（0、0.25、0.5、1 mg·mL^-1）对鸡胚存活率无显著影响，但0.25 mg·mL^-1 PM_2.5组鸡胚肺组织出现炎性细胞浸润现象，0.5 mg·mL^-1和1 mg·mL^-1 PM_2.5组鸡胚肺组织出现肺水肿现象，表明随着PM_2.5浓度增加，鸡胚肺部炎症损伤加重。在TBHQ对PM_2.5诱导鸡胚肺损伤的缓解试验中，各组对鸡胚胚胎重量、肺脏重量、胚蛋比和肺胚比均无影响；与PM_2.5组相比，TBHQ和PM_2.5共处理组的鸡胚肺组织中炎性细胞浸润现象明显减少，MDA水平下降；RT-PCR结果显示：与对照组相比，TBHQ组Caspase-1的表达显著上升（P<0.01），PM_2.5组IL-1β（P<0.01）、RIPK1（P<0.01）的表达显著上升；与PM_2.5组相比，TBHQ和PM_2.5共处理组IL-18（P<0.01）、IL-1β（P<0.01）、RIPK1（P<0.01）、MLKL（P<0.01）的表达显著下调，Caspase-1（P<0.01）、RIPK3（P<0.05）的表达显著上升。【结论】TBHQ能够通过抑制氧化应激，降低细胞焦亡和细胞程序性坏死相关基因的表达，缓解鸡舍PM_2.5引起的鸡胚肺组织炎症损伤。

关键词: TBHQ, 细胞焦亡, 细胞程序性坏死, 细颗粒物, 鸡胚, 氧化应激

Abstract:

【Objective】 The aim of this study was to investigate the protective effect and mechanism of tert-butylhydroquinone (TBHQ) on chicken embryo lung injury induced by fine particulate matter (PM_2.5) in chicken house, so as to provide a theoretical basis for prevention and mitigation of chicken respiratory health problems caused by PM_2.5 pollution in chicken house. 【Method】 14-day-old chicken embryos were selected as the research model in this study. Firstly, a model of lung tissue damage induced by PM_2.5 from chicken houses was established, and then the alleviating effects of TBHQ on lung injury of chicken embryos was studied. In the experiment of establishing the model of chicken embryo lung tissue injury induced by PM_2.5 from chicken houses, different concentrations of PM_2.5 (0, 0.25, 0.5, and 1 mg·mL^-1) were injected into egg albumens. After 5 days, the survival rate and lung tissues of chicken embryos were observed, and the appropriate concentration of PM_2.5 (0.25 mg·mL^-1) was selected to establish the model of chicken embryo lung injury. In the experiment of the effects of TBHQ on PM_2.5-induced lung injury of chicken embryos, chicken embryos were divided into control group (saline), PM_2.5 group (0.25 mg·mL^-1 PM_2.5), TBHQ group (0.1 μg·mL^-1 TBHQ) and PM_2.5+TBHQ group (0.25 mg·mL^-1 PM_2.5+0.1 μg·mL^-1 TBHQ). The experiment lasted for 5 days. The lung tissues were collected and the egg weight, embryo weight and lung tissue weight were recorded. The expressions of MDA, T-SOD, T-AOC and pyroptosis-related genes (NLRP3, Caspase-1, IL-18, IL-1β) and necroptosis-related genes (RIPK1, RIPK3, MLKL) in lung tissues of chicken embryos were detected. 【Result】 The results suggested that in the experiment to establish a model for PM_2.5-induced lung tissue damage in chicken embryos, different concentrations of PM_2.5 (0, 25, 50, and 100 μg·mL^-1) from chicken houses did not significantly affect the survival of chicken embryos, but the lung tissues of chicken embryos in the 0.25 mg·mL^-1 PM_2.5 group showed inflammatory cell infiltration. The lung tissues of chicken embryos in 0.5 mg·mL^-1 and 1 mg·mL^-1 PM_2.5 groups showed pulmonary edema. In the experiment on the effects of TBHQ on PM_2.5-induced lung damage in chicken embryos, there was no effect on embryo weight, lung tissue weight and related indices in all groups. Compared with the PM_2.5 group, the inflammatory cell infiltration and MDA levels were reduced in the lung tissues of chicken embryos co-treated with TBHQ and PM_2.5. RT-PCR results showed that the expression of the Caspase-1 gene in the TBHQ group was significantly increased compared with the control group (P<0.01), as were the expressions of IL-1β (P<0.01) and RIPK1 (P<0.01) genes in the PM_2.5 group. Compared with the PM_2.5 group, the expressions of IL-18 (P<0.01), IL-1β (P<0.01), RIPK1 (P<0.01), and MLKL (P<0.01) genes were significantly down in the TBHQ and PM_2.5 co-treatment groups. On the contrary, the expressions of Caspase-1 (P<0.01), RIPK3 (P<0.05) genes in the TBHQ and PM_2.5 co-treatment groups were significantly increased compared with the PM_2.5 group. 【Conclusion】TBHQ could alleviate inflammatory damage in chicken embryo lung tissues induced by PM_2.5 from chicken houses by inhibiting oxidative stress and reducing the gene expressions of pyroptosis and necroptosis.

Key words: TBHQ, pyroptopsis, necroptosis, PM_2.5, chicken embryos, oxidative stress

郗蒙雪, 沈丹, 石一凡, 李春梅. TBHQ对鸡舍PM_2.5诱导鸡胚肺组织细胞焦亡、坏死和炎症损伤的影响[J]. 中国农业科学, 2023, 56(4): 779-787.

XI MengXue, SHEN Dan, SHI YiFan, LI ChunMei. Effects of TBHQ on Pyroptosis, Necroptosis and Inflammatory Damage of Chicken Embryonic Lung Tissues Induced by PM_2.5 from Chicken Houses[J]. Scientia Agricultura Sinica, 2023, 56(4): 779-787.

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引物名称 Gene	引物序列 Sequence (5′ to 3′)
Caspase-1	TTCCTTCAACACCATCTACG / GGTGAGCTTCTCTGGTTTTA
IL-1β	ACTGGGCATCAAGGGCTACA / GCTGTCCAGGCGGTAGAAGA
IL-18	AGTTGCTTGTGGTTCGTCCA / TCCACTGCCAGATTTCACCTC
NLRP3	AGCTACCACACATCTAGGAT / GGTGTCCAAATCCTCAATCT
PERK	TCGAGCTGCTTTACCCTTTC / CTCATTGTCCGTGACCTCTG
CHOP	CAGGAAGAAGAGCTGGCCCCACT / TGCTGTGCTCGCCGTGCTGT
IRE1	CCCAAAGCATCAAACCATTC / CAACGTCGCGGTTATCAAAT
ATF6	CGTCGTCTGAACCACTTACTGA / CCTTCTTTCCTAACAGCCACAC
GRP78	GAATCGGCTAACACCAGAGGA / CGCATAGCTCTCCAGCTCATT
RIPK1	CTTCAACCAGCGCCATTAGC / TTGAGTCTTCTGTATCCGTGTCT
RIPK3	ACATCCTTCGCTCACAGCAA / ACCTGTGCTGCCTTCTCTCC
MLKL	AAGGTGGACTGGATGCAAGG / TAGAGGTCGTAGCGCTCAGT
β-actin	TGTTACCAACACCCACACCC / TCCTGAGTCAAGCGCCAAAA

浓度 Concentration (mg·mL^-1)	胚数Embryo number		总数 Total	鸡胚存活率 Survival rate of chicken embryo (%)
浓度 Concentration (mg·mL^-1)	死亡Death	存活Survival	总数 Total	鸡胚存活率 Survival rate of chicken embryo (%)
0	0	5	5	100
0.25	0	5	5	100
0.5	0	5	5	100
1	0	5	5	100

项目 Item	对照组 Control group	TBHQ组 TBHQ group	PM_2.5组 PM_2.5 group	PM_2.5+ TBHQ组 PM_2.5+ TBHQ group	P值 P value
绝对重量Absolute weight (g)
种蛋重量 Egg weight	42.83 ± 1.33	41.53 ± 0.96	43.05 ± 1.08	40.41 ± 0.58	0.45
胚胎重量 Embryo weight	24.13 ± 0.75	22.52 ± 0.81	23.66 ± 1.19	23.47 ± 0.37	0.44
肺组织重量 Lung tissue weight	0.18 ± 0.02	0.17 ± 0.01	0.20 ± 0.02	0.17 ± 0.01	0.14
相对重量Relative weight
胚蛋比 The ratio of embryo and egg (g·g^-1)	0.57 ± 0.02	0.54 ± 0.02	0.55 ± 0.03	0.58 ± 0.01	0.94
肺胚比 The ratio of lung and embryo (mg·g^-1)	0.73 ± 0.09	0.75 ± 0.04	0.84 ± 0.08	0.74 ± 0.05	0.95

基因 Gene	对照组 Control group	TBHQ组 TBHQ group	PM_2.5组 PM_2.5 group	PM_2.5 + TBHQ组 PM_2.5 and TBHQ group
NLRP3	1.00 ± 0.19ab	0.62 ± 0.09 b	1.29 ± 0.13 a	1.56 ± 0.18a
Caspase-1	1.00 ± 0.10 c	1.63 ± 0.10 b	1.35 ± 0.08 bc	2.44 ± 0.16a
IL-18	1.00 ± 0.13 ab	0.72 ± 0.14 bc	1.14 ± 0.03a	0.57 ± 0.0a5c
IL-1β	1.00 ± 0.12b	0.57 ± 0.12bc	1.62 ± 0.14a	0.37 ± 0.03c
RIPK1	1.00 ± 0.17b	0.75 ± 0.06b	1.66 ± 0.09a	1.07 ± 0.14b
RIPK3	1.00 ± 0.07b	1.44 ± 0.21 ab	1.20 ± 0.23b	1.92 ± 0.12a
MLKL	1.00 ± 0.30ab	0.37 ± 0.10b	1.74 ± 0.26a	0.42 ± 0.08b