冷刺激对APP感染仔猪肺损伤的影响及其作用机制研究

doi:10.16431/j.cnki.1671-7236.2025.03.039

摘要/Abstract

摘要： 【目的】探讨冷刺激对猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae ,APP)感染所致仔猪肺损伤的影响，以及沉默信息调节因子1(silent information regulator 1,SIRT1)在此过程中发挥的作用。【方法】选用健康、体重相近(8.0 kg±0.5 kg)的4周龄雄性断奶长白仔猪12头，随机分为4组：对照组(Control)、冷刺激组(Cold)、APP感染组(APP)、冷刺激下APP感染组(Cold＋APP)，每组3头。预饲1周后，Control组仔猪在室温条件下进行饲养；Cold组仔猪在(4±1) ℃环境中连续接受冷刺激6 h；APP组仔猪通过滴鼻方式感染APP (2×10⁹ CFU/mL)，左右鼻孔各滴入1.5 mL；Cold＋APP组仔猪先按照APP组方法进行感染，随后在(4±1) ℃环境中连续暴露6 h。试验结束后，屠宰仔猪并采集肺脏组织样本。通过苏木素-伊红(HE)染色观察肺脏组织病理变化、Masson染色观察肺脏组织纤维化情况；利用ELISA法检测肺脏组织中丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)含量；通过实时荧光定量PCR检测肺脏中炎症因子白细胞介素-6(interleukin-6,IL-6)、IL-1β及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)基因mRNA表达水平；用Western blotting检测核因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、Kelch样ECH关联蛋白1(Kelch-like ECH-associated protein 1,Keap1)、血红素氧合酶1(heme oxygenase-1,HO-1)、SIRT1以及乙酰化核因子κB亚基p65(acetyl-nuclear factor κB p65,Ace-NF-κB p65)蛋白表达水平。【结果】HE染色结果显示，与APP组相比，Cold＋APP组仔猪肺脏组织中存在大量炎性细胞浸润，毛细血管出现扩张和充血。Masson染色结果显示，与APP组相比，Cold＋APP组仔猪肺脏组织中出现大量弥散性分布的胶原纤维沉积。ELISA检测结果显示，与APP组相比，Cold＋APP组仔猪肺脏组织中MDA含量显著升高(P＜0.05)，GSH含量显著下降(P＜0.05)。实时荧光定量PCR检测结果显示，与APP组相比，Cold＋APP组仔猪肺脏组织中IL-6、IL-1β和TNF-α基因mRNA相对表达量均显著升高(P＜0.05)。Western blotting检测结果显示，与APP组相比，Cold＋APP组仔猪肺脏组织中Nrf2、HO-1和SIRT1蛋白相对表达量均显著降低(P＜0.05)，Keap1和Ace-NF-κB p65蛋白相对表达量均显著升高(P＜0.05)。【结论】冷刺激会增强APP感染诱导的仔猪肺脏组织的氧化应激和炎症反应，SIRT1能通过调控NF-κB p65的乙酰化加剧冷刺激下仔猪APP感染后肺损伤。

关键词: 冷刺激; 猪胸膜肺炎放线杆菌(APP); 沉默信息调节因子1(SIRT1); 肺损伤

Abstract: 【Objective】 This experiment was to investigate the effect of cold stimulation on lung injury of piglets induced by Actinobacillus pleuropneumoniae (APP) infection and the role of silent information regulator 1 (SIRT1) in this process.【Method】 12 healthy 4-week-old male weaned Landrace piglets with similar body weight (8.0 kg±0.5 kg) were randomly divided into 4 groups:Control group (Control),cold stimulation group (Cold),APP infection group (APP) and cold stimulation with APP infection group (Cold＋APP),with 3 piglets in each group.After 1 week of pre-feeding,piglets in Control group were fed at room temperature.Piglets in Cold group were subjected to continuous cold stimulation at (4±1) ℃ for 6 h.Piglets in APP group were infected with APP by nasal drip (2×10⁹ CFU/mL),and 1.5 mL was injected into each of the left and right nostrils.Piglets in Cold＋APP group were first infected according to the APP group method,and then exposed at (4±1) ℃ for 6 h.After the experiment,the piglets were slaughtered and lung tissue samples were collected.The pathological changes of lung tissue were observed by hematoxylin-eosin (HE) staining and the fibrosis of lung tissue was observed by Masson staining.The contents of malondialdehyde (MDA) and glutathione (GSH) in lung tissues were determined by ELISA.The mRNA expression level of the inflammatory factors interleukin-6 (IL-6),IL-1β,and tumor necrosis factor-α (TNF-α) genes in lung was detected by Real-time quantitative PCR.Western blotting was used to detect the expression levels of nuclear factor E2-related factor 2 (Nrf2),Kelch-like ECH-associated protein 1 (Keap1),heme oxygenase-1 (HO-1),SIRT1,and acetylated nuclear factor κB subunit p65 (Ace-NF-κB p65) proteins. 【Result】 HE staining results showed that compared with APP group,there was a large number of inflammatory cell infiltration in lung tissues,capillary dilation and congestion in piglets in Cold＋APP group.Masson staining showed that,compared with APP group,a large number of diffuse collagen fiber deposition appeared in the lung tissues of piglets in Cold＋APP group.ELISA results showed that compared with APP group,MDA content in lung tissues of piglets in Cold＋APP group was significantly increased (P＜0.05),and GSH content was significantly decreased (P＜0.05).The results of Real-time quantitative PCR showed that compared with APP group,the mRNA relative expression levels of IL-6,IL-1β and TNF-α genes in lung tissues of piglets in Cold＋APP group were significantly increased (P＜0.05).Western blotting test results showed that compared with APP group,the relative expressions of Nrf2,HO-1 and SIRT1 proteins in lung tissues of piglets in Cold＋APP group were significantly decreased (P＜0.05)，the relative expression levels of Keap1 and Ace-NF-κB p65 proteins were significantly increased (P＜0.05).【Conclusion】 Cold stimulation could enhance the oxidative stress and inflammatory response of piglet lung tissues induced by APP infection.SIRT1 could aggravate the lung injury of piglets after APP infection under cold stimulation by regulating the acetylation of NF-κB p65.

Key words: cold stimulation; Actinobacillus pleuropneumoniae(APP); silent information regulator 1 (SIRT1); lung injury

中图分类号:

S858.2

陈鑫鹏, 夏榕鸽, 盖新燕, 逯静静, 徐彬. 冷刺激对APP感染仔猪肺损伤的影响及其作用机制研究[J]. 中国畜牧兽医, 2025, 52(3): 1383-1392.

CHEN Xinpeng, XIA Rongge, GAI Xinyan, LU Jingjing, XU Bin. Effects and Mechanism of Cold Stimulation on Lung Injury of APP-infected Piglets[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1383-1392.

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