C5a-C5aR轴在鸭疫里氏杆菌感染致鸭肝损伤中的作用

doi:10.11843/j.issn.0366-6964.2022.10.036

摘要/Abstract

摘要： 本试验旨在阐明C5a-C5aR轴在鸭疫里氏杆菌（Riemerella anatipestifer）感染致雏鸭肝损伤中的作用。将保存的鸭疫里氏杆菌复苏培养，同时制备鸭抗凝血，将鸭疫里氏杆菌与鸭抗凝血混匀，分别与抗C5a抗体和抗C5aR抗体孵育后进行全血试验，利用ELISA检测C5a及各炎性因子表达水平；利用鸭疫里氏杆菌感染雏鸭，同时分别用抗C5a抗体和抗C5aR抗体处理进行动物试验，采集主要组织进行病理组织学检查；采集雏鸭外周血和肝组织进行细菌计数；采集外周血分离血清后，进行血清酶活性检测，应用ELISA检测血清C5a及炎性因子水平；采集肝组织，利用荧光定量PCR检测主要炎性因子mRNA转录水平。结果显示，在全血试验和动物试验中，与R.anatipestifer组相比，R.anatipestifer +anti-C5a组和R.anatipestifer +anti-C5aR组C5a、TNF-α、IL-6和IL-1β表达水平显著降低（P<0.05）；阻断C5a-C5aR轴可显著降低鸭疫里氏杆菌感染雏鸭的发病率和死亡率，同时减轻雏鸭心、肝和脾组织损伤；阻断C5a-C5aR轴能显著降低鸭疫里氏杆菌感染雏鸭外周血和肝细菌数及谷丙转氨酶（alanine aminotransferase，ALT）和谷草转氨酶（aspartate aminotransferase，AST）水平；荧光定量PCR检测发现，与R.anatipestifer组相比，R.anatipestifer +anti-C5a和R.anatipestifer +anti-C5aR组C5aR、Sphk1、FGL2、TNF-α和IL-1β mRNA转录水平显著降低（P<0.05）。本研究成功证实C5a-C5aR轴激活有助于鸭疫里氏杆菌感染雏鸭，阻断C5a-C5aR轴可显著减轻雏鸭组织损伤和炎症反应，为进一步研究抗鸭疫里氏杆菌感染药物提供新思路。

关键词: C5a-C5aR轴, 鸭疫里氏杆菌, 补体系统, 肝损伤

Abstract: This experiment aimed to clarify the role of C5a-C5aR axis in the liver injury of ducklings caused by Riemerella anatipestifer (R. anatipestifer) infection. The preserved R. anatipestifer was resuscitated and duck anticoagulant blood was also prepared. The whole blood experiments were conducted by mixing R. anatipestifer with duck anticoagulant blood and then incubating with anti-C5a antibody and anti-C5aR antibody, respectively. ELISA was used to detect the expression levels of C5a and various inflammatory factors. Then the ducklings were infected with R. anatipestifer and treated with anti-C5a antibody and anti-C5aR antibody, respectively to conduct the animal experiments. The main tissues of ducklings were collected to conduct the histopathological examination. Peripheral blood and liver tissues of ducklings were collected for bacterial counts. Peripheral blood was collected to separate the serum for serum enzyme activity measurement. ELISA was used to detect the expression levels of C5a and inflammatory factors in the serum. The liver tissues were collected to detect the mRNA transcriptional levels of main inflammatory factors by fluorescent quantitative PCR. The results showed that in the whole blood experiments and animal experiments, compared with R. anatipestifer group, the expression levels of C5a, TNF-α, IL-6 and IL-1β in R. anatipestifer + anti-C5a group and R. anatipestifer + anti-C5aR group were significantly decreased (P<0.05). Blocking the C5a-C5aR axis could significantly reduce the morbidity and mortality of ducklings infected with R. anatipestifer, and meanwhile alleviated the tissue damage of the heart, liver and spleen. Blocking the C5a-C5aR axis could significantly reduce the number of bacteria in the peripheral blood and liver tissues, and decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the ducklings infected with R. anatipestifer. The results of fluorescence quantitative PCR showed that compared with R. anatipestifer group, the mRNA transcriptional levels of C5aR, Sphk1, FGL2, TNF-α and IL-1β in R. anatipestifer + anti-C5a and R. anatipestifer + anti-C5aR groups were significantly decreased (P<0.05). The study successfully confirmed that the activation of C5a-C5aR axis could help R. anatipestifer infect ducklings, and blocking the C5a-C5aR axis could significantly alleviate the tissue damages and inflammatory responses of ducklings, and provided new ideas for further research on anti-R. anatipestifer infection drugs.

Key words: C5a-C5aR axis, Riemerella anatipestifer, complement system, liver injure

中图分类号:

S855.1

李德龙, 王思媛, 杨云川, 刘泓邑, 何忆箫, 刘锦琳, 高继业, 李继祥, 陈思怀. C5a-C5aR轴在鸭疫里氏杆菌感染致鸭肝损伤中的作用[J]. 畜牧兽医学报, 2022, 53(10): 3654-3666.

LI Delong, WANG Siyuan, YANG Yunchuan, LIU Hongyi, HE Yixiao, LIU Jinlin, GAO Jiye, LI Jixiang, CHEN Sihuai. Role of C5a-C5aR Axis in Liver Injury of Ducklings Caused by Riemerella anatipestifer Infection[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3654-3666.

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