绿原酸对FAdV-4的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响

doi:10.16431/j.cnki.1671-7236.2021.12.040

中国畜牧兽医 ›› 2021, Vol. 48 ›› Issue (12): 4718-4724.doi: 10.16431/j.cnki.1671-7236.2021.12.040

绿原酸对FAdV-4的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响

崔淹鸽^1,2, 李京帅¹, 李银聚¹, 张春杰¹

1. 河南科技大学, 动物疫病与公共安全重点实验室, 洛阳 471023;
2. 洛阳市动物疫病预防控制中心, 洛阳 471003

收稿日期:2021-05-20 出版日期:2021-12-20 发布日期:2021-12-02
通讯作者: 李银聚 E-mail:lyj6511@126.com
作者简介:崔淹鸽(1986-),女,河南郑州人,硕士,兽医师,研究方向:动物疫病防控技术,E-mail:cuiyange_1986@163.com
基金资助:
河南省科技攻关项目（132102110117）

Inhibitory Effect of Chlorogenic Acid on FAdV-4 and Its Effects on the Expression of Inflammatory Factors and Signal Molecules in Chicken Embryo Liver

CUI Yange^1,2, LI Jingshuai¹, LI Yinju¹, ZHANG Chunjie¹

1. The Key Laboratory of Animal Disease and Public Security, Henan University of Science and Technology, Luoyang 471023, China;
2. Luoyang Animal Disease Prevention and Control Center, Luoyang 471003, China

Received:2021-05-20 Online:2021-12-20 Published:2021-12-02

摘要/Abstract

摘要： 试验旨在研究绿原酸对禽腺病毒4型(FAdV-4)的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响。将9日龄SPF鸡胚随机分为7组:对照组、5个不同浓度的绿原酸组和FAdV-4感染组。绿原酸组经尿囊腔注射不同浓度的绿原酸,48 h后与FAdV-4感染组均经尿囊腔接种0.2 mL ELD₅₀为1.36×10⁷拷贝的FAdV-4病毒,对照组注射等体积的生理盐水。用MTT法检测绿原酸的安全浓度(IC₅₀),计算每枚鸡胚接种绿原酸的含量;接种病毒后72 h后,剖检并无菌收集鸡胚肝脏进行病理观察,实时荧光定量PCR法检测病毒载量,ELISA法测定肝脏细胞因子白介素-1β(IL-1β)、IL-6和肿瘤坏死因子-α(TNF-α)及信号分子磷脂酰肌醇-3-激酶(PI3K)、核转录因子-κB(NF-κB)和NF-κBp65含量。MTT法测定结果表明,绿原酸对鸡胚成纤维细胞的IC₅₀为28.6 μg/mL,每枚鸡胚接种0.2 mL的绿原酸含量分别为858、429、214.5(≈215)、107.25(≈107)和53.125(≈53) μg。FAdV-4病毒接种72 h后,FAdV-4感染组鸡胚肝脏边缘钝圆、肿大,变脆,呈土黄色或黄色,甚至出现坏死;随着绿原酸浓度增加,绿原酸组鸡胚肝脏肿胀渐不明显,坏死灶和出血瘀点减少;肝脏内病毒明显增殖,载量为7.8 log₂拷贝/mg,107 μg绿原酸即可显著抑制FAdV-4在鸡胚肝脏中的增殖(P<0.05)。炎性细胞因子检测结果表明,与对照组相比,FAdV-4可极显著增加鸡胚肝脏中IL-1β、IL-6和TNF-α的含量(P<0.01);53 μg绿原酸组IL-1β和TNF-α含量极显著增加(P<0.01),107 μg绿原酸组IL-6和TNF-α的含量均显著增加(P<0.05)。与FAdV-4感染组相比,107 μg绿原酸组IL-1β和TNF-α的含量均极显著降低(P<0.01),215和429 μg绿原酸组IL-6的含量显著降低(P<0.05),858 μg绿原酸组IL-6的含量极显著降低(P<0.01)。细胞信号分子检测结果表明,与对照组相比,FAdV-4可极显著增加PI3K、NF-κB和NF-κBp65的含量(P<0.01);53 μg绿原酸组PI3K、NF-κB和NF-κBp65的含量均显著增加(P<0.05)。与FAdV-4感染组相比,53 μg绿原酸PI3K和NF-κBp65的含量均显著降低(P<0.05);215 μg绿原酸组NF-κB的含量显著降低(P<0.05),NF-κBp65的含量极显著降低(P<0.01);当绿原酸达429 μg时,PI3K、NF-κB和NF-κBp65的含量均极显著降低(P<0.01)。本试验结果表明,绿原酸能够抑制FAdV-4的增殖,且剂量依赖性地抑制炎性因子和信号分子的表达,说明绿原酸可用于鸡抗病毒和炎性相关疾病的防控。

关键词: 绿原酸; 禽腺病毒血清4型(FAdV-4); 炎性因子; 信号分子; 鸡胚

Abstract: The purpose of this experiment was to study the inhibitory effect of chlorogenic acid on fowl adenovirus type 4 (FAdV-4) and its effect on the expression of inflammatory factors and signal molecules in chicken embryo liver.9-day-old SPF chicken embryos were randomly divided into 7 groups:Control group, 5 chlorogenic acid groups with different concentrations and FADV-4 infection group.Chlorogenic acid groups were injected with different concentrations of chlorogenic acid through the allantoic cavity.48 h after hatching, chlorogenic acid groups and FAdV-4 infection group were inoculated with 0.2 mL FAdV-4, of which the ELD₅₀ was 1.36×10⁷ copies, and the control group was injected with equal volume of normal saline.The safe concentration (IC₅₀) of chlorogenic acid was detected by MTT method, and the content of chlorogenic acid inoculated into each chicken embryo was calculated.The liver of chicken embryos was collected for pathological observation 72 h after inoculation with virus, viral load was detected by Real-time quantitative PCR, the contents of liver cytokine interleukin-1β (IL-1 β), IL-6 and tumor necrosis factor-α (TNF-α) as well as signal molecules phosphatidylinositol-3-kinase (PI3K), nuclear transcription factor-κB(NF-κB) and NF-κBp65 were measured by ELISA method.The MTT results showed that the IC₅₀ of chlorogenic acid to chick embryo fibroblast was 28.6 μg/mL.Based on the IC₅₀, chlorogenic acid treated groups were inoculated with 0.2 mL of different doses of chlorogenic acid per egg containing 858, 429, 214.5(≈215), 107.25(≈107) and 53.125(≈53) μg, respectively.72 h after FADV-4 inoculation, the liver edge of chicken embryo in FADV-4 infection group was blunt, round, swollen, brittle, earthy yellow or yellow, and even necrotic.In chlorogenic acid group, with the increase of chlorogenic acid concentration, liver swelling became less obvious, necrotic foci and bleeding petechiae decreased.The virus proliferated significantly in liver, and the viral load was 7.8 log₂copies/mg, 107 μg chlorogenic acid could significantly inhibit the proliferation of FAdV-4 in chicken embryo liver (P<0.05).The detection results of inflammatory factors showed that compared with control group, FADV-4 could extremely significantly increase the content of IL-1β, IL-6 and TNF-α in chicken embryo liver (P<0.01), the contents of IL-1β and TNF-α were extremely significantly increased in 53 μg chlorogenic acid group (P<0.01), the contents of IL-6 and TNF-α were significantly increased in 107 μg chlorogenic acid group (P<0.05).Compared with FAdV-4 infection group, the contents of IL-1β and TNF-α were extremely significantly increased in 107 μg chlorogenic acid group (P<0.01), the content of IL-6 was significantly decreased in 215 and 429 μg chlorogenic acid group (P<0.05), and the content of IL-6 was extremely significantly decreased in 858 μg chlorogenic acid group (P<0.01).Signal molecules determination results showed that compared with control group, FAdV-4 could extremely significantly increase the contents of PI3K, NF-κB and NF-κBp65 (P<0.01), the contents of PI3K, NF-κB and NF-κBp65 were significantly increased in 53 μg chlorogenic acid group (P<0.05).Compared with FAdV-4 infection group, the contents of PI3K and NF-κBp65 were significantly decreased in 53 μg chlorogenic acid group (P<0.05), the content of NF-κB was significantly decreased (P<0.05) and the content of NF-κBp65 was extremely significantly decreased in 215 μg chlorogenic acid group (P<0.01).When chlorogenic acid reached 429 μg, the contents of PI3K, NF-κB and NF-κBp65 were extremely significantly decreased (P<0.01).These findings suggested that chlorogenic acid could inhibit the proliferation of FAdV-4, and inhibit the expression of inflammatory factors and signal molecules in a dose-dependent manner, which indicated that chlorogenic acid might be used for the prevention and control of antiviral and inflammatory diseases in chickens.

Key words: chlorogenic acid; fowl adenovirus serotype-4 (FAdV-4); inflammatory factors; signal molecules; chicken embryo

中图分类号:

S831.7

崔淹鸽, 李京帅, 李银聚, 张春杰. 绿原酸对FAdV-4的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响[J]. 中国畜牧兽医, 2021, 48(12): 4718-4724.

CUI Yange, LI Jingshuai, LI Yinju, ZHANG Chunjie. Inhibitory Effect of Chlorogenic Acid on FAdV-4 and Its Effects on the Expression of Inflammatory Factors and Signal Molecules in Chicken Embryo Liver[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4718-4724.

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绿原酸对FAdV-4的抑制作用及其对鸡胚肝脏炎性因子和信号分子表达的影响

Inhibitory Effect of Chlorogenic Acid on FAdV-4 and Its Effects on the Expression of Inflammatory Factors and Signal Molecules in Chicken Embryo Liver

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