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