RNA-seq analysis of chlorogenic acid intervention in duck embryo fibroblasts infected with duck plague virus
Authors: Yang Yunyun , Zhang Qiandong , Cai Haiqing , Feng Yi , Wen Anlin , Yang Ying , Wen Ming 
Published: October 2023

Abstract
Introduction Chlorogenic acid is the main active ingredient of honeysuckle, with anti-inflammatory and antiviral effects. At present, duck plague virus causes a large number of waterfowl disease, seriously affecting the development of the breeding industry, there is an urgent need for the prevention and treatment of drugs with unique efficacy.
Methods In order to investigate the effect of chlorogenic acid on duck embryo fibroblasts infected with duck plague virus, in this study, maintenance medium containing different final concentrations of chlorogenic acid (0, 0.125, 0.250, 0.500, 1.000, 2.000, and 4.000 mg/mL) was added to 96-well plates, and the plates were incubated for 24 h, and cellular activity was measured by using a CCK-8 kit in order to determine the safe concentration of the drug; cell control group, chlorogenic acid control group, virus-infected group, and chlorogenic acid intervention group were set up. Cell control group, chlorogenic acid control group, virus-infected group and chlorogenic acid intervention group were set up. Cell samples were collected at 24 h, 36 h and 48 h after treatment. qRT-PCR was used to detect the viral content, and transcriptomic sequencing was used to screen the differentially expressed genes, and GO functional classification and KEGG signaling pathway analysis were utilized; In addition, 90 ducks were randomly divided into blank control group, virus infection group and chlorogenic acid intervention group. Ducks in virus infection group and chlorogenic acid intervention group were injected with DPV (0.2 mL/only) and chlorogenic acid intervention group were manually dosed with 0.6±0.02 g chlorogenic acid capsule at the same time until the end of the experiment in 5 d. Clinical symptoms were observed every day and 10 ducks were killed in each group at 24 h, 36 h and 48 h after the infection. At 24 h, 36 h and 48 h after infection, 10 ducks were killed, and samples of each immune organ were collected aseptically for the preparation of pathological sections and the detection of DPV load by fluorescence quantitative PCR. Ducks were observed for the clinical symptoms every day, and 10 ducks were dissected and killed in each group at 24 h, 36 h and 48 h, respectively, and the samples of each immune organ were collected in aseptic condition. The samples were collected aseptically from each immune organ for the preparation of pathological sections and the load of DPV was detected by fluorescence quantitative PCR.
Results CCK-8 assay showed that the highest cellular activity was observed at a chlorogenic acid concentration of 0.250 mg/mL. qRT-PCR assay showed that DPV load in DEF cells after chlorogenic acid intervention showed a significant decrease compared with that of the DEV-infected group. IL-16, CHCHD10, ROR2 and GNG10 were down-regulated, while TNFSF15 expression was up-regulated, and it was basically consistent with the qRT-PCR validation results; GO and KEGG analyses showed that chlorogenic acid inhibited DPV proliferation in DEF cells by up-regulating the activity of the Toll-like receptor signaling pathway, the TNF signaling pathway and by down-regulating the NF-κB signaling pathway. CCK-8 assay showed that the safe concentration of chlorogenic acid drug was 0.250 mg/mL; qRT-PCR assay showed that DPV load in DEF cells after chlorogenic acid intervention appeared to be significantly decreased from the DPV-infected group (P<0.05); transcriptomics sequencing showed that there were 8 differential genes in the chlorogenic acid intervention group, among which TNFAIP2, IFNAR1, CCL26, IL-16, CHCHD10, ROR2 and GNG10 expression was down-regulated, while TNFSF15 expression was up-regulated, and it was basically consistent with the results of qRT-PCR validation; GO and KEGG analyses showed that chlorogenic acid inhibited DPV proliferation in DEF cells through up-regulation of the Toll-like receptor signaling pathway, the activity of the TNF signaling pathway, and down-regulation of the NF-κB signaling pathway Histopathological observation showed that compared with the DPV-infected group, the damage of all immune organs in the ducks in the chlorogenic acid intervention group was reduced to different degrees, and the lesions such as cellular nuclear consolidation and nuclear fragmentation were alleviated; in vivo fluorescence quantitative PCR results showed that compared with the DPV-infected group, the DPV load in all immune organs in the ducks in the chlorogenic acid intervention group was decreased.
Conclusions This study provides a theoretical basis for the screening of targeted anti-DPV drugs and a scientific basis for the clinical guidance of drug administration.