Baicalin Mitigates NIBV Infection-induced Spleen Injury Via Inhibiting Mitophagy And Promoting M1 Macrophage Polarization In Chicken

The aim of this study is to investigate the correlation between mitophagy,macrophage polarization and the antiviral activity of baicalin in the spleen of Nephropathogenic Infectious Bronchitis Virus（NIBV）-infected chickens.28-day-old Hy-Line brown chicks were randomly divided into four groups:Control group（no NIBV inoculation,basic diet）,Control+Baicalin group（no NIBV inoculation,basic diet+10mg/kg baicalin）,Disease group(inoculation of SX9 10^-5 ELD50/0.2 m L,basic diet),Disease+Baicalin group(inoculation of SX9 10^-5ELD50/0.2 m L,basic diet+10 mg/kg baicalin).Spleen tissues were weighed and collected at 3,7,and 11 days post infection（dpi）.Spleen injury,mitophagy and macrophage polarization in chicks were evaluated by HE-stained pathological tissue sections,transmission electron microscopy,immunofluorescence detection,enzyme-linked immunoassay（ELISA）,RT-q PCR and western blot techniques.The results showed that NIBV infection caused splenomegaly and weight loss;HE staining showed obvious enlargement and hemorrhage of white pulp,and the boundary between white pulp and red pulp was blurred;In addition,NIBV infection led to the destruction of mitochondrial cristae in splenocytes,the appearance of mitophagosomes in the cytoplasm,the increase of the m RNA levels of PINK1,Parkin,LC3A,and LC3B,and the enrichment of PINK1,Parkin,lipidated LC3 proteins in mitochondria,and promoted the colocalization of LC3 protein with Hsp60,inhibited the type of I interferon responses（P<0.05 or P<0.01）.In addition,NIBV infection promoted the expression of M1 macrophage-related genes（i NOS,TNF-α,IL-1β,IL-6）and proteins,suppressed M2-related genes（ARG2,IL-4,IL-10,Pparg）and protein expression,enhanced the fluorescence signal intensity of i NOS（P<0.05 or P<0.01）.However,baicalin could attenuate the spleen injury induced by NIBV infection,increase PINK1/Parkin-mediated mitophagy and type I interferon levels,and promote the polarization of M1 macrophages at 3 dpi,7dpi and the polarization of M2 macrophages at 11 dpi,significantly reduce the number of virus copies in the spleen after NIBV infection（P<0.05 or P<0.01）.In conclusion,baicalin can resist NIBV infection in the spleen of Hy-Line brown chicks by inhibiting PINK1/Parkin-mediated mitophagy and promoting M1 macrophage polarization.