Skip to main content
NCBI home page
Poultry Science logoLink to Poultry Science
. 2025 Mar 16;104(5):105054. doi: 10.1016/j.psj.2025.105054

Oxidative stress in the liver of chicken during fowl adenovirus serotype 4 infection

Jiayu Sun a,1, Xu Cao a,1, Yufeng Li b, Kexiang Yu b, Yanfang Cong c, Qing Pan a, Yanbo Yin a, Jianlin Wang a,⁎
PMCID: PMC11987656  PMID: 40120244

Abstract

Hepatitis is a significant pathological manifestation of fowl adenovirus serotype-4 (FAdV-4) infection, which is a crucial factor contributing to the mortality of chickens. The pathophysiology of liver disease is rooted in oxidative stress. The present study aims to investigate the presence of oxidative stress during the liver lesion process in FAdV-4 infection. Specifically, one-day-old specific pathogen-free (SPF) chickens were allocated into three groups, the control group, the infection group, and the quercetin group. The quercetin group received daily oral administration of quercetin. At the age of 12 days, the chickens belonging to both the infection and quercetin groups were subjected to intramuscular injection of FAdV-4 (0.3 mL103TCID50/mL). Samples were collected from each group at 2, 4, and 6 days post-infection (dpi), and sera were collected to measure the levels of ALT and AST. A portion of liver tissue was fixed to examine the histological changes, cell apoptosis, and mitochondrial morphology, while another portion was homogenized and mitochondria were isolated. The levels of MDA, SOD, H2O2, and GSH-Px in the homogenate supernatants of livers and isolated mitochondria were measured, and the viral load in the liver was studied. And Cyt C levels in the mitochondria and cytosolic supernatant were recorded. The results showed that AST and ALT in the serum of chicken in the infection group were significantly higher than those in the control and quercetin group at 6 dpi. Obvious swelling, steatosis, necrosis, and inflammatory cell infiltration were observed in the liver of the infection group. Administered with quercetin can significantly decrease the viral load in the liver at 4 and 6 dpi. H2O2 in the liver, and MDA, H2O2, GSH and SOD levels in mitochondria in the hepatocyte of the infection group were significantly higher than those in the control and quercetin groups. Cyt C in the mitochondria of the hepatocyte of infection and quercetin groups were significantly lower than those in the control group at 2 dpi. Cyt C in the cytoplasm of the liver in chicken in the quercetin group was significantly higher than those in the control and infection groups. It was found that the outer mitochondrial membrane in hepatocytes was fractured in the infection group. The proportion of apoptotic cells in the liver in the infection groups was significantly higher than those in the control and quercetin group at 4 dpi, and that in the control group was significantly lower than in the infection and quercetin group. The results suggested that during liver injury induced by FAdV-4 infection, oxidative damage occurred obviously in the liver and mitochondria, and hepatocyte apoptosis was observed. Quercetin, as an antioxidant, can inhibit virus replication to some extent, and alleviate oxidative damage, liver damage, and the mortality caused by FAdV-4 infection.

Keywords: Fowl adenovirus serotype 4, Oxidative stress, Liver damage, Mitochondria, Apoptosis

Introduction

Fowl adenovirus (FAdVs) are members of Aviadenovirus belonging to Adenoviridae, and non-enveloped double-stranded DNA viruses. FAdVs can be divided into 5 species (FAdV-A to FAdV-E) containing 12 serotypes (FAdV-1 to 8a and -8b to 11), according to molecular criteria, restriction enzyme digest pattern and serum cross-neutralization test (Hess, 2000). FAdVs have a worldwide distribution, but different serotypes or genotypes were discovered in different geographic regions. In China, the most prevalent FAdVs in recent years has been FAdV-4 (Ye et al., 2016), and related diseases in chickens are hepatitis-hydropericardium syndrome (HHS). HHS is characterized by the accumulation of clear, straw-colored fluid in the pericardial sac, as well as multifocal areas of necrosis in the liver and hepatitis at necropsy, and with a relatively high mortality of 30 %-70 % in the epidemiological studies (Kim et al., 2008).

Hydropericardium is the main characteristic of HHS, but it was indicated that the heart was not the target organ of FAdV-4, and the virus may not directly lead to the occurrence of lesion of cardiomyocytes (Niu et al., 2019). FAdV-4 was detected in all of the organs of the chicken infected, and extensive antigen staining and the highest viral load were found in the livers by immunohistochemistry (IHC) analysis and quantitative real-time PCR (qRT-PCR) respectively (Niu et al., 2016). The appearance of liver lesions in the organs was consistent with the viral copy numbers, indicating that virus replication in livers closely correlated with disease progression (Wu et al., 2020). The liver is a major target organ of FAdV-4, and acute hepatic necrosis may cause marked hepatic circulatory failure, which may lead to circulatory failure, hydropericardium, and, ultimately, death (Niu et al., 2016). Therefore, the mechanism of liver lesions is the focus of the pathogenesis of FAdV-4 infection. FAdV-4 induces hepatic steatosis by activating the LXR-α signaling pathway (Yuan et al., 2021) and leads to the dysfunction of liver function and metabolism. FAdV-4 induced liver injury via severe inflammatory response (Niu et al., 2018), and arginine protects hepatocytes against inflammation induced by FAdV-4 through JAK2/STAT3 signaling pathway (Xiang et al., 2022). FAdV-4 induced liver injury via apoptosis in the hepatocytes (Niu et al., 2018), and PX, a structural protein of FAdV-4, as an apoptosis-inducer plays an important role in inducing Leghorn Male Hepatocellular (LMH) cell apoptosis (Zhao et al., 2020), which facilitating FAdV-4 replication in host cells (Haiyilati et al., 2022). FAdV-4 induced liver injury via autophagy in the hepatocytes by activating the endoplasmic reticulum stress-related unfolded protein response pathway (Niu et al., 2018; Ma et al., 2022).

Oxidative stress is a consequence of an imbalance in the amount of reactive oxygen species (ROS), commonly called free radicals (Silveira et al., 2018). ROS are byproducts of normal cellular metabolism. Low and moderate amounts of ROS have beneficial effects on several physiological processes including the killing of invading pathogens, wound healing, and tissue repair processes. However, a disproportionate generation of ROS poses a serious problem to bodily homeostasis and causes oxidative tissue damage (Bhattacharyya et al., 2014). Once infected, viruses interact with the host complicated. Oxidative stress has been implicated in the pathogenesis of many viruses (Camini et al., 2017). Beyond its role in the cellular antiviral response, induction of oxidative stress has emerged as a common strategy employed by many viruses to promote their replication (Foo et al., 2022). Oxidative stress has been proven involved in many viral diseases of poultry, such as Newcastle disease, avian influenza, infectious bronchitis, infectious bursal disease. Redox state constitutes an important background of numerous liver disorders, oxidative stress underlies the pathophysiology of liver disease, especially viral hepatitis, such as hepatitis B virus infection, hepatitis C virus infection, and duck hepatitis A virus (DHAV) 1 infection (Chen et al., 2014; Seen, 2021). Within the cell, mitochondria play a key role in energy production and are involved in diverse cellular processes such as metabolism, production of ROS, and apoptotic regulation (Spinelli et al, 2018). Liver injury is a key process in the pathogenesis of FAdV-4 infection, in which whether or not oxidative stress is involved, has not been proved.

The present study was to investigate the impact of FAdV-4 infection on oxidative stress in chicken liver, mitochondrial injury, and hepatocyte apoptosis. Quercetin, a known antioxidant, was employed to assess the extent of oxidative damage in the liver and to mitigate the disease. The findings of this study may serve as a foundation for further exploration of the mechanisms underlying FAdV-4 infection, and offer insights into the prevention and treatment of FAdV-4 infections.

Materials and methods

The animal experiment was approved and performed in accordance with the guidelines of Ethics and Animal Welfare Committee of Qingdao Agricultural University.

FAdV-4 strain used in the study (GenBank Accession No. KU981149) was isolated from liver sample of broiler with HHS outbreak in China. Quercetin (purity≥98 %) was purchased from Xi'an Dewei Biotechnology Co., LTD, China.

Experiment 1 survival rate of chicken

Seventy two 1-day-old specific pathogen-free (SPF) leghorn chickens (Shandong Haotai Experimental Animal Breeding Co., LTD, China) were raised in separated negative -pressure isolators and randomly divided into three groups, respectively. Chickens in the infection group and quercetin group were inoculated with FAdV-4 by intramuscular injection with a dose 0.3mL(103TCID50/mL) at 12 days of age. Chicken in the control group was inoculated with 0.3mL DMEM/F12 basic medium (Gibco, Australia). Chickens in the quercetin group were orally administered with quercetin 15mg per chicken, once a day throughout the experiment. All chickens were monitored daily and the survival rate of chicken in every group was calculated till 6 days post infection (dpi).

Experiment 2 animal experimental design

One hundred and fifty 1-day-old SPF leghorn chickens (Shandong Haotai Experimental Animal Breeding Co., LTD, China) were raised and randomly divided into three groups, and treated as experiment 1. Up to seven birds per group were weighed, collected blood,and humanely euthanized (intravenous injection of pentobarbitone sodium,125 mg/kg body weight) at 2, 4, and 6 dpi. Birds were necropsied, and gross lesions in the liver was documented. Some of the liver was fixed in 4 % neutral-buffered formalin and 2.5 % glutaraldehyde, and some of the liver was prepared to extract the mitochondria with sucrose buffer, the remaining liver samples were stored at -80°C.

Biochemical assays for liver damage

Immediately before euthanasia, blood was collected from the jugular vein of each bird. Then it was transferred into procoagulant-coated tubes to separate the serum and stored at -20°C. And aspartate transaminase (AST) and alanine aminotransferase (ALT) activity in the serum was investigated according to the instruction of kit (Nanjing Jiancheng Bioengineering Institute, China) by enzymatic colorimetry using an ultraviolet photometer (UV2501, Japan).

Histological, mitochondrial morphology, and cell apoptosis analyses

Liver samples fixed in 4 % neutral-buffered formalin were embedded in paraffin blocks, cut into 4-μm sections, and stained with hematoxylin and eosin, and examined under a light microscope for lesions. The in situ labeling of apoptotic cells was performed using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay with commercial kits (Biological Technology Co. Ltd, Wuhan, China). Six fields (original magnification, 400 ×) were randomly selected for each slide, and the percentage of positive cells in each field to the total number of cells was calculated. Liver samples fixed in 2.5 % glutaraldehyde at 6 dpi were used to observe the morphology of mitochondria by transmission electron microscopy (TEM).

Viral load measurement

Liver samples were collected from birds of every group and stored at -20°Cfor quantification of viral load. DNA extraction was performed with DNeasy Kit (Accurate, China) according to the manufacturers protocol, and the DNA was subsequently analyzed by qRT-PCR according to the method of Günes et al (2012).

Isolation of mitochondria and cytosolic supernatant in liver

According to the study of Baev et al (2017) and Boulares et al (2001), mitochondria and cytosolic supernatant were isolated from the liver of birds by differential centrifugation. Fresh liver samples were homogenized using a Teflon-glass homogenizer and resuspended in 50 mL of isolation buffer (300 mM sucrose, 2 mM EDTA, and 5 mM Tris- HCl, 0.5 mg/mL bovine serum albumin, pH 7.4) on ice. Nuclei and whole cells were centrifuged at 2000 × g for 10 min °C. The supernatant was collected and spun at 6000 × g for 20 min °C. The resulting pellet was re-suspended in 30 mL of the isolation buffer without EDTA and bovine serum albumin and spun at 7500g for 20 min °C, and then the pellet (mitochondria) resuspended in 0.5 mL of the isolation buffer without EDTA and bovine serum albumin and put on the ice-bath. The resulting supernatant was then centrifuged at 12000×g for 15min at 4°C, samples of the final (cytosolic) supernatant were collected and put in the ice bath.

Evaluation indices of the oxidative stress

Liver samples were homogenated with a phosphate buffer solution (PBS, pH 7.4) in an ice bath. Levels of the malondialdehyde (MDA), superoxidedismutase (SOD), hydrogen peroxide (H2O2), and glutathione peroxidase (GSH-Px) in homogenate supernatants of livers and isolated mitochondria were measured by corresponding kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions by an ultraviolet photometer (UV2501, Japan).

Measurement of cytochrome C level

According to the method of Pharmacopoeia of the People's Republic of China (2005 Edition) and Skemiene et al (2013), the Cytochrome C (Cyt C) level in the mitochondria and cytosolic supernatant was recorded with an ultraviolet photometer (UV2501, Japan). Prepared samples were added to PBS for constant volume, and then added dithionite. The absorption peak height at 550 nm wavelength with an interval of 0.5nm was taken. The content of Cyt C was calculated according to the absorption coefficient of Cyt C of 23.0. And the linearity range is 0.1-10 mg/mL.

Statistical analysis

The results are shown as the means ± standard deviation (SD). The significance of the variability between groups was analyzed using Student's t-test in GraphPad software (version 5.0). A probability (P) value less than 0.05 and 0.01 is considered statistically significant and extremely significant respectively.

Results

Survival rate of chicken in different groups

The dynamic survival rate of chickens in different groups is illustrated in Fig. 1. The dynamic death of chicken in the infection group was similar to that of the quercetin group, death began from 2dpi, and death peak occurred between the 3dpi and the 5dip. The survival rates of SPF chicken in the infection, quercetin, and control group were 37.5 %, 45.8 % and 100 % at 6 dpi. And no deaths recorded in the chicken of quercetin group at 5-6 dpi.

Fig. 1.

Fig. 1

Open in a new tab

Dynamic survival rate of chicken in different groups.

Body weight of chicken in different groups

Fig. 2 shows the body weight of SPF chicken in different groups. Compared to the control group, the body weight in the infection and quercetin group decreased significantly at 4 to 6 dpi (P < 0.05 or P < 0.01). Body weight in the querctein group was significantly lower than that in the infection group at 5dpi, but there was no significant difference at 6dpi, and combined with the survival rate above, it suggested that the protective effect of quercetin was realized after 5 dpi.

Fig. 2.

Fig. 2

Open in a new tab

Dynamic body weight of SPF chicken in different groups.

Biochemical assays results for liver damage

As shown in Fig. 3, ALT in the serum of chicken in the infection group were significantly higher than those in the control group in the whole experiment (P < 0.05 or P < 0.01), and significantly higher than those in the quercetin group at 2 and 6 dpi (P < 0.01) (Fig. 3A). AST in the serum of chicken in the infection group was significantly higher than those in the control group at 4 and 6 dip (P < 0.01), and AST in the quercetin group was significantly higher than that in the control group at 4 dpi and lower than that in the infection group at 6 dpi (Fig. 3B).

Fig. 3.

Fig. 3

Open in a new tab

ALT and AST in the serum of SPF chicken in different groups (A. ALT; B.AST).

Gross, histological, mitochondrial morphology, and cell apoptosis in liver analyses

At necropsy, the livers of chicken in the infection group were slightly swollen and bleeding at 2dpi, visibly swollen and yellow at 4dpi, and there was severe necrosis on the surface at 6dpi. The livers of chicken in the quercetin group were slightly swollen at 2dpi, slightly yellow at 4dpi, and there was focal necrosis on the surface at 6dpi. No obvious change was observed in the liver of the chicken in the control group (Fig. 4).

Fig. 4.

Fig. 4

Open in a new tab

Gross pathological changes in the liver of SPF chicken in different groups (A. liver of SPF chicken in the infection group; B. liver of SPF chicken in the quercetin group; C. liver of SPF chicken in the control group. 1. liver of SPF chicken at 2 dpi; 2. liver of SPF chicken at 4 dpi; 3. liver of SPF chicken at 6 dpi.).

Histological study showed that hepatocytes of chicken in the infection group were swollen at 2dpi, fatty degeneration and necrosis in hepatocytes at 4dpi, and the presence of intranuclear inclusion bodies in degenerated hepatocytes and large inflammatory cell infiltration at the necrotic foci at 6dpi. Hepatocytes of chicken in the quercetin group were swollen at 2dpi, mild congestion and inflammatory cell infiltration at 4dpi, and the presence of intranuclear inclusion bodies and focal inflammatory cell infiltration at 6dpi. No obvious change was observed in the liver of the chicken in the control group (Fig. 5).

Fig. 5.

Fig. 5

Open in a new tab

Histopathology of the liver of SPF chicken in different groups (A. liver of SPF chicken in the infection group; B. liver of SPF chicken in the quercetin group; C. liver of SPF chicken in the control group. 1. liver of SPF chicken at 2 dpi; 2. liver of SPF chicken at 4 dpi; 3. liver of SPF chicken at 6 dpi.).

Ultrastructure of mitochondria in hepatocytes was observed, the number of mitochondria decreased, the inner and outer membranes were broken or absent, and the cristae in mitochondria were sparse, broken, indistinct, or even disappeared in the liver of the infection group chicken. And there were no obvious changes in the mitochondria of hepatocytes in the control group chicken (Fig. 6).

Fig. 6.

Fig. 6

Open in a new tab

Ultrastructure of mitochondria in hepatocytes of SPF chicken in different groups at 6 dpi (A. liver of SPF chicken in the infection group; B. liver of SPF chicken in the quercetin group; C. liver of SPF chicken in the control group.).

Proportion of apoptotic cells in the liver of chicken in the infection group was significantly higher than those in the control group at 4 and 6dpi (P < 0.01), in the quercetin group at 2 and 4dpi (P < 0.01 or P < 0.05), and proportion of apoptotic cells in the liver of chicken in the quercetin group were significantly higher than those in the control group at 4 and 6 dpi (P < 0.01) (Figs. 7 and 8).

Fig. 7.

Fig. 7

Open in a new tab

Apoptosis in the liver of SPF chicken in different groups (A. liver of SPF chicken in the infection group; B. liver of SPF chicken in the quercetin group; C. liver of SPF chicken in the control group. 1. liver of SPF chicken at 2 dpi; 2. liver of SPF chicken at 4 dpi; 3. liver of SPF chicken at 6 dpi.).

Fig. 8.

Fig. 8

Open in a new tab

Proportion of apoptotic cells in the liver of SPF chicken in different groups.

Viral load measurement results

Viral load in the liver of chicken in the infection group was significantly increased at 4 and 6 dpi (P < 0.01), and administered with quercetin can significantly decrease the viral load in the liver at 4 and 6 dpi (P < 0.01) (Fig. 12).

Fig. 12.

Fig. 12

Open in a new tab

Viral load in the liver of SPF chicken in different groups.

Results of the oxidative stress in liver

MDA in the liver increased after infection of FAdV-4, and was significantly higher than those in the control group at 4 and 6dpi (P < 0.01). Compared with the infection group, MDA in the liver of the quercetin group was decreased, and with a significant difference at 4dpi (P< 0.01) (Fig. 9A).

Fig. 9.

Fig. 9

Open in a new tab

Oxidation and antioxidation indexes in the liver of SPF chicken in different groups (A. MDA; B. H2O2; C. GSH; D. SOD).

H2O2 in the liver of chicken in the infection group was significantly higher than those in the control group in the whole experiment (P < 0.05 or P < 0.01), and higher than those in the quercetin group at 2 and 4dpi (P < 0.05) (Fig. 9B).

GSH in the liver of chicken in the infection group was significantly lower than those in the control group at 4 and 6 dpi (P < 0.05). GSH in the quercetin group was higher than those in the infection group, but there was no significant difference, and lower than those in the control group at 6 dpi (P < 0.05) (Fig. 9C).

There was no significant difference among all the groups of SOD in the liver of chicken at 2 and 4 dpi, and those were significantly higher in the infected and quercetin group than in the control group at 6dpi (P < 0.01) (Fig. 9D).

Results of the oxidative stress in mitochondria

H2O2 levels in the mitochondria of the liver of chicken in the infection group were significantly higher than those in the control and quercetin group (P < 0.01) (Fig. 10B).

Fig. 10.

Fig. 10

Open in a new tab

Oxidation and antioxidation indexes in the mitochondria of hepatocytes of SPF chicken in different groups (A. MDA; B. H2O2; C. GSH; D. SOD).

MDA level in the mitochondria of hepatocytes of chicken in the infection group was significantly higher than those in the control group (P < 0.01). MDA in the quercetin group was significantly higher than those in the control group and lower than those in the infection group at 4 and 6 dpi (P < 0.01) (Fig. 10A).

GSH in the mitochondria of the liver of chicken in the infection group was significantly higher than those in the control and quercetin group at 4 and 6 dpi (P < 0.01). GSH in the quercetin group was significantly lower than those in the control group at 2dpi (P < 0.05) (Fig. 10C).

SOD in mitochondria of the liver of chicken in the infection group was significantly higher than those in the control group in the whole experiment (P < 0.01 or P < 0.05), and significantly higher than those in the quercetin group at 2 and 4 dpi (P < 0.01) (Fig. 10D).

Cytochrome C level in the mitochondria and cytoplasm

Cyt C in the mitochondria in the liver of the infection group was lower than those in the control group in the whole experiment, and Cyt C in the control and quercetin group were significantly lower than those in the control group at 2 dpi (P < 0.01 or P < 0.05) (Fig. 11A).

Fig. 11.

Fig. 11

Open in a new tab

Cyt C content in the mitochondria and cytoplasm in the hepatocytes of SPF chicken in different groups (A. mitochondria; B. cytoplasm).

Cyt C in the cytoplasm of the liver in chicken in the quercetin group was significantly higher than those in the control and infection group (P < 0.01), and Cyt C in the infection group significantly higher than those in the control group at 6dpi (P < 0.01) (Fig. 11B).

Disscussion

In the study, FAdV-4 infection significantly affected the weight gain of SPF chickens, and with a mortality of 62.5 %, these are similar to the cases in the wild field. ALT and AST are common in the cytoplasm of hepatocytes. Under normal conditions, the activity of these two enzymes in serum was very low. When cells are damaged, membrane permeability increases, and ALT and AST were released into the blood and resulting in an increase in blood (Liumeij, 2008). So the serum ALT and AST levels, as the biomarkers of the hepatic injury (Niu et al., 2018) were quantified in the study. The results found that ALT and AST in chickens of the infection group were significantly higher than those in the control group, and suggested that the liver function of chickens was seriously impaired by FAdV-4 infection. Both the gross inspection and histopathological observation demonstrated distortion of hepatic architecture, the degeneration and necrosis of hepatocytes, and inflammatory response in the liver. From the results of dysfunction and morphological damage of hepatocytes, it was concluded that the liver of the chicken was seriously injured by FAdV-4 infection.

The term “oxidative stress” refers to a disturbance in the oxidant-antioxidant balance, leading to potential cellular damage. This imbalance could result from a lack of antioxidant capacity or an overabundance of reactive species (Camini et al., 2017). In most liver diseases, including those of metabolic and viral origin, oxidant stress acts as an important determining factor in the development of diseases (Seen, 2021). There are two ways to measure oxidative stress, by evaluating the concentrations of both by-products and promoters of the oxidative damage; which provides the dimensions of the damage; and by the antioxidant capacity of the medium in response to oxidative stress which determines the adaptation the organism to the unfavorable environment (Dotan et al., 2004; Silveira et al., 2018). The concentration of H2O2 in tissue is an indicator of oxidative damage and MDA is a commonly used marker for assessing lipid damage by oxidation (Silveira et al., 2018). In the study, H2O2 and MDA contents in the liver infected significantly increased and suggested that there was obvious oxidative damage to the liver of chicken infected with FAdV-4. During oxidative stress, cellular antioxidant capacity was suppressed by depleting antioxidant systems, such as GSH and SOD (Poprac et al., 2017). GSH is an important nucleophilic scavenger and an enzyme-catalyzed antioxidant, with a major role in protecting from oxidative tissue injury (Soto et al., 2020). GSH content reduction is the main mechanism of redox imbalance in viral-infected cells (De et al., 2020). In the study, GSH in the liver of chicken infected with FAdV-4 was significantly reduced and suggested that the antioxidant capacity of the liver was significantly decreased. SOD, as an enzymes that scavenge superoxide free radicals (Jacobs et al., 2021) was significantly increased in the later stage of this study. This suggested that SOD compensatory increased to remove free radicals, and GSH reduced by excessive consumption in the liver of chicken infected with FAdV-4. In this condition, free radicals unable to be effectively neutralized or oxidative damage unable to be repaired, indicating increased oxidative stress and antioxidant system imbalance, thereby triggering inflammation or cell damage. This phenomenon has also been reported in the studies of Qadri et al (2004) and Jacoby et al (1994), it has been reported that during HCV replication, oxidative stress was observed to a 5-fold increase, and levels of MnSOD (manganese-superoxide dismutase) were significantly increased, and H3N2 influenza virus infection in human airway epithelial cells increased SOD. One viral approach to control the excessive production of ROS is to increase the antioxidant capacity of the cell, implying that a fine balance between induction of oxidative stress and antioxidant cellular response is needed for the timely release of viral progeny (Sheyn et al., 2016). To prove the effect of oxidative stress on liver damage, quercetin, an antioxidant, was used to inhibit oxidative stress. It was found that quercetin significantly inhibited the FAdV-4-induced increase of MDA and H2O2 in the liver and AST and ALT in the serum, and decrease of GSH in the liver. These results revealed that oxidative stress occurred in the liver infected with FAdV-4, and antioxidant capacity was overwhelmed by oxidative damage leading to hepatocyte dysfunction and injury.

Mitochondria are membrane-bound organelles found inside most eukaryotic cells. Referred to as the powerhouse of the cell, the primary function of a mitochondrion is to generate energy in the form of ATP which is synthesized as electrons shuttle through the mitochondrial electron transport chain (ETC), and the ETC represents a major source of ROS within the cell (Foo et al., 2022). In the study, the mitochondria in the livers of chicken infected were damaged and the number was significantly decreased. The concentration of H2O2, MDA, SOD, and GSH in mitochondria of hepatocytes in chicken infected was significantly increased and was all significantly inhibited by quercetin. These results showed that the large amounts of ROS generated in the mitochondria, and the increase of SOD and GSH may be a compensatory response to the increase of antioxidant capacity in mitochondria. ROS can directly attack mitochondrial DNA, oxidize mitochondrial proteins, especially key enzymes in the electron transport chain, affecting mitochondrial function. In addition, ROS can attack mitochondrial membrane lipids, resulting in decreased membrane fluidity and permeability changes, affecting mitochondrial integrity. When mitochondria are damaged, the damaged electron transport chain increases electron leakage and reacts with oxygen to produce more ROS, reduced ATP production and insufficient cell energy further induce ROS production and reduced the activity of antioxidant enzymes further impairs the ability to clear ROS. The generation of ROS and damaging the mitochondria forms a vicious cycle (Filomeni et al., 2015). In contrast to the liver, GSH levels in the mitochondria were increased in the study, which may be related to the fact that GSH is the major antioxidant and is produced in the cytosol but is also required by mitochondria, transport into the mitochondria is needed (Von, 2022). The results of the study indicated that oxidative stress in hepatocytes and mitochondria was induced by FAdV-4 infection, and quercetin inhibited oxidative stress, it was proved that mitochondrial oxidative stress caused by FAdV-4 infection was the main source of oxidative stress in hepatocytes.

As the report of Niu et al (2018), a large number of positive hepatocytes was observed in the liver of chicken infected with FAdV-4 by the assay of TUNEL in this study. For further analysis, hepatocytes apoptosis was quantified, and found that the apoptosis in chicken infection group was significantly higher than that in the control group. These results suggested that hepatocyte apoptosis was induced by FAdV-4 infection. But the mechanism of apoptosis induced by FAdV-4 is not clear. Cyt C is primarily known for its function in the mitochondria as a key participant in the life-supporting function of ATP synthesis. However, when a cell receives an apoptotic stimulus, Cyt C is released into the cytosol and triggers programmed cell death through apoptosis (Ow et al., 2008). In this study, it was found that the concentration of Cyt C in the mitochondria was significantly decreased and that in the cytoplasm was increased. After Cyt C release into the cytosol, the protein binds to apoptosis protease activating factor-1, activates pro-caspase 9, and triggers an enzymatic cascade leading to cell death (Santucci et al., 2019). Therefore, it is presumed that the release of Cyt C stimulated by oxidative stress is one of the factors leading to hepatocytes apoptosis in chicken infected with FAdV-4.

To prove the effect of oxidative stress on hepatocytes apoptosis induced by FAdV-4, quercetin, an antioxidant, was used to inhibit oxidative stress. It was found that hepatocytes apoptosis induced by FAdV-4 was significantly decreased after the application of quercetin, indicating that oxidative stress was involved in the process of apoptosis. It was reported that oxidative stress has been well established as one of the stimuli that initiate the intrinsic apoptotic pathway (Foo et al., 2022), which is triggered by the release of Cyt C from the mitochondria into the cytoplasm (Ow et al., 2008). It should be noted, however, that when quercetin was used in this study, the decreased mitochondrial Cyt C by FAdV-4 infection was increased, but the cytosolic Cyt C was significantly further increased. And compared with the FAdV-4 infection group, the increase of cytosolic Cyt C was not accompanied by the increase of apoptosis in the quercetin group. This may be related to the multiple other functions of Cyt C, such as the key participant in the life-supporting function of ATP synthesis, ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import (Kalpage et al., 2019) and also may be related to the function and mechanism of quercetin. This needs to be explored.

Oxidative stress plays distinct roles in different viral infections and influences viral infectivity (Khan et al., 2021). In the case of human immunodeficiency virus infection, oxidative stress is induced and leads to activation of NF-κB-dependent virus transcription (Choi et al., 2004). ROS has been demonstrated to interfere with the formation of replication complexes leading to suppression of hepatitis C virus replication (Choi et al., 2004). Despite the distinct roles, studies have increasingly reported that ROS-mediated modulation of host signaling pathways has been exploited by viruses to thrive in their host (Foo et al., 2022). Enterovirus-A71 as a prime example was proved to exploit the oxidative stress response for its replication and release of its viral progeny (Tung et al., 2011; Cheng et al., 2014). This claim is mostly based on the observation that antioxidant treatments resulted in lower viral titers in vitro in the culture supernatants (Foo et al., 2022). Similar to the report, viral loads in the liver of chicken treated with quercetin were significantly decreased in the study, these suggested that increased ROS levels benefited the replication of FAdV-4. One viral approach to control excessive production of ROS is to increase the antioxidant capacity of the cell, implying that a fine balance between induction of oxidative stress and antioxidant cellular response is needed for the timely release of viral progeny (Sheyn et al., 2016). To defend against oxidative injury, cells themselves have evolved defenses primarily dependent upon antioxidant enzymes, supply of their substrates, and repair of injury (Forman et al., 2021). In the study, the concentration of some antioxidants in the liver or in the mitochondria of hepatocytes was increased after infection of FAdV-4. Increased antioxidant capacity to counteract ROS, which has to do with the direct action of hepatocytes for survival? or /and with the indirect action of FAdV-4 evolved to counteract ROS to keep the cellular environment favorable for viral replication, this mechanism needs to be explored.

In summary, this study firstly revealed that FAdV-4 infection caused oxidative stress in the liver and hepatocytes mitochondrial of chicken and that oxidative stress-induced apoptosis by altering the content of Cyt C in the mitochondria and cytoplasm. Quercetin, as an antioxidant, could significantly reduce mitochondrial oxidative damage, liver oxidative damage, hepatocytes apoptosis, and virus replication, but it could not completely prevent liver damage, weight loss, and mortality, the results suggested that oxidative damage is an important factor in liver injury infected by FAdV-4 infection, but not the only one.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Natural Science Foundation of Shandong Province (grant number ZR2023MC030).

References

  1. Baev A.Y., Negoda A., Abramov A.Y. Modulation of mitochondrial ion transport by inorganic polyphosphate - essential role in mitochondrial permeability transition pore. J. Bioenerg. Biomembr. 2017;49:49–55. doi: 10.1007/s10863-016-9650-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bhattacharyya A., Chattopadhyay R., Mitra S., Crowe S.E. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol. Rev. 2014;94:329–354. doi: 10.1152/physrev.00040.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boulares A.H., Zoltoski A.J., Yakovlev A., Xu M., Smulson M.E. Roles of DNA fragmentation factor and poly (ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis. J. Biol. Chem. 2001;276:38185–38192. doi: 10.1074/jbc.M100629200. [DOI] [PubMed] [Google Scholar]
  4. Camini F.C., da S.C.C.C., Almeida L.T., de B.M.C.L. Implications of oxidative stress on viral pathogenesis. Arch Virol. 2017;162:907–917. doi: 10.1007/s00705-016-3187-y. [DOI] [PubMed] [Google Scholar]
  5. Chen Y., Xiong W., Zeng L., Wang Y., Zhang S., Xu M., Song M., Wang Y., Du H., Liu J., Wang D., Wu Y., Hu Y. Bush Sophora Root polysaccharide and its sulfate can scavenge free radicals resulted from duck virus hepatitis. Int. J. Biol. Macromol. 2014;66:186–193. doi: 10.1016/j.ijbiomac.2014.02.038. [DOI] [PubMed] [Google Scholar]
  6. Cheng M.L., Weng S.F., Kuo C.H., Ho H.Y. Enterovirus 71 induces mitochondrial reactive oxygen species generation that is required for efficient replication. PLoS. One. 2014;9 doi: 10.1371/journal.pone.0113234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choi J., Lee K.J., Zheng Y., Yamaga A.K., Lai M.M., Ou J.H. Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells. Hepatology. 2004;39:81–89. doi: 10.1002/hep.20001. [DOI] [PubMed] [Google Scholar]
  8. De F.S., Balansky R., La M.a S. Rationale for the use of N-acetylcysteine in both prevention and adjuvant therapy of COVID-19. FASEB. J. 2020;34:13185–13193. doi: 10.1096/fj.202001807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dotan Y., Lichtenberg D., Pinchuk I. Lipid peroxidation cannot be used as a universal criterion of oxidative stress. Prog. Lipid. Res. 2004;43:200–227. doi: 10.1016/j.plipres.2003.10.001. [DOI] [PubMed] [Google Scholar]
  10. Filomeni G., De Z.D., Cecconi F. Oxidative stress and autophagy: the clash between damage and metabolic needs. Cell. Death. Differ. 2015;22:377–388. doi: 10.1038/cdd.2014.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Foo J., Bellot G., Pervaiz S., Alonso S. Mitochondria-mediated oxidative stress during viral infection. Trends. Microbiol. 2022;30:679–692. doi: 10.1016/j.tim.2021.12.011. [DOI] [PubMed] [Google Scholar]
  12. Forman H.J., Zhang H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat. Rev. Drug. Discov. 2021;20:689–709. doi: 10.1038/s41573-021-00233-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Günes A., Marek A., Grafl B., Berger E., Hess M. Real-time PCR assay for universal detection and quantitation of all five species of fowl adenoviruses (FAdV-A to FAdV-E) J. Virol. Methods. 2012;183:147–153. doi: 10.1016/j.jviromet.2012.04.005. [DOI] [PubMed] [Google Scholar]
  14. Haiyilati A., Zhou L., Li J., Li W., Gao L., Cao H., Wang Y., Li X., Zheng S.J. Gga-miR-30c-5p enhances apoptosis in fowl Adenovirus serotype 4-infected leghorn male hepatocellular cells and facilitates viral replication through myeloid cell leukemia-1. Viruses. 2022;14:990. doi: 10.3390/v14050990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hess M. Detection and differentiation of avian adenoviruses: a review. Avian. Pathol. 2000;29:195–206. doi: 10.1080/03079450050045440. [DOI] [PubMed] [Google Scholar]
  16. Jacobs P.J., Oosthuizen M.K., Mitchell C., Blount J.D., Bennett N.C. Oxidative stress in response to heat stress in wild caught Namaqua rock mice, Micaelamys namaquensis. J. Therm. Biol. 2021;98 doi: 10.1016/j.jtherbio.2021.102958. [DOI] [PubMed] [Google Scholar]
  17. Jacoby D.B., Choi A.M. Influenza virus induces expression of antioxidant genes in human epithelial cells. Free. Radic. Biol. Med. 1994;16:821–824. doi: 10.1016/0891-5849(94)90198-8. [DOI] [PubMed] [Google Scholar]
  18. Kalpage H.A., Bazylianska V., Recanati M.A., Fite A., Liu J., Wan J., Mantena N., Malek M.H., Podgorski I., Heath E.I., Vaishnav A., Edwards B.F., Grossman L.I., Sanderson T.H., Lee I., Hüttemann M. Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis. FASEB. J. 2019;33:1540–1553. doi: 10.1096/fj.201801417R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khan N.A., Kar M., Panwar A., Wangchuk J., Kumar S., Das A., Pandey A.K., Lodha R., Medigeshi G.R. Oxidative stress specifically inhibits replication of dengue virus. J. Gen. Virol. 2021;102:1–28. doi: 10.1099/jgv.0.001596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim J.N., Byun S.H., Kim M.J., Kim J., Sung H.W., Mo I.P. Outbreaks of hydropericardium syndrome and molecular characterization of Korean fowl adenoviral isolates. Avian. Dis. 2008;52:526–530. doi: 10.1637/8178-112207-Case. [DOI] [PubMed] [Google Scholar]
  21. Lumeij J. In: Avian Clinical Biochemistry. Pages 839–872 in the Clinical Biochemistry of Domestic Animals. 6th edn. Kaneko J.J., Harvey J.W., Bruss M.L., editors. Academic Press; Cambridge: 2008. [Google Scholar]
  22. Ma H., Ding Y., Du K., Chang K., Niu Y. FAdV-4 induce autophagy via the endoplasmic reticulum stress-related unfolded protein response. Vet, Microbiol. 2022;269 doi: 10.1016/j.vetmic.2022.109388. [DOI] [PubMed] [Google Scholar]
  23. Niu Y., Sun Q., Liu X., Liu S. Mechanism of fowl adenovirus serotype 4-induced heart damage and formation of pericardial effusion. Poult Sci. 2019;98:1134–1145. doi: 10.3382/ps/pey485. [DOI] [PubMed] [Google Scholar]
  24. Niu Y., Sun Q., Zhang G., Liu X., Shang Y., Xiao Y., Liu S. Fowl adenovirus serotype 4-induced apoptosis, autophagy, and a severe inflammatory response in liver. Vet. Microbiol. 2018;223:34–41. doi: 10.1016/j.vetmic.2018.07.014. [DOI] [PubMed] [Google Scholar]
  25. Niu Y.J., Sun W., Zhang G.H., Qu Y.J., Wang P.F., Sun H.L., Xiao Y.H., Liu S.D. Hydropericardium syndrome outbreak caused by fowl adenovirus serotype 4 in China in 2015. J. Gen. Virol. 2016;97:2684–2690. doi: 10.1099/jgv.0.000567. [DOI] [PubMed] [Google Scholar]
  26. Ow Y.P., Green D.R., Hao Z., Mak T.W. Cytochrome c: functions beyond respiration. Nat. Rev. Mol. Cell. Biol. 2008;9:532–542. doi: 10.1038/nrm2434. [DOI] [PubMed] [Google Scholar]
  27. Pharmacopoeia Commission of the People's Republic of China . Chemical Industry Press; Beijing, China: 2005. Page 370 in Pharmacopoeia of The People's Republic of China. [Google Scholar]
  28. Poprac P., Jomova K., Simunkova M., Kollar V., Rhodes C.J., Valko M. Targeting free radicals in oxidative stress-related Human diseases. Trends. Pharmacol. Sci. 2017;38:592–607. doi: 10.1016/j.tips.2017.04.005. [DOI] [PubMed] [Google Scholar]
  29. Qadri I., Iwahashi M., Capasso J.M., Hopken M.W., Flores S., Schaack J., Simon F.R. Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1. Biochem. J. 2004;378:919–928. doi: 10.1042/BJ20031587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Santucci R., Sinibaldi F., Cozza P. Polticelli F, Fiorucci L. Cytochrome c: an extreme multifunctional protein with a key role in cell fate. Int. J. Biol. Macromol. 2019;136:1237–1246. doi: 10.1016/j.ijbiomac.2019.06.180. [DOI] [PubMed] [Google Scholar]
  31. Seen S. Chronic liver disease and oxidative stress-a narrative review. Expert Rev. Gastroenterol. Hepatol. 2021;15:1021–1035. doi: 10.1080/17474124.2021.1949289. [DOI] [PubMed] [Google Scholar]
  32. Sheyn U., Rosenwasser S., Ben-Dor S., Porat Z., Vardi A. Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean. ISME. J. 2016;10:1742–1754. doi: 10.1038/ismej.2015.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Silveira A.S., Aydos R.D., Ramalho R.T., Silva I.S., Caldas R.A., Santos N.A.T.D., Rodrigues C.T. Oxidative stress effects in the uterus, placenta and fetus of pregnant rats submitted to acute and chronic stress. Acta. Cir. Bras. 2018;33:806–815. doi: 10.1590/s0102-865020180090000009. [DOI] [PubMed] [Google Scholar]
  34. Skemiene K., Rakauskaite G., Trumbeckaite S., Liobikas J., Brown G.C., Borutaite V. Anthocyanins block ischemia-induced apoptosis in the perfused heart and support mitochondrial respiration potentially by reducing cytosolic cytochrome c. Int. J. Biochem. Cell. Biol. 2013;45:23–29. doi: 10.1016/j.biocel.2012.07.022. [DOI] [PubMed] [Google Scholar]
  35. Soto M.E., Guarner-Lans V., Soria-Castro E., Manzano P.L., Pérez-Torres I. Is antioxidant therapy a useful complementary measure for Covid-19 treatment? An algorithm for its application. Medicina (Kaunas). 2020;56:386. doi: 10.3390/medicina56080386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spinelli J.B., Haigis M.C. The multifaceted contributions of mitochondria to cellular metabolism. Nat. Cell. Biol. 2018;20:745–754. doi: 10.1038/s41556-018-0124-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tung W.H., Hsieh H.L., Lee I.T., Yang C.M. Enterovirus 71 induces integrin β1/EGFR-Rac1-dependent oxidative stress in SK-N-SH cells: role of HO-1/CO in viral replication. J. Cell. Physiol. 2011;226:3316–3329. doi: 10.1002/jcp.22677. [DOI] [PubMed] [Google Scholar]
  38. Von B.U.H.O. Controlling glutathione entry into mitochondria: potential roles for SLC25A39 in health and (treatment of) disease. Signal. Transduct. Target. Ther. 2022;7:75. doi: 10.1038/s41392-022-00928-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wu N., Yang B., Wen B., Li W., Guo J., Qi X., Wang J. Pathogenicity and immune responses in specific-pathogen-free chickens during fowl adenovirus serotype 4 infection. Avian. Dis. 2020;64:315–323. doi: 10.1637/aviandiseases-D-20-00004. [DOI] [PubMed] [Google Scholar]
  40. Xiang S., Huang R., He Q., Xu L., Wang C., Wang Q. Arginine regulates inflammation response-induced by Fowl Adenovirus serotype 4 via JAK2/STAT3 pathway. BMC. Vet. Res. 2022;18:189. doi: 10.1186/s12917-022-03282-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ye J., Liang G., Zhang J., Wang W., Song N., Wang P., Zheng W., Xie Q., Shao H., Wan Z., Wang C., Chen H., Gao W., Qin A. Outbreaks of serotype 4 fowl adenovirus with novel genotype. China. Emerg. Microbe. Infect. 2016;25:e50. doi: 10.1038/emi.2016.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yuan F., Hou L., Wei L., Quan R., Wang J., Liu H., Liu J. Fowl Adenovirus serotype 4 induces hepatic steatosis via activation of liver X receptor-α. J. Virol. 2021;95:e01938. doi: 10.1128/JVI.01938-20. -20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhao M., Duan X., Wang Y., Gao L., Cao H., Li X., Zheng S.J. A novel role for PX, a structural protein of Fowl Adenovirus serotype 4 (FAdV4), as an apoptosis-inducer in Leghorn male hepatocellular cell. Viruses. 2020;12:228. doi: 10.3390/v12020228. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Poultry Science are provided here courtesy of Elsevier

RESOURCES