Andrographolide Alleviates Oxidative Damage and Inhibits Apoptosis Induced by IHNV Infection via CTSK/BCL2/Cytc Axis

Abstract

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen that causes significant economic losses to salmon trout farming. Although vaccines have been invented for the treatment of IHNV, findings from our previous survey show that breeding enterprises and farmers require effective oral drugs or immune enhancers. However, studies on the development of oral drugs are limited. In the present study, we used bioinformatics methods to predict the protein targets of andrographolide (Andro) in IHNV. Cells were infected with IHNV, and the effect of andrographolide was explored by evaluating the expression levels of genes implicated in oxidative stress, activities of antioxidant enzymes, and the expression of genes implicated in apoptosis and necrosis. In the present study, cells were divided into NC, IHNV, IHNV+10 μM andrographolide, and IHNV+20 μM andrographolide groups. qRT-PCR was performed to determine the expression level of genes, and an antioxidant enzyme detection kit was used to evaluate the activities of antioxidant enzymes. Fluorescent staining was performed using a reactive oxygen species detection kit (ROS) and Hoechst 33342/PI double staining kit, and the mechanism of alleviation of apoptosis and oxidative stress andrographolide after IHNV infection was determined. The results indicated that andrographolide inhibits viral growth by binding to the NV protein of IHNV and increasing the antioxidant capacity of the body through the CTSK/BCL2/Cytc axis, thereby inhibiting the occurrence of IHNV-induced apoptosis. This is the first study to explore the antagonistic mechanism of action of andrographolide in alleviating IHNV infection. The results provide valuable information on alternative strategies for the treatment of IHNV infection during salmon family and provide a reference for the use of andrographolide as an antioxidant agent in agricultural settings.

Keywords: IHNV; andrographolide; antiviral drug; apoptosis; oxidative stress.

Figure 1

Titers and growth curves of IHNV incubated with EPC cells at different time points. (a) Viral titers in EPC cells at different time points; (b) Growth curves of IHNV growing in EPC cells at different time points.

Figure 2

Effect of different concentrations of andrographolide on IHNV infected/uninfected cells. (a) Effect of different concentrations of andrographolide on cell viability. (b) Antiviral effects of different concentrations of andrographolide on IHNV. The scale is 200 μm.

Figure 3

Effect of andrographolide and IHNV infection on the antioxidant capacity of EPC cells. (a) Effect of andrographolide and IHNV infection on cell viability and the expression levels of antioxidant enzymes. * Represents significant differences compared with the NC group (p < 0.05). # Represents significant differences between IHNV and IHNV+20 Μm groups (p < 0.05); (b) The effects of IHNV infection and andrographolide treatment on intracellular ROS levels as determined by fluorescent staining. The white arrows represent cells with positive staining. The scale is 200 μm; (c) The statistical analysis results of ROS fluorescence staining, * representing a significant difference compared with the NC group (p < 0.05); # represents significant differences between groups (p < 0.05).

Figure 4

Effect of andrographolide and IHNV infection on cell death. (a) Apoptotic cells are colored in bright blue, and necrotic cells are colored in red. The white arrows represent cells with positive staining. The scale is 200 μm. (b) The statistical analysis results of Hoechst33342/PI fluorescence staining, * representing a significant difference compared with the NC group (p < 0.05).

Figure 5

Prediction of the binding between andrographolide and IHNV viral proteins. The interactions were assessed by the numerical analysis of binding energy (Etotal), whereby Etotal < 0 indicated binding between the ligand and protein target. A smaller Etotal value indicates a higher possibility of binding. The structures formed by the red-green globules represent andrographolide, and the 3D structure composed of the remaining color pellets is the viral protein of IHNV.

Figure 6

Prediction of interactions between host proteins and NV proteins of IHNV. The binding efficiency was determined by the numerical analysis of the Etotal value, with Etotal < 0 indicating high binding affinity. A smaller value of Etotal indicates a higher binding affinity between the ligand and target.

Figure 7

Effect of IHNV and andrographolide on expression levels of oxidative stress, apoptosis, and necrosis pathway genes. (a) Effect of andrographolide on genes coded IHNV virus proteins. (b) Effect of andrographolide on the expression of genes involved in necroptosis pathway. (c) Effect of andrographolide on the expression of genes related to oxidative stress. (d) Effect of andrographolide on the expression of genes involved in apoptosis. * Represents differentially expressed genes compared with the NC group (p < 0.05).