Exploration of the effect and mechanism of indirubin on infectious bovine rhinotracheitis virus based on network pharmacology

Abstract

Background

Our team’s previous research indicates that infectious bovine rhinotracheitis virus (IBRV) induces mitochondrial damage in bovine kidney cells (MDBK). Indirubin, a bisindole alkaloid, can alleviate mitochondrial damage. However, it is currently unclear whether indirubin can regulate mitochondrial damage caused by IBRV in MDBK cells.

Results

This study predicted the anti-IBRV effect of indirubin through network pharmacology, verified its anti-IBRV activity through experiments, and confirmed that indirubin can alleviate mitochondrial damage caused by IBRV. Network pharmacological results show that 81 potential targets of indirubin in infectious bovine rhinotracheitis (IBR) have been screened through network analysis. The analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) shows that the anti-IBR activity of indirubin involves multiple signaling pathways. Genetic ontology (GO) analysis reveals that its anti-IBR effect encompasses a variety of biological functions, with 15 target genes enriched in mitochondria. Virus suppression experiments have shown that indirubin can inhibit IBRV virus replication in MDBK cells. Flow cytometry showed that indirubin can reduce the elevated levels of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential (MMP) caused by IBRV infection. The molecular docking results confirm that indirubin exhibits a strong binding affinity with 15 targets.

Conclusions

Our research shows that indirubin may exert anti-IBRV properties by regulating the production of ROS and inhibiting MMP depolarization.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-026-05325-x.

Background

Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus 1 (BoHV-1, commonly referred to as infectious bovine rhinotracheitis virus, IBRV), is a prevalent respiratory disease affecting cattle populations worldwide. Infected adult cattle may exhibit symptoms such as high fever and cough, leading to significant economic losses in the livestock industry [1]. The virus belongs to the subfamily Alphaherpesvirinae of the family Orthoherpesviridae. IBRV has an icosahedral capsid, contains double-stranded DNA and a glycoprotein envelope, and can survive in a low-temperature environment for a long time [2]. Research has shown that IBRV infection of MDBK cells activates the NF-κB and caspase pathways [3], inducing cell apoptosis and promoting virus release [3]. Other studies have shown that the bovine infectious rhinotracheitis virus not only induces epithelial cell apoptosis, but also leads to immunosuppression, including the down-regulation of the interferon (IFN) response. These processes will cause respiratory inflammation and secondary bacterial infection, which will eventually lead to typical symptoms such as fever and a runny nose [4]. At present, the prevention and treatment methods for IBRV are still limited to symptomatic treatment. The fundamental reason is that the pathogenic mechanism of IBRV is not clear enough. Our team’s previous research has confirmed that IBRV can induce mitochondrial damage in MDBK cells [5], which manifests as morphological alterations and decreased membrane potential [5]. This finding provides a key starting point for clarifying the pathogenic mechanism of IBRV, and is expected to provide new ideas and foundations for the breakthrough of IBRV prevention and treatment technology.

Due to the complex structure of mitochondria, they are more susceptible to damage than other organelles. Mitochondrial damage is mainly manifested as structural and functional abnormalities. Structurally, mitochondrial swelling is an iconic feature, manifested as volume expansion, matrix transparency, and ridge structure destruction [6]. The key indicators of functional abnormalities include abnormal mitochondrial permeability transition pore (mPTP) [7], changes in mitochondrial membrane potential (MMP) [7], and disruption of calcium ion (Ca²⁺) homeostasis [7]. These factors often have a synergistic effect, jointly exacerbating mitochondrial damage [8, 9]. Previous research by our team has shown that IBRV infection with host cells directly leads to an abnormal increase in the level of ROS in cells and significantly reduces the MMP [5]. These two factors work together to disrupt the structural integrity and functional stability of mitochondria, thereby inducing mitochondrial damage. This discovery provides key clues for further elucidating the pathogenic mechanism of IBRV.

MMP is a key indicator of mitochondrial function and is crucial to cell energy generation. The asymmetric distribution of protons and ions on both sides of the mitochondrial membrane is the basis for the formation of MMP, among which potassium ions (K⁺) and sodium ions (Na⁺) play a central role in maintaining the stability of MMP. The destruction of their balance will lead to abnormal MMP, which directly damages the energy metabolism of cells [10]. Reactive oxygen species (ROS) are the main cause of mitochondrial damage. Excessive ROS can trigger oxidative stress, oxidizing mitochondrial membrane lipids, proteins, and other biomolecules, damaging mitochondrial membrane structure, protein function, and mitochondrial DNA, ultimately leading to a cellular energy crisis [11]. At the same time, ROS can induce the opening of mPTP and change mitochondrial permeability, which leads to the collapse of MMP. This forms a vicious cycle of ROS accumulation → mitochondrial permeable pore opening → MMP reduction → mitochondrial dysfunction [7].

Indirubin is an isomer of indigo. It is a kind of diindoline alkaloid and one of the main active ingredients of the Chinese herbal banlan root and Qingdai [12]. Recent studies have indicated that indirubin exhibits multiple pharmacological properties, for example, anti-inflammatory, anti-tumor, and antiviral activity [13–17]. Indirubin demonstrates anti-inflammatory effects by activating the aryl hydrocarbon receptor (AHR) pathway and inhibiting inflammasomes [13], improve the condition of colon tissue damage and pro-inflammatory factors [13]. Indirubin’s antitumor effects help inhibit chronic myeloid leukemia by suppressing the Bcr-Abl fusion protein [14]. Indirubin’s derivative, E804, also downregulates the Nrf2-HO-1/GPX4 pathway to induce ferroptosis in lung cancer A549 cells [18]. Owing to its antiviral effects, indirubin significantly suppresses hepatitis B virus (HBV) antigen secretion and DNA replication by downregulating poly(d, l)-pyrimidine-2,6-tetraeste-1,3,5,7-tetraol (PTB) in host cells [15]. Indirubin has been shown to enhance the expression of type I interferon (IFN-β) and interferon-induced transmembrane protein 3 (IFITM3) by modulating the mitochondrial antiviral signaling pathway (MAVS), thereby reducing susceptibility to the influenza A (H1N1) virus in stressed mice and mitigating lung injury [19]. Indirubin exhibits direct inactivating effects against the Japanese encephalitis virus (JEV), exerting antiviral activity by blocking the adsorption of the virus onto host cells [16]. In addition, indirubin can alleviate mitochondrial damage through various pathways, such as activating the Nrf2 pathway to alleviate oxidative damage [20] and exerting antiviral effects by regulating the MAVS pathway to reduce susceptibility to influenza [21]. However, there have been no reports on the intervention effect of indirubin on IBRV induced mitochondrial damage.

In summary, this study aims to further elucidate the role of indirubin in alleviating IBRV-induced mitochondrial damage and inhibiting viral replication. Firstly, network pharmacology was employed to identify therapeutic targets for indirubin in treating IBRV-induced mitochondrial damage. Subsequently, experimental validation confirmed indirubin’s restorative effects on mitochondrial function and its inhibitory action on viral replication. This research seeks to provide novel candidate molecules for the development of anti-IBRV therapeutics.

Methods

Drug target prediction

The secondary structure file for indirubin was sourced from the PubChem (https://pubchem.ncbi.nlm.nih.gov/) [22]. This file was then imported into multiple databases, including PharmMapper [22] (https://lilab-ecust.cn/pharmmapper/), SwissTargetPrediction [23] (https://swisstargetprediction.ch/), TCMSP [24] (https://tcmsp.91medicine.cn/), SEA [25] (https://sea.bkslab.org/), and Herb [26] (http://herb.ac.cn), to identify the target genes of indirubin. Subsequently, merge the target data obtained from the five databases and remove duplicates.

Screening of potential targets of IBRV

Based on prior research findings, we established criteria of count ≥ 100 and P < 0.5 for screening differentially expressed mRNAs from high-throughput sequencing data. High-throughput sequencing data from the Mock group(normal cell culture for 24 h) and IBRV group (IBRV was inoculated at a multiplicity of infection (MOI) of 1.5 and cultured for 24 h) were uploaded to the Omicsmart online platform to identify differentially expressed mRNAs, which were then utilised as disease targets [27]. The intersection targets of indirubin and IBR targets and the Venn diagram were obtained by using Venny 2.1.0, a Venn diagram–generating online site. Indirubin’s potential target sites for treating IBR were acquired for subsequent analyses.

Construction of the protein-protein interaction (PPI) network

Based on the prospect therapeutic target molecules of indirubin for IBRV, we constructed a PPI network using the STRING database (https://string-db.org/) [23]. We set the functional score threshold to < 0.400 and the p-value threshold for PPI enrichment analysis to < 1.0 × 10^⁽⁻⁶⁾ [28]. After constructing the PPI network, the degree centrality (DC) value of each target was analyzed using Cytoscape [23]. Visualize the PPI network using Cytoscape software (Cytoscape_v3.10.0) [29].

Genontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis of potential therapeutic targets

To enhance our understanding of the mechanism of action of indirubin on IBRV, we carried out enrichment analyses of GO terms (including BP, CC, and MF) and KEGG pathways on potential targets [22]. The conversion of crossover genes to gene-stable IDs and subsequent analyses were completed using the Omicsmart online platform. For GO analysis, we selected the top 10 genes; for KEGG analysis, the top 20 genes, with a screening threshold set at p < 0.05. KEGG classification diagrams were created via the bioinformatics platform (http://www.bioinformatics.com.cn/) [22]. Finally, Cytoscape software was employed to construct a “drug-disease-target-signaling pathway” network and to reveal the relationship between them.

Sourcing of cell lines, virus, and other chemical reagents

In this study, MDBK cells were acquired via the Chinese Academy of Sciences Cell Bank (Shanghai, China), and the IBRV AV21 strain was obtained from the China Veterinary Drug Control Institute (Beijing, China). IBRV primers and probes were fabricated by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China; Table S1). The ROS assay kit, MMP assay kit (JC-1), and CCK-8 assay kit were commercially purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Jiangsu, China).

Medication preparation

Indirubin and acyclovir (ACV) were acquired via the Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Indirubin was stored at − 20 °C after dissolution in dimethyl sulfoxide (DMSO) to a concentration of 15 mM [30]. ACV was diluted in DMEM to a Storage concentration of 100 µM and stored at 4 °C [31].

Cytotoxicity assay

Cultivate MDBK cells in a 96-well plate at 37 °C and 5% CO₂ for 12 h. The cells were then treated with increasing concentrations of indirubin (20 µM, 40 µM, 60 µM, 80 µM, 100 µM, 120 µM, and 140 µM) and subjected to incubation for 24 h. After the initial incubation, CCK-8 reagent was added to the cells according to the manufacturer’s instructions and incubated for 2.5 h. Absorbance was measured using a multifunction microplate tester (BioTek Instruments, Inc., Vermont, USA). Evaluate the relative cell survival rate.

Real-time fluorescence quantitative PCR

After digestion with trypsin, MDBK cells were inoculated into a 6-well culture plate at 1 × 10⁵ cells/mL and allowed to adhere overnight(2 mL/well). When cell confluence reached 80%–90%, treatments were administered according to the following groups: indirubin group: First pre-treated with 120 µM indirubin solution (2 mL/well) for 2 h; After discarding the supernatant, infected with 1.5 mL of a mixture containing 120 µM indirubin and 1.5 MOI virus for 2 h; Subsequently, replace with 2 mL of 120 µM indirubin solution and continue culture. Positive drug group (ACV): Except for replacing 120 µM indirubin with 10 µM ACV, the treatment steps are identical to the indirubin group. Virus control group: The treatment procedure was identical to the indirubin group, except that the drug solution was replaced with cell maintenance medium. After the final drug replacement step, all groups were placed in a 37 °C, 5% CO₂ incubator. Samples were collected after 6, 12, 24, 36, and 48 h of incubation. Total cellular DNA was isolated with the Tiangen DNA Extraction Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. The viral copy number was quantified by real-time fluorescence quantitative PCR (QuantReady K9600, Hangzhou Suizhen Biotechnology Co., Ltd., China).

Flow cytometry

Perform cell plating as previously described. After the cells reached the desired 80%–90% fusion, DMEM and 120 µM of indirubin solution were added to the model group and the indirubin-treated wells, respectively. IBRV at an MOI of 1.5 was added to each well. Once the virus infection was completed. Then, 120 µM of indirubin solution was added to the indirubin red–treated wells, and 2% DMEM was added to the model. Subsequently, Incubation was continued for 24 h, after which the cells were collected, and a flow cytometer (CytoFLEX A00-1-1102, Beckman Coulter Biotechnology Suzhou Co., Ltd., China) was used to detect intracellular ROS and MMP levels.

Determination of intracellular ROS levels

Intracellular ROS levels were determined via flow cytometry following the ROS detection kit’s instructions. (Biyuntian, Jiangsu, China). (DCFH-DA itself is non-fluorescent and freely permeates the cell membrane. Upon entering the cell, it is hydrolyzed by intracellular esterases to form DCFH. Intracellular ROS oxidize the non-fluorescent DCFH into fluorescent DCF. Higher detected fluorescence intensity indicates higher intracellular ROS levels.)

Determination of intracellular MMP levels

Determine changes in cell membrane potential using a flow cytometer with the JC-1 MMP Assay Kit (Jiangsu Biyuntian, China). (JC-1 monomers (green fluorescence) and JC-1 aggregates (red fluorescence) represent low and high MMP states, respectively. The red/green fluorescence intensity ratio decreasing indicates mitochondrial depolarization.)

Experimental design of different drug administration timings

Cell seeding was conducted as previously detailed. For the full-process drug administration, When cell confluence reaches 80%–90%, cells are first pretreated with 120 µM indirubin solution (2 mL/well) for 2 h; After removing the supernatant, add 1.5 mL of a mixture containing 120 µM indirubin and 1.5 MOI virus for a 2 h infection; subsequently replace with 2 mL of 120 µM indirubin solution and maintain this until the detection endpoint. For the pretreatment procedure, cells were incubated with 120 µM indirubin for 2 h before infection. For the co-treatment procedure, IBRV was incubated with 120 µM indirubin for 2 h. For the post-treatment procedure, 2 h after infection, the culture medium in the indirubin-treated group was replaced with 120 µM indirubin. For all of the abovementioned studies, cells were collected at 24 h, and the levels of ROS and MMP were detected using flow cytometry. For a visual assessment of MMP, fluorescence microscopy observation was additionally performed with the following procedure: For MMP detection, the cells were treated with the identical protocol. Prior to cell collection, the manufacturer’s instructions of the kit were followed for cell processing. Post-treatment, the distribution of red and green fluorescence was observed under a fluorescence microscope.

Molecular docking

Molecular docking studies were conducted on indirubin and its core target proteins. First, indirubin was processed by downloading its 3D structure from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Using PyMOL software (version 2.5.4), the 3D structure was converted to PDB format and saved. Subsequently, the core target protein was processed by downloading the protein PDB format file acquired from the official Protein Data Bank (PDB;https://www.rcsb.org). Using PyMOL software (version 2.5.4), the protein was processed by removing water molecules, ligands, and small molecules before saving in PDB format. The ADFR suite software (version 1.0) was employed to identify the active binding pocket of the core protein, which was saved as a PDBQT file. Finally, molecular docking analyses were carried out using AutoDock Vina software (version 1.1.2) to determine the free binding energy between indirubin and the core protein. PyMOL software (version 2.5.4) was used to visualize the docking results [22].

Statistical analysis

All experiments included three biological replicates. Data analysis was performed with GraphPad Prism 8.0.2 software, and data were expressed as mean ± standard error of the mean (SEM). Statistical significance was defined as follows: P > 0.05 (non-significant), P < 0.05 (significant), and P < 0.01/P < 0.001 (highly significant), respectively.

Results

Prediction of indirubin drug targets

To compile the indirubin (Fig. 1) targets, we searched five databases: PharmMapper, SwissTargetPrediction, TCMSP, Herb, and SEA. We identified 899 indirubin drug targets using the PharmMapper database (Table S2). Using the SwissTargetPrediction database, the selected genes, which have a greater than zero probability of being an indirubin target, were selected, and 52 target points were obtained (Table S3). In TCMSP, 27 target points were predicted (Table S4), and 30 target genes were predicted in the Herb database (Table S5). Through the SEA analysis, 38 target points were obtained (Table S6). The target points from the five databases were collated and integrated, and duplicates were removed. Ultimately, 960 indirubin-specific drug target points were obtained. After cross-referencing and calibrating the 960 target genes with bovine-sourced targets from the UniProt database (Table S7), 211 target points were obtained (Table S8).

Fig. 1.

Structure of indirubin

Interaction network relationships between indirubin and IBRV proteins

From high-throughput sequencing data, we identified 6,610 differentially expressed mRNAs (count ≥ 100 and P ≤ 0.05; Table S9), among which 2,967 were upregulated, and 3,643 were downregulated (Fig. 2a and b). By intersecting genes associated with disease that have potential indirubin targets, a total of 81 overlapping targets were identified by our team(Table S10), which were considered potential therapeutic targets for indirubin in the therapy for IBRV (Fig. 3a). The interrelationships are shown in Fig. 3b.

Fig. 2.

Clustering and heatmap analysis of messenger RNAs (mRNAs). a Clustering and Heatmap Analysis of Differentially Expressed mRNAs. Red to green indicates expression levels from high to low. b The IBRV group includes 2,967 upregulated mRNAs and 3,643 downregulated mRNAs

Fig. 3.

Identification of potential therapeutic targets of indirubin for IBRV. a A Venn diagram to identify the intersection of genes as potential therapeutic targets. b PPI network of indirubin targets against IBRV. The colors of the nodes reflect the degree of connectivity, with redder colors indicating higher degrees

Integrative analysis of GO and KEGG enrichment and drug–disease networks

We analyzed KEGG and GO pathways using the Omicsmart online platform. KEGG results revealed 20 significantly enriched pathways (Fig. 4b), primarily including the PI3K-Akt, AMPK, and p53 pathways. During GO enrichment analysis, we focused on three primary categories: CC, BP, and MF (Fig. 4a). In cellular components (CC), the top-ranked entries include “mitochondria,” “cytoplasmic matrix,” and “protease inhibitor complexes. In the category of biological processes (BP), prominent entries include “metabolic processes,” “cellular responses to oxygen-containing compounds,” and “cellular responses to endogenous stimuli. MF involves functions such as “transferase activity”, “phosphotransferase activity”, and “enzyme binding”. Based on the GO analysis results, we selected “mitochondrial enrichment” as the core target for anti-IBRV activity. Finally, a comprehensive network diagram integrating GO, KEGG results, and indirubin was constructed (Fig. 4c).

Fig. 4.

GO and KEGG enrichment analyses of the 81 intersecting genes were performed using the Omicsmart online platform. a In the GO enrichment analysis, the top 10 significantly enriched terms from the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) categories were selected for display. b In the KEGG enrichment analysis, the top 20 significantly enriched pathways are shown. c Comprehensive network diagram of drug–disease functional annotation and signaling pathways. Red represents indirubin; pink represents 81 potential indirubin targets; purple, green, and blue represent the top 10 terms in the BP, CC, and MF categories of the GO analysis, respectively. Orange and blue represent the top 10 diseases and top 20 pathways, respectively, in the KEGG analysis

Toxicity of indirubin in MDBK cells

The CCK-8 data of MDBK cells show that the cytotoxicity of indirubin is enhanced with the increase of drug concentration, and the maximum safe concentration is 120 µM (Fig. 5).

Fig. 5.

Indirubin exhibited low cytotoxicity in MDBK cells treated with different concentrations of indirubin at 24 h and subjected to cell viability analysis. The red dashed line indicates cell viability (% of control) at 90%. The results represent the means ± SD of triplicate samples from three independent experiments

Effect of indirubin on IBRV DNA replication

We investigated the direct inhibitory effect of indirubin on viral DNA replication using qPCR technology. The results showed that indirubin at a concentration of 120 µM significantly reduced IBRV replication and significantly reduced virus copy number from 6 to 36 h after infection. This reduction was most significant within 6 to 24 h of early infection (Fig. 6).

Fig. 6.

Indirubin inhibited the DNA replication of IBRV. The IBRV group represents MDBK cells infected with IBRV and cultured at 37 °C in a 5% carbon dioxide incubator for 6, 12, 24, 36, and 48 h. IBRV+Indirubin denotes treatment with Indirubin, while IBRV + ACV denotes treatment with ACV. The horizontal axis (h) represents hours post-infection. The results represent the means ± SD of triplicate samples from three independent experiments. ns: nonsignificant *: P < 0.05, **: P < 0.01, ***: P < 0.001

Indirubin suppresses IBRV-Induced ROS formation in MDBK cells

Next, we investigated the relationship between the antiviral effect of indirubin on IBRV and its ability to clear ROS. Intracellular ROS levels were measured using the DCFH-DA assay. Continuous treatment with indirubin significantly suppressed the IBRV-induced increase in ROS levels in MDBK cells (P < 0.001; Fig. 7a and b). The results of different indirubin treatment regimens showed that all three treatment methods significantly suppressed IBRV-induced ROS production. The effectiveness is ranked from highest to lowest as follows: co-treatment (P < 0.001), post-treatment (P < 0.05), and pretreatment (P < 0.01); see Figs. 7c–h.

Fig. 7.

Indirubin inhibits IBRV-induced ROS formation in MDBK cells. a Changes in ROS levels in MDBK cells treated with full-process drug administration. b Quantification of ROS in MDBK cells treated with full-process drug administration. c Changes in ROS levels in pretreated cells. d Quantification of ROS in pretreated cells. e Changes in ROS levels in co-treated cells. f Quantification of ROS in co-treated cells. g Changes in ROS levels in post-treated cells. h Quantification of ROS in post-treated cells. All quantifications (b, d, f, h) show the mean ± SD of three biological replicates (n = 3). *: P < 0.05, **: P < 0.01, ***: P < 0.001

The effect of indirubin on the IBRV-Induced membrane potential depolarization level in MDBK cells

For the purpose of exploring the influence of indirubin on membrane potential depolarization in IBRV-induced MDBK cells, we compared the full-process handling group to the model group. The results showed a highly significant decrease in membrane potential depolarization in the full-process handling group relative to the model group (P < 0.01; Fig. 8a and b). All three treatment methods significantly suppressed the membrane potential depolarization level. The effectiveness is ranked from highest to lowest as follows: co-treatment (P < 0.001), post-treatment (P < 0.05), and pretreatment (P < 0.01). See Figs. 8b–m. Complementary to the above quantitative data, the qualitative analysis of MMP based on JC-1 fluorescence images further validated the protective effect of indirubin: Among them, panels i–m are the fluorescence images of MMP. Red fluorescence represents a high membrane potential, which is attributed to JC-1 J-aggregates, while green fluorescence represents a low membrane potential, which is derived from JC-1 monomers. As observed in the figure, the strong green fluorescence in the IBRV-infected Untreated Group (Fig. 8i) indicates severe mitochondrial damage. In contrast, the green fluorescence intensity in all other treatment groups was lower than that in the IBRV-infected Untreated Group, suggesting that different indirubin treatment regimens can alleviate IBRV-induced mitochondrial damage.

Fig. 8.

Effect of indirubin on JC-1 expression in IBRV-induced MDBK cells. a Changes in MMP in MDBK cells treated with full-process drug administration. b Quantification of MMP in MDBK cells treated with full-process drug administration. c Changes in MMP in pretreated cells. d Quantification of MMP in pretreated cells. e Changes in MMP in co-treated cells. f Quantification of MMP in co-treated cells. g Changes in MMP of post-treated cells. h Quantification of MMP of post-treated cells. (i) IBRV-infected Untreated Group; (j) IBRV-infected + full-process indirubin treatment; (k) IBRV-infected + indirubin pre-treatment; (l) IBRV-infected + indirubin co-treatment; (m) IBRV-infected + indirubin post-treatment. All quantifications (b, d, f, h) show the mean ± SD of three biological replicates (n = 3). *: P < 0.05, **: P < 0.01, ***: P < 0.001

Binding affinity of indirubin to core target sites

We employed molecular docking technology to calculate binding energy scores between indirubin and its core genes. Usually, if the binding energy value is less than − 5.0 kcal/mol, it means that the affinity is moderate, while if it is less than − 7.0 kcal/mol, it means that the affinity is high [22]. Molecular docking revealed strong binding affinities between indirubin and the proteins encoded by the 14 core genes (Table 1; Fig. 9a-o). For instance, the most stable conformation with DHODH (Fig. 9a) exhibited a binding free energy (ΔG) of -10.0 kcal/mol. In this pose, indirubin forms a key hydrogen bond between its carbonyl oxygen and the catalytic residue ARG-153. The labeled distance for this bond is 2.6 Å (Fig. 9a), which is within the optimal range for a strong hydrogen bond. This specific, short-range electrostatic interaction is a primary factor contributing to the highly favorable (negative) binding energy calculated for this complex. The fact that indirubin docks favorably into the putative active sites of these proteins suggests that it may exert its antiviral effects by modulating their functions.

Table 1.

Binding affinity between genes enriched in mitochondria and indirubin

name	affinity (kcal/mol)
DHODH	-10
GLUD1	-9
ADH5	-8.9
GOT2	-8.2
HK1	-8.2
CDK1	-8.1
OAT	-8.1
PARP1	-8.1
AK3	-8
SORD	-8
PPP1CC	-7.7
CLPP	-7.6
BCAT2	-7.4
SOD2	-6.6
AKT1	-4

Fig. 9.

Detailed view of molecular docking poses for indirubin with representative target proteins (see Table 1 for full list). Hydrogen bonds are shown as yellow dashed lines with labeled distances (Å). Key interacting amino acid residues are displayed as sticks. (a) DHODH, (b) GLUD1, (c) ADH5, (d) GOT2, (e) HK1, (f) CDK1, (g) OAT, (h) PARP1, (i) AK3, (j) SORD, (k) PPP1CC, (l) CLPP, (m) BCAT2, (n) SOD2, (o) AKT1

Discussion

The global prevalence of IBR (an OIE Category B notifiable disease) caused by IBRV imposes a significant socio-economic burden on the cattle industry, rendering it a serious public health threat [32, 33]. The current main prevention and control measures mainly rely on vaccines, but this method has significant limitations and is difficult to contain the spread of the virus [34]. In addition, there is an extreme shortage of specific therapeutic drugs for IBRV. The current clinical management only relies on symptomatic treatment (using antibiotics to prevent secondary bacterial infection) to relieve symptoms [35]. These methods cannot directly inhibit or remove IBRV. The current situation that vaccines cannot control diseases and drugs cannot effectively treat them further highlights the urgency of developing effective anti-IBRV antiviral drugs.

Given that indirubin has antiviral, antibacterial, antioxidant, and anti-inflammatory properties [13, 36, 37]. This study aims to explore the potential application value of indirubin in the treatment of IBRV infection. In vitro pharmacological experiments showed that indirubin (120 µM) significantly inhibited DNA replication of IBRV in MDBK cells, especially in the early stages of infection (6–24 h), with the most significant reduction in virus copy number. However, no inhibitory effect was observed during the later stages of infection (48 h), suggesting that its mechanism of action may be time-dependent, primarily targeting the early stages of viral replication. This finding aligns with the previously reported early-stage inhibition pattern in adenovirus models [38], further expanding the antiviral application potential of indirubin within the Herpesviridae family. Our findings indicate that indirubin may exert its antiviral effect by inhibiting viral genome replication. Supporting this mechanism, previous studies have shown that indirubin and its derivatives can indirectly interfere with viral transcription and replication by modulating the host CDK pathway, leading to a reduction in RNA polymerase II CTD phosphorylation levels [38, 39]. Future investigations should explore why its efficacy diminishes during later stages of infection and whether potential mechanisms exist for directly targeting viral proteins. This would provide new avenues for refining its antiviral theory and developing highly effective derivatives.

To verify whether indirubin exerts its anti-IBRV effect by improving mitochondrial damage, we validated its repairing effect on IBRV induced mitochondrial damage using flow cytometry. ROS and MMP were selected as markers of detection, as prior studies have established them as key indicators of mitochondrial damage [7, 40]. Furthermore, reports indicate that viral infection can cause mitochondrial damage, leading to ROS accumulation and MMP depolarisation [5, 41]. The present findings further confirm that IBRV infection of MDBK cells induces ROS accumulation and MMP depolarisation, while indirubin treatment significantly alleviates both abnormalities, suggesting its role in repairing mitochondrial damage. This aligns with prior studies demonstrating that indirubin significantly inhibits virus- or stress-induced MMP depolarization and restores mitochondrial function [19, 21]. Furthermore, reports indicate that indirubin can inhibit ATP-mediated ROS production within macrophages [42]. These findings are similar to our research findings, suggesting that indirubin may exert antiviral effects by improving mitochondrial function.

According to relevant research reports, drugs can play an antiviral role by repairing mitochondria with targeted proteins. For example, Sheng et al. found that SIRT3 maintains mitochondrial structure and metabolic homeostasis through the fatty acid β - oxidase ACAA2, thereby inhibiting cytomegalovirus replication [43]. Similarly, Lin et al.‘s studies confirmed that MOTS-c can stimulate mitochondrial biogenesis and activate the MAVS signaling pathway, effectively inhibiting hepatitis B virus replication [44]. In order to investigate the pathways and targets through which indirubin repairs mitochondrial damage and exerts its anti-IBRV effect, we conducted KEGG and GO enrichment analysis on the potential therapeutic targets of indirubin. The KEGG-enriched signaling pathways mainly include the PI3K/Akt and p53 pathways. This suggests that indirubin may exert its anti-IBRV effect by regulating mitochondrial-related pathways. GO enrichment analysis revealed that 15 target proteins are mitochondrial-related, and molecular docking results showed that 14 target proteins have strong binding affinity with indirubin. Among them, AK3 (binding energy − 8.0 kC/mol) is responsible for maintaining the mitochondrial ATP/AMP balance and catalyzing the AMP + ATP → 2ADP reaction [45]. PARP1 (binding energy − 6.6 kC/mol) can respond to DNA damage and activate the repair mechanism; its excessive activation will lead to NAD+ depletion, which is closely related to cell death [46]. SOD2 (binding energy − 6.6 kcal/mol) is a key enzyme in the antioxidant defense system, and its active activation can effectively clear excess ROS and inhibit oxidative stress damage [47]. CLPP (binding energy − 7.6 kcal/mol) is a core participant in the mitochondrial unfolded protein reaction, responsible for clearing toxic protein aggregates [48]. These findings suggest that indirubin may exert antiviral effects by regulating mitochondrial damage through a multi-target mechanism.

Conclusions

Overall, this study confirms that indirubin is an effective inhibitor of IBRV, capable of regulating mitochondrial damage induced by IBRV and reducing IBRV replication. However, this study has the following limitations: the mechanism of action of indirubin has not been fully elucidated, and further in-depth research is needed. The specific correlation between core targets and downstream pathways still needs to be verified through experiments such as protein overexpression. Nevertheless, this study has laid a solid foundation for the antiviral research of indirubin and provided important evidence for its development as a clinical candidate drug for IBRV.

Abbreviations

IBRV

Infectious bovine rhinotracheitis virus

IBR

Infectious bovine rhinotracheitis

KEGG

Kyoto Encyclopedia of Genes and Genomes

GO

Genetic ontology

MDBK

Bovine kidney cells

MMP

Mitochondrial membrane potential

ROS

Reactive oxygen species

mPTP

Mitochondrial permeability transition pore

ACV

Acyclovir

HBV

Hepatitis B virus

PTB

poly(d,l)-pyrimidine-2,6-tetraeste-1,3,5,7-tetraol

IFN-β

Type I interferon

IFITM3

Interferon-Induced Transmembrane Protein 3

MAVS

Mitochondrial antiviral signaling pathway

H1N1

Influenza A

JEV

Japanese encephalitis virus

Authors’ contributions

MX, LH have made substantial contributions to the conception and design of the work, and was a major contributor in writing the manuscript.LH, LX, and LJ have made substantial contributions to the acquisition, analysis, YG, ZQ, LN, JQ, MY and. LX have made substantial contributions to substantively revise it. All authors read and approved the final manuscript.”

Funding

This research was supported by the project “National Natural Science Foundation of China” (Grant number 32460870) .”

Data availability

All data generated or analysed during this study are included in this published article (and its supplementary information files).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.