Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2025 Aug 28;10(35):40258–40270. doi: 10.1021/acsomega.5c05364

Selenomethionine Inhibited RSV Infection-Induced Apoptosis and Inflammatory Response through ROS-Mediated Signaling Pathway

Chuqing Li 1, Yuqun Wei 1, Jiali Li 1, Jia Lai 1, Yuhui Zeng 1, Bing Zhu 1, Yinghua Li 1,*, Mingqi Zhao 1,*
PMCID: PMC12423889  PMID: 40949232

Abstract

Respiratory syncytial virus (RSV) is the leading pathogen of acute lower respiratory tract infections in children under 5 years of age worldwide. Respiratory syncytial virus pneumonia in infants and children causes more deaths than influenza each year due to the high rate of severe illness. There is a lack of safe and effective antiviral drugs, and the development of novel antirespiratory syncytial virus drugs is of great clinical importance. Selenomethionine (SeMet), as the main ingredient in commercially available selenium supplements, exerts excellent antioxidant, antiviral, immunomodulatory, and other physiological functions mainly in the form of selenoprotein. The antiviral mechanism of SeMet anti-RSV was explored by detecting the apoptotic state, the degree of DNA damage, cytokine and reactive oxygen species (ROS) secretion levels, and the mitochondrial membrane potential. Meanwhile, this study screened the affinity of SeMet for common RSV target proteins and explored the dynamic interactions between SeMet and the screened viral target proteins.Conclusions: SeMet inhibited apoptosis and inflammatory responses by regulating the ROS-mediated PARP/Bcl-2, NF-κB/JAK1-STAT3 signaling pathways. Meanwhile, SeMet formed a stable interaction with RSV polymerase and may bind to key amino acid residues of RSV polymerase mainly through hydrogen bonding.

graphic file with name ao5c05364_0012.jpg

graphic file with name ao5c05364_0010.jpg

1. Introduction

Respiratory syncytial virus is globally estimated to be the leading cause of acute lower respiratory tract infections in infants and children. For infants through the first year of life, 70% of children are infected with respiratory syncytial virus, and almost 100% of children are infected with respiratory syncytial virus by the age of 2 years. Respiratory syncytial virus (RSV) infection has become a great challenge in respiratory infections in infants and children worldwide. Respiratory syncytial viruses are enveloped single-stranded negative-stranded RNA viruses with a diameter of 150 nm. The clinical manifestations of RSV infection are related to the immune status of the host. Mild infections show some self-limitation and resolve spontaneously within 1–2 weeks. Infants and young children or immunocompromised adults are prone to progress to severe respiratory syncytial virus pneumonia, with clinical manifestations of severe capillary bronchitis and even respiratory failure. , In addition, RSV infection may lead to viral myocarditis, heart failure, etc. RSV infection activates the host’s innate immune response, with alveolar macrophages and mast cells releasing NF-κB and IRF, and increased expression of pro-inflammatory cytokines (IL-6, IL-8, TNF-α, etc.). All of these disrupt the alveolar epithelial-endothelial barrier, leading to pulmonary edema and respiratory insufficiency. It can further develop into severe pneumonia, and in severe cases, ARDS and sepsis. The nonstructural proteins NS1 and NS2 of RSV inhibit the production of type I IFN, and IFN signaling is important for pro-inflammatory factors and antiviral gene expression. RSV resists antiviral immunity in humans by inhibiting type I IFN signaling. There is no specific anti-RSV therapy, and the main treatment is still symptomatic support, including maintaining airway patency, ensuring oxygen demand, and nutritional supportive care. Ribavirin is a nucleoside broad-spectrum antiviral drug for which RSV infection is a clear indication. However, its use has been limited by its efficacy, potential teratogenic toxicity, and the need for long-term aerosol administration. It is not recommended by the American Academy of Pediatrics. Ziresovir (AK-0529), a novel fusion protein inhibitor being tested in clinical trials, is a direct-acting antiviral that holds promise as a future therapeutic option. Palizumab is a humanized murine monoclonal antibody that inhibits viral spread by binding to fusion proteins of RSV. Its prophylactic administration can reduce RSV infection in high-risk children, but this treatment must be given throughout the RSV epidemic. It also has the disadvantages of low cure rates and high costs.

Selenium is an essential trace element for the human body. Selenium in the human body is mainly categorized into organic and inorganic selenium. Organic selenium includes seleno-substituted amino acids (selenomethionine and selenocysteine) and selenoproteins. Inorganic selenium is a tetravalent or hexavalent selenium salt. Selenomethionine is mainly derived from dietary sources, such as grains and animal offal. Selenomethionine is the storage form of selenium, while selenocysteine is the active center of GPX, which is directly involved in antioxidant and metabolic regulation. − Selenium exerts its biological functions in the organism mainly in the form of selenoproteins, which have the role of scavenging free radicals and protecting the structure and function of cell membranes. −

Oxidative stress is a process in which an imbalance between the oxidative and antioxidant systems in the body leads to an excessive accumulation of free radicals, such as reactive oxygen species, which triggers cellular damage. Many studies have demonstrated that oxidative stress plays a key role in the pathogenesis of inflammatory diseases associated with respiratory syncytial virus infection. Oxidative stress mechanisms are involved in many respiratory diseases, including viral pneumonia, asthma, chronic obstructive pulmonary disease, and bronchiectasis. RSV infection produces reactive oxygen species (ROS)-induced oxidative stress and enhances it through virus-induced transcriptional inhibition of antioxidant enzymes. , This triggers a massive release of inflammatory cytokines and chemokines, exacerbating lung injury. Antioxidants have been shown to ameliorate clinical disease and limit RSV infection in mouse models. , This paper focuses on the signaling pathways involved in the inhibition of respiratory syncytial virus-induced apoptosis and inflammatory response by selenomethionine.

2. Materials and Methods

2.1. Materials

Hep2 cells used in this study were obtained from ATCC, and RSV viruses were isolated and cultured from clinical specimens of pharyngeal swabs from the central laboratory of Guangzhou Medical University Women’s and Children’s Medical Center. Fetal bovine serum (FBS), trypsin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (California). CCK-8 Cell Activity Assay Kit, JC-1 Mitochondrial Membrane Potential Assay Kit, Annexin–V-PI Co-Staining Kit, and One-Step TUNEL Apoptosis Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was purchased from Sigma-Aldrich (St. Louis). Aimplex Cytokine Detection Kit was purchased from Kuangbo Biological Company (Beijing, China), and Bcl-2, BAX, C-Caspase-3, Caspase-9, NF-κB, PARP, P65, JAK1, and STAT3 antibodies were purchased from Cell Signaling Technology (Boston). Selenomethionine was kindly donated by Chen Tianfeng’s group at Jinan University.

2.2. TCID50 Assay for RSV

Hep2 cells were spread in 96-well plates, and Hep2 cells were infected with RSV. The viral stock solution was diluted 10-fold with serum-free DMEM from 10–1 to 10–8, and 100 μL of virus dilution was added to each well. The virus was incubated at 37 °C in a 5% CO2 incubator for 2 h to promote virus adsorption and then replaced with a virus maintenance solution containing 1% FBS DMEM. The TCID50 values of the viruses were calculated by the Reed–Muench method.

2.3. CCK-8 Method

CCK-8 assay is a common method to detect cell proliferation or toxicity based on cell metabolic activity, which is based on the principle that water–soluble tetrazolium salts are reduced to orange–yellow metazoan by dehydrogenase in the mitochondria, and the depth of the color is related to cell viability. Hep2 cells were spread in 96-well plates at a suitable density and cultured in a 37 °C, 5% CO2 incubator for 12–24 h until the cells were adherent to the wall. DMEM was used to dedilute the 5 mM SeMet stock solution to prepare SeMet with a concentration gradient ranging from 32 to 256 μM. 100 μL of different gradient SeMet solutions was added to each well and incubated at 37 °C, 5% CO2 for 48 h. The intervention was carried out by adding 100 μL of different drug gradients to the plates and incubating at 37 °C with 5% CO2 for 48 h. Mix the CCK-8 working solution and DMEM in the ratio of 1:9 and then add 100 μL of the mixture to each well. The 96-well plate was continued to be incubated in a 37 °C incubator protected from light for 30 min–2 h, and the absorbance of each well was detected at 450 nm using a microplate reader. To test the antirespiratory syncytial virus efficiency of SeMet, we added SeMet after 2 h of RSV addition and then carried out the absorbance measurement as described before.

2.4. Annexin V/PI Double Staining Method

Annexin V is a Ca2+-dependent phospholipid-binding protein with a high affinity that binds specifically to phosphatidylserine (PS). In normal cells, PS is predominantly distributed on the inner side of the cell membrane, but in the early stages of apoptosis, PS is ectopically translated to the outer side of the cell membrane. Annexin V labels early apoptotic cells by binding to externally turned PS. Propidium iodide (PI) is a nucleic acid dye; necrotic and late apoptotic cells have a loss of cell membrane integrity, and PI enters the cell and binds to intracellular DNA. Hep2 cells were spread in six-well cell culture plates at a density of 7 × 104 cells/mL, and the control, SeMet, RSV, and RSV + SeMet groups were set up. Cells after four groups of different treatments were collected with pancreatic enzyme and DMEM. 195 μL Annexin V-FITC binding solution was added and mixed well, followed by 5 μL Annexin V-FITC staining solution and finally 10 μL PI staining solution. Incubate for 20 min away from light. Staining results were observed using a BD FACS Canto II flow cytometer. Similarly, double staining can be performed in situ in cell culture plates, and the staining results were observed by fluorescence microscopy.

2.5. TUNEL/DAPI Double Staining Method

During apoptosis, endogenous nucleic acid endonucleases are activated, and chromosomal DNA breaks produce 3′-OH ends. TUNEL staining, a deoxyribonucleotide end-transferase-mediated dUTP nick end-labeling, allows for apoptotic cells to be specifically labeled. DAPI is a fluorescent dye that binds specifically to DNA and is used to label the nuclei of cells to help locate the cell position and observe changes in nuclear morphology. Hep2 cells were inoculated into cell crawls in 12-well plates, treated into four groups as before, fixed in 4% paraformaldehyde for 30 min, washed, and then added with an immunopenetrating solution of 0.3% TritonX-100 and incubated for 5 min. Add 50 μL TUNEL assay solution, incubate for 1 h away from light, and then add 10 μL 10 μg/mL DAPI staining for 5 min. The degree of DNA damage in the nucleus of Hep2 cells was observed by using fluorescence microscopy.

2.6. Mitochondrial Membrane Potential Detection

JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential, which is characterized by aggregating in the mitochondrial matrix and forming multimers to emit red fluorescence when the membrane potential is high and dispersing in the cytoplasm as monomers when the membrane potential is low, with a change in fluorescence color or intensity. By detecting the change of fluorescence signal, it can reflect the change of mitochondrial membrane potential of Hep2 cells. First, the JC-1 staining working solution. Dilute 25 μL of JC-1 (200×) with 4 mL of ultrapure water, vortex vigorously, and mix well. Wait until JC-1 is fully dissolved before adding 2 mL of JC-1 staining buffer (5×). Add 1 mL of prepared JC-1 staining solution to each well of the six-well plate, mix well, and incubate for 20 min in an incubator under light. Finally, the plates were washed with an appropriate amount of precooled JC-1 staining buffer. The results of JC-1 probe staining on Hep2 cells were observed under a fluorescence microscope.

2.7. Intracellular ROS Assay

The principle of the ROS Assay Kit is mainly to use specific fluorescent probes to react with ROS, and quantitatively or qualitatively detect the level of intracellular ROS through the change of fluorescence intensity. In this study, a nonfluorescent DCFH-DA probe was used that was hydrolyzed by esterase into DCFH after entering the cell, which was retained in the cell. When ROS are present, DCFH is oxidized to DCF, emitting green fluorescence. Cells from different treatment groups were digested and collected for cell counting. DCFH-DA staining working solution was prepared in a ratio of 1:1000. Each group was added 500 μL DCFH-DA staining working solution and incubated for 30 min, and then the culture plate was washed with PBS. Finally, the intracellular ROS levels in Hep2 cells were observed by a semiquantification of ROS levels by a microplate reader and fluorescence microscope.

2.8. Cytokine Detection by Cytometric Bead Array Method

The cytometric bead array (CBA) method uses microspheres of different sizes and fluorescence intensities encapsulated with capture antibodies to specific cytokines, and then quantifies the cytokines by detecting the fluorescence intensity of the microspheres by flow cytometry. Collect the cell culture supernatants of different treatment groups, use the gradient dilution of Aimplex Cytokine Detection Kit standards to make a standard curve, add the capture microspheres, cell culture supernatant, and antihuman detection antibody into the same reaction tube, incubate for 2–3 h under light, wash and centrifuge to remove impurities, and then detect the fluorescence signal of the microspheres by flow cytometry.

2.9. Western Blot

Hep2 cells infected with RSV were treated with SeMet for 48 h. Different treatment groups were washed with PBS three times, and 100 μL of cell lysate was added to each group. The supernatant was collected and centrifuged to obtain the protein samples after the lysis was completed. The protein concentration was determined using the BCA method, and the protein concentration of different groups was adjusted to 2.5 μg/L by diluting with Loading buffer. 10% separating gel and 5% concentrating gel were prepared, and 8 μL of protein samples and prestained Protein Marker were added to each well. The electrophoresis could be terminated by running at 70 V the separating gel and then at 110 V the bottom layer of the gel. After activating the PVDF membrane with methanol and making a membrane transfer “sandwich”, the membrane was transferred by Bio-RAD electrophoresis at a constant pressure of 110 V for 75 min. The PVDF membrane was washed with TBST three times and then put into the rapid containment solution for 15 min at room temperature on a shaking table, washed again, and then added with the corresponding primary antibody on a shaking table at 4 °C for the rest of the night. The membrane was washed with TBST, followed by incubation with the secondary antibody for 2 h. The results were exposed by using a chemiluminescence developer after the preparation of the ECL chemiluminescent solution.

2.10. Molecular Docking

Molecular docking is a theoretical simulation method used to study intermolecular interactions and predict their binding modes and affinities. After reviewing the literature to screen for antirespiratory syncytial virus targets, in order to explore the potential antirespiratory syncytial virus targets of SeMet, the present study used molecular docking for preliminary screening. The steps are as follows: First, the appropriate protein is selected from the Protein Database (RCSB) and the PDB file is downloaded; then, the protein is dehydrated and hydrogenated in AutoDock and saved as a PDBQT protein receptor file. The molecular structure of the drug was downloaded from the PubChem database and also processed in AutoDock to export the PDBQT ligand file. Docking of SeMet and screened antiviral target molecules was done by AutoDock Vina. The docking parameters are as follows: energy_range = 5, exhaustiveness = 13, and num_modes = 20. The final results were visualized using PyMol 2.5 and Discovery Studio 2019.

2.11. Molecular Dynamics Simulation

Based on the experimental results of molecular docking, we performed 100 ns MD simulations of the SeMet-RSV polymerase complex using Gromacs v2022.03 software. The topology file of the molecular dynamics system of SeMet and RSV polymerase was first prepared. The three-point transferable intermolecular potential (TIP3P) water model was used to solubilize the SeMet-RSV polymerase complex, while avoiding boundary effects and ensuring electroneutrality. Energy minimization was performed by using the steepest descent algorithm. The complex was then equilibrated for 100 ps in a constant temperature and constant volume (NVT) and constant temperature and pressure (NPT) system, with the temperature and pressure of the complex and solvent system. Finally, we analyze the complex RMSD, RMSF, Rg, SASA, and hydrogen bonding trends based on MD simulation trajectories. Combining the RMSD and Rg data files, we calculated the Gibbs free energy using the “g_sham” and “xpm2txt.py” scripts within the Gromacs v2022.03 software. In addition, we ran the “MMPBSA.py v.16.0” script to calculate the binding free energy of SeMet complexed with RSV polymerase using molecular dynamics/Poisson–Boltzmann surface area method, and the binding energy was analyzed in terms of energy decomposition and residue contribution using the GROMACS plug-in.

2.12. Data Processing and Statistical Analysis

We used BioRender software to draw the mechanism and schematic diagrams. Graphs were drawn using GraphPad Prism 8.0, data were analyzed using SPSS 22.0, and data between groups were analyzed for differences using ANOVA. p < 0.05 (*) was considered statistically different.

3. Results and Discussion

3.1. CCK-8 Assay for Detection of Optimal Antiviral Concentration for Low Toxicity

SeMet has a protective effect on Hep2 cells at low concentrations for reasons that may be related to its involvement in the synthesis of antioxidant enzymes, such as glutathione peroxidase, but high concentrations may trigger cytotoxicity. The cytotoxicity of SeMet on Hep2 cells was determined by CCK-8 assay at 32–256 μM. As shown in Figure A, SeMet was essentially nontoxic and even protective to Hep2 cells in the range of 32–256 μM compared with the control group. As shown in Figure B, the survival of Hep2 cells after SeMet intervention was increased in the range of 32–256 μM, with cell survival reaching a maximum of 128 μM. After the pre-experimentation to find out the approximate effective SeMet antiviral concentration range, combined with the drug having a dual effect on Hep2 cells, the lower concentration of SeMet was selected for the experiment to understand its antiviral efficiency. Compared with the control group, Hep2 cells in the viral group had typical syncytium formation, and some cells were rounded (Figure C). After the intervention of different concentrations of SeMet, these cytopathic effects were significantly reduced, and the antiviral effect of SeMet at 128 μM was the best, and the survival rate of Hep2 cells reached 87.15% (Figure D). The results of this experiment indicated that SeMet significantly inhibited RSV-induced Hep2 cell infection. In addition, 128 μM was the concentration with low cytotoxicity and the best antiviral effect, and 128 μM SeMet was used for subsequent experiments. The titer of the RSV used in this experiment was 10–4.5.

1.

1

Open in a new tab

Optimal antiviral concentration of SeMet against RSV in Hep2 cells. (A) Changes in the virulence of SeMet on Hep2 cells were observed under a light microscope. (B) CCK-8 assay to detect the survival of Hep2 cells after the intervention of 32–256 μM SeMet. (C) Changes in morphology and number of Hep2 cells after RSV infection and infections with different concentration gradients of SeMet intervention were observed under a light microscope. (D) The survival rate of RSV-infected Hep2 cells after SeMet intervention at 64–256 μM was detected by CCK-8 assay. The data was the mean standard error (N = 3), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****), indicating statistically significant differences.

3.2. SeMet Inhibits DNA Damage to Alleviate RSV Infection-Induced Apoptosis

The Annexin V/PI double staining assay is based on the biological characteristics of cell membrane integrity and phosphatidylserine ectopia and is able to distinguish between normal, early apoptotic, late apoptotic, and necrotic cells. Figure C flow results showed that RSV infection significantly induced apoptosis in Hep2 cells, and the early and late apoptotic cells were 7.9 and 9.0%, respectively. After the addition of SeMet treatment, the early and late apoptotic cells decreased to 0.8 and 3.0%, respectively. The fluorescence and morphological changes of the Hep2 cells were observed by fluorescence microscopy. Compared with the control group, the intensity of green fluorescence and red fluorescence in the viral group was significantly enhanced, and the cells in the viral group fused into clusters in large numbers, forming typical syncytia. Cell membrane phosphatidylserine ectopics lead to a disruption of cell membrane integrity. After adding SeMet treatment, the intensity of green fluorescence and red fluorescence was obviously weakened, and the cell morphology tended to be normalized (Figure A). The experimental results indicated that SeMet might effectively inhibit RSV-induced early and late apoptosis. In the present study, we confirmed by TUNEL/DAPI double staining that RSV infection significantly induced chromosomal DNA damage in Hep2 cells, which was accompanied by changes, such as nuclear consolidation and nuclear fragmentation (Figure B). In contrast, the intensity of green fluorescence of the cells was significantly reduced after SeMet treatment, and the proportion of abnormal nuclear morphology was reduced. This suggests that SeMet can attenuate RSV infection-induced chromosomal DNA damage in Hep2 cells.

2.

2

Open in a new tab

Inhibitory effect of SeMet on RSV infection-induced apoptosis in Hep2 cells. (A) fluorescence microscopy observation of Hep2 cell apoptosis. (B) Fluorescence microscopy observation of Hep2 cell DNA damage and changes in cell nucleus morphology. (C) Quantification of the percentage of apoptosis in Hep2 cells by flow cytometry.

3.3. SeMet Repairs RSV Infection-Induced Mitochondrial Dysfunction and Oxidative Stress in Hep2 Cells

Changes in mitochondrial membrane potential are a hallmark indicator of apoptosis, indicating loss of mitochondrial function. The results in Figure A show that control and pure SeMet-treated cells showed strong red fluorescence; however, significant green and weaker red fluorescence were detected in RSV-treated cells, suggesting that RSV infection can lead to mitochondrial damage. We observed an increase in red fluorescence and a concomitant decrease in green fluorescence upon the addition of SeMet to RSV-infected cells, suggesting that SeMet repairs RSV infection-induced mitochondrial membrane potential in Hep2 cells.

3.

3

Open in a new tab

SeMet repair of RSV infection-induced mitochondrial membrane potential and reduced ROS production in Hep2 cells. (A) Fluorescence microscopy observation of SeMet repair of RSV infection-induced mitochondrial membrane potential changes in Hep2 cells. (B) Fluorescence microscopy observation of DCF-excited fluorescence in different treatment groups. (C) Semiquantification of ROS levels by a microplate reader. The data was the mean standard error (N = 4), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****), indicating statistically significant differences.

To assess the level of oxidative stress in RSV-infected Hep2 cells, ROS levels were detected in this experiment using the DCFH-DA probe. As shown in Figure B, the intensity of green fluorescence in RSV-infected cells was significantly increased compared to the control group, and the intensity of cellular fluorescence was significantly weakened by the addition of SeMet. As shown in Figure C, we calculated the average fluorescence intensity of each cell by using a microplate reader. The intracellular ROS fluorescence intensity in RSV-infected cells was 518% of that in control cells. However, the RSV + SeMet group was only 282% of it, indicating that SeMet significantly reduced RSV infection-induced ROS production in Hep2 cell lines. The protective effect of SeMet may be through the inhibition of RSV-induced ROS overproduction.

3.4. SeMet Attenuates RSV Infection-Induced Inflammatory Response

The degree of inflammation was assessed by detecting cytokine levels in Hep2 cell culture supernatants. As shown in Figure , relevant pro-inflammatory factors such as IL-1β, IL-6, IL-8, and TNF-α were significantly elevated in the viral group. All of these inflammatory factors were reduced to different degrees after dosing treatment, which may be due to the inhibition of related inflammatory pathways by SeMet. At the same time, SeMet was able to maintain a high expression of IFN-α, realizing the balance between antiviral and anti-inflammatory.

4.

4

Open in a new tab

SeMet regulates cytokine secretion in the RSV-infected Hep2 cells. The concentrations of IL-1β, IL-6, IL-8, TNF-α, and IFN-α in the cell culture supernatants of each group were measured by flow fluorescence. The concentration used for SeMet in the experiments was 128 μM. The data was the mean standard error (N = 3), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), or p < 0.0001 (****), indicating statistically significant differences.

3.5. Regulation of ROS-Mediated Mitochondrial Apoptotic Pathway as well as Inflammatory Pathway by SeMet

Mitochondrial dysfunction is closely related to the balance between ROS production and clearance. ROS are reactive oxygen molecules produced during cellular metabolism, and they have certain physiological functions under normal physiological conditions. As shown in Figure B, RSV infection led to the production of large amounts of ROS in Hep2 cells, resulting in an increased level of oxidative stress. Mitochondria are the main source of ROS production, and their dysfunction is often considered to be a key factor leading to the excessive accumulation of ROS. Excessive accumulation of ROS also directly damages the mtDNA of mitochondria, leading to abnormal function of the respiratory chain complex, further generating more ROS, forming a vicious cycle, and ultimately accelerating apoptotic cell death. As shown in Figure , this is consistent with our experimental results. During the oxidative stress-induced mitochondrial apoptotic pathway, mitochondrial outer membrane permeabilization (MOMP) is crucial, which is regulated by the Bcl-2 family and ultimately affects the release of the apoptotic factor cytochrome C. , Cytochrome C is released into the cytoplasm and binds to Apaf-1 to form apoptotic bodies, which activate Caspase-9 and Caspase-3 leading to apoptosis (Figure A).

5.

5

Open in a new tab

SeMet regulates apoptosis and inflammatory response. (A) Conceptual diagram of SeMet regulation of ROS-mediated mitochondrial apoptotic pathway as well as inflammatory pathway. (B) SeMet is involved in the regulation of PARP, the Bcl-2-signaling pathway to inhibit apoptosis, and NF-κB/JAK1-STAT3 to inhibit the inflammatory pathway.

Oxidative stress not only affects mitochondria alone but more seriously may affect DNA damage in the nucleus. It has been reported that reactive substances produced during oxidative stress can directly oxidize biomolecules, including membrane lipids, structural proteins, enzymes, and nucleic acids, leading to abnormal cellular function and death. We hypothesize that oxidative stress generated during viral infection may lead to DNA damage in host cells and activate a series of apoptotic pathways. PARP is activated to participate in DNA repair when the cell is stimulated to undergo DNA damage. PARP specifically recognizes and binds to damaged DNA strands, recruiting proteins and repair factors to repair DNA. Caspase-3 is a major effector protein in the execution of apoptosis, and its activation triggers the typical features of apoptosis, such as DNA breaks and cell membrane rupture. RSV may induce Caspase-3 activation through the mitochondrial pathway, which in turn activates endogenous nuclease to cleave DNA into fragments. Meanwhile, Caspase-3 is able to cleave the DNA damage repair protein PARP, leading to the loss of DNA repair capacity and promoting apoptosis. ,

During RSV infection, excessive cytokine production exacerbates lung tissue damage. Elevated levels of cytokines and chemokines are associated with disease severity and clinical progression, so it is essential to detect cytokine levels after RSV infection. Cytokines (e.g., IL-6, IL-1β, TNF-α) trigger receptor dimerization and activation of JAK1 kinase by binding to their specific receptors. Activated JAK1 phosphorylates the receptor tyrosine residues, providing a docking site for the STAT3 protein, which is phosphorylated by JAK1 to form a homodimer that enters the nucleus and binds to the promoter region of the target gene to regulate NF-κB, which in turn regulates inflammatory factor expression. , The key subunit of the NF-κB family is the P65 protein. It plays a central role in inflammatory response, immune response, apoptosis, and tumorigenesis. Inflammatory factors, oxidative stress, and DNA damage signals can activate the IKK complex. This leads to phosphorylation and down-regulation of IκB to release a heterodimer of P50/P65, which subsequently translocates to the nucleus to initiate transcription of pro-inflammatory genes (e.g., TNF-α, IL-6). The data in Figure B demonstrate that SeMet can attenuate RSV-induced inflammation by inhibiting the NF-κB/JAK1-STAT3 pathway in Hep2 cells.

Interferons are cytokines secreted by host cells in response to pathogen and tumor stimulation, and their effects cover a wide range of aspects, including antiviral, immunomodulatory, antitumor, and cell proliferation regulation. When humans are infected with viruses, type I IFN is a major part of the innate immune response. The binding of type I IFN to its receptor activates the JAK family, which in turn phosphorylates the transcription factors STAT1 and STAT2, ultimately leading to enhanced antiviral immunity in the body. In the present study, SeMet was found to significantly increase the secretion of IFN-α in Hep2 cells and exert its antiviral effects.

3.6. Prediction of Targets for Direct Antiviral Effects of SeMet

The target proteins obtained from the screening were docked with SeMet, respectively, and the binding energies of the complexes were calculated to further investigate the antiviral mechanism of SeMet. The docking results showed that the binding energy of SeMet to RSV RNA polymerase was < −5.0 kcaL/moL, indicating that SeMet and it have better activity and binding ability. The docking results are shown in Figure A–C, and SeMet and RSV RNA polymerase have strong binding activity, mainly through hydrogen bonding, van der Waals force, alkyl interaction, electrostatic interaction, etc. The binding energies of SeMet and the RSV nuclear protein, RSV fusion protein, RSV nonstructural protein 1, and RSV nonstructural protein 2 are relatively weak (Figure D). SeMet forms hydrogen bonding interactions with amino acid residues ARG1339, HIS1338, THR1267, and GLN1386, respectively, all of which play important roles in maintaining the stability of the complex. RSV polymerase is a key target for antiviral drug development, which is mainly involved in the replication of the viral genome and in the transcription of mRNA generation. The experimental results suggest that SeMet may exert direct antiviral effects through RSV polymerase targets.

6.

6

Open in a new tab

Molecular docking results of SeMet with respiratory syncytial virus protein targets. (A) Location of SeMet and RSV RNA polymerase docking activity pockets. (B) 3D schematic visualization of SeMet and RSV RNA polymerase docking results. (C) SeMet and RSV RNA polymerase binding interaction force 2D Schematic diagram. (D) Optimal binding conformation of SeMet binding to RSV nuclear protein, RSV fusion protein, RSV nonstructural protein 1, and RSV nonstructural protein 2.

3.7. Molecular Dynamics Simulation of the SeMet-RSV Polymerase Complex

3.7.1. Stability Analysis of SeMet-RSV Polymerase Complexes

Based on the molecular docking results, we further performed 100 ns molecular dynamics simulations to explore the dynamic stability of the SeMet-RSV polymerase complex. The root-mean-square deviation (RMSD) is a measure of how much the molecule deviates from its initial conformation relative to its initial conformation during the simulation process. The lower the RMSD value, the higher the protein–ligand stability. As shown in Figure A, the RMSD value of the complex fluctuates considerably at the beginning and then stabilizes and remains around 0.3 nm. As shown in Figure B, the RMSD of SeMet varied considerably throughout the simulation, with a maximum value close to 0.2 nm, suggesting that the ligand may not be fully stabilized in the complex and that structural rearrangements may exist. The radius of gyration (R g) measures the compactness of the molecular structure by calculating the average distance of all atoms of a macromolecule concerning the center of mass defined by the rotational coordinate axis. As shown in Figure C, the R g values exhibited relatively small fluctuations (approximately between 3.38 and 3.42 nm) during the simulation. This indicates that the overall structure of the complexes maintained a relatively stable compactness during the simulation. The solvent-accessible surface area (SASA) provides information about the average surface area of all amino acid residues exposed to the solvent as well as the density of the system. The smaller the SASA, the smaller the contact area of the macromolecule with the solution; the more tightly the protein is folded, the less susceptible it is to thermal denaturation. The results show that the SASA value fluctuates slightly throughout the simulation, between approximately 560–590 nm2. The results in Figure D show that the SASA values fluctuated slightly throughout the simulation, between approximately 560–590 nm2, indicating that the surface contact solvent area of the complexes remained relatively constant. Root-mean-square fluctuation (RMSF) can help us assess how much each atom or residue of a protein fluctuates during the simulation. As shown in Figure E, the RMSF plot shows the volatility of individual residues, and certain regions with less volatility may be critical amino acid residues for complex binding. In addition, hydrogen bonding acts as a strong noncovalent interaction and contributes to ligand binding recognition with precise interactions at the active site. As shown in Figure F, a large number of hydrogen bonds are formed throughout the simulation. The system’s stability was measured according to the changes of key parameters such as RMSD, RMSF, R g, and SASA, indicating that the complexes became stable after initial structural adjustment during the simulation. Meanwhile, SeMet formed more hydrogen bonds with RSV polymerase, suggesting hydrogen bonding may be the key force for its stable binding.

7.

7

Open in a new tab

100 ns molecular dynamics simulation analysis of the complex. (A) RMSD curve of the complex, (B) RMSD curve of the SeMet small molecule, (C) R g curve of the complex, (D) SASA curve of the complex, (E) RMSF curve of the RSV polymerase protein, and (F) hydrogen bonding change curve of the complex.

3.7.2. Gibbs Free Energy Analysis of SeMet-RSV Polymerase Complexes

Gibbs free energy mapping is designed to provide insight into the conformational behavior of protein–ligand interactions. The wider and dark blue area indicates a more stable conformation with minimal energy, while the darker dispersion represents the conformational flexibility of the protein–ligand structure. As shown in Figure A, the 3D plot shows that the free energy shape map presents smooth and single energy clusters, suggesting that the complexes form strong and stable interactions. Regions with low RMSD values (0.22–0.25 nm) and low Gyrate (3.35–3.38 nm) values have lower free energies, indicating that this region is the most conformationally stable. As the RMSD values increase, the free energy rises rapidly, indicating a decrease in the stability of the system. The 2D plot further clearly demonstrates the relationship between RMSD and Gyrate. These regions represent molecular structures that are similar and more compact to the initial conformation, and the system is more stable. Based on the Gibbs free energy landscape map, we found the frame number corresponding to the minimum energy extracted from the PDB file with the minimum energy for visualization (Figure B), and SeMet formed hydrogen bonding, carbon–hydrogen bonding, van der Waals forces, alkyl interaction forces, and π–alkyl interactions with the RSV polymerase, which led to a more stable complex conformation.

8.

8

Open in a new tab

Gibbs free energy analysis of complexes. (A) Complex Gibbs free energy topography. (B) 2D and 3D plots of the visualization of the complex structure corresponding to a Gibbs free energy of 0 kJ/mol.

3.7.3. Binding Free Energy Analysis of SeMet-RSV Polymerase Complexes

The binding free energy is a thermodynamic parameter describing the energy change of the system during intermolecular interactions, and its central role is to quantitatively assess the feasibility and stability of the binding process. The MM/PBSA algorithm divides the binding free energy into molecular mechanics free energy (ΔG gas) and solvation free energy (ΔG solvation). Among them, ΔG gas is divided into van der Waals interaction energy (ΔVDWAALS) and electrostatic energy (ΔE elec); ΔG solvation is divided into polar solvation energy (ΔE GB) and nonpolar solvation energy (ΔE surf). As shown in Figure A, the total binding free energy of the complex was negative (−23.36 kcal/mol), indicating that the binding of the complex was spontaneous. While solvation and polarization effects contribute positive energy, electrostatic interactions and van der Waals interactions provide a larger negative energy, resulting in a negative total free energy overall.

9.

9

Open in a new tab

Binding free energy analysis of the SeMet-RSV polymerase complex. (A) MM/PBSA method to calculate the average binding free energy of SeMet-RSV polymerase. (B) Plot of the contribution of RSV polymerase amino acid residues to the total free energy.

A plot of the contribution of amino acid residues to the total free energy of RSV polymerase (Figure B) shows that the ΔG > −1 kcal/mol of these residues, SER:1266, THR:1267, HIS:1338, and GLN:1386, contribute significantly to the binding energy. It is worth mentioning that the ΔG of GLN:1386 reaches −3.105 kcal/mol, suggesting that it may be a key binding site for SeMet.

We further analyzed the role of these key residues in complex binding (Table ), with SER:1266, THR:1267, and HIS:1338 contributing mainly to van der Waals forces and electrostatic interactions, and GLN:1386 contributing mainly to electrostatic interactions. When electrostatic interactions dominate in the binding energy, hydrogen bonding tends to be the main intermolecular force because it is essentially an electrostatic attraction between highly electronegative atoms and a partially positively charged H. The hydrogen bond is the main intermolecular force in the binding energy. So these key residues maintain the stability of the complex mainly through the formation of hydrogen bonding interactions.

1. Binding Free Energy Decomposition Range of Key Amino Acid Residues.
interactions (kcal/mol) SER:1266 THR:1267 HIS:1338 GLN:1386
ΔVDWAALS –0.627 ± 0.927 –0.636 ± 1.443 –1.814 ± 1.230 0.285 ± 2.591
ΔE elec –1.901 ± 2.891 –2.050 ± 3.002 –1.143 ± 2.118 –4.663 ± 2.982
ΔE GB 1.129 ± 1.883 0.910 ± 1.747 2.151 ± 1.439 1.318 ± 1.262
ΔE surf –0.050 ± 0.040 –0.085 ± 0.071 –0.215 ± 0.040 –0.044 ± 0.047
ΔTOTAL –1.449 ± 4.830 –1.861 ± 4.412 –1.021 ± 4.579 –3.105 ± 5.625
Open in a new tab

4. Conclusions

In summary, SeMet is characterized by low toxicity and high antiviral efficiency. It enhances the body’s antioxidant capacity by integrating it into selenoproteins. Thus, it scavenges RSV infection-induced ROS and attenuates the damage of immune cells by oxidative stress; SeMet regulates apoptosis by participating in the ROS-mediated PARP and Bcl-2 signaling pathways, regulates inflammatory responses through NF-κB/JAK1-STAT3, and ultimately exerts an indirect anti-RSV effect. We investigated the interaction between SeMet and anti-RSV targets by a molecular docking technique. On this basis, a molecular dynamics simulation technique was performed to verify that SeMet formed more and more stable interactions with RSV polymerase. We found that SeMet may bind to key amino acid residues of the RSV polymerase mainly through hydrogen bonding. We will further validate the clearer mechanism of direct antiviral action of SeMet to provide a scientific basis for the development of anti-RSV drugs.

Acknowledgments

We gratefully acknowledge Chen Tianfeng’s group at Jinan University for giving the SeMet. Furthermore, we extend our heartfelt thanks to the technology planning projects of Guangzhou (202201020655) and the Open Project of State Key Laboratory of Respiratory Disease (SKLRD-OP-202608) for financial support.

Data sharing does not apply to this article as no data sets were generated or analyzed during the current study.

†.

C.L., Y.W., and J.L. contributed equally to this work. C.L. and Y.W. designed the study protocol, were responsible for the main experimental part, and drafted the manuscript. J.L. drafted the manuscript and analyzed the data. J.L. and B.Z. conducted the experiments. M.Z. and Y.L. were responsible for refining the manuscript and communicating and coordinating. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.

References

  1. Debessai H., Jones J. M., Meaney-Delman D., Rasmussen S. A.. What U.S. Obstetricians Need to Know About Respiratory Syncytial Virus. Obstet. Gynecol. 2024;143(3):e54–e62. doi: 10.1097/AOG.0000000000005492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Hu M., Bogoyevitch M. A., Jans D. A.. Impact of Respiratory Syncytial Virus Infection on Host Functions: Implications for Antiviral Strategies. Physiol. Rev. 2020;100(4):1527–1594. doi: 10.1152/physrev.00030.2019. [DOI] [PubMed] [Google Scholar]
  3. Nam H. H., Ison M. G.. Respiratory Syncytial Virus Infection in Adults. BMJ. 2019:l5021. doi: 10.1136/bmj.l5021. [DOI] [PubMed] [Google Scholar]
  4. Krishnamoorthy N., Walker K. H., Brüggemann T. R., Tavares L. P., Smith E. W., Nijmeh J., Bai Y., Ai X., Cagnina R. E., Duvall M. G., Lehoczky J. A., Levy B. D.. The Maresin 1–LGR6 Axis Decreases Respiratory Syncytial Virus-Induced Lung Inflammation. Proc. Natl. Acad. Sci. USA. 2023;120(2):e2206480120. doi: 10.1073/pnas.2206480120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Zhang X.-L., Zhang X., Hua W., Xie Z.-D., Liu H.-M., Zhang H.-L., Chen B.-Q., Chen Y., Sun X., Xu Y., Shu S.-N., Zhao S.-Y., Shang Y.-X., Cao L., Jia Y.-H., Lin L.-N., Li J., Hao C.-L., Dong X.-Y., Lin D.-J., Xu H.-M., Zhao D.-Y., Zeng M., Chen Z.-M., Huang L.-S.. Expert Consensus on the Diagnosis, Treatment, and Prevention of Respiratory Syncytial Virus Infections in Children. World J. Pediatr. 2024;20(1):11–25. doi: 10.1007/s12519-023-00777-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Saravanos G. L., King C. L., Deng L., Dinsmore N., Ramos I., Takashima M., Crawford N., Clark J. E., Dale R. C., Jones C. A., Wood N. J., Britton P. N.. Respiratory Syncytial Virus–Associated Neurologic Complications in Children: A Systematic Review and Aggregated Case Series. J. Pediatr. 2021;239:39–49.e9. doi: 10.1016/j.jpeds.2021.06.045. [DOI] [PubMed] [Google Scholar]
  7. Shang Z., Tan S., Ma D.. Respiratory Syncytial Virus: From Pathogenesis to Potential Therapeutic Strategies. Int. J. Biol. Sci. 2021;17(14):4073–4091. doi: 10.7150/ijbs.64762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gu X., Zhou F., Wang Y., Fan G., Cao B.. Respiratory Viral Sepsis: Epidemiology, Pathophysiology, Diagnosis and Treatment. Eur. Respir. Rev. 2020;29(157):200038. doi: 10.1183/16000617.0038-2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Efstathiou C., Abidi S. H., Harker J., Stevenson N. J.. Revisiting Respiratory Syncytial Virus’s Interaction with Host Immunity, towards Novel Therapeutics. Cell. Mol. Life Sci. 2020;77(24):5045–5058. doi: 10.1007/s00018-020-03557-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kirsebom F. C. M., Kausar F., Nuriev R., Makris S., Johansson C.. Neutrophil Recruitment and Activation Are Differentially Dependent on MyD88/TRIF and MAVS Signaling during RSV Infection. Mucosal Immunol. 2019;12(5):1244–1255. doi: 10.1038/s41385-019-0190-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Rahman F., Libre C., Oleinikov A., Tcherniuk S.. Chloroquine and Pyrimethamine Inhibit the Replication of Human Respiratory Syncytial Virus A. J. Gen. Virol. 2021;102(8):001627. doi: 10.1099/jgv.0.001627. [DOI] [PubMed] [Google Scholar]
  12. Attaianese F., Guiducci S., Trapani S., Barbati F., Lodi L., Indolfi G., Azzari C., Ricci S.. Reshaping Our Knowledge: Advancements in Understanding the Immune Response to Human Respiratory Syncytial Virus. Pathogens. 2023;12(9):1118. doi: 10.3390/pathogens12091118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sun J., Xie X., Song Y., Sun T., Liu X., Yuan H., Shen C.. Selenomethionine in Gelatin Methacryloyl Hydrogels: Modulating Ferroptosis to Attenuate Skin Aging. Bioact. Mater. 2024;35:495–516. doi: 10.1016/j.bioactmat.2024.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goularte P. D. S., Imperador C. H. L., Piedade F. H., Guerin A. N., Martins K. D. T., Boreski D., Gabriel E. A., Gabriel S. A., Tiezzi D. G., Chin C. M.. Copper, Zinc, and Selenium Levels During Pregnancy and Their Relationship with Pre-Eclampsia Risk: A Systematic Review. Nutrients. 2025;17(6):1038. doi: 10.3390/nu17061038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Obeme-Nmom J. I., Abioye R. O., Reyes Flores S. S., Udenigwe C. C.. Regulation of Redox Enzymes by Nutraceuticals: A Review of the Roles of Antioxidant Polyphenols and Peptides. Food Funct. 2024;15(22):10956–10980. doi: 10.1039/D4FO03549F. [DOI] [PubMed] [Google Scholar]
  16. Jing J., Zeng H., Shao Q., Tang J., Wang L., Jia G., Liu G., Chen X., Tian G., Cai J., Kang B., Che L., Zhao H.. Selenomethionine Alleviates Environmental Heat Stress Induced Hepatic Lipid Accumulation and Glycogen Infiltration of Broilers via Maintaining Mitochondrial and Endoplasmic Reticulum Homeostasis. Redox Biol. 2023;67:102912. doi: 10.1016/j.redox.2023.102912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li Y., Liu T., Zheng R., Lai J., Su J., Li J., Zhu B., Chen T.. Translational Selenium Nanoparticles Boost GPx1 Activation to Reverse HAdV-14 Virus-Induced Oxidative Damage. Bioact. Mater. 2024;38:276–291. doi: 10.1016/j.bioactmat.2024.04.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li Y., Chen D., Su J., Chen M., Chen T., Jia W., Zhu B.. Selenium-Ruthenium Complex Blocks H1N1 Influenza Virus-Induced Cell Damage by Activating GPx1/TrxR1. Theranostics. 2023;13(6):1843–1859. doi: 10.7150/thno.83522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hu M., Schulze K. E., Ghildyal R., Henstridge D. C., Kolanowski J. L., New E. J., Hong Y., Hsu A. C., Hansbro P. M., Wark P. A., Bogoyevitch M. A., Jans D. A.. Respiratory Syncytial Virus Co-Opts Host Mitochondrial Function to Favour Infectious Virus Production. eLife. 2019;8:e42448. doi: 10.7554/eLife.42448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Su J., Lai J., Li J., Li C., Liu X., Wang C., Zhu B., Li Y.. Selenium Nanoparticles Control H1N1 Virus by Inhibiting Inflammatory Response and Cell Apoptosis. Molecules. 2023;28(15):5920. doi: 10.3390/molecules28155920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hu M., Bogoyevitch M. A., Jans D. A.. Subversion of Host Cell Mitochondria by RSV to Favor Virus Production Is Dependent on Inhibition of Mitochondrial Complex I and ROS Generation. Cells. 2019;8(11):1417. doi: 10.3390/cells8111417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liang Y.-y., Li K., Niu F., Li Y., Wei H., Dai Y., Wang Y., Zhou C., Wan X.. Salvia Plebeia R. Br. Polysaccharides (SPP) against RSV (Respiratory Syncytial Virus) Infection: Antiviral Effect and Mechanisms of Action. Biomed. Pharmacother. 2021;141:111843. doi: 10.1016/j.biopha.2021.111843. [DOI] [PubMed] [Google Scholar]
  23. Centonze M., Di Conza G., Lahn M., Fabregat I., Dituri F., Gigante I., Serino G., Scialpi R., Carrieri L., Negro R., Pizzuto E., Giannelli G.. Autotaxin Inhibitor IOA-289 Reduces Gastrointestinal Cancer Progression in Preclinical Models. J. Exp. Clin. Cancer Res. 2023;42(1):197. doi: 10.1186/s13046-023-02797-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lin K., Ramesh S., Agarwal S., Kuo W., Kuo C., Chen M. Y., Lin Y., Ho T., Huang P., Huang C.. Fisetin Attenuates Doxorubicin-induced Cardiotoxicity by Inhibiting the Insulin-like Growth Factor II Receptor Apoptotic Pathway through Estrogen Receptor-α/-β Activation. Phytother. Res. 2023;37(9):3964–3981. doi: 10.1002/ptr.7855. [DOI] [PubMed] [Google Scholar]
  25. Erfu C., Li L., Weiting Q., Tao C., Liwei M., Hemin Y., Junkun L.. Matrine Attenuating Cardiomyocyte Apoptosis in Doxorubicin-Induced Cardiotoxicity through Improved Mitochondrial Membrane Potential and Activation of Mitochondrial Respiratory Chain Complex I Pathway. Biomed. Pharmacother. 2024;173:116464. doi: 10.1016/j.biopha.2024.116464. [DOI] [PubMed] [Google Scholar]
  26. Chi L., Shan Y., Cui Z.. N-Acetyl-L-Cysteine Protects Airway Epithelial Cells during Respiratory Syncytial Virus Infection against Mucin Synthesis, Oxidative Stress, and Inflammatory Response and Inhibits HSPA6 Expression. Anal. Cell. Pathol. 2022;2022:4846336. doi: 10.1155/2022/4846336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Chen W.-F., Tsai S.-C., Zhang Y.-H., Chang H.-M., Wu W.-J., Su J.-H., Wu B.-N., Chen C.-Y., Lin M.-Y., Chen H.-L., Lee C.-H.. Rhopaloic Acid A Triggers Mitochondria Damage-Induced Apoptosis in Oral Cancer by JNK/BNIP3/Nix-Mediated Mitophagy. Phytomedicine. 2024;132:155855. doi: 10.1016/j.phymed.2024.155855. [DOI] [PubMed] [Google Scholar]
  28. Bai R., Guo J., Ye X.-Y., Xie Y., Xie T.. Oxidative Stress: The Core Pathogenesis and Mechanism of Alzheimer’s Disease. Ageing Res. Rev. 2022;77:101619. doi: 10.1016/j.arr.2022.101619. [DOI] [PubMed] [Google Scholar]
  29. Xiang S., Yang D. K.. Aerial Yam Bulbils Protect Against APAP-Induced Hepatotoxicity by Inhibiting Oxidative Stress and Mitochondrial Dysfunction Through Nrf2 Activation. Nutrients. 2025;17(6):966. doi: 10.3390/nu17060966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kaarniranta K., Uusitalo H., Blasiak J., Felszeghy S., Kannan R., Kauppinen A., Salminen A., Sinha D., Ferrington D.. Mechanisms of Mitochondrial Dysfunction and Their Impact on Age-Related Macular Degeneration. Prog. Retinal Eye Res. 2020;79:100858. doi: 10.1016/j.preteyeres.2020.100858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Flores-Romero H., Dadsena S., García-Sáez A. J.. Mitochondrial Pores at the Crossroad between Cell Death and Inflammatory Signaling. Mol. Cell. 2023;83(6):843–856. doi: 10.1016/j.molcel.2023.02.021. [DOI] [PubMed] [Google Scholar]
  32. Liu X., Lai J., Su J., Zhang K., Li J., Li C., Ning Z., Wang C., Zhu B., Li Y., Zhao M.. Selenadiazole Inhibited Adenovirus-Induced Apoptosis through the Oxidative-Damage-Mediated Bcl-2/Stat 3/NF-κB Signaling Pathway. Pharmaceuticals. 2023;16(10):1474. doi: 10.3390/ph16101474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Winter J. M., Yadav T., Rutter J.. Stressed to Death: Mitochondrial Stress Responses Connect Respiration and Apoptosis in Cancer. Mol. Cell. 2022;82(18):3321–3332. doi: 10.1016/j.molcel.2022.07.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Forman H. J., Zhang H.. Targeting Oxidative Stress in Disease: Promise and Limitations of Antioxidant Therapy. Nat. Rev. Drug Discovery. 2021;20(9):689–709. doi: 10.1038/s41573-021-00233-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang X., Zeng X., Li D., Zhu C., Guo X., Feng L., Yu Z.. PARP Inhibitors in Small Cell Lung Cancer: The Underlying Mechanisms and Clinical Implications. Biomed. Pharmacother. 2022;153:113458. doi: 10.1016/j.biopha.2022.113458. [DOI] [PubMed] [Google Scholar]
  36. Prashanth T., Avin B. R. V., Thirusangu P., Ranganatha V. L., Prabhakar B. T., Sharath Chandra J. N. N., Khanum S. A.. Synthesis of Coumarin Analogs Appended with Quinoline and Thiazole Moiety and Their Apoptogenic Role against Murine Ascitic Carcinoma. Biomed. Pharmacother. 2019;112:108707. doi: 10.1016/j.biopha.2019.108707. [DOI] [PubMed] [Google Scholar]
  37. Akpolat M., Oz Z. S., Gulle K., Hamamcioglu A. C., Bakkal B. H., Kececi M.. X Irradiation Induced Colonic Mucosal Injury and the Detection of Apoptosis through PARP-1/P53 Regulatory Pathway. Biomed. Pharmacother. 2020;127:110134. doi: 10.1016/j.biopha.2020.110134. [DOI] [PubMed] [Google Scholar]
  38. Zheng R., Chen D., Su J., Lai J., Wang C., Chen H., Ning Z., Liu X., Tian X., Li Y., Zhu B.. Inhibition of HAdV-14 Induced Apoptosis by Selenocystine through ROS-Mediated PARP and P53 Signaling Pathways. J. Trace Elem. Med. Biol. 2023;79:127213. doi: 10.1016/j.jtemb.2023.127213. [DOI] [PubMed] [Google Scholar]
  39. Major J., Crotta S., Llorian M., McCabe T. M., Gad H. H., Priestnall S. L., Hartmann R., Wack A.. Type I and III Interferons Disrupt Lung Epithelial Repair during Recovery from Viral Infection. Science. 2020;369(6504):712–717. doi: 10.1126/science.abc2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mahmudpour M., Roozbeh J., Keshavarz M., Farrokhi S., Nabipour I.. COVID-19 Cytokine Storm: The Anger of Inflammation. Cytokine. 2020;133:155151. doi: 10.1016/j.cyto.2020.155151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xue C., Yao Q., Gu X., Shi Q., Yuan X., Chu Q., Bao Z., Lu J., Li L.. Evolving Cognition of the JAK-STAT Signaling Pathway: Autoimmune Disorders and Cancer. Signal Transduction Targeted Ther. 2023;8(1):204. doi: 10.1038/s41392-023-01468-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ezeonwumelu I. J., Garcia-Vidal E., Ballana E.. JAK-STAT Pathway: A Novel Target to Tackle Viral Infections. Viruses. 2021;13(12):2379. doi: 10.3390/v13122379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Li S., Du Y., Chen G., Mao Y., Zhang W., Kang M., Zhu S., Wang D.. Protocatechuic Acid Attenuates Inflammation in Macrophage-like Vascular Smooth Muscle Cells in ApoE–/– Mice. Nutrients. 2025;17(6):1090. doi: 10.3390/nu17061090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yu H., Lin L., Zhang Z., Zhang H., Hu H.. Targeting NF-κB Pathway for the Therapy of Diseases: Mechanism and Clinical Study. Signal Transduction Targeted Ther. 2020;5(1):209. doi: 10.1038/s41392-020-00312-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Gödecke N., Riedel J., Herrmann S., Behme S., Rand U., Kubsch T., Cicin-Sain L., Hauser H., Köster M., Wirth D.. Synthetic Rewiring and Boosting Type I Interferon Responses for Visualization and Counteracting Viral Infections. Nucleic Acids Res. 2020;48(20):11799–11811. doi: 10.1093/nar/gkaa961. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data sharing does not apply to this article as no data sets were generated or analyzed during the current study.

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES