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. 2025 Sep 10;104(11):105810. doi: 10.1016/j.psj.2025.105810

Naringenin's rescue of broiler spleen from LPS-triggered pyroptosis and inflammation: Decoding the AMPK/PINK1/Parkin-driven mitophagy pathway☆

Yu Xia a,c, Yidan Wang a, Jiahui Xue a, Jiahong Chu a, Yanhe Zhang a, Fuze She a, Huijie Chen b, Shu Li a,⁎
PMCID: PMC12466259  PMID: 40961773

Highlights

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    Network pharmacology uncovers effect of Nar on anti-inflammation.

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    Nar promoted mitophagy through the interaction with AMPK.

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    Nar restored mitochondrial dysfunction and oxidative stress induced by LPS.

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    Nar alleviated pyroptosis and inflammation through regulation of mitophagy pathway.

Key words: Naringenin, AMPK, Mitophagy, Pyroptosis, Broiler spleen

Abstract

Naringenin (Nar) is known to maintain mitochondrial homeostasis, antioxidation and anti-inflammation. Damaged mitochondria can promote excessive Reactive oxygen species (ROS) production, triggering pyroptosis and inflammation process in immune tissues. PTEN induced putative kinase 1 (PINK1)/E3 ubiquitin ligase PARK2 (Parkin)-mediated mitophagy contributes to removing damaged mitochondria. This study aims to investigate detailed mechanism of Nar against Lipopolysaccharide (LPS)-induced injury in broiler spleens and the role of mitophagy in this process. We used LPS as a stimulus and treated with Nar to establish relevant models in vivo and in vitro. Our findings demonstrated Nar increased the expression levels of Phospho-Adenosine 5′-monophosphate (AMP)-activated protein kinase (p-AMPK)/AMPK, PINK1, Parkin and Microtubule-Associated Protein 1A/1B Light Chain 3 (LC3), and reduced the level of Sequestosome 1 (P62), leading to a reduction in levels of factors associated with mitochondrial fission, mtDNA release, pyroptosis, and inflammation. Conversely, Nar treatment enhanced the levels of factors related to mitochondrial fusion, energy metabolism, and anti-inflammatory response. Moreover, an increase was observed in ΔΨm, ATP content, and ATPase activity. Molecular docking analysis and cellular thermal shift assay (CETSA) supported the interaction between Nar and AMPK. In summary, Nar enhanced LPS-induced mitophagy and alleviated mitochondrial homeostasis imbalance and oxidative stress in broiler spleens through its interaction with AMPK, resulting in alleviating pyroptosis and inflammation.

Graphical abstract

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Introduction

Naringenin (Nar), a natural flavonoid compound abundant in fruits and vegetables, is recognized for its potent antioxidant, anti-inflammatory, and immunomodulatory effects (Chang et al., 2024; Chen, et al., 2024c). These properties suggest that Nar displays significant potential in enhancing immune function to alleviate injury in the spleen, bursa of Fabricius, and thymus. It can reduce the release of mtROS by regulating mitochondrial dynamics and the respiratory chain, thereby mitigating ferroptosis and immune deficiencies in the chicken thymus (Yu, et al., 2024). In mice with experimental autoimmune encephalomyelitis, Nar protects the blood-brain barrier, curtails dendritic cell accumulation and maturation, and obstructs their chemotactic and antigen-presenting functions, demonstrating an immunoprotective role (Niu, et al., 2021). Nar effectively protects the organism from pyroptosis and inflammation damage. It prevents microgravity-induced pyroptosis in osteoblasts via the Nuclear factor erythroid 2-related factor 2/Heme oxygenase-1 (Nrf2/HO-1) signaling pathway (Cao, et al., 2024). Zhang et.al demonstrated that treatment with Nar promoted AMP-activated protein kinase (AMPK) phosphorylation, triggering autophagy in macrophages to improve collagen-induced arthritis (Zhang, et al., 2023). Chen et al. discovered that oral administration of Nar counteracted retinal degeneration by regulating mitochondrial dynamics and autophagy process (Chen et al., 2022b). Pyroptosis, a programmed cell death involving Cysteinyl aspartate specific proteinase-1 (Caspase-1) activation and the release of inflammatory cytokines, such as Interleukin-1β (IL-1β) and Interleukin-18 (IL-18), amplifies the inflammatory response. In a sepsis-induced acute lung injury model, Nar significantly alleviates inflammation progression by inhibiting the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome (Chen, et al., 2019).

AMPK is regarded as a critical regulator of mitochondrial homeostasis, modulating various aspects of the mitochondrial life cycle, including mitophagy (Iorio, et al., 2021). Mitophagy specifically targets and degrades damaged mitochondria (Chen, et al., 2024a; Wang, et al., 2024). When mitochondria are impaired, PTEN-induced putative kinase 1 (PINK1) recruits E3 ubiquitin ligase PARK2 (Parkin) to the outer membrane, leading to the aggregation of Sequestosome 1 (p62) (Narendra, et al., 2008). The p62 interacts with microtubule-associated protein light chain 3 (LC3), promoting mitophagosome formation and hydrolase degradation (Pankiv, et al., 2007). PINK1/Parkin-mediated mitophagy is vital in response to various injuries (Zhao, et al., 2024). Dapagliflozin promotes PINK1/Parkin-mediated mitophagy to remove the damaged mitochondria through the activation of AMPK, providing protection against myocardial ischemia-reperfusion injury (Zuo, et al., 2024). Likewise, exogenous H2S mitigates senescence in glomerular mesangial cells by upregulating mitophagy through the AMPK/ULK1/PINK1/Parkin pathway, thus reducing neurotoxicity from environmental pollutants PBDE-47 (E, et al., 2023).The elimination of damaged mitochondria is critical for maintaining mitochondrial homeostasis to alleviate pyroptosis and inflammation (Fu, et al., 2025). Jiao et al. demonstrated that PINK1/Parkin-mediated mitophagy could eliminate damaged mitochondria to maintain mitochondrial homeostasis, improving inflammation associated with cerebral ischemia-reperfusion (Jiao, et al., 2024). Liu et al. revealed that allicin promoted mitophagy and reduced excessive Reactive oxygen species (ROS) production, further inhibiting NLRP3 inflammasome activation to alleviate LPS-induced pyroptosis in THP-1 cells (Liu, et al., 2022). Zhang et al. revealed that Gs-Rb1 facilitated the removal of damaged mitochondria and decreased intracellular ROS production, thus alleviating 3-MCPD-induced pyroptosis in renal cells (Zhang et al., 2024).

AMPK, as a crucial regulator of mitochondrial homeostasis, is capable of effectively clearing damaged mitochondria by activating the PINK1/Parkin-mediated mitophagy pathway (Herzig and Shaw, 2018), thereby maintaining the stability of mitochondrial function. This mechanism aids in ameliorating mitochondrial dysfunction and inhibits the activation of the NLRP3 inflammasome, thus mitigating inflammation responses induced by harmful substances (Lv, et al., 2024). Nar typically exhibits immunomodulatory functions that protect immune tissues from damage (Alimohammadi, et al., 2022). However, the regulatory role of Nar in mitophagy and its mechanism in alleviating inflammatory injury in immune tissues remain unclear. Therefore, this study was conducted to elucidate the role of Nar in mitigating LPS-induced pyroptosis and inflammatory injury in spleens, as well as the involvement of mitophagy in this process. To explore the underlying mechanisms, we combined network pharmacology and selected 14-day-old broilers and MSB-1 cells as experimental models. Histopathological and ultrastructural alterations were observed using HE staining and transmission electron microscopy in spleen tissues. Oxidative stress indicators were evaluated by measuring ROS levels using DHE staining in chicken spleen tissue and DCFH-DA staining in MSB-1 cells. IF, qRT-PCR and WB were utilized to examine the expression levels of factors related to mitophagy, mitochondrial dynamics, mtDNA release, energy metabolism, pyroptosis, and inflammation. Additionally, we assessed mitochondrial membrane potential, Adenosine Triphosphate (ATP) content and Adenosine Triphosphatase (ATPase) activity. This study significantly enhances the potential applications of Nar, establishing a solid theoretical foundation for its practical production and efficient utilization.

Materials and methods

Animal preparation and sample collection

All procedures used in this study were approved by the Animal Protection and Use Committee of Northeast Agricultural University (NEAUEC20230308, January seventh, 2023). Fourty broilers (2-week-old) were randomly divided into 4 groups, including the Control, LPS, Nar (the chemical structure was shown as Fig. S1) and Nar+LPS groups. In the Nar group and the Nar+LPS group, Nar (40 mg/kg BW, purity ≥ 98 %, N486154, Aladdin, China) (Yu et al., 2024) was administered by oral gavage for 7 days. The Control and LPS groups were given corn oil intragastric administration for 7 days. LPS (0.15 mg/kg BW, BS904, Biosharp, China) (Wang, et al., 2023) was intraperitoneally injected for 12 h, and other groups were intraperitoneally injected normal saline. The spleens were collected and stored at −80°C for biochemical measurements.

Antioxidant function detection

Chicken spleen tissue was rinsed with saline and tissue homogenate was obtained after centrifugation at 2000 rpm for 10 min. Content of malondialdehyde (MDA, A003-2-2), glutathione peroxidase (GSH-Px, A005-1-1), superoxide dismutase (SOD, A001-2-2) and Catalase (CAT, A007-1-1) was detected according to the kit instructions (Nanjing Jiancheng, China).

Hematoxylin-eosin (HE) staining

Spleen tissues were fixed in 4 % Paraformaldehyde (P0099, Beyotime, China) solution at room temperature for 24 h, and then the wax blocks were embedded and cut into 5 μm slices. After that, the slices were dehydrated, stained with hematoxylin and eosin, soaked in xylene for 2 h and dried until transparent. Finally, the slices were mounted with neutral resin and under a microscope (Nikon, Japan).

Transmission electron microscopy (TEM)

The tissue was cut into 1 mm3 and fixed in 2.5 % glutaraldehyde for overnight. Then, the sample was re-fixed with 1 % osmium tetroxide, dehydrated with gradient acetone, embedded with epoxy resin, and cut into 50 nm thick slices. Finally, section was stained with uranyl acetate for 10-15 min, and taken images using a transmission electron microscope (Hitachi H7650, Tokyo, Japan).

Immunofluorescence (IF)

IF was used to assess the GSDMD and IL-6 levels in broiler spleen and MSB-1 cells. Briefly, tissue sections were deparaffinized and rehydrated for antigen extraction. The sections were then incubated with GSDMD (WL05686, Wanleibio, China) and IL-6 (WL02841, Wanleibio, China) primary antibody at 4°C overnight. After washing, the sections were incubated with secondary antibody (Dylight594, Abbkine) for 1 h. Finally, the nuclei were stained with DAPI (G1012, Servicebio, China) to visualize the nuclei. Images were captured using a laser confocal microscope (NIKON, Japan).

Cell culture and treatment

MSB-1 cells were suspended in RPMI-1640 media (Gibco, China) supplemented with 10 % fetal bovine serum (BS1101, Opcell, China) and 1 % penicillin-streptomycin (Beyotime, China). The cells were cultured in an incubator maintained at 37°C and 5 % CO₂ (Thermo, USA).

Cell viability assay

MSB-1 cells were inoculated into 96-well plates. The cell density reached 70 % at the next day, LPS (2.5, 5, 10, 20, 30, 40, 50, 60 μM), Nar (2.5, 5, 10, 20, 40, 60, 80 μM), AMPK inhibitor Compound C (CC, M3946, AbMole, China, at a concentration of 1, 2, 4 μM) and mitophagy inhibitor Cyclosporine (CsA, M1831, AbMole, China, at a concentration of 1, 2, 4 μM) was added to wells for 24 h. Then, 10 μL CCK-8 solution (MA0218, Meilun, China) was added to the experimental wells and cultured at 37°C for 1 h. Finally, the absorbance was detected at 450 nm using a microreader (Aglient, USA).

Dihydroethidium (DHE) detection in spleen tissue

Fresh tissue samples were rinsed with pre-cooled physiological saline and sliced into thin sections. The sections were then fixed in 4 % paraformaldehyde for 1 to 2 h, followed by washing with PBS. Subsequently, a staining solution containing approximately 10 μM DHE (G1904-100T, Servicebio, China) was prepared, and the tissue sections were incubated in this solution at 37°C in the dark for 30 min. After incubation, the sections were washed three times with PBS, with each wash lasting 5 to 10 min. Finally, the tissue sections were placed on glass slides, and an anti-fade mounting medium was applied before covering with a coverslip. The samples were then observed and imaged under a fluorescence microscope.

Detection of ROS production

The detection of ROS was performed with a commercial kit provided by Nanjing Jiancheng Bioengineering Institute following the manufacturer’s instructions. Briefly, the supernatant of treated cells was removed, and the cells were washed with PBS for twice time. Then, the cells were stained with 20 μM DCFH-DA for 30 min. Finally, ROS level was observed using a fluorescence microscope (Olympus, Japan).

Detection of ATP content and ATPase activity

Adenosine triphosphate (ATP) content (A091-1-1) and ATPase activity (A070-1-2) were detected using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng, China). The experimental results were measured using a microplate reader (Olympus, Japan).

Detection of mitochondrial membrane potential

Mitochondrial membrane potential was detected with a JC-1 commercial kit (D-9113, Bioss, China) kit. Cells were stained with JC-1 probe for 30 min, and then cells were rinsed with JC-1 dye buffer for twice. Finally, the cells were observed with a fluorescence microscope (Olympus, Japan). Using the same cell treatment method as described above, analysis was performed with a flow cytometer (BD FACSCanto, USA), and the results were analyzed using FlowJo software.

Total RNA isolation and qRT-PCR

Total RNA was obtained using Trizol reagent (15596026, Thermo, USA). Then, NanoDrop ND-2000 (Thermo, USA) instrument was used to determine the ratio of OD260 to OD280 and the concentration of total RNA. After that, cDNA was synthesized according to the first strand cDNA synthesis kit (AU341, TransGen, China) and qRT-PCR was performed using SYBR Green (AQ311, TransGen, China) to measure expression levels of mRNA in different groups (Xia, et al., 2025; Zhang, et al., 2024). The detailed primer sequences were presented in Table S1 and data were calculated using 2−ΔΔCt method.

Protein extraction and western Blot analysis

Total protein was extracted from cells of different groups with RIPA lysis and PMSF solution (P0013, Beyotime, China) and the concentration of total protein was measured with a BCA commercial kit. Total protein was loaded to SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked in 5 % skimmed milk powder solution, incubated with primary antibody at 4 °C overnight and incubated with secondary antibody (bs-0311P-HRP, Bioss, China) at 37 °C for 1 h. Finally, the target protein was captured using Chemiluminescent imaging instrument provided by Tanon (Shanghai, China). The detailed primary antibody information was presented in Table S2 and the results were analyzed using Image J software.

Network pharmacology analysis

The targets of Nar were identified through the Swiss Target Prediction database (http://www.swisstargetprediction.ch/) and the Super-PRED database (https://prediction.charite.de/index.php). Inflammatory targets were screened based on the GeneCards database (https://www.genecards.org/), the OMIM database (https://www.omim.org/), and the TTD database (https://db.idrblab.net/ttd/). Subsequently, the STRING database (https://www.string-db.org/) was employed to construct the protein-protein interaction (PPI) network of the common targets. Network topological analysis was conducted using the CytoScape software, and GO and KEGG enrichment analyses were performed by utilizing the DAVID database (https://david.ncifcrf.gov/).

Molecular docking

The molecular docking approach was employed to evaluate the affinity and analyze the interactions between AMPK and Nar. The 3D structure of AMPK is selected from PDB (https://www.rcsb.org/) database and the 3D structure of Nar is downloaded from PubChem (https://pubchem.ncbi.nlm). Use vina-2.0 integrated in the pyrx software for molecular docking, calculate the binding energy and output the result file. Finally, use the Discovery Studio 2020 Client (https://discover.3ds.com/discovery-studio-visualizer-download) software for result visualization. Furthermore, the CB-dock2 database (https://cadd.labshare.cn/cb-dock2) and SWISSDOCK database (https://www.swissdock.ch/) were used to forecast the binding potential existing between Nar and AMPK.

Cell thermal shift assay (CETSA)

MSB-1 cells were divided into control and Nar groups. Cells were collected when the cell density reached 80 %−90 %. After being washed with PBS, the cells were resuspended. The cell suspensions of the two groups were respectively placed at different temperatures (37, 41, 45, 49, 53, 57, and 61°C). Subsequently, cell lysis was carried out using liquid nitrogen and two repeated freeze - thaw cycles. The cells were centrifuged at 12000 rpm for 15 min, and the supernatant was collected. 5×SDS-PAGE protein loading buffer was added to the supernatant, and the mixture was boiled at 100°C for 10 min to denature the proteins. The denatured protein samples were using for WB analysis.

Statistical analysis

Data analysis was performed using GraphPad Prism version 8.0.2 software and the means±SD was used to express all results. Differences between groups were compared by conducting one-way ANOVO and P < 0.05 was considered to be a significant difference.

Results

Nar alleviated LPS-induced pyroptosis and inflammation in broiler spleens

To observe the histopathological and ultrastructural changes in the spleens of broilers (Fig. 1A), we collected samples for HE staining and TEM observation. HE staining showed that the structure was normal in the Control and Nar groups, with distinct boundaries between the red and white pulp and visible central arteries. In contrast, the LPS group exhibited a significant expansion of the white pulp area, looser arrangement of splenic cells (orange arrows), an increase in the number of red blood cells within the splenic sinuses (red arrows), and inflammatory cell infiltration (yellow arrows). However, these pathological changes were notably reduced in the Nar+LPS group (Fig. 1B). The TEM observations revealed that the cellular morphology was normal in the Control and Nar groups, with clear mitochondrial cristae and distinct cell boundaries. In the LPS group, the ultrastructure exhibited typical features of pyroptosis, including swollen or vacuolated mitochondria (green arrows), ruptured and lysed cell membranes with cytoplasmic content leakage, and extensive vesicle formation (blue arrows), along with severe nuclear deformation was observed (yellow arrows). Conversely, the cell membranes were relatively intact, and the mitochondrial cristae partially returned to normal after treatment with Nar (Fig. 1C). These findings may suggest that LPS stimulation trigger pyroptosis and inflammation, and Nar has an effective protection against LPS-induced damage in broiler spleens. Following HE and TEM observation, we conducted IF detection for GSDMD and IL-6 in each group. The results revealed a significant increase in GSDMD and IL-6 in the LPS group compared to the control group, while treatment with Nar effectively inhibited the expression levels of GSDMD and IL-6 (P < 0.05, Fig. 1D–1E). The results indicated that Nar alleviated LPS-induced pyroptosis and inflammatory damage in broiler spleens.

Fig. 1.

Fig 1

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Nar alleviated LPS-induced pyroptosis and inflammation in broiler spleens. (A) A schematic design of experiment in broilers treated with Nar and LPS, alone or both. (B) Histopathological changes (n = 6, scale bar, 50 µm). (C) Ultrastructural observation (n = 6, scale bar, 1 µm). (D) IF results for GSDMD (n = 6, scale bar, 200 µm). (E) IF results for IL-6 (n = 6, scale bar, 200 µm). Data are presented as means±SD. Bars with different letters are considered to have significant differences (P < 0.05).

Nar promoted mitophagy via the AMPK pathway

We measured the mRNA and protein levels of factors related to the AMPK pathway and mitophagy in spleen tissues. The results demonstrated that LPS exposure upregulated the levels of p-AMPK/AMPK and the expression levels of mitophagy-related factors such as PINK1, Parkin, P62 and LC3. Moreover, Nar could further promote the activation of AMPK, increase the expression of PINK1, Parkin, and LC3 and simultaneously decrease the expression of P62 (P < 0.05, Fig. 2A–2C). This reflects the ability of Nar to facilitate the complete occurrence of mitophagy by activating AMPK. We screened 16,234 genes from the pathological database, and then intersected them with the target database of Nar, obtaining 200 intersecting genes (Fig. 2D). Subsequently, the above-mentioned overlapping targets were uploaded to the STRING database to generate a PPI network diagram, and topological analysis was carried out (Fig. 2E–2F). It was found that among the 200 intersecting targets obtained above, AMPK was present. GO analysis revealed that the gene-related BPs mainly included protein phosphorylation, cell differentiation, and the cell-surface receptor protein tyrosine kinase signaling pathway. The CCs were mainly related to the cytoplasm, lysosomes, and protein-containing complexes and the MFs mainly involved ATP binding, signal-receptor binding, and transmembrane receptor protein tyrosine kinase activity (Fig. 2G). These findings suggest that Nar may interfere with the inflammatory response through these biological processes. KEGG pathway enrichment analysis identified 33 significantly enriched pathways (P < 0.05). The results of the KEGG pathway enrichment analysis indicated that the targets of Nar were predominantly associated with processes such as nitrogen metabolism, the calcium signaling pathway, autophagy, the NOD-like receptor signaling pathway, and the mTOR signaling pathway (Fig. 2H). In addition, molecular docking results showed that Nar had potential interactions with AMPK at Glutamic acid (GLU)-234, Leucine (LEU)-232, Alanine (ALA)-230, Threonine (THR)-267, Proline (Pro)-233, Tyrosine (TYR)-265, Tryptophan (TRP)-258, Valine (VAL)-266, and Isoleucine (ILE)-218, with a mean binding energy of −6.2 ± 0.3 kcal/mol (Fig. 2I). Meanwhile, the results of the CETSA revealed that after the addition of Nar, the thermal stability curve of AMPK shifted to the right, further validating the interaction between Nar and AMPK (Fig. 2J). To further investigate the mechanism, we conducted the experiments using MSB-1 cells, Based on CCK8 assay, we treated cells with 30 µg/mL LPS, 10 µM Nar, 2 µM CC, and 2 µM CsA, alone or in combination (Fig. S2A-S2D). The mRNA and protein expression levels of p-AMPK/AMPK, and mitophagy-related factors were significantly increased in the LPS group (P < 0.05) compared to the Control group. Moreover, treatment with Nar significantly increased the expression levels of p-AMPK/AMPK, PINK1, Parkin, and LC3, while decreasing the expression level of P62 (P < 0.05). After adding CC and CsA, the expression levels of AMPK, p-AMPK and mitophagy-related factors were detected. The results showed that the promoting effect of Nar on mitophagy was reversed (P < 0.05, Fig. 2K–2P). These results suggested that Nar promoted the expression of the AMPK pathway through a direct interaction, thereby promoting mitophagy.

Fig. 2.

Fig 2

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Nar promoted mitophagy via AMPK. (A-C) Protein and mRNA expression levels of AMPK, p-AMPK and mitophagy-related factors in spleen tissue (n = 6). (D) Venn diagram of the intersection between the targets of Nar and the inflammatory targets. (E-F) PPI network diagram. (G) GO Analysis of Intersection Targets. (H) KEGG Enrichment Analysis of Intersection Targets. (I) Molecular docking results between Nar and AMPK. (J) Cellular thermal shift assays to verify interaction between Nar and AMPK. (K-M) Protein and mRNA expression levels of the AMPK, p-AMPK in MSB-1 cells (n = 3). (N-P) Protein and mRNA expression levels of mitophagy-related factors in MSB-1 cells (n = 3). Data are presented as means±SD. Bars with different letters are considered to have significant differences (P < 0.05).

Nar ameliorated LPS-induced mitochondrial dysfunction by promoting mitophagy via AMPK

Mitophagy can effectively remove damaged mitochondria and maintain mitochondrial homeostasis. Therefore, we examined the mRNA and protein expression levels of factors related to mitochondrial dynamics in broiler spleens. The results showed that the expression level of the DRP1 was significantly increased and the expression levels of Opa1, Mfn1, and Mfn2 were significantly decreased in the LPS group (P < 0.05). Notably, these factor expression levels were reversed after treatment with Nar (P < 0.05, Fig. 3A–3C), indicating Nar treatment significantly alleviated LPS-induced mitochondrial dynamic imbalance. Furthermore, we conducted in vitro using MSB-1 cells. Our findings elucidated that Nar treatment reversed the expression levels of these factors related to mitochondrial dynamics (P < 0.05, Fig. 3D–3F). Mitochondrial homeostasis imbalance can rupture mitochondrial membranes, releasing mtDNA and triggering downstream reactions. We detected the release level of mtDNA and experimental results revealed that the levels of ND4, ND1, 16S, 12S, and d-LOOP were significantly enhanced after exposure to LPS compared with the Control and Nar groups and these levels were evidently decreased after treatment with Nar compared with the LPS group in vivo and in vitro. Whereas, treatment with CC and CsA antagonized the inhibitory effect of Nar on LPS-induced mtDNA release ((P < 0.05, Fig. S3A-3B). Mitochondrial membrane potential (ΔΨm) is also an important indicator reflecting the functional state of mitochondria. Thus, we measured the ΔΨm in different groups of MSB-1 cells using JC-1 staining. Experimental results showed that the Red/Green fluorescence ratio in the LPS group was significantly reduced compared to the Control group and treatment with Nar promoted the Red/Green fluorescence ratio (P < 0.05). However, in the Nar+LPS+CC and Nar+LPS+CsA groups, the therapeutic effect of Nar was significantly inhibited (P < 0.05, Fig. 3G). The outcomes of JC-1 flow cytometry are presented in Fig. 3H. Following treatment with LPS, a marked depolarization of the mitochondrial membrane potential was observed. In contrast, subsequent treatment with Nar led to a partial restoration of the membrane potential. However, upon the addition of two inhibitors, the efficacy of Nar in restoring the mitochondrial membrane potential was abrogated. These results indicated that Nar treatment ameliorated LPS-induced mitochondrial dysfunction by promoting mitophagy via AMPK.

Fig. 3.

Fig 3

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Nar ameliorated LPS-induced mitochondrial dysfunction by promoting mitophagy via AMPK. (A-C) Protein and mRNA expression levels of mitochondrial dynamics-related factors (Drp1, Opa1, mfn1 and mfn2) in spleen tissue (n = 6). (D-F) Protein and mRNA expression levels of mitochondrial dynamics-related factors (Drp1, Opa1, mfn1 and mfn2) in MSB-1 cells (n = 3). (G) JC-1 staining (n = 3, scale bar, 100 µm) in MSB-1 cells and quantitative analysis. (H) Flow cytometry results of JC-1-stained MSB-1cell. Results are expressed as means±SD. Bars with different letters are considered to have statistically significant differences (P < 0.05).

Nar alleviated LPS-induced energy metabolism disruption by promoting mitophagy via AMPK

Mitochondria are the primary site for ATP production, and mitochondrial dysfunction inevitably leads to energy metabolism disruption. Therefore, we examined the expression levels of key enzymes involved in energy metabolism. Compared with the Control group, the mRNA and protein levels of pyruvate kinase M2 (PKM2), isocitrate dehydrogenase 1 (IDH1), and succinate dehydrogenase complex subunit B (SDHB) were significantly reduced in the LPS group (P < 0.05). In contrast, the mRNA and protein expression levels of PKM2, IDH1, and SDHB in the Nar+LPS group were significantly increased compared to the LPS group (P < 0.05, Fig. 4A–4C). The detection of ATP content and ATPase activity revealed a sharp decline following LPS exposure in the spleen tissues of each group (P < 0.05), which was reversed by the addition of Nar (P < 0.05, Fig. 4D–4E). In vitro (Fig. 4F), the mRNA and protein expression levels of PKM2, IDH1, and SDHB were significantly reduced in the LPS group and treatment with Nar significantly promoted the mRNA and protein expression levels of PKM2, IDH1, and SDHB (P < 0.05). However, the effects of Nar treatment were inhibited by the addition of CC or CsA to the MSB-1 cells (P < 0.05, Fig. 4G–4I). Additionally, the detection of ATP content and ATPase activity showed a significant decrease in the LPS group compared to the Control group (P < 0.05) and those were significantly enhanced in the Nar+LPS group (P < 0.05). However, the effects of Nar were suppressed after adding CC or CsA to the MSB-1 cells (P < 0.05, Fig. 4J–4K). These results suggested that Nar alleviated LPS-induced energy metabolism disruption by AMPK/PINK1/Parkin-mediated mitophagy.

Fig. 4.

Fig 4

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Nar alleviated LPS-induced energy metabolism disruption by promoting mitophagy via AMPK. (A-C) Protein and mRNA expression levels of energy metabolism-related factors (PKM2, IDH1 and SDHB) in spleen tissue (n = 6). (D) ATP concentration in spleen tissue (n = 6). (E) ATPase activity in spleen tissue (n = 6). (F) An overview image of treated cells. (G-I) Protein and mRNA expression levels of energy metabolism-related factors (PKM2, IDH1 and SDHB) in MSB-1 cells (n = 3). (J) ATP concentration in MSB-1 cells (n = 3). (K) ATPase activity in MSB-1 cells (n = 3). Results are expressed as means±SD. Bars with different letters are considered to have statistically significant differences (P < 0.05).

Nar attenuated LPS-induced oxidative stress via AMPK/PINK1/Parkin-mediated mitophagy

Impaired mitochondrial disrupts antioxidant defense system, triggering excessive ROS production. DHE staining results showed a significant increase in red fluorescence intensity in the LPS group compared to the Control group, which decreased after Nar treatment (P < 0.05, Fig. 5A). Furthermore, the activities of SOD, CAT, and GSH-Px, along with MDA content, were measured. The results showed that compared with the Control group, the activities of SOD, CAT, and GSH-Px were significantly decreased in the LPS group, while MDA content was significantly increased (P < 0.05). In the Nar+LPS group, the activities of SOD, CAT, and GSH-Px in the spleen tissue were significantly increased, and MDA content was significantly reduced (P < 0.05, Fig. 5B). These findings indicated that Nar treatment reduced LPS-induced oxidative stress. In addition, we detected ROS levels in MSB-1 cells, as well as MDA content and the activities of SOD, CAT, and GSH-Px. The results showed that compared with the Control group, LPS treatment caused a burst of ROS in MSB-1 cells (P < 0.05), with a significant decrease in the activities of SOD, CAT, and GSH-Px (P < 0.05) and a significant increase in MDA content (P < 0.05). The ROS levels and MDA content in the Nar+LPS group were significantly lower than those in the LPS group (P < 0.05), and the activities of SOD, CAT, and GSH-Px were significantly higher (P < 0.05). However, the effects of Nar were reversed after adding inhibitors CC or CsA (P < 0.05, Fig. 5C–5D). Collectively, these results indicated that Nar alleviated oxidative stress through the AMPK/PINK1/Parkin-mediated mitophagy.

Fig. 5.

Fig 5

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Nar attenuated LPS-induced oxidative stress via AMPK pathway regulation of mitophagy. (A) DHE staining (scale bar, 50 µm) in spleen tissue (n = 6). (B) Levels of MDA, GSH-px, SOD, and CAT in spleen tissue (n = 6). (C) Levels of MDA, GSH-px, SOD, and CAT in MSB-1 cells (n = 3). (D) DCFH-DA staining (n = 3, scale bar, 100 µm) in MSB-1 cells. Results are expressed as means±SD. Bars with different letters are considered to have statistically significant (P < 0.05).

Nar alleviated LPS-induced pyroptosis by promoting mitophagy via AMPK

We examined the mRNA and protein expression levels of factors related to pyroptosis in each group. Experimental results indicated that the expression levels of NLRP3, GSDMD, ASC, and Caspase-1 were significantly elevated in the LPS group, and Nar significantly reversed the expression levels of above factors (P < 0.05, Fig. 6A–6C). Pyroptosis can activate caspase-1 via the inflammasome, release pro-inflammatory factors such as IL-1β, and trigger an inflammatory response. Therefore, we detected the mRNA and protein levels of inflammation-related factors in the spleen tissues of each group. The results showed that compared with the LPS group, the mRNA and protein expression levels of IL-1β, IL-18, IL-6, IFN-γ, and COX2 were significantly reduced after treatment with Nar, while the mRNA and protein expression level of IL-10 was significantly increased (P < 0.05, Fig. 6D–6F). These results indicated that Nar alleviated LPS-induced pyroptosis and inflammation in broiler spleens. To investigate the underlying mechanisms, we performed IF detection on the pyroptosis marker factor GSDMD of MSB-1 cells in each group. The results showed that the fluorescence signal intensity in the LPS group was significantly higher than that in the control group, and the fluorescence signal of Nar combined with LPS was weakened, indicating treatment with Nar suppressed the expression level of GSDMD (P < 0.05). However, after the addition of CC and CsA, the effect of Nar was blocked (P < 0.05, Figure. 6G). We also assessed pyroptosis and inflammation-related factors in MSB-1 cells, finding the expression levels of GSDMD, Caspase-1, ASC, and NLRP3 were significantly elevated in the LPS group compared to the Control group (P < 0.05). Nar treatment significantly reduced these factors' expression levels (P < 0.05). In cells treated with CC or CsA, the expression levels of pyroptosis-related factors were reversed compared to the Nar+LPS group, indicating that the protective effect of Nar was inhibited (P < 0.05, Fig. 6H–6J). In addition, the results of detecting the mRNA and protein expression levels of inflammation-related factors revealed that that compared with the LPS group, treatment with Nar significantly reduced the mRNA and protein expression levels of IL-1β, IL-18, IL-6, IFN-γ, and COX2 in MSB-1 cells and increased the expression level of IL-10 (P < 0.05). However, after treating MSB-1 cells with CC and CsA, the effects of Nar were offset (P < 0.05, Fig. 6K–6M). These results suggested that Nar alleviated LPS-induced pyroptosis and inflammation by promoting mitophagy via the AMPK pathway.

Fig. 6.

Fig 6

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Nar alleviated LPS-induced pyroptosis and inflammation by promoting mitophagy via AMPK. (A-C) Protein and mRNA expression levels of pyroptosis-related factors (GSDMD, ASC, Caspase 1 and NLRP3) in spleen tissues (n = 6). (D-F) Protein and mRNA expression levels of inflammation-related factors (IL-10, IL-6, IL-18, IL-1β, COX2 and IFN-γ) in spleen tissue (n = 6). (G) IF results for GSDMD in MSB-1 cells (n = 3, scale bar, 200 µm). (H-J) Protein and mRNA expression levels of pyroptosis-related factors (GSDMD, ASC, Caspase 1 and NLRP3) in MSB-1 cells (n = 3). (K-M) Protein and mRNA expression levels of inflammation-related factors (IL-10, IL-6, IL-18, IL-1β, COX2 and IFN-γ) in MSB-1 cells (n = 3). Results are expressed as means±SD. Bars with different letters are considered to have statistically significant difference (P < 0.05).

Bioinformatics analysis of AMPK, mitophagy, mitochondrial fission/fusion, energy metabolism, pyroptosis and inflammation after Nar treatment

We employed PPI and Metascape to conduct a comprehensive analysis of genes associated with AMPK, mitophagy (PINK1, Parkin, LC3 and P62), mitochondrial dynamics (DRP1, MFN1, MFN2 and Opa1), energy metabolism (SDHB, PKM2 and IDH1), pyroptosis (NLRP3, GSDMD, ASC and Caspase1), and inflammation (IL-10, IL-6, IL-18, IL-1β, COX2 and IFN-γ). This analysis unveiled the intricate correlations among diverse proteins, as depicted in Fig. 7A and 7B. The clustering heatmap presenting the mRNA expression levels of these genes across different treatment groups is illustrated in Fig. 7C. Notably, treatment with Nar led to a significant up-regulation of the mRNA expression levels of AMPK, PINK1, Parkin, LC3, Opa1, MFN1, MFN2, SDHB, PKM2, IDH1 and IL-10. Conversely, the mRNA expression levels of Drp1, P62, NLRP3, GSDMD, ASC, Caspase1, IL-18, IL-1β, IL-6, COX2 and IFN-γ were significantly down-regulated. The variations in the mRNA expression levels of genes within each treatment group were effectively differentiated by color, and genes exhibiting similar trends of change were readily clustered together. A correlation analysis of these factors demonstrated significant inter - relationships among them, as presented in Fig. 7D.

Fig. 7.

Fig 7

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Bioinformatics analysis. (A) The PPI analysis. (B) The Metascape analysis. (C) Clustered heatmap of the expression level changes of AMPK, mitophagy, mitochondrial dynamics dysfunction, energy metabolism disorder, pyroptosis and inflammation. (D) Pearson's correlation analysis.

Discussion

Naringenin (Nar) is a natural flavonoid compound mainly found in citrus fruits, known for its strong anti-inflammatory, antioxidant, and immune-regulating effects that protect the immune system of poultry (Salehi, et al., 2019). AMPK serves as an important regulator of mitochondrial homeostasis, modulating the PINK1/Parkin signaling pathway that affects mitophagy (Cao, et al., 2021). This process is crucial for maintaining the balance of mitochondrial ROS by removing damaged mitochondria, thus suppressing the activation of NLRP3 to alleviate pyroptosis in organisms (Luo et al., 2023). Therefore, this study aimed to investigate the protective effect of Nar on LPS-induced pyroptosis and inflammation in the spleen tissues of broiler chickens, with a focus on effects of mitophagy process. Through the network pharmacological analysis, it was found that PRKAA1, a key catalytic subunit of AMPK, was included in the intersection targets of Nar. Subsequently, through molecular docking analysis and CETSA experiments, we found that Nar had a strong interaction with AMPK. Treatment with Nar promoted the expression levels of factors related to the AMPK signaling pathway, mitophagy, energy metabolism, mitochondrial fusion, and anti-inflammation, as well as the activities of antioxidant enzymes after LPS stimulation. It reduced the expression levels of factors related to mitochondrial fission, mtDNA release, pyroptosis, and inflammation, as well as the content of MDA and ROS level. In addition, Nar treatment significantly elevated ΔΨm, the concentration of ATP, and the activity of ATPase compared to the LPS group.

AMPK is described as a primary sensor of stress in eukaryotic cells (Chen et al., 2024b). It can modulate multiple cellular processes, containing energy metabolism, cell growth, lipid synthesis, and autophagy. Recent research also revealed that it could participate in maintaining mitochondrial homeostasis through the regulation of PINK1-Parkin-mediated mitophagy. In heart failure, fatty liver, osteoarthritis, and diabetic nephropathy, AMPK activates the PINK1/Parkin-dependent mitophagy pathway through the phosphorylation of PINK1. This process restores mitochondrial homeostasis by eliminating dysfunctional mitochondria, thereby protecting the organism form injury (Chen, et al., 2022a). Loss of PINK1 or Parkin reduced mitophagy, impairing the clearance of damaged mitochondria and diminishing protection against endothelial injury (Wu, et al., 2015). Wang et al. discovered that the process of mitophagy inhibition exacerbated acetaminophen-induced liver injury, supporting the protective role of PINK1/Parkin-mediated mitophagy (Wang, et al., 2019). Moreover, enhancing this pathway alleviates hippocampal neuronal injury following cerebral ischemia-reperfusion (Mao, et al., 2022). In this study, our findings revealed that LPS exposure led to an increase in the expression levels of factors associated with mitophagy, resulting in the association of damaged mitochondria with autophagolysosomes and preventing their normal metabolic degradation. Nar, a naturally occurring and versatile compound, exhibits extensive regulatory activities within organisms, including the regulation of mitophagy (Ahsan, et al., 2020). Therefore, we propose that Nar may be a candidate therapeutic agent that contributes to maintaining cellular health by promoting autophagy to remove damaged mitochondria. Our findings indicated that the expression levels of PINK1, Parkin and LC3 were significantly increased, while the expression level of p62 was decreased after treatment with Nar, suggesting that Nar promotes mitophagic flux, allowing p62-bound substrates to be more effectively degraded by autophagolysosomes. and the expression levels of above factors were suppressed after treatment with an AMPK inhibitor, indicating that Nar promoted mitophagy through AMPK.

Mitochondria, as key organelles that respond to stress and metabolic demands, orchestrate a wide range of cellular processes. Safeguarding their integrity is vital for preserving cellular homeostasis (Ahsan, Sharma, Wani and Chopra, 2020). Guo et al. demonstrated that activating PINK1/Parkin-mediated mitophagy maintained mitochondrial homeostasis, alleviating immune suppression in the spleen of mice induced by Aflatoxin B1 (Guo, et al., 2022). Wu et al. demonstrated that hexadecanoic acid promoted the mitophagy to preserve mitochondrial function through the PINK1/Parkin signaling pathway, alleviating metabolic stress-induced endothelial injury (Wu, et al., 2015). Maintaining mitochondrial homeostasis is crucial for preserving the balance of mitochondrial dynamics and ensuring the normal functioning of energy metabolism. Mitochondria exhibited a continuous process of fusion and fission, which referred to as mitochondrial dynamics, involving in regulating morphology of mitochondrial (Huang, et al., 2023). Research demonstrated that alterations in the expression levels of proteins involved in mitochondrial fusion and fission, signified an imbalance in mitochondrial dynamics (Chan, 2020). In addition, ATP is fundamental energy molecule of cells and also serves as a critical energy supplier for all cellular activities. Mitochondria are the hub of cellular energy metabolism, primarily producing ATP through oxidative phosphorylation. Evidence from studies suggested that mitochondrial damage affected the energy metabolism of cells and, consequently, the entire organism (Li, et al., 2024). In this study, our findings revealed that Nar treatment antagonized LPS-induced imbalances in mitochondrial dynamics and disorders of energy metabolism. In addition, the effects of Nar were inhibited following treatment with AMPK or mitophagy inhibitors, suggesting that Nar alleviated mitochondrial dysfunction through AMPK/PINK1/Parkin signaling pathway.

Mitochondria, as the site of ROS production and scavenging, profoundly influence the redox balance of the organism, thereby affecting cellular function and survival. Angelica polysaccharides ameliorated 5-fluorouracil-induced spleen injury through the inhibition of excessive ROS production (Du, et al., 2023). Xi et al. demonstrated that paeoniflorin can maintain mitochondrial homeostasis and suppress oxidative stress by activating mitochondrial autophagy, thereby improving the development of mouse ovaries (Xi, et al., 2024). In our study, we revealed that Nar protected against mitochondrial damage and oxidative stress through AMPK/PINK1/Parkin-mediated mitophagy. Under conditions of oxidative stress, ROS can act as second messengers to trigger pyroptosis (Zhang, et al., 2022). Luteolin inhibited excessive ROS production to prevent pyroptosis in THP-1 cells (Zou, et al., 2021). Zhang et al. demonstrated that Gs-Rb1 promoted mitophagy to eliminate damaged mitochondria, reducing excessive ROS production, thereby mitigating 3-MCPD-induced renal cell pyroptosis (Zhang, et al., 2024). The process of pyroptosis can amplify inflammatory responses by releasing pro-inflammatory cytokines such as IL-1β and IL-18. Bromo-and extraterminal (BET) proteins inhibitor JQ1 protects hippocampal blood-brain barrier and neurons by attenuating neuroinflammation through the inhibition of the canonical inflammasome-dependent pyroptosis pathway induced by LPS in mice (Zhong, et al., 2022). Li et al. elucidated that GRb1 activated mitophagy to reduce ROS and pyroptosis in astrocytes, thereby reducing inflammation, increasing synaptic plasticity, and improving depression-like behaviors in rats (Li, et al., 2023). This study found that Nar treatment alleviated LPS-induced pyroptosis and inflammatory injury by suppressing oxidative stress through the activation of the AMPK pathway to promote mitophagy.

Conclusion

In summary, our findings elucidated the molecular mechanisms of Nar in mitigating LPS-induced pyroptosis and inflammatory injury in broiler spleens. Specifically, Nar promoted mitophagy through its interaction with AMPK, eliminating damaged mitochondria and alleviating mitochondrial dysfunction and oxidative stress, which alleviates cell pyroptosis and inflammation. This research broadened the biological function of Nar and underscored its potential therapeutic applications.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 32202875), the Science Research Project of Hebei Education Department (ZC2025031), and the Research Fund for the Doctoral Program of Hebei North University (BSJJ202504).

Ethics statement

The experiments were approved by the Institutional Animal Care and Use Committee of the Northeast Agricultural University (NEAUEC20230308).

Data availability

Data that support the findings of this study are available on reasonable request.

CRediT authorship contribution statement

Yu Xia: Writing – original draft, Methodology, Formal analysis, Data curation, Conceptualization. Yidan Wang: Investigation, Formal analysis, Conceptualization. Jiahui Xue: Methodology, Investigation, Formal analysis. Jiahong Chu: Methodology, Data curation, Conceptualization. Yanhe Zhang: Methodology, Data curation. Fuze She: Visualization, Software, Investigation. Huijie Chen: Investigation, Formal analysis. Shu Li: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Disclosures

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

Acknowledgement

The authors thank the Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and Treatment, College of Veterinary Medicine, Northeast Agricultural University for providing conditions.

Footnotes

☆

All authors have read the manuscript and have agreed to submit the manuscript in its current form for consideration for publication in this journal.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2025.105810.

Appendix. Supplementary materials

mmc1.docx (5.1MB, docx)

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