Hydroxytyrosol Restrains NLRP3 Inflammasome Activation and Pyroptosis Through the TLR4/NF-κB Pathway to Prevent Mycoplasma gallisepticum-Inflammation

Abstract

Mycoplasma gallisepticum (MG), a significant pathogen, causes chronic respiratory disease (CRD) in poultry, characterized by coughing, wheezing, and dyspnea. This widespread disease severely impedes the healthy development of the poultry industry. Hydroxytyrosol (HT), a plant-derived monomeric compound, possesses diverse bioactivities, including antibacterial, antioxidant, and cardiovascular protective properties. This study aimed to investigate HT's protective effect against MG-induced inflammatory damage in HD11 cells. The findings revealed that MG infection activated the TLR4/NF-κB signaling pathway and the NLRP3 inflammasome, resulting in macrophage accumulation and inflammatory damage in the lung tissue of chicks. HT mitigated MG-induced inflammatory damage in HD11 cells, inhibited the M1 phenotype shift, and reduced the secretion of pro-inflammatory factors. Molecular docking and molecular dynamics simulations demonstrated HT's stable, spontaneous binding to NLRP3. Further investigations showed that HT alleviates oxidative stress-induced cellular damage, diminishes pathological apoptosis and pore formation, and concurrently reduces NLRP3 activation by regulating the TLR4/NF-κB pathway, thereby downregulating the expression of key pyroptosis factors. In summary, HT reduces NLRP3 inflammasome activation by specifically inhibiting the TLR4/NF-κB signaling axis, consequently alleviating inflammation-induced damage, oxidative stress, and pyroptosis in HD11 cells caused by MG infection.

Introduction

Mycoplasma gallisepticum (MG) is a minute prokaryote characterized by the absence of a cell wall, being enclosed solely by a cell membrane (Kreizinger et al., 2017). MG is widespread, with high incidence rates reported in regions such as the United States (Staley et al., 2018), Europe (Michiels et al., 2016; Felice et al., 2020), and Asia (Limsatanun et al., 2022). In China, MG infection was detected in 938 samples, showing an average infection rate of 75.04% (Wei et al., 2023). Furthermore, the disease's incidence exhibits seasonal variations, with fall and winter identified as high-risk periods (Wan et al., 2025). MG infection represents a major chronic respiratory disease threatening intensive poultry farming (Zhao et al., 2025). Affected chickens develop tracheitis, airsacculitis, and pneumonia, accompanied by coughing, wheezing, and dyspnea. These respiratory symptoms lead to feeding difficulties and nutritional deficiencies, resulting in stunted growth and weight loss, manifesting as a chronic, debilitating process (Barbour et al., 2000). MG colonizes the respiratory epithelium via adhesion proteins, impairs ciliary function, and triggers epithelial hyperplasia, leading to the accumulation of mucosal secretions, vascular swelling, and epithelial thickening (Nguyen et al., 2021). Simultaneously, MG stimulates macrophages and other cells to secrete pro-inflammatory factors (Wang et al., 2024), inducing persistent pulmonary inflammation.

Macrophages, the primary effector cells of the innate immune system in poultry (Han et al., 2026), are strategically distributed across various tissues, including the lungs, spleen, and respiratory tract mucosa. These cells are crucial for host defense, orchestrating inflammatory responses, and maintaining homeostasis through functions such as phagocytosis, antigen presentation, and the secretion of inflammatory factors (Peng et al., 2018). Macrophages detect pathogen invasion via pattern recognition receptors (PRRs). Specifically, pathogen-associated lipoproteins activate the TLR2/TLR4 signaling cascade, which triggers NF-κB nuclear translocation and the subsequent transcriptional expression of pro-inflammatory genes. This process can lead to excessive or sustained polarization of macrophages towards the classical M1 phenotype (Sanjukta and K., 2015), resulting in the overproduction of pro-inflammatory cytokines and reactive oxygen species (ROS). Such an imbalance exacerbates inflammation and induces oxidative stress-related tissue damage (Ciraci and Lamont, 2011). Furthermore, excessive stimulation of the TLR4/NF-κB pathway by pathogens also leads to the significant intracellular accumulation of NLRP3. This protein then assembles with ASC and pro-Caspase-1 to form an inflammasome (Balahura Stămat and Dinescu, 2024). This complex cleaves and activates Caspase-1, which in turn promotes the release of the N-terminal region of GSDMD. GSDMD then oligomerizes and forms pores in the cell membrane (Munkong et al., 2025), disrupting its integrity and leading to osmotic lysis. This process releases pro-inflammatory factors such as IL-1β and IL-18 (Shen et al., 2021), thereby inducing pyroptosis in macrophages. Pyroptosis continuously amplifies the inflammatory response and exacerbates pneumonia-related damage (Chen et al., 2024).

The escalating and widespread antibiotic resistance among MG strains presents significant challenges, including the risk of drug residues (Zhang et al., 2026). The current overreliance on antibiotics further exacerbates this resistance problem (Zubair et al., 2025), highlighting an urgent need for innovative control strategies (Kamal et al., 2025). Consequently, exploring novel antimicrobial compounds derived from natural products has emerged as a key research focus for the prevention and control of MG infections. Hydroxytyrosol (HT), a polyphenolic compound formed from the hydrolysis of oleuropein in olive products, exhibits a broad spectrum of biological activities, including antioxidant (Zhao et al., 2021), anti-inflammatory (Cecci et al., 2026), and neuroprotective effects (Tantipongpiradet et al., 2026). Research by Montoya et al. demonstrated that HT reduced NO and pro-inflammatory cytokine levels, alongside COX-2 and iNOS expression, in lipopolysaccharide (LPS)-stimulated mouse macrophages (Montoya et al., 2018). Similarly, Yu et al. showed that HT promoted the polarization of LPS-stimulated macrophages from the M1 to the M2 phenotype and reduced pro-inflammatory factor levels by inhibiting the ERK pathway (Yu et al., 2020).

However, it remains unclear whether HT exerts an anti-MG effect and mitigates inflammatory damage during MG infection. This study employs various experimental techniques, with a specific focus on the TLR4/NF-κB pathway, to investigate whether the natural compound HT regulates NLRP3 inflammasome-mediated pyroptosis in HD11 cells. The aim is to determine if HT can alleviate inflammatory damage to pulmonary macrophages caused by MG infection, thereby offering new insights for the development of natural targeted drugs to treat Mycoplasma pneumonia.

Materials and methods

Experiment 1

Fifteen healthy 7-day-old SPF male chicks were acclimatized for 7 days and then randomly divided into 3 equal groups: Control, MG and TAK-242+MG. An MG infection model was established in the MG and TAK-242+MG groups by air-sac inoculation (MG F strain at a concentration of 1×10⁹ CCU/mL) at a dose of 0.2 mL per bird. The Control group received intranasal and ocular instillation of saline once daily for 21 consecutive days. Starting on day 7 post-infection, 2 h after intranasal and ocular administration of MG, chicks in the TAK-242+MG group (3 mg/kg, TLR4 inhibitor, HY-11109, MedChemExpress, USA) received subcutaneous injections of TAK-242, while chicks in the Control and MG groups received subcutaneous injections of a comparable volume of saline solution, once daily for 14 consecutive days. 12 h after the final infection, chick serum and lung tissue was harvested for subsequent experiments.

Experiment 2

HD11 chicken macrophages (YS2279C, Yaji Biotechnology Co., Ltd., Shanghai, China) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (KS01, Biocomma, Shenzhen, China) in a humidified incubator at 37°C and 5% CO₂. The HD11 cells were divided into 3 groups: Control, MG, and TAK-242+MG, with 3 replicates in each group. After pretreating the TAK-242+MG group with 10 μM TAK-242 for 24 h, both the MG and TAK-242+MG groups were incubated with a MG bacterial suspension at a MOI of 400 for 12 h. MOI = (MG bacterial suspension concentration × MG bacterial suspension volume) / number of HD11 cells. The cell supernatants and pellets were then collected for subsequent experiments.

Experiment 3

HD11 cells were divided into 5 groups: Control, MG, HT+MG, MCC950+MG, and HT, with 3 replicates per group. The HT+MG and HT groups were treated with 100 μM HT (H811041, aladdin Biochemical Technology Co., Ltd., Shanghai, China), while the MCC950+MG group was treated with 10 μM MCC950 (NLRP3 inhibitor, HY-12815A, MedChemExpress, USA). After 24 h of pretreatment, the MG, HT+MG, and MCC950+MG groups were incubated with a MG bacterial suspension at a MOI of 400 for 12 h. The cell supernatants and precipitates were then collected for subsequent experiments.

H&E

Chicken lung tissues were fixed with 4% paraformaldehyde, processed into paraffin blocks, and then sectioned at a thickness of 6 μm. Staining was performed according to the instructions for H&E staining (BL700A, Biosharp, Anhui, China). HD11 cells were cultured in 12-well plates (1 × 10⁵ cells per well), fixed and stained according to the H&E staining protocol.

TEM

Chicken lung tissues were fixed with 2.5% glutaraldehyde, the samples were sent to Lilai Biotechnology Co., Ltd., (Chengdu, China) for the preparation of ultrathin sections, and images were acquired and analyzed using a transmission electron microscope.

Immunohistochemistry

Frozen sections of chicken lungs were prepared from each group and processed according to the instructions for the UltraSensitive™ SP Immunohistochemistry Kit (SP9710, Maxin Biotechnology Co., Ltd., Fuzhou, China). The sections were incubated overnight with the macrophage marker KUL01 antibody (1:100, 8420-01, Southern Biotechnology Associates, Inc., USA) and the NLRP3 antibody (1:500, WL02635, Wanleibio Co., Ltd., Shenyang, China) for overnight incubation, followed by secondary antibody incubation.

qPCR

Total RNA was extracted from the lungs and HD11 cells of each group using the Trizol method. After reverse transcribing the RNA into cDNA according to the instructions for the All-In-One 5X RT MasterMix with gDNA Removal (G592, Applied Biological Materials Inc., Vancouver, Canada) reverse transcription kit, amplification was performed using the TB Green® Premix Ex Taq™ II kit (RR820Q, Takara Bio, Dalian, China) in a 20 μL reaction system, with β-actin serving as the endogenous control. The relative expression levels of TLR4, NF-κB, NLRP3, Caspase-1, IL-1β, IL-6, MG pvpA, IL-18, iNOS, Bax, Bcl-2, Caspase-3, Caspase-8, ASC, and GSDMD mRNA using the 2^−ΔΔCt method. The primers were synthesized by Sangon Biotechnology (Shanghai, China). Their sequences are shown in Table 1.

Table 1.

Specific primers of target genes.

Gene	ID	Primer (5’ -3’)	Fragment length(bp)
TLR4	NC_052548.1	F-CCATCCCAACCCAACCAC	122
		R-ACCCACTGAGCAGCACCA
NF-κB	NM_001396395.1	F-TCACGCCGCTGACATGG	130
		R-ATACGGATCCTCTCCTGCCA
NLRP3	NM_001348947.2	F-AGCTCACTCCAGAGCCTCA	139
		R-TCACACCATCATCACCCAGC
Caspase-1	XM_015295935.4	F-TTTCCCCACCCCTGAAGTGA	140
		R-GTCCACAGGGATCCACCCTT
IL-1β	NM_204524.2	F-CTGCCTGCAGAAGAAGCCT	135
		R-CGCAGCAGTTTGGTCATGG
IL-6	NM_204628.2	F-GAGGTTGGGCTGGAGGAGGAG	130
		R-TCTCGCACACGGTGAACTTCTTG
MG pvpA	NC_018406.1	F-CTAGCAATCAAGCTGCAGGTACT	149
		R-TGGTCCGTGTTGTGGATGAC
IL-18	NM_204608.3	F-GATGATGAGCTGGAATGCGATG	150
		R-ACTGCCAGATTTCACCTCCTG
iNOS	NM_204961.2	F-TGGGTGGAAGCCGAAATA	241
		R-GTACCAGCCGTTGAAAGGAC
Bax	XM_040693909.2	F-GTACGTCAATGTGGTCACCC	211
		R-TGGGATAATGCTGGGGTTGA
Bcl-2	XM_046910476.1	F-ACCATGAATGAAACCGTGCC	181
		R-TTGTCGTAGCCTCTTCTCCC
Caspase-3	XM_046915477.1	F-ATTGAAGCAGACAGTGGACCAGATG	111
		R-TGCGTTCCTCCAGGAGTAGTAGC
Caspase-8	NM_204592.4	F-CCATTTGGCTGGCATCAT	149
		R-CACTGCTTCCCTGGCTTT
ASC	NM_001040467.1	F-ACGGGTGACTCGGAGTTCTA	141
		R-GTACTGCTCTGGGGTGCAAT
GSDMD	NM_001006361.2	F-TCACCTCCGCTGTCTGTTTC	139
		R-GCCTTCACAGAGGCGAGTAA
β-action	NM_205518.2	F-CGAACGCCCCCAAAGTTCTA	125
		R-GCCTTCACAGAGGCGAGTAA

CCK-8

When the cells (5 × 10⁴ cells per well) have grown to approximately 80% confluence, treat HD11 cells with MG bacterial suspensions at MOIs of 0, 50, 100, 200, 400, 800, and 1000 for 2, 4, 8, 12, and 24 h; treat HD11 cells with HT solutions at concentrations of 0, 10, 25, 50, 100, and 200 μM HT solutions were applied to HD11 cells for 6, 12, 24, and 48 h, respectively. CCK-8 (SB-CCK8, Share Bio Co., Ltd, Shanghai, China) was added, after which OD₄₅₀ values were measured using a microplate reader. The calculation formulas are as follows: cell viability = (OD of experimental well – OD of blank well) / (OD of control well – OD of blank well) × 100%.

Molecular docking and molecular dynamics modeling

Download the 3D structure of HT from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Using the NLRP3 structure from Swiss-Model (https://swissmodel.expasy.org/), 20 docking simulations were performed using Autodock Vina. The HT-TLR4 and HT-NLRP3 complexes with the lowest binding energies were selected, and the results were visualized using PyMOL 4.0 software.

To further assess the stability of the association between HT and NLRP3, the HT-NLRP3 complex with the lowest binding energy was solvated using the Amber99sb-ildn force field and the charged water (SPC) model, placed in a 1.5 nm³ cubic simulation box. Following energy minimization and a 100 ps NVT/NPT equilibration, a 100 ns molecular dynamics simulation was performed at ambient temperature and pressure using GROMACS 2025.2. The trajectory files were analyzed for root-mean-square deviation (RMSD), gyration radius (Rg), residual mean square fluctuation (RMSF), and solvent-accessible surface area (SASA). To further assess the conformational stability of the complex, two-dimensional (2D) and three-dimensional (3D) free energy landscapes (FELs) were constructed based on the molecular dynamics trajectories, and the binding free energy (ΔGbind) was determined using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method.

Immunofluorescence

After fixation (5 × 10⁴ cells per well), perforation and blocking, add diluted NF-κB (1:500, 80979-1-RR, Proteintech Group Inc., Wuhan, China), NLRP3 (1:300, WL02635, Wanleibio Co., Ltd.), and Caspase-1 (1:100, 84752-1-RR, Proteintech Group Inc.) antibodies. Follow the instructions in the FlexAble 2.0 Antibody Labeling Kit for Rabbit IgG (KFA501, KFA509, Proteintech Group Inc.). After incubation in the dark, DAPI (G1012, Servicebio Technology Co., Ltd., Wuhan, China) was added. Images were acquired using a fluorescence microscope (Olympus CX43,Tokyo,Japan) andlaser scanning confocal microscope (Leica TCS SP8, Wetzlar, Germany), and analyzed using Image ProPlus 6.0 software.

NO detection

Follow the instructions for the nitric oxide assay kit (S0021S, Beyotime Biotechnology Co., Ltd., Shanghai, China), measure the absorbance (5 × 10⁴ cells per well) at OD₅₄₀ using a microplate reader (K3 TOUCH, Thermo Fisher Scientific, Massachusetts, USA), and calculate the NO concentration in the cell supernatant for each group.

ELISA

Following the manufacturer's instructions for the ELISA kits, the concentrations of TNF-α, IL-6, and IL-1β were measured in the cell culture supernatant (2 × 10⁵ cells per well) using the corresponding kits (JYM0033Ch, JYM0028Ch, JYM0041Ch, Genome Biotech Co. Ltd., Wuhan, China), with absorbance read at OD₄₅₀.

DCFH-DA

After preparing the cell smears, follow the instructions in the DCFH-DA manual (G1706, Servicebio) and incubate the cells with DAPI.

Oxidative stress biomarker detection

Following the kit instructions, we measured the total antioxidant capacity—including Glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), Malondialdehyde (MDA), Total antioxidant capacity (T-AOC), Catalase (CAT), and Hydrogen peroxide (H₂O₂) in 2 × 10⁵ cells per well (A005-1-1, A001-1-1, A003-4-1, A015-1-1, A007-1-1, A064-1-1, Jiancheng Biotechnology Research Institute Co., Ltd., Nanjing, China).

JC-1

Cells were seeded (5 × 10⁴ cells per well). After treating the experimental groups, cell smears were prepared; the JC-1 fluorescent probe (C2006, Beyotime Biotechnology) was added and incubated.

Western blot

Cells from each group were collected and treated with a high-efficiency lysis buffer. Proteins were quantified utilizing the BCA Assay kit. The antibodies used included Bax (1:20,000, 50599-2lg, Proteintech Group Inc), Bcl-2 (1:2,000, 12789-1-AP, Proteintech Group Inc), TLR4 (1:2,000, WL00, Wanleibio Co., Ltd.), NF-κB p65 (1:1,000, 80979-1-RR, Proteintech Group Inc), phosphorylated NF-κB p65 (1:2,000, 82335-1-RR, Proteintech Group Inc.), Caspase-1 (1:1,000, 84752-1-RR, Proteintech Group Inc), GSDMD (1:5,000, 20770-1-AP, Proteintech Group Inc.), and GAPDH (1:5,000, AC001, ABclonal Technology Co., Ltd., Wuhan, China).

Hoechst 33342/PI

Cells were seeded (5 × 10⁴ cells per well) cells per well. After grouping the experimental treatments, Hoechst 33342 (C0030, Solarbio Science Technology Co., Ltd., Beijing, China) and PI staining solution (G1021, Servicebio) were added to the respective groups. Images were captured using a fluorescence microscope, and the PI-positive rate was calculated (PI-positive rate = number of red-fluorescent cells / total number of cells × 100%).

Data Analysis

Data were analyzed using SPSS 27.0. Results are presented as mean ± SD (x̄ ± s). Comparisons between groups were performed using Student’s t-test. A P > 0.05 was considered not statistically significant, a P < 0.05 indicated statistical significance, and a P < 0.01 indicated high statistical significance.

Results

MG infection activated the TLR4/NF-κB pathway, which subsequently triggered NLRP3 activation, leading to lung damage in chicks

Chicks were inoculated with a MG bacterial suspension via the air sac to establish a model of MG infection (Fig. 1A). Histopathological examination using H&E staining revealed that the Control group exhibited well-formed alveoli with normal alveolar wall structure. Conversely, the MG group displayed widespread inflammatory cell infiltration, thickened alveolar walls, and abnormal alveolar morphology and structure. However, following intervention with TAK-242, these pathological lesions showed improvement (Fig. 1B). Further observation of lung ultrastructure via TEM revealed that cells in the Control group exhibited regular morphology, intact cell membranes, and normal organelles. In contrast, the MG group displayed cellular swelling, uneven chromatin distribution in the nuclei, irregularly shaped nucleoli, swollen mitochondria with fractured cristae, dilated rough endoplasmic reticulum cisternae, and ribosomal detachment, among other ultrastructural pathological phenomena; a small number of MG cells were also observed. Following treatment with TAK-242, these pathological changes were alleviated (Fig. 1C).

Fig. 1.

The effects of TAK-242 on MG-infected chicks. (A) A schematic diagram showing the experimental groupings for chicks. (B) Representative H&E staining images of lungs. (C) Representative electron micrographs of lungs (Nu: cell nucleus, Mi: mitochondria, MG: Mycoplasma gallisepticum). (D) IHC detection of KUL01. (E) IOD values for KUL01. (F-I) The relative mRNA expression levels of TLR4, NF-κB, NLRP3, and IL-1β in lungs. (J) IHC examination of NLRP3. (K) IOD values for NLRP3.

Macrophages are closely associated with inflammation. IHC assessment of the pulmonary macrophage marker KUL01 revealed an increased number of macrophages in the MG group (P < 0.01). After administration of TAK-242, macrophage aggregation was relatively reduced (Fig. 1D). Further analysis using qPCR to detect the relative transcriptional levels of TLR4, NF-κB, NLRP3, and IL-1β mRNA in the lungs revealed that the relative transcriptional levels of these factors were significantly upregulated in the MG group (P < 0.01). Pretreatment with TAK-242 significantly downregulated the mRNA transcriptional levels of these genes (P < 0.01) (Fig. 1E-I). Subsequent IHC analysis of NLRP3 activation in the lungs revealed increased NLRP3 protein expression in the MG group (P < 0.01), while treatment with TAK-242 reduced NLRP3 protein levels (Fig. 1J–K). These findings suggest that MG infection triggers activation of the TLR4/NF-κB pathway, which in turn activates the NLRP3 inflammasome, leading to pathological damage in the lungs of chicks.

MG upregulated the TLR4/NF-κB/NLRP3 pathway in HD11 cells

To assess the effect of MG on HD11 cell viability, an HD11 cell infection model was conducted using MG at a multiplicity of 400 (MOI), and 10 μM/mL of a TLR4 inhibitor was added as an intervention (Fig. 2A). H&E staining was performed to explore cellular damage; the Control and HT groups exhibited normal cell morphology with no signs of cell swelling. It was observed that cells in the MG group were significantly swollen, particularly the nuclei and pale, unevenly stained cytoplasm (Fig. 2B). Further qPCR analysis revealed a significant increase in the relative transcriptional levels of TLR4, NF-κB, NLRP3, IL-1β, and IL-6 mRNA in the MG group (P < 0.01) (Fig. 2C). These findings indicate that MG infection of HD11 cells activates the TLR4/NF-κB/NLRP3 pathway, leading to inflammatory cell damage.

Fig. 2.

The impact of MG on the TLR4/NF-κB pathway and NLRP3 activation in HD11 cells. (A) CCK8 screening to determine the MG infection concentration. (B) H&E staining of cells. (C) Relative mRNA expression levels of TLR4, NF-κB, NLRP3, IL-1β, and IL-6.

Molecular docking and molecular dynamics Simulations of the HT-NLRP3 complex stability

Our molecular docking analysis revealed a strong binding affinity between HT and NLRP3, with the HT-NLRP3 complex exhibiting a binding energy of -24.6856 kJ/mol (Fig. 3A). To further assess the stability of this complex, we performed 100 ns MD simulations. Despite fluctuations in RMSD values between 0.30 and 0.50 nm (3-5 Å), the overall structure of the complex remained stable throughout the simulation (Fig. 3B-D). The radius of gyration (Rg) also remained relatively stable, fluctuating within a narrow range of less than 0.1-0.2 nm, indicating that HT binding did not induce significant conformational changes in NLRP3 (Fig. 3E). Furthermore, the RMSF values for most residues were below 0.15-0.20 nm (1.5-2.0 Å) (Fig. 3F). The solvent-accessible surface area (SASA) values also remained generally stable, fluctuating between 330 and 380 Å, with only minor variations and no significant changes observed throughout the simulation (Fig. 3G). Free energy landscape (FEL) analysis further supported the stability of the complex, revealing a distinct and concentrated low-free-energy valley, indicative of a deep-valley structure and high conformational clustering (Fig. 3H-I). Collectively, these findings suggest that HT maintains a stable conformational state with NLRP3 throughout the 100 ns simulation.

Fig. 3.

MD dynamics Simulation of the HT-NLRP3 Interaction. (A) Schematic diagram showing the HT-NLRP3 protein docking and local magnification. (B) RMSD of NLRP3. (C) RMSD of HT. (D) XVG comparison. (E) Radius of gyration. (F) RMS fluctuation. (G) Solvent-accessible surface. (H) 3D Gibbs energy landscape. (I) 2D Gibbs energy landscape.

HT mitigated inflammatory damage induced by MG infection in HD11 cells

An HD11 cell infection model was established using a bacterial suspension of MG at a concentration of 400 MOI. Intervention involved adding either 10 μM/mL of the NLRP3 inhibitor MCC950 or 100 μM/mL of HT (Fig. 4A-B). The successful establishment of this cellular infection model was analyzed using qPCR and H&E staining. Results showed that neither the Control group nor the HT group expressed the MG-specific gene pvpA, and cell morphology remained normal, characterized by spindle-shaped cells without signs of cellular swelling. In contrast, the MG group displayed significant pvpA expression, accompanied by cellular swelling and cells that were oval or round with pale cytoplasm. The severity of these cytopathological changes was notably reduced following pretreatment with HT or MCC950, thereby verifying the model's successful establishment (Fig. 4C-D). IF analysis of NF-κB protein expression in the cells revealed a significant increase in the MG group compared to the Control group (P < 0.01). Pretreatment with HT and MCC950 significantly downregulated NF-κB protein expression (P < 0.01) (Fig. 4E-F). MG infection is known to cause the accumulation of pulmonary macrophages, leading to the release of pro-inflammatory cytokines and an amplification of the inflammatory response. Further analysis focused on the M1 phenotype polarization of HD11 cells induced by MG infection. The results indicated that in the MG group, the levels of NO, TNF-α, IL-1β, and IL-6, as well as the relative mRNA transcription levels of iNOS, IL-1β, IL-6 and IL-18, were significantly increased (P < 0.01). Pretreatment with either MCC950 or HT significantly reduced the levels of these factors and their mRNA transcription levels (P < 0.01) (Fig. 4G-H). These findings suggest that pretreatment with HT can inhibit the M1-type polarization of MG-infected HD11 cells, reduce the expression of NF-κB and pro-inflammatory factors, and alleviate the extent of macrophage damage.

Fig. 4.

The impact of HT on inflammatory damage in MG-infected HD11 cells. (A) CCK8 screening to determine the maximum concentration of HT. (B) Schematic diagram showing the cell experiment groupings. (C) Relative transcriptional levels of pvpA. (D) H&E staining. (E) NF-κB protein expression. (F) Mean fluorescence intensity of NF-κB. (G) Measurements of NO, TNF-α, IL-1β and IL-6 in the cell culture supernatant. (H) Relative transcriptional levels of iNOS, IL-1β, IL-6 and IL-18 mRNA.

HT reduced cell apoptosis

JC-1 staining results demonstrated that MG infection led to weakened red fluorescence and enhanced green fluorescence (P < 0.01). This indicates a reduction in mitochondrial membrane potential in HD11 cells, thereby inducing early apoptosis. Both MCC950 and HT pretreatment significantly reversed these fluorescence changes and inhibited MG-induced early apoptosis (P < 0.01) (Fig. 5A-B). Western blotting and qPCR were employed to detect the expression of apoptosis-related factors. The analysis revealed that pretreatment with MCC950 and HT significantly reduced MG-induced Bax protein expression and Caspase-3 and Caspase-8 mRNA transcription levels in HD11 cells, while Bcl-2 protein expression and mRNA transcription levels increased significantly (P < 0.01) (Fig. 5C-I). These results suggest that HT can reduce the pathological apoptosis in HD11 cells caused by MG infection.

Fig. 5.

The effects of HT on apoptosis in HD11 cells. (A-B) Representative JC-1 staining images and red-to-green fluorescence ratio. (C-E) Bax and Bcl-2 protein expression levels. (F-I) Relative mRNA expression levels of Bax, Bcl-2, Caspase-3, and Caspase-8.

HT alleviated MG-induced oxidative stress damage in HD11 cells

To investigate the effects of HT on markers of oxidative stress in HD11 cells infected with MG, ROS activity was initially measured using the DCFH-DA probe method. The results demonstrated that MG infection led to excessive ROS production (P < 0.01). Notably, both the MCC950+MG and HT+MG groups successfully restored ROS levels to normal (P < 0.01) (Fig. 6A–B), indicating that HT exhibits an intervention effect comparable to that of the NLRP3 inhibitor. Further investigation into HT's impact on endogenous antioxidant enzymes revealed a significant reduction in the activities of GSH-PX, SOD, T-AOC, and CAT in the MG group (P < 0.01). Concurrently, MDA and H₂O₂ levels were significantly elevated (P < 0.01). Following treatment with MCC950 and HT, all these indicators improved significantly (P < 0.01) (Fig. 6C-H). Given that oxidative stress is a key factor in the activation of the NLRP3 inflammasome during pathogen infection. IF analysis of NLRP3 protein expression was conducted. This analysis showed elevated NLRP3 protein expression in the MG group (P < 0.01), which was subsequently reduced in the MCC950 and HT groups (P < 0.01) (Fig. 6I-J). These findings collectively suggest that HT exerts an antioxidant effect and reduces NLRP3 protein expression.

Fig. 6.

Effects of HT on MG-induced oxidative stress in HD11 cells. (A) IF analysis of intracellular ROS activity. (B) The mean fluorescence intensity of ROS. (C-H) Results of GSH-PX, SOD, MDA, T-AOC, CAT, and H₂O₂ measurements in the supernatant. (I) NLRP3 protein expression. (J) Mean fluorescence intensity of NLRP3.

HT downregulated the expression of a key factor in the NLRP3 pathway, thereby alleviating pyroptosis

The NLRP3 inflammasome is known to activate the Caspase-1 precursor. Activated Caspase-1, in turn, promotes the release of downstream substrates and pro-inflammatory factors, thereby triggering the classic process of pyroptosis. Confocal microscopy was employed to examine the colocalization of Caspase-1 and NLRP3 in HD11 cells. The results indicated that, compared with the MG group, pretreatment with HT and MCC950 significantly downregulated the expression of both Caspase-1 and NLRP3 proteins (P < 0.01) (Fig. 7A-B). A core feature of pyroptosis is a significant change in cell membrane permeability and the formation of membrane pores. To observe these changes in HD11 cells, Hoechst 33342/PI double staining was used. The results showed a significantly elevated PI-positive rate in the MG group compared to the Control group (P < 0.01), indicating that MG can lead to increased cell membrane permeability. In contrast, the PI-positive rate was significantly reduced in both the HT+MG and MCC950+MG groups (P < 0.01) (Fig. 7C–D). Western blotting and qPCR were used to detect changes in pyroptosis-related downstream factors. The results demonstrated that pretreatment with MCC950 and HT significantly reduced the protein expression of TLR4, NF-κB, NLRP3, Caspase-1, and GSDMD in HD11 cells (P < 0.01) (Fig. 7E-J). Furthermore, the relative transcriptional levels of ASC mRNA were significantly reduced (P < 0.01) (Fig. 7K). These results collectively suggest that HT reduces MG-induced pyroptosis in HD11 cells by downregulating the expression of key factors in the NLRP3 pyroptosis pathway, thereby mitigating membrane pore formation.

Fig. 7.

Effect of HT on NLRP3-mediated pyroptosis in HD11 cells. (A) Representative fluorescent colocalization images of Caspase-1 (red) and NLRP3 (green). (B) Fluorescent colocalization analysis of Caspase-1 and NLRP3. (C) Hoechst 33342 (blue)/PI (red) dual fluorescence staining. (D) PI-positive rate. (E-J) Protein expression levels of TLR4, p-p65, NLRP3, Caspase-1, and GSDMD. (K) The relative mRNA expression levels of TLR4, NF-κB, NLRP3, Caspase-1, ASC, and GSDMD.

Discussion

In recent years, the poultry industry has increasingly embraced intensive production methods and ecological sustainability. MG stands out as a primary pathogen responsible for chronic respiratory disease in commercial poultry farming. MG adheres to the mucous membranes of the chicken trachea, compromising the protective barrier and subsequently entering the lungs and terminal bronchioles before migrating to the lung tissue. While pro-inflammatory factors are crucial for pathogen clearance, they also initiate an inflammatory response characterized by vasodilation and increased blood flow. This process recruits immune cells, such as monocytes and macrophages, to the infection site, accompanied by tissue fluid exudation, ultimately inducing inflammation. The TLR4/NF-κB pathway is pivotal in recognizing pathogen invasion and initiating both inflammatory and immune responses. Specifically, TLR4 identifies foreign pathogens, and its downstream factor, NF-κB, undergoes phosphorylation and translocates to the cell nucleus. There, it contributes to the activation of the NLRP3 inflammasome and the expression of various inflammatory factors, including IL-1β, IL-6, and TNF-α. However, excessive activation of this pathway can lead to chronic inflammation (Qiao et al., 2025). Our research team previously observed that during MG infection in chick lungs, ultrastructural pathological changes occur, such as thickening of alveolar walls, proliferation of connective tissue, inflammatory cell infiltration, an increase in macrophage numbers, and mitochondrial swelling. Furthermore, MG infection severely damaged HD11 cells, upregulating the expression of factors associated with the TLR4/NF-κB pathway and leading to over-activation of the NLRP3 inflammasome. These findings suggest that MG infection activates the NLRP3 inflammasome via the TLR4/NF-κB signaling pathway, thereby driving an inflammatory response that damages both lung tissue and macrophages.

Macrophages are essential immune cells that perform critical functions such as phagocytosis, pathogen clearance, antigen presentation, adaptive immunity activation, inflammation regulation, cytokine secretion, tissue homeostasis, and repair. Their phenotype and function can be significantly altered by pathogenic factors like lipopolysaccharide and interferon-γ (Wang et al., 2024). Specifically, macrophages can differentiate into the pro-inflammatory M1 phenotype, leading to increased secretion of iNOS, TNF-α, IL-6, and IL-1β, which in turn amplifies the inflammatory response (Chen et al., 2024). NF-κB is a central transcriptional regulator for M1 polarization and a key player in inflammation (Xiang et al., 2024). The release of pro-inflammatory factors by M1 macrophages further activates NF-κB signaling in themselves and surrounding macrophages, mediating the body's inflammatory response (Ren et al., 2024). This study revealed that MG infection activates NF-κB, promotes M1 polarization of macrophages, increases inflammatory factor expression, and accelerates inflammatory damage progression in HD11 cells. Previous research by Fan et al. demonstrated that HT mitigates septicemic lung injury by inhibiting hepatocyte autophagy, thereby preventing inflammation and oxidative stress (Fan et al., 2024). Additionally, HT exhibits anti-inflammatory effects by suppressing pro-inflammatory cytokine release and macrophage infiltration in fungal-induced keratitis in mice via the Nrf2/HO-1 pathway, thus protecting corneal tissue (Gu et al., 2025). Consistent with these findings, this study found that HT pretreatment reduced NF-κB expression in HD11 cells, inhibited their transition to the M1 phenotype, and decreased the release of TNF-α, IL-1β, IL-6, and IL-18, thereby alleviating MG-induced pathological damage in HD11 cells.

HT is a primary polyphenolic component in olive oil (Rubio-Senent et al., 2026) and a potent natural antioxidant (Liu et al., 2025). Its hydroxyl group forms stable hydrogen bonds with phenoxy groups, effectively scavenging exogenous and endogenous free radicals and oxidants (Prevete et al., 2025). NLRP3, as a core effector platform of the innate immune system (Shen and Zhang, 2025) and a crucial recognition receptor capable of detecting danger signals and activating the body's immune response (Willingham et al., 2007). Its abnormal activation is closely linked to the pathological progression of various infectious diseases (Li et al., 2025). MD simulations indicate that HT exhibits a binding affinity for NLRP3, maintaining a stable overall conformation over 100 ns with a thermodynamically favorable conformation. This study demonstrated that MG infection leads to elevated ROS levels, decreased antioxidant enzyme activity, and abnormal accumulation of H₂O₂ in HD11 cells, disrupting redox homeostasis (Ding et al., 2021). Under oxidative stress, macrophages experience disrupted redox reactions and metabolic homeostasis, resulting in elevated ROS levels (Yuan et al., 2025). Excessive ROS production is a critical upstream signal for NLRP3 activation, promoting NLRP3 assembly, upregulating Bax, and downregulating Bcl-2 expression, thereby inducing apoptosis (Li et al., 2025). Concurrently, the inflammatory cascade downstream of NLRP3 can further exacerbate oxidative damage (Huang et al., 2025), creating a bidirectional regulatory loop between oxidative stress and the NLRP3 inflammasome (Zhang et al., 2026). Chen et al. found that HT, when administered to rat dermal papilla cells treated with H₂O₂, reduced ROS levels, apoptotic markers, and oxidative stress-induced inflammatory responses (Chen et al., 2019). Research by Fang et al. showed that HT reduces malondialdehyde levels and increases catalase and glutathione levels, thereby mitigating oxidative damage and lowering inflammatory factor expression in alcohol-induced fatty liver in mice (Fang et al., 2023). This study revealed that HT increases GSH-PX, SOD, CAT, antioxidant enzyme activity, and T-AOC, while reducing MDA, and H₂O₂ levels, and decreasing NLRP3 protein expression. These findings indicate that HT can reduce MG-induced oxidative stress damage, lower NLRP3 expression, and decrease the occurrence of pathological apoptosis.

Pyroptosis is a lytic and highly inflammatory form of regulated cell death (RCD) characterized by gasdermin-mediated plasma membrane pore formation (Fu et al., 2025). Upon activation, the NLRP3 inflammasome (NLRP3/ASC/pro-Caspase-1 complex) cleaves Caspase-1, and activated Caspase-1 subsequently cleaves GSDMD and pro-inflammatory factor precursors (Ruan et al., 2024). The N-terminal fragment of GSDMD (GSDMD-NT) oligomerizes on the plasma membrane to form nonselective pores, which upregulates cell membrane permeability and facilitates the release of IL-1β and IL-18 into the extracellular space, thereby enhancing intercellular inflammatory signaling and exacerbating the inflammatory response (Li et al., 2025). During pyroptosis in macrophages, GSDMD-NT oligomerizes on the plasma membrane to form pores, increasing cell permeability and enhancing elevated ion gradients and osmotic pressure, ultimately leading to cell swelling and lysis. The release of pro-inflammatory factors as damage-associated molecular patterns activates neighboring macrophages, amplifying the inflammatory cascade (Shan et al., 2023). This study found that the NLRP3 inflammasome and GSDMD-driven pyroptosis play key roles in MG-induced damage to HD11 cells. HT inhibits the expression of key pro-inflammatory factors in the TLR4/NF-κB pathway and inhibits the expression of key factors in the pyroptosis pathway following NLRP3 inflammasome assembly. It may exert a dual effect by modulating upstream TLR4 signaling and targeting downstream NLRP3, functionally confirming that HT downregulates the NLRP3-dependent pyroptosis pathway. Therefore, targeted regulation of the TLR4/NF-κB signaling axis to inhibit NLRP3 inflammasome activation and thereby alleviate pyroptosis holds promise as an effective strategy in anti-MG infection and anti-inflammatory therapy.

Conclusion

This study elucidates the protective mechanism of HT against lung injury caused by MG infection. MG infection activates the TLR4/NF-κB pathway in chicks lungs, which in turn activates the NLRP3 inflammasome, leading to inflammatory damage, macrophage accumulation, and ultrastructural changes. HT reduces M1 differentiation and the release of pro-inflammatory factors, and decreases pathological apoptosis. By alleviating MG-induced oxidative stress damage and downregulating the TLR4/NF-κB pathway, HT regulates NLRP3 inflammasome assembly and GSDMD-mediated pyroptosis. This establishes a multi-step regulatory protective mechanism (Fig. 8), offering a theoretical and experimental foundation for using HT to treat MG infection and TLR4/NF-κB-driven pyroptosis.

Fig. 8.

A schematic diagram showing MG infection of HD11 cells. MG infects HD11 cells, activates the TLR4/NF-κB signaling pathway, and enhances the activation of the NLRP3 inflammasome and pyroptosis.

Authorship contributions

Shunxing Liu designed the study, carried out the experiments, analyzed the data, performed the data visualization, and wrote the initial draft of the manuscript. Yanling Xiong carried out the experiments, confirmed the results, and compiled and organized the data. Zhentao Cheng conceived and designed the study, provided research resources, and contributed to funding acquisition. Anchun Cheng conceived and designed the study, provided research resources, and contributed to funding acquisition. Rutao Lin provided scientific guidance on the experimental design and the interpretation of the findings. Hui Li provided scientific guidance on the experimental design and the interpretation of the findings.Yao Huang provided technical support for the methods and contributed equally to data curation, investigation, and visualization.Binlan Zhou provided technical support for the methods and contributed equally to data curation, investigation, and visualization.Ting Yang provided technical support for the methods and contributed equally to data curation, investigation, and visualization.Chunlan Shan, the corresponding author, designed and supervised the study, coordinated the research project, obtained financial support, provided research resources, and revised and refined the manuscript.All authors critically reviewed the manuscript for important intellectual content, read and approved the final version of the manuscript before submission, and agree to be personally and publicly accountable for all aspects of the work, ensuring that any questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding

This research work was financially supported by the Guizhou Provincial Science and Technology Plan Projects (QKHZC 2026-284), National Natural Science Foundation of China (Grant No. 32460877), Hundred Thousand Million Leading Talent Teams of Guizhou Province (BQW2025-004), and Guizhou University Natural Sciences Project for Special Post (Leading Group2024-12).

Ethical statements

This study procedures were approved by the Laboratory Animal Ethics Committee of Guizhou University (Guiyang, China). All procedures were conducted according to the guidelines for the ethical review of laboratory animals for animal welfare (EAE-GZU-2026-T099), which are consistent with the guidelines approved by the Institutional Animal Care and Use Committee.

Data availability

The data and materials used in this study are available from the corresponding author on reasonable request.

Disclosures

The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted “HT Restrains NLRP3 Inflammasome Activation and Pyroptosis Through the TLR4/NF-κB Pathway to Prevent MG-Inflammation”.