3’-hydroxypuerarin mitigates LPS-induced acute lung injury by inhibiting TLR4 activation-mediated NF-κB p65/NLRP3/GSDMD signaling

Abstract

Introduction

Acute lung injury (ALI) is a common critical respiratory illness. 3’-Hydroxypuerarin (3HP), a beneficial isoflavone from Pueraria lobata (Willd.) Ohwi, possesses significant pharmacological activities, but its effects on ALI remain limited.

Methods

To investigate the role of 3HP in mitigating ALI, in vivo lipopolysaccharide (LPS)- induced ALI in mice and in vitro LPS-induced RAW264.7 macrophage inflammatory injury were carried out using ELISA kits, RT-qPCR, immunofluorescence, Western blotting, molecular docking, and molecular dynamics simulation analyses.

Results

3HP significantly reduced IL6 and TNF-α levels in bronchoalveolar lavage fluid (BALF) and serum, attenuated pulmonary edema, inhibited the mRNA levels of chemokines and inflammatory factors in lung tissues, and suppressed the expression of IL1β, IL6, TNF-α, HMGB1, TLR4, MyD88, p-IκBα (S32/S36), p-NF-κB p65 (S536), COX2, iNOS, ICAM1, VCAM1, NLRP3, ASC, Caspase-1, Cleaved Caspase-1 (Ala317) p10, NEK7, Caspase-8, IL18, GSDMD, and GSDMD N-terminal both in lung tissues and in RAW264.7 cells, indicating that 3HP inhibited LPS-stimulated TLR4 activation, thereby reducing IκBα phosphorylation and degradation, and preventing NF-κB p65 nuclear translocation to mediate the transcription and expression of inflammatory mediators. It also inhibited NLRP3 inflammasome activation, decreased Caspase-1 cleavage of GSDMD, and lowered the release of the pore-forming GSDMD N-terminal structural domain, blocking the immune response to pyroptosis. Moreover, the NLRP3 inhibitor MCC950 enhanced the effect of 3HP on acute cellular inflammatory injury. Notably, molecular docking and dynamics simulation revealed that 3HP stably bound to TLR4. Resatorvid (TAK-242), a selective TLR4 signaling inhibitor, significantly enhanced 3HP’s inhibitory effect on TLR4, further indicating that 3HP may block the NF-κB p65/NLRP3/GSDMD signaling pathway by inhibiting TLR4 activation in response to LPS stimulation.

Conclusions

Our results demonstrated that 3HP mitigates LPS-induced ALI by inhibiting TLR4 activation-mediated NF-κB p65/NLRP3/GSDMD signaling. These findings provide scientific evidence for the clinical treatment of ALI and present new insights into the pharmacological role of 3HP in mitigating acute lung inflammatory diseases.

1. Introduction

Acute lung injury (ALI) is a severe respiratory disease caused by endogenous and exogenous pathogenic factors, clinically characterized by manifestation of acute hypoxic respiratory failure with bilateral pulmonary infiltrates and noncardiogenic hypoxemia, with a worldwide prevalence (1, 2). It mainly presents as an uncontrolled inflammatory response, oxidative stress, pulmonary edema, and the presence of inflammatory cells, eventually progressing to acute respiratory distress syndrome (ARDS) (3). Although mechanical ventilation effectively alleviates inflammatory lung injury, the mortality rate of ALI/ARDS remains high at 40 to 60% (4, 5). Therefore, clarifying ALI’s specific pathogenesis and pathophysiological mechanisms and exploring effective therapeutic options is crucial.

Macrophages, a vital part of the innate immune system, combat infections, eliminate inflammatory responses, and facilitate tissue repair (6, 7). Numerous studies have demonstrated that infection with lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, triggers overactivation of macrophages, promoting lung inflammation and damage, thereby exacerbating the onset and progression of ALI (8). Consequently, LPS is widely used to model ALI/ARDS in rodents (9). Common induction routes include intratracheal instillation (typically 2 to 10 mg/kg), intraperitoneal injection (5 to 20 mg/kg), and intravenous injection (10). LPS stimulation triggers a series of intracellular signaling events, including binding to toll-like receptor 4 (TLR4), activating the myeloid differentiation primary response protein 88 (MyD88)-nuclear factor-kappa B (NF-κB) signaling pathway, and promoting the nuclear translocation of NF-κB. This process regulates the transcription of inflammatory mediators and the release of pro-inflammatory cytokines and chemokines, such as interleukin 6 (IL6), tumor necrosis factor-α (TNF-α), and interleukin 1β (IL1β) (11, 12). Additionally, LPS exposure triggers the assembly of the macrophage NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and activates a subsequent inflammatory cascade response (13, 14). Indeed, the activated downstream protease, Caspase-1, cleaves the substrate GSDMD, releasing its N-terminal domain and triggering membrane pore formation and the immune response associated with cellular pyroptosis (15). Furthermore, activated Caspase-1 cleaves the precursor inflammatory cytokines pro-IL1β and pro-IL18 to release mature IL1β and IL18 (16, 17), which are released through molecular pores in the cell membrane and expelled from the cell, thereby worsening the inflammatory cascade response and influencing the progression of ALI (18, 19). Thus, pharmacological inhibition of macrophage NF-κB signaling and NLRP3 inflammasome activation represents a viable approach to alleviate ALI.

Traditional Chinese medicine (TCM) has a rich history of effectively preventing and treating various illnesses with few side effects (20). Classical TCM strategies for ALI primarily focus on clearing heat-toxin, resolving phlegm, and relieving lung obstruction. They often use formulas such as Yupingfeng San (which regulates immunity and protects the lung barrier) and Gegentang (derived from Pueraria lobata and exerting anti-inflammatory effects), along with single herbs such as Scutellaria baicalensis and Pueraria lobata that target inflammatory pathways. These approaches integrate syndrome differentiation with modern pharmacological evidence to mitigate excessive inflammation and promote lung tissue repair (21, 22). Pueraria lobata (Kudzu) root, a traditional herbal medicine from the root of Pueraria lobata (Willd.) Ohwi, is effective in relieving lung dryness and cough. It is commonly used in clinical TCM to treat upper respiratory tract infections caused by heat and pathogens, pneumonia, and other illnesses (23–25), including reducing lung inflammation, combating pulmonary fibrosis, and providing antioxidant effects (26–28). Studies have shown that Pueraria lobata (Kudzu) root contains various bioactive compounds and is a notable source of unique and beneficial isoflavones (29, 30). Among them, Puerarin has been reported to have pharmacological protective effects against ALI (31, 32). 3’-Hydroxypuerarin (3HP), a key isoflavonoid active ingredient in Pueraria lobata with a high content, is a derivative of Puerarin, has higher lipid solubility (33), and exhibits significant activities in scavenging ONOO (–), NO·, and total ROS than Puerarin (34). In our preliminary experiments, we found that 3HP exhibits significant anti-inflammatory effects in vitro. However, studies on the pharmacoprotective effects of 3HP on acute pulmonary inflammatory responses or ALI remain limited. Therefore, based on the commonly used LPS-induced ALI model and dosage, as well as our previous research (14, 35), the current study employed an LPS (5 mg/kg) intraperitoneally injected mouse model of ALI and an in vitro LPS (0.5 μg/mL)-induced inflammatory damage model in RAW264.7 macrophages to evaluate the molecular pharmacologic mechanisms of 3HP. The results showed that 3HP effectively inhibited TLR4 activation, thereby suppressing NF-κB p65 signaling-mediated inflammatory cascade response and NLRP3/GSDMD signaling-mediated pyroptosis, thereby mitigating ALI. These findings provide scientific evidence for the clinical treatment of ALI and present new insights into the pharmacological role of 3HP in mitigating acute lung inflammatory diseases.

2. Materials and methods

2.1. Chemicals and reagents

3’-Hydroxypuerarin (CAS No.: 117076-54-5, Purity > 98.0%) was supplied by Weikeqi Biological Technology Co., Ltd (Sichuan, China). Resatorvid (TAK-242) (Cat No. HY-11109) and MCC950 (Cat No. HY-12815) was purchased from MedChemExpress (Shanghai, China). LPS (Cat No. L2880) was obtained from Sigma-Aldrich (Shanghai, China). Anti-IL6 (Cat No. DF6087), Anti-IκBα (Cat No. AF5002), anti-Phospho-IκBα (S32/S36) (Cat No. AF2002), anti-Caspase-8 (Cat No. AF6442), anti-ICAM1 (Cat No. AF6088), anti-VCAM1 (Cat No. DF6082), anti-COX2 (Cat No. AF7003), anti-iNOS (Cat No. AF0199), anti-Cleaved-Caspase 1 (Ala317), p10 (Cat No. AF4022), anti-GSDMD N-terminal (Cat No. DF13758), and anti-Phospho-NF-κB p65 (S536) (Cat No. AF2006) were purchased from Affinity (Affinity Biosciences, Changzhou, Jiangsu, China). Anti-TNF-α (Cat No. PY19810) was supplied by Abmart (Shanghai, China). Anti-TLR4 (Cat No. 19811-1-AP) and anti-MyD88 (Cat No. 67969-1-Ig) were obtained from Proteintech (Proteintech Group, Wuhan, Hubei, China). Anti-IL1β (Cat No. YT5201), anti-IL18 (Cat No. YN1926), anti-NF-κB p65 (Cat No. YT3108), anti-NEK7 (Cat No. YT3034), anti-NLRP3 (Cat No. YT5382), anti-ASC (Cat No. YT0365), anti-Caspase-1 (Cat No. YT5743), anti-GSDMD N-terminal (Cat No. YT7991), and anti-β-actin (Cat No. YM8343 and YM3028) were purchased from ImmunoWay (ImmunoWay Biotechnology Company, Plano, TX, USA).

2.2. Cell culture and cell viability

RAW264.7 macrophages were cultured at 37 °C in a 5% CO₂ atmosphere using Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). To assess cell viability, RAW264.7 cells (2 × 10^4/well) were seeded into 96-well plates for 24 hours, after which the cells were treated with varying concentrations of 3HP for another 24 hours. Cell viability was determined using the CCK-8 kit (GK10001) according to the manufacturer’s instructions (GLPBIO, Montclair, CA, USA).

2.3. Measurement of NO

RAW264.7 macrophages were plated in a 6-well plate at a density of 1 × 10^6 cells/well at 37 °C for 24 hours, followed by incubation with 3HP (25, 50, and 100 μM) and LPS (0.5 μg/ml) for an additional 24 hours. The supernatant was analyzed for NO (Cat No. S0021S) following the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China).

2.4. Animal experiment

In vivo experiments adhere to the 3R principles of animal welfare (Replacement, Reduction, Refinement). Twenty-four specific pathogen-free male C57BL/6 mice (6 to 8 weeks old, weighing 18 to 22 g) were supplied by the Guangdong Medical Laboratory Animal Center. They were housed in cages and provided free access to food and water, then used after a one-week acclimatization period. The Ethics Committee of Guangzhou University of Chinese Medicine approved all animal care and experimental procedures (No. 20240322007). Mice were randomly placed into four groups (6 mice per group): Control group (Con), LPS group (LPS), LPS + 3HP 50 mg/kg group, and LPS + 3HP 100 mg/kg group. After 1 week of oral administration of the 3HP intervention, the mice were injected intraperitoneally with LPS (5 mg/kg) to induce ALI, except in the Con group. After 24 hours of observation, serum, bronchoalveolar lavage fluid (BALF), and lung tissues were collected for further analysis.

2.5. Measurement of BALF protein concentration

The concentration of BALF protein was determined using the enhanced bicinchoninic acid (BCA) protein assay kit (Cat No. P0010) following the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China).

2.6. Enzyme-linked immunosorbent assay analysis

The levels of IL-6 (Cat No. 88-7064-86) and TNF-α (Cat No. 88-7324-86) in BALF, serum of mice and RAW264.7 macrophage supernatants were determined using ELISA kits following the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).

2.7. Giemsa staining

The BALF was centrifuged to collect the supernatant, and the precipitated cells were resuspended in PBS and stained with Wright-Giemsa (Cat No. C0131). Images were acquired using a digital microscope scanner.

2.8. Hematoxylin and eosin staining

Lung tissues were fixed in 4% paraformaldehyde for 48 hours, dehydrated through graded ethanol solutions, embedded in paraffin blocks, and then sectioned into 5-μm-thick slices. Sections were deparaffinized, rehydrated, stained with H&E reagent (Biosharp, Hefei, China), and finally dehydrated, cleared, and mounted in neutral resin for microscopic examination. Images were acquired using a digital camera scanner.

2.9. Measurement of the lung wet-to-dry ratio

The W/D ratio was used to assess the severity of pulmonary edema. The left lung was collected and weighed to determine its wet (W) weight. The dry (D) weight was recorded after drying at 60 °C for 48 hours. Subsequently, the W/D ratio was then calculated via the following formula.

$$\text{W}/\text{D ratio}=\text{Wet weight }(\text{g})/\text{Dry weight }(\text{g})$$

2.10. Measurement of superoxide dismutase and malondialdehyde

SOD (Cat No. A001-3-2) and MDA (Cat No. A003-1-2) levels in lung tissues were determined using commercial assay kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.11. qPCR analysis

Total RNA was extracted from lung tissues with Trizol reagent (Invitrogen™, Shanghai, China). A cDNA transcription kit (Takara Biomedical Technology Co., Ltd., Dalian, China) was used for reverse transcription. Subsequently, qPCR was performed using the Bio-Rad CFX Connect Real-Time System and the TB Green™ Premix ExTaq™ kit (Takara Biomedical Technology Co., Ltd., Dalian, China). The relative mRNA levels of target genes were computed using the 2-ΔΔCt method. Primers for the genes are listed in Supplementary Table 1.

2.12. Western blotting

Lung tissues or RAW264.7 macrophages were lysed using RIPA buffer containing protease and phosphatase inhibitors for protein extraction. The protein concentration was determined using the BCA method. For immunoblotting, equal amounts of protein were separated using SDS-PAGE and subsequently transferred to a PVDF membrane. The membranes were incubated overnight at 4 °C with the specific primary antibody and for 1 hour at room temperature with the secondary antibody. Signals were detected using either the Molecular Imager^® System (Bio-Rad, Hercules, CA, USA) or the Amersham ImageQuant™ 800 System (Cytiva, Uppsala, Sweden) with ECL reagents. Quantification was performed using ImageJ (NIH, Bethesda, MD, USA).

2.13. Immunofluorescence

Lung tissue sections were deparaffinized and gradient-hydrated, incubated with 3% H₂O₂ prior to antigen retrieval, and then blocked with 1% BSA. For RAW264.7 macrophages, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 at room temperature, and then blocked with 1% BSA. All experimental samples were incubated overnight at 4 °C with primary antibodies, followed by an hour at room temperature with secondary antibodies, and then re-stained cell nuclei with DAPI. Images were obtained using a digital camera scanner.

2.14. Molecular docking analysis

The structures of the TLR4 (PDB ID: 3VQ2) target protein was acquired from the RCSB database (https://www.rcsb.org/) and processed using Schrödinger’s Protein Preparation Wizard for energy minimization and geometry optimization. The structure of 3HP was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), imported into Chem3D, optimized, and energy-minimized using the MM2 module, and saved as an SDF file for use as a ligand in molecular docking. Molecular docking was performed using the Glide module in Schrödinger Maestro (Version: 2019.01), with TLR4 binding sites predicted using the Binding Site Detection module. Docking scores of 3HP were evaluated to hypothesize its active effects.

2.15. Molecular dynamics simulation

To further explore how 3HP binds to the TLR4 protein at the molecular level, we used the Gromacs software (Version: 2020.6) package to perform energy minimization, heating, equilibration, and production molecular dynamics simulations of the receptor protein-small molecule complex. Protein was modeled using the AMBER99SB-ILDN force field parameters. Small-molecule ligands were constructed using the sobtop program for topology generation, with charge fitting performed via RESP. For the molecular dynamics simulation workflow: first, the heavy atoms of the protein and small molecule were constrained to perform 10,000 steps of energy minimization on the water molecules; then, the constraints were released, and another 10,000 steps of energy minimization were carried out on the entire system. Finally, the system underwent 100 ns of molecular dynamics simulations under the NPT ensemble. Trajectory data were saved every 20 ps and processed with the trjconv module. The binding free energies of the ligands and proteins were calculated using the gmxMMPBSA method in Gromacs 2020.6.

2.16. Statistical analysis

All data were expressed as mean ± SEM and analyzed with GraphPad Prism 9.0 software (San Diego, CA, USA). For data sets that satisfied the assumptions of normality and homogeneity of variances, comparisons between groups were made using one-way ANOVA. For data that were normally distributed but had unequal variances, Dunnett’s T2 test was applied. If the data did not meet the normality assumption, non-parametric tests were employed. P-values less than 0.05 were regarded as statistically different.

3. Results

3.1. 3HP decreased the infiltration and exudation of pro-inflammatory cells in ALI mice

3HP, a key isoflavonoid active ingredient in Pueraria lobata with a high content (Figure 1A), has higher lipid solubility (33). To investigate the pharmacological effect of 3HP on LPS-induced ALI in mice (Figure 1B), we determined the protein concentrations in BALF. As shown in Figure 1C, LPS stimulation increased protein leakage in the lung tissues, while 3HP intervention significantly reversed this phenomenon. We also determined the levels of inflammatory cytokines in BALF using an ELISA kit and found that 3HP significantly reduced IL6 and TNF-α levels (Figures 1D, E), demonstrating excellent anti-inflammatory capacity. Additionally, Giemsa staining results confirmed the effectiveness of 3HP in decreasing neutrophils (light purple-red, arrows: black) and macrophages (blue-purple, arrows: red) in BALF (Figures 1F–H), indicating that 3HP positively affects the reduction of pro-inflammatory cell infiltration and exudation in ALI mice.

Figure 1.

3HP reduced infiltration and exudation of pro-inflammatory cells in ALI mice. (A) 3HP from Pueraria lobata (Kudzu) root. (B)In vivo experimental procedure. (C) BALF protein level (n = 6). (D, E) IL6 and TNF-α levels in BALF (n = 6). (F) Representative Giemsa staining in BALF, neutrophils (light purple-red, arrows: black) and macrophages (blue-purple, arrows: red), original magnification, 4.00× and 20.00×. (G, H) Relative neutrophils and macrophages count in BALF (n = 3). Data were expressed as mean ± SEM. Compared to the Con group, *p < 0.05, **p < 0.01, and ****p < 0.001. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001.

3.2. 3HP alleviated LPS-induced ALI in mice by suppressing inflammatory cascade responses

The immune cell-mediated inflammatory response plays a crucial role in regulating the progression of ALI (36). LPS-induced lung tissues in ALI mice displayed significant infiltration of inflammatory cells and pulmonary edema, while prophylactic administration of 3HP notably reduced lung inflammatory injury (Figure 2A). The lung W/D weight ratio clearly confirmed this result (Figure 2B). Additionally, we analyzed serum inflammatory cytokine levels using the ELISA assay and found that 3HP significantly reduced IL-6 and TNF-α levels (Figures 2C, D). Further qPCR analysis indicated that LPS stimulation increased the levels of chemokines and inflammatory cytokines, while 3HP significantly reduced mRNA levels of Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl1, Cxcl2, Cxcl9, Cxcl10, IL1α, IL1β, IL6, and TNF-α (Figures 2E–I). Interestingly, we also found that 3HP significantly increased SOD activity and reduced MDA levels (Figures 2J, K), indicating its ability to resist oxidative stress. Moreover, the impressive anti-inflammatory potential of 3HP was reaffirmed by immunoblotting results, which demonstrated that 3HP markedly inhibited the expression of IL1β, IL6, TNF-α, and HMGB1 proteins in the lung tissues of ALI mice (Figures 2L–P). All these findings suggested that 3HP attenuated ALI by inhibiting LPS-stimulated acute inflammatory responses in the lungs.

Figure 2.

3HP alleviated LPS-induced ALI in mice by suppressing inflammatory cascade responses. (A) Representative H&E staining in lung tissues, original magnification, 5.00× and 20.00×. (B) W/D radio of the lungs (n = 6). (C, D) IL6 and TNF-α levels in serum (n = 6). (E) mRNA levels of Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl1, Cxcl2, Cxcl9, and Cxcl10 in lung tissues (n = 6). (F–I) mRNA levels of IL1α, IL1β, IL6, and TNF-α in lung tissues (n = 6). (J, K) SOD and MDA levels in serum (n = 6). (L–P) Representative immunoblotting and quantification of IL1β, IL6, TNF-α, and HMGB1 in lung tissues (n = 3). Data were expressed as mean ± SEM. Compared to the Con group, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.00. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001.

3.3. 3HP alleviated LPS-induced ALI in mice by inhibiting TLR4/NF-κB p65 signaling

It has long been established that NF-κB signaling plays a crucial role in the pathogenesis of ALI (37). To investigate the effect of 3HP on LPS-activated NF-κB signaling, we examined the expression levels of the core components using immunoblotting. As shown in Figures 3A–E, 3HP significantly reduced the expression levels of LPS-stimulated TLR4, MyD88, p-IκBα (S32/S36), and p-NF-κB p65 (S536). Additionally, immunofluorescence analysis further confirmed the inhibitory effect of 3HP on LPS-activated TLR4 and MyD88 (Figures 3F, G). Notably, NF-κB signaling activation and transactivation significantly regulated the transcription of inflammatory mediators, thereby exacerbating the inflammatory cascade response. Thus, we further analyzed biomarkers of the inflammatory response and found that 3HP markedly inhibited the expression of COX2, iNOS, ICAM1, and VCAM1 (Figures 3H–L). Additionally, qPCR analysis indicated that 3HP significantly down-regulated the mRNA levels of F4/80 and IFN-γ (Figures 3M, N), reaffirming its strong anti-inflammatory pharmacological effects. Overall, these results demonstrated that 3HP alleviated LPS-induced ALI in mice by inhibiting TLR4/NF-κB p65 signaling pathway.

Figure 3.

3HP alleviated LPS-induced ALI in mice by inhibiting TLR4/NF-κB p65 signaling. (A–E) Representative immunoblotting and quantification of TLR4, MyD88, p-IκBα (S32/S36), IκBα, p-NF-κB p65 (S536), and NF-κB p65 in lung tissues (n = 3). (F, G) Representative immunofluorescence images and intensity of TLR4-positive and MyD88-positive in lung tissues (n = 3). Scale bars: 25 μm. (H–L) Representative immunoblotting and quantification of COX2, iNOS, ICAM1, and VCAM1 in lung tissues (n = 3). (M, N) mRNA levels of F4/80 and IFN-γ in lung tissues (n = 6). Data were expressed as mean ± SEM. Compared to the Con group, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.000. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001.

3.4. 3HP alleviated LPS-stimulated ALI in mice by inhibiting NLRP3 inflammasome activation

To further explore the molecular mechanisms of 3HP in alleviating ALI, we concentrated on assessing the pharmacological effects of 3HP on NLRP3 signaling. As illustrated in Figures 4A–E, LPS stimulation activated the NLRP3 inflammasome, leading to the upregulation of NLRP3, ASC, Caspase-1, and Cleaved Caspase-1 p10. Interestingly, 3HP significantly reversed this phenomenon. Additionally, qPCR results confirmed the inhibitory effect of 3HP on the mRNA levels of the NLRP3, ASC, and IL18 (Figures 4F–H). Notably, we also found that 3HP significantly reduced the expression of NEK7, Caspase-8, and IL18 (Figures 4I–L). Immunofluorescence confirmed the pharmacological inhibition of NLRP3 activation by 3HP (Figures 4M–O). Indeed, the NLRP3 inflammasome activates Caspase-1, which cleaves GSDMD. In this study, we found that 3HP significantly inhibited the expression of GSDMD and GSDMD N-terminal (Figures 4P–R), highlighting the substantial inhibitory effect of 3HP on NLRP3 signaling. Taken together, these findings suggested that 3HP mitigated LPS-induced ALI in mice by inhibiting the NLRP3/GSDMD-mediated cellular pyroptosis.

Figure 4.

3HP alleviates LPS-stimulated ALI in mice by inhibiting NLRP3 inflammasome activation. (A–E) Representative immunoblotting and quantification of NLRP3, ASC, Caspase-1, and Cleaved Caspase-1 p10 in lung tissues (n = 3). (F–H) mRNA levels of NLRP3, ASC, and IL18 in lung tissues (n = 6). (I–L) Representative immunoblotting and quantification of NEK7, Caspase-8, and IL18 in lung tissues (n = 3). (M–O) Representative immunofluorescence images and intensity of NLRP3-positive and ASC-positive in lung tissues (n = 3). Scale bars: 25 μm. (P–R) Representative immunoblotting and quantification of GSDMD and GSDMD N-terminal in lung tissues (n = 3). Data were expressed as mean ± SEM. Compared to the Con group, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.000.

3.5. 3HP attenuated LPS-stimulated acute inflammatory injury in RAW264.7 macrophages by inhibiting inflammatory cascade responses

Research indicates that pathogens enter lung tissues and activate alveolar macrophages, leading to the release of significant amounts of inflammatory factors, which consequently trigger an inflammatory cascade response (38). In this study, we utilized an in vitro LPS-stimulated RAW264.7 macrophage model of acute inflammatory injury to evaluate the pharmacological effects of 3HP (Figure 5A). As shown in Figure 5B, the CCK-8 assay demonstrated that various concentrations of 3HP (6.25, 12.5, 25, 50, 100, 200, and 400 μM) exhibited no toxic effects on RAW264.7 macrophages. Interestingly, it also showed a slight promotional impact on cell proliferation. Further immunoblotting assays showed that 3HP (25, 50, and 100 μM) significantly reduced the expression of inflammatory cytokines IL1β, IL6, TNF-α, and HMGB1 stimulated by LPS (Figures 5C–G). Additionally, we found that 3HP significantly reduced IL6, TNF-α, and NO levels in LPS-stimulated RAW264.7 macrophage supernatants (Figures 5H–J), reaffirming its strong anti-inflammatory response capabilities.

Figure 5.

3HP attenuated LPS-stimulated acute inflammatory injury in RAW264.7 macrophages by inhibiting inflammatory cascade responses. (A)In vitro experimental procedure. (B) Relative cell viability. (C–G) Representative immunoblotting and quantification of IL1β, IL6, TNF-α, and HMGB1 in RAW264.7 macrophages. (H–J) IL6, TNF-α, and NO levels in supernatants of RAW264.7 macrophages. Data were expressed as mean ± SEM (n = 3). Compared to the Con group, *p < 0.05, **p < 0.01, and ****p < 0.0001. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001.

3.6. 3HP attenuated LPS-stimulated inflammatory injury in RAW264.7 macrophages by inhibiting TLR4/NF-κB p65 signaling

LPS, a key element of the outer membrane of Gram-negative bacteria, activates intracellular signaling pathways, such as MyD88 and interleukin-1 receptor-associated kinase (IRAK), by binding to the TLR4 receptor of the host cell. This ultimately activates the NF-κB transcription factor, which initiates the production and release of inflammatory factors (11). Based on the findings above, we reanalyzed the effect of 3HP on the TLR4/NF-κB pathway in an in vitro model of acute inflammatory injury. Our analysis revealed that 3HP significantly inhibited the fluorescence expression of TLR4 and MyD88 in LPS-stimulated RAW264.7 macrophages (Figures 6A–D). Further immunoblot analysis demonstrated that 3HP significantly reduced the expression of key components of NF-κB signaling, including TLR4, MyD88, p-IκBα (S32/S36), and p-NF-κB p65 (S536) (Figures 6E–I). Furthermore, we also discovered that 3HP significantly inhibited the expression of the inflammatory mediators COX2, iNOS, ICAM1, and VCAM1 (Figures 6J–N). In fact, the translocation of p65/p50 into the nucleus is an important factor in the activation of the classic NF-κB signaling. Therefore, we analyzed this phenomenon using immunofluorescence analysis. The results revealed that 3HP suppressed NF-κB p65 translocation from the cytoplasm to the nucleus (Figures 6O, P). Overall, these results indicated that 3HP inhibited LPS-stimulated TLR4 activation, thereby reducing IκBα phosphorylation and degradation, and blocking the translocation of the active NF-κB p65 transcription factor into the nucleus, thereby inhibiting target gene expression. Interestingly, these findings aligned with the results of the in vivo experiments mentioned above, further emphasizing 3HP’s pharmacological inhibition of NF-κB signaling in ameliorating ALI.

Figure 6.

3HP attenuated LPS-stimulated inflammatory injury in RAW264.7 macrophages by inhibiting TLR4/NF-κB p65 signaling. (A–D) Representative immunofluorescence images and intensity of TLR4-positive and MyD88-positive in RAW264.7 macrophages, Scale bars: 100 μm. (E–I) Representative immunoblotting and quantification of TLR4, MyD88, p-IκBα (S32/S36), IκBα, p-NF-κB p65 (S536), and NF-κB p65 in RAW264.7 macrophages. (J–N) Representative immunoblotting and quantification of COX2, iNOS, ICAM1, and VCAM1 in RAW264.7 macrophages. (O–P) Representative immunofluorescence images and intensity of NF-κB p65 translocation into the nucleus. Data were expressed as mean ± SEM (n = 3). Compared to the Con group, *p < 0.05, **p < 0.01, and ****p < 0.0001. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^####p < 0.0001.

3.7. 3HP alleviated LPS-stimulated acute inflammatory injury in RAW264.7 macrophages by inhibiting NLRP3/GSDMD signaling-mediated cellular pyroptosis

The above in vivo experiments have revealed that 3HP exerts a pharmacological inhibitory effect on the activation of NLRP3 inflammasome in lung tissues of ALI mice. To further confirm the ameliorative effect of 3HP on acute inflammatory injury, we analyzed key proteins involved in NLRP3 inflammasome activation by immunoblotting in LPS-stimulated RAW264.7 macrophages. As shown in Figures 7A–H, 3HP significantly decreased the expression of NLRP3, Caspase-1, Cleaved Caspase-1 p10, NEK7, Caspase-8, and IL18. Interestingly, we also found that 3HP significantly inhibited the cleavage of the substrate GSDMD by Caspase-1 and decreased the membrane-pore-forming activity of the GSDMD N-terminal domain, thereby reducing cellular pyroptosis (Figures 7I–K). Further immunofluorescence results indicated that 3HP significantly suppressed NLRP3 expression (Figure 7L), reaffirming its pharmacological inhibitory effect on NLRP3 inflammasome activation. Additionally, we selected MCC950, a specific inhibitor of NLRP3 inflammasome activation, to further evaluate the effects of 3HP and MCC950. As shown in Figures 7M, N, MCC950 enhanced the LPS-stimulated pharmacological inhibition of NLRP3 by 3HP, emphasizing 3HP’s protective effect against LPS-stimulated cellular acute inflammatory injury. All these findings indicated that 3HP could reduce ALI by inhibiting NLRP3/GSDMD signaling-mediated pyroptosis.

Figure 7.

3HP alleviated LPS-stimulated acute inflammatory injury in RAW264.7 macrophages by inhibiting NLRP3/GSDMD signaling-mediated cellular pyroptosis. (A–D) Representative immunoblotting and quantification of NLRP3, Caspase-1, and Cleaved Caspase-1 p10 in RAW264.7 macrophages. (E–H) Representative immunoblotting and quantification of NEK7, Caspase-8, and IL18 in RAW264.7 macrophages. (I–K) Representative immunoblotting and quantification of GSDMD and GSDMD N-terminal in RAW264.7 macrophages. (L) Representative immunofluorescence images of NLRP3-positive in RAW264.7 macrophages, Scale bars: 25 μm. (M, N) Representative immunoblotting and quantification of NLRP3 after MCC950 and 3HP intervention in RAW264.7 macrophages. Data were expressed as mean ± SEM (n = 3). Compared to the Con group, *p < 0.05, and **p < 0.01. Compared to the LPS group, ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001. Compared to the 3HP group, ^$p < 0.05.

3.8. 3HP might ameliorate ALI by inhibiting NF-κB/NLRP3 signaling through targeting TLR4

The pharmacological effects of 3HP in inhibiting TLR4/NF-κB and NLRP3/GSDMD signaling to alleviate LPS-induced ALI have been demonstrated. Indeed, in LPS-stimulated activation of the classical NF-κB signaling pathway, upstream TLR4 activation plays a crucial role. To investigate the pharmacological molecular mechanism by which 3HP inhibits TLR4, we conducted molecular docking analysis. As shown in Figure 8A, the molecular binding energy of 3HP to TLR4 was -6.968 kcal/mol (Less than -5.0 kcal/mol), indicating that 3HP has a strong affinity for TLR4 and can bind stably to exert pharmacological inhibitory activity.

Figure 8.

3HP bound to TLR4 stably. (A) The binding mode of TLR4 with 3HP. (The 3D structure of complex. The electrostatic surface of protein. The detail binding mode of complex. The backbone of protein was rendered in tube and colored in green. Compound is rendering by green). (B) The hydrogen bond number of 3HP with TLR4. (C) The RMSD of 3HP with TLR4. (D) The RMSF of 3HP with TLR4. (E) The Rg of 3HP with TLR4. (F) The SASA of 3HP with TLR4. (G) The energy decomposition of binding free energy for TLR4-3HP. (H–J) levels of IL6, TNF-α, and NO (n = 3). (K, L) Representative immunofluorescence images and intensity of TLR4-positive in RAW264.7 macrophages (n = 3). Scale bars: 50 μm.

To gain a more intuitive understanding of TLR4 binding to 3HP, we used molecular dynamics simulations to track changes in the number of hydrogen bonds between the protein and the small molecule throughout the simulation. As shown in Figure 8B, 3HP was able to form about five hydrogen bonding interactions with the TLR4 pocket amino acids (100% hydrogen bond occupancy), and these hydrogen bonds played a key role in stabilizing the 3HP-TLR4 binding. Furthermore, the average root mean square deviation (RMSD) of the complexes were all below 0.3 nm, and they generally reached dynamic equilibrium around 40 ns. Interestingly, we observed that the fluctuations of 3HP were significantly smaller than the RMSD fluctuations of the TLR4 protein, suggesting that the compounds could consistently remain in the protein’s binding site (Figure 8C). Meanwhile, the root mean square fluctuation (RMSF) results indicated that a few amino acids in the complex formed by the interaction between TLR4 and 3HP experienced significant conformational changes, especially near the C-terminal and N-terminal sites. This was mainly because these regions are at the ends of the protein, making their conformation more prone to specific changes during the simulation (Figure 8D). To examine the relative densities and stability of complexes formed by primary and secondary site binding, we measured the radius of gyration (Rg) of the target proteins and observed that TLR4 Rg initially increased slightly before decreasing. However, binding of TLR4 to 3HP enhanced the formation of more effective interactions within the protein, thereby increasing the stability of the complexes (Figure 8E). Moreover, the solvent-accessible surface area (SASA) begins to decrease after the kinetic adjustment, which is beneficial for complex stability (Figure 8F). Notably, the binding free energy is a key tool for analyzing changes in the ligand binding mode by measuring the ligand’s thermodynamic properties. Negative values of the binding free energy (ΔGbinding energy) indicate system stability, while positive values suggest instability. As shown in Supplementary Table 2, electrostatic interactions play a significant role in stabilizing small molecule compounds, followed by van der Waals forces. The binding free energy of 3HP with TLR4 protein was -33.48 ± -5.71 kcal/mol, mainly due to hydrogen-bonding interactions between the compounds and the protein pockets, making electrostatic interactions a key contributor to the compounds’ stability (-85.01 ± -5.4 kcal/mol). Additionally, van der Waals forces also significantly contribute (-22.46 ± 0.87 kcal/mol), indicating that 3HP can remain stably in TLR4 pocket with strong van der Waals interactions with surrounding residues. We further examined the binding free energies and found that the amino acids HIS:158, ASP:180, SER:182, TYR:183, ASP:208, SER:210, LEU:211, PHE:262, and ASP:264 play a significant role in stabilizing 3HP (Figure 8G). In summary, 3HP shows strong affinity for TLR4. These affinities can help form small molecules into stable complexes with the protein, thereby exerting active inhibitory effects.

To further evaluate the pharmacological effects of 3HP on TLR4, we analyzed the therapeutic impact of TAK-242, a TLR4 inhibitor. Results revealed that TAK-242 potentiated the pharmacological inhibition of TLR4 by 3HP, as evidenced by significantly reduced levels of NO, IL6, and TNF-α (Figures 8H–J), along with decreased TLR4 expression (Figures 8K, L). This indicates that 3HP partially exerts its inhibitory effect on downstream inflammatory cascades by modulating TLR4.

4. Discussion

ALI, a prevalent respiratory critical illness characterized by high morbidity and mortality rates, is caused by various pathogenic factors both within and outside the lungs (1, 2). An imbalance in the inflammatory response cascade caused by infection and trauma is the root cause of ALI progression (39, 40). While significant breakthroughs have been achieved in ALI/ARDS research, no effective drug therapies currently exist. Therefore, exploring pharmacological treatments for ALI/ARDS is urgently needed. LPS, a significant component of the outer membrane of Gram-negative bacteria, is an endotoxin commonly used to induce various inflammatory models, such as ALI. Once inside the host, LBP recognizes LPS and binds to transport to the surface of myeloid-derived cells to bind to mCD14, forming the LPS-LBP-CD14 triplex, which is subsequently transported to the TLR4-MD2 protein complex. The triplex complex binds to TLR4 with the assistance of MD-2 and activates TLR4, thereby activating intracellular classical NF-κB signaling pathway, leading to the release of inflammatory mediators such as IL1β, IL6, TNF-α, and NO, and ultimately triggering an inflammatory cascade response. It is important to note that NF-κB signaling has long been recognized as a significant factor in the progression of ALI (37). Thus, pharmacologically inhibiting the core target proteins of NF-κB signaling is an essential way to ameliorate ALI. In fact, natural compounds have consistently inspired drug development, with many natural products demonstrating potential therapeutic effects for ALI/ARDS.

TCM, a healthcare system that has evolved over thousands of years for the Chinese people, has been extensively utilized to treat various diseases (41). An increasing number of active ingredients and formulations of TCM have been demonstrated to have significant therapeutic effects in treating ALI, which provides some guidance for clinical practice (22). Pueraria lobata (Kudzu) root, a traditional Chinese herb mentioned in the TCM, is known for its effectiveness in clearing heat, generating fluids, quenching thirst, and alleviating lung dryness and cough. It treats upper respiratory tract infections caused by heat and pathogens, pneumonia, and various other illnesses (23–25). Modern pharmacological studies have confirmed its ability to reduce lung inflammation, combat pulmonary fibrosis, and exert an anti-oxidative stress effect (26–28). Pueraria lobata (Kudzu) root is rich in isoflavonoid active compounds, such as puerarin and 3’-hydroxypuerarin (3HP). 3HP is a derivative of puerarin, has higher lipid solubility than puerarin (33), and exhibits significant ONOO (-), NO-, and total ROS scavenging activity (34). Studies have consistently shown that puerarin alleviates ALI by suppressing the inflammatory response and regulating NLRP3 inflammasome-induced cellular pyroptosis (31, 32). However, the effects and mechanisms of 3HP in alleviating acute inflammatory lung injury remain unclear.

In the current study, we evaluated the pharmacological benefits of 3HP through an in vivo LPS-induced ALI in mice and an in vitro LPS-induced acute inflammatory injury in the RAW264.7 macrophage model. Previous extensive research has demonstrated that intratracheal or intraperitoneal LPS stimulation can significantly induce acute inflammatory injury in mouse lungs (9). Concurrently, LPS-induced inflammatory injury in RAW 264.7 cells serves as a commonly used in vitro model for investigating ALI (10). This cell line produces a robust, prototypical inflammatory response, particularly upon stimulation with agents such as lipopolysaccharide (a TLR4 agonist). TLR4 stimulation activates intracellular signaling cascades, mobilizing nuclear transcription factors like nuclear factor-κB, ultimately leading to the production of inflammatory mediators including NO, TNF-α, ILβ, and IL6. As expected, we found that 3HP, an active isoflavone ingredient derived from the root of Pueraria lobata (Kudzu), significantly inhibited endotoxin LPS-stimulated TLR4 activation, decreased the expression of MyD88, a key transducer protein in intrinsic immune signaling, reduced downstream NF-κB pathway signaling, suppressed IκBα protein phosphorylation and protease degradation, and blocked the translocation of active NF-κB p65 into the nucleus. This ultimately inhibited the expression of target genes, including chemokines such as Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl1, Cxcl2, Cxcl9, Cxcl10, and inflammatory factors such as IL1α, IL1β, IL6, TNF-α, F4/80, and IFN-γ. These findings first highlight the remarkable ability of 3HP to reduce the inflammatory damage caused by endotoxin infection, indicating that it can attenuate LPS-induced ALI both in vitro and in vivo by inhibiting the inflammatory cascade response mediated by the classical TLR4/NF-κB p65 pathway signaling.

Indeed, the endotoxin-activated TLR4/NF-κB signaling pathway enhances the transcription and translation of NLRP3 and cytokines, triggers changes in the post-translational modifications of NLRP3, and thereby mediates the assembly of inflammasomes (42). NLRP3, a pattern recognition receptor (PRR) that triggers an inflammatory cascade response by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), serves as a critical modulator of innate immune defense (43). Activating the NLRP3 inflammasome is crucial in the progression of ALI (18, 19). Previous studies have demonstrated that Natural product derived phytochemicals or herbal formulations can alleviate ALI by decreasing the inhibition of NLRP3 inflammasome activation (44). For instance, dihydromyricetin alleviates sepsis-induced acute lung injury through Inhibiting NLRP3 inflammasome-dependent pyroptosis in mice model (45). Sinensetin ameliorates LPS-induced acute lung injury by suppressing Txnip/NLRP3/Caspase-1/GSDMD signaling-mediated inflammatory responses and pyroptosis (14). In this study, we found that 3HP significantly inhibited NLRP3 inflammasome assembly and activation, demonstrating consistent pharmacological inhibition of NLRP3 inflammasome activation. Notably, NEK7 is a crucial mediator of NLRP3 activation (46). Intracellular potassium ion efflux triggers JNK kinase to phosphorylate NEK7, which regulates NLRP3 oligomerization and activation (47, 48). 3HP significantly downregulates NEK7 expression, further substantiating its pharmacological inhibition on NLRP3. Interestingly, we also found that 3HP markedly reduced the level of caspase-8. Previous studies have confirmed that in the presence of caspase-1, caspase-8 positively regulates the NLRP3/caspase-1 signaling cascade, which drives IL-1β production and cellular pyroptosis (49) and plays a crucial part in TLR4-mediated activation of the NLRP1/NLRP3 inflammasome (50). In light of these findings, our study confirmed the significant pharmacological inhibitory effect of 3HP on the LPS-stimulated NLRP3 inflammasome activation-mediated inflammatory cascade response. Additionally, to further evaluate the effect of 3HP on cellular pyroptosis, we examined the GSDMD protein family, which is closely associated with pyroptosis. In fact, the activation of the NLRP3 inflammasome induces specific cleavage of the GSDMD-N structural domain at the amino-terminal end of GSDMD by inflammatory caspase-1, resulting in pore formation at the cell membrane and initiating the release of inflammatory cytokines and cellular pyroptosis (51). In the current study, 3HP significantly inhibited the expression of Caspase-1 and Cleaved Caspase-1 p10, thus hindering the formation of pores in the membrane by the N-terminal structural domain of GSDMD and decreasing the pro-inflammatory cytokines, such as IL1β and IL18, from crossing the membrane pores to trigger the inflammatory cascade response, thereby inhibiting the cellular pyroptosis. MCC950, a potent and specific NLRP3 antagonist, directly targets the ATP hydrolysis motif of NLRP3 to inhibit the inflammasome (52). To further evaluate the protective effect of 3HP against NLRP3 activation, we conducted an in vitro experimental assay and found that MCC950 significantly enhanced the pharmacological inhibition of NLRP3 by 3HP. However, whether 3HP directly targets NLRP3 for pharmacological effects similar to MCC950 needs to be confirmed through more in-depth studies.

Considering TLR4 as a crucial upstream molecule for NF-κB/NLRP3 signaling activation, we investigated the interaction between 3HP and TLR4 through molecular docking and molecular dynamics simulations. Notably, 3HP exhibits stable binding with TLR4 within the pocket. Further analysis reveals that 3HP stabilizes interactions with TLR4 amino acid residues through electrostatic interactions, van der Waals forces, and hydrogen bonding, thereby exerting effective inhibitory effects. Meanwhile, pharmacologic inhibition results comparing the TLR4 selective inhibitor TAK-242 with 3HP indicate that 3HP may partially target TLR4 activation, thereby inhibiting downstream NF-κB/NLRP3 signaling and mitigating the onset and progression of ALI.

It is undeniable that the anti-inflammatory mechanism of dexamethasone, a glucocorticoid currently used for ALI treatment, may differ from that of 3HP. Therefore, in this study exploring the pharmacological mechanism by which 3HP improves ALI, we did not use dexamethasone or similar agents as positive controls. This represents a limitation in terms of the lack of comparative efficacy data between the two agents. Moreover, although RAW264.7 macrophages, as a cell line for in vitro classical models of inflammation, provide a useful research basis for understanding the pharmacological mechanisms of 3HP in alleviating ALI in this study. However, considering its inherent limitations, to better understand the molecular pharmacological mechanism of 3HP, we plan to use bone marrow-derived macrophages, THP-1 cells, and lung epithelial cells (MLE12) to explore the communication between macrophages and lung epithelial cells more deeply in our future work. We also aim to reveal cell-cell interactions in lung tissues through single-cell sequencing analyses, with the goal of providing additional experimental insights. Meanwhile, with ongoing innovation and advances in modern molecular pharmacology tools, such as activity-based protein profiling (ABPP), a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins (53), will help reveal the direct targeting molecules of 3HP to alleviate ALI and provide a new basis for clinical ALI treatment. While 3HP exhibits higher lipid solubility than puerarin (33), the oral bioavailability of isoflavones is generally limited by poor gastrointestinal absorption and first-pass metabolism (54).Our study utilized oral gavage, which is effective in mice but may require optimization for human application.We currently lack data on 3HP’s lung tissue concentration, a critical parameter for ALI treatment, as natural products often suffer from low tissue penetration and 3HP’s ability to reach the lung at therapeutic concentrations awaits verification; To address these challenges, future research can focus on the following directions: developing targeted delivery systems to improve 3HP’s oral bioavailability and lung tissue accumulation (55). Conducting in vivo pharmacokinetic analyses to clarify its absorption, distribution, metabolism, and excretion profiles, particularly lung tissue concentration-time curves; evaluating 3HP’s efficacy in large animal ALI models to more closely mimic human physiology; and exploring the synergistic effects of 3HP with low-dose dexamethasone to reduce adverse effects while enhancing therapeutic efficacy. Overall, our findings highlight the potent anti-inflammatory effects of 3HP and preliminarily reveal its significant inhibition of TLR4 activation-mediated NF-κB p65/NLRP3/GSDMD signaling in mitigating the onset and progression of ALI.

5. Conclusion

Taken together, our results demonstrated that 3HP mitigated LPS-induced ALI by inhibiting TLR4 activation-mediated NF-κB p65/NLRP3/GSDMD signaling (Figure 9). These findings provide scientific evidence for the clinical treatment of ALI and present new insights into the pharmacological role of 3HP in mitigating acute lung inflammatory diseases.

Figure 9.

Pharmacological mechanism of 3HP in alleviating LPS-induced ALI in the current study.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This research was funded by the project of Hainan Medical University, grant number RZ2300007534.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Ethics statement

The animal study was approved by the Ethics Committee of Guangzhou University of Chinese Medicine approved all animal care and experimental procedures (No. 20240322007). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

HH: Investigation, Methodology, Writing – original draft. KW: Investigation, Writing – original draft. YQ: Investigation, Writing – original draft. ZX: Investigation, Writing – original draft. YC: Investigation, Writing – original draft. YH: Visualization, Writing – original draft. JH: Investigation, Methodology, Visualization, Writing – review & editing. ZL: Conceptualization, Investigation, Methodology, Visualization, Writing – review & editing.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2026.1701778/full#supplementary-material