Integrated Molecular Docking and Dynamic Simulations Reveal Glycyrrhizic Acid Alleviates Allergic Rhinitis in Rats by Inhibiting the TLR4/NF‐κB/IL‐1β Pathway
Aug 2026· Journal of Immunological Research· Vol 2026· 0 citations· 35 references
Medicine
TL;DR
Structural and functional evidence is provided supporting GA as a promising targeted therapeutic candidate for AR by directly binding to core proteins of the TLR4/NF‐κB/IL‐1β pathway, inhibiting pathway activation, restoring Th1/Th2 immune balance, and suppressing inflammatory responses.
Abstract
Background Allergic rhinitis (AR) is a prevalent chronic inflammatory nasal disorder with suboptimal current therapies. Glycyrrhizic acid (GA), a bioactive triterpenoid from Glycyrrhiza uralensis, exerts well‐documented antiinflammatory effects, and preliminary evidence indicates its efficacy in alleviating airway inflammation in AR models. However, two critical knowledge gaps remain unaddressed: whether GA directly binds to core proteins of the toll‐like receptor 4 (TLR4)/nuclear factor‐kappa B (NF‐κB)/IL‐1β inflammatory pathway, and whether this binding mediates GA’s therapeutic effects in AR have not been systematically verified by integrated computational and in vivo experiments. Methods An ovalbumin (OVA)‐induced AR rat model was established. GA was administered intranasally for seven consecutive days. Behavioral observations, histopathological examination, and serum cytokine detection were performed to evaluate AR‐related symptoms and immune imbalance. Molecular docking was employed to assess the binding affinity between GA and four key proteins in the TLR4 pathway (TLR4, myeloid differentiation primary response 88 [MyD88], NF‐κB, and IL‐1β). About 100 ns molecular dynamics (MD) simulations were further conducted to validate the stability of the GA–protein complexes. Immunohistochemistry and RT‐qPCR were used to verify the expression of pathway‐related proteins and cytokines in nasal mucosal tissues. Results GA significantly reduced sneezing, rhinorrhea, and nasal mucosal pathological damage in AR rats. It restored the T helper 1 (Th1)/Th2 immune balance by suppressing Th2 cytokines (IL‐4 and IL‐13) and enhancing Th1 cytokines (interferon‐gamma [IFN‐γ] and IL‐2). Molecular docking results showed that GA bound strongly to all four target proteins, with the highest affinity for IL‐1β (−9.3 kcal/mol) and TLR4 (−7.6 kcal/mol). MD simulations confirmed the stable conformational dynamics of the GA–IL‐1β and GA–TLR4 complexes. In vivo experiments further demonstrated that GA significantly downregulated the expression of TLR4, MyD88, NF‐κB, and IL‐1β in nasal mucosa and reduced the levels of downstream proinflammatory cytokines (TNF‐α and IL‐6). Conclusions GA alleviates AR symptoms by directly binding to core proteins of the TLR4/NF‐κB/IL‐1β pathway, inhibiting pathway activation, restoring Th1/Th2 immune balance, and suppressing inflammatory responses. This study provides structural and functional evidence supporting GA as a promising targeted therapeutic candidate for AR.
Chronic rhinosinusitis (CRS) frequently co-occurs with depressive disorders. TCF4 aberrant expression is strongly linked to CRS with depression-like behaviors (CRSDB), highlighting the urgent need for TCF4-targeted drugs to treat this complex comorbidity. This study validated the reproducibility and stability of the previously developed mouse model of CRSDB by assessing nasal and hippocampal histopathology via H&E, PAS, and Nissl staining. ELISA determined inflammatory cytokine levels. TCF4’s role was explored through lentivirus-mediated in vivo and in vitro microglial knockdown. TLR4/NF-κB pathway regulation by TCF4 was confirmed via immunofluorescence, Western Blot, and RT-qPCR. Metformin’s therapeutic effect was tested, with its TCF4 targeting verified by molecular docking and in vitro experiments. The study comprehensively links TCF4 to neuroinflammation in CRSDB, highlighting metformin’s potential as a therapeutic agent. The CRSDB model exhibited excellent reproducibility and time-dependent exacerbation of depression-like behaviors, cognitive deficits, and concurrent nasal and hippocampal inflammation. Mechanistically, CRSDB induced TCF4 upregulation, which activated the TLR4/NF-κB signaling pathway, leading to microglial activation and neuroinflammation. Knockdown of TCF4 significantly alleviated behavioral impairments, suppressed microglial activation, and mitigated peripheral inflammation. Furthermore, We identified TCF4 as a direct target of metformin, through which it inhibits the TLR4/NF-κB pathway and subsequent microglial activation. TCF4 has been confirmed as a key regulatory mediator of CRSDB, and it has been demonstrated that metformin exerts its antidepressant effect by specifically targeting TCF4 in microglia to inhibit the TLR4/NF-κB axis.
ETHNOPHARMACOLOGICAL RELEVANCE
Jiu-Wei-Yong-An (JWYA) decoction is a clinical traditional Chinese medicine prescription for atopic dermatitis (AD). Its anti-inflammatory and anti-pruritic activities have been previously confirmed, yet the underlying molecular mechanism remains unclear.
AIM OF STUDY
This work aimed to identify JWYA's bioactive compounds and clarify its therapeutic mechanisms against AD.
MATERIALS AND METHODS
We established MC903-induced AD mouse models and TNF-α/IFN-γ-stimulated HaCaT keratinocytes to assess JWYA's efficacy. LC-MS/MS combined with bioinformatics, public microarray datasets, and compound-target prediction was used to screen active components and core pathways. In vivo dermatitis severity and epidermal thickness were quantified after 14-day JWYA oral administration. Western blotting detected pathway protein expression in vitro and in vivo. TLR4 inhibitor TAK-242 rescue assays verified TLR4-dependent regulation. Molecular docking, molecular dynamics (MD) simulations, DARTS and CETSA assays further validated the direct binding of candidate compounds to TLR4/MyD88/NF-κB.
RESULTS
Thirteen absorbable ingredients of JWYA were identified, with the TLR4/MyD88/NF-κB cascade screened as the core anti-AD pathway. JWYA alleviated AD-like lesions, reduced IgE and proinflammatory cytokines, and suppressed TLR4/MyD88/NF-κB overactivation in mice and keratinocytes; TAK-242 assays confirmed JWYA's effects rely on intact TLR4 signaling. Phylliroside and Suspenoidside B exhibited high affinity toward core targets, with direct physical binding validated by MD, DARTS and CETSA.
CONCLUSIONS
JWYA ameliorates AD through multi-component, multi-target modulation of the TLR4/MyD88/NF-κB inflammatory axis. Phylliroside and Suspenoidside B are key bioactive substances that directly bind TLR4/MyD88/NF-κB to inhibit NF-κB hyperactivation, relieving AD-related inflammation. This study provides experimental basis for JWYA's clinical application and subsequent monomer drug development.
Han-Wen Zhang, Qinwufeng Gu, Yuanyuan Meng et al.· Journal of Ethnopharmacology· 0 citations
Rheumatoid arthritis (RA) is a chronic immune-mediated disease in which persistent inflammation and oxidative stress contribute to progressive joint damage. This study evaluated the prophylactic effects of sabinene in complete Freund's adjuvant (CFA)-induced arthritis and explored potential molecular associations using integrated in vivo and computational approaches. Rats were allocated to vehicle control, arthritic control, piroxicam, or sabinene groups (15, 30, or 60 mg/kg; n = 6/group); sabinene was administered orally beginning 30 minutes before CFA injection and continued for 28 days. Paw swelling, hematological and biochemical parameters, inflammatory and oxidative-stress markers, and gene expression were assessed, followed by network pharmacology, enrichment analysis, and molecular docking. Sabinene dose-dependently attenuated CFA-induced paw swelling and body-weight loss and improved hematological, hepatic, renal, and inflammatory parameters. Treatment also enhanced antioxidant defenses and reduced lipid peroxidation, prostaglandin E₂, 5-lipoxygenase, and anti-cyclic citrullinated peptide antibody levels. Sabinene reduced messenger RNA expression of nuclear factor kappa B (NF-κB), toll-like receptor 4 (TLR4), NOD-like receptor family pyrin domain-containing 3 (NLRP3), caspase-1, gasdermin D, and other pro-inflammatory genes while increasing interleukin-4 and interleukin-10 expression. Network pharmacology identified 133 overlapping sabinene-RA targets, and molecular docking predicted interactions with tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), signal transducer and activator of transcription 3 (STAT3), and interferon gamma (IFN-γ). These findings support a prophylactic protective effect of sabinene against CFA-induced arthritis associated with reduced inflammation, improved antioxidant status, and modulation of inflammasome-related gene expression. Further protein-level, functional, and post-induction studies are required to confirm the proposed mechanisms and therapeutic relevance.
Kanwal Asif, Ambreen Malik Uttra, Arham Shabbir et al.· Biomolecules & biomedicine· 0 citations
Allergic rhinitis (AR) is a prevalent inflammatory disorder of the upper airways, and exposure to environmental endocrine-disrupting chemicals such as bisphenol A (BPA) and bisphenol F (BPF) has been implicated in its pathogenesis, yet the underlying molecular mechanisms remain poorly understood. We integrated network toxicology, bioinformatics, and machine learning approaches to identify key target genes linking BPA/BPF exposure to AR, followed by molecular docking, molecular dynamics simulations, and surface plasmon resonance (SPR) to evaluate binding affinities. In vitro experiments using human nasal epithelial cells (HNEpC) were conducted to validate the effects of BPA/BPF on cell viability, apoptosis, inflammatory cytokines, senescence-associated secretory phenotype (SASP), and the NF-κB signaling pathway, with further functional assessment via MMP9 overexpression. Four common genes (MAPT, MMP9, CHRM3, ESR2) were identified for BPA and three (MMP9, CHRM3, ESR2) for BPF. SPR confirmed direct binding of both bisphenols to MMP9 with KD values of 3.46 μM (BPA) and 9.47 μM (BPF). BPA/BPF treatment inhibited cell viability, suppressed MMP9, promoted apoptosis, upregulated IL-4, IL-6, IL-8, IL-13, CCL2, TNF-α, and IL-1β, downregulated MMP1, and suppressed the NF-κB pathway. MMP9 overexpression reversed these effects. Our findings demonstrate that BPA and BPF promote AR pathogenesis through MMP9 suppression, SASP activation, and NF-κB inhibition, identifying MMP9 as a potential therapeutic target for pollutant-exacerbated AR.
Min Liu, Tianxin Zhao, Yi-Jun Liu et al.· Toxicology and Applied Pharm...· 0 citations
ETHNOPHARMACOLOGICAL RELEVANCE
Euphorbium, the dried latex from Euphorbia resinifera, is classic traditional medicinal material used to treat various diseases in Uyghur medicine and has demonstrated significant efficacy in the intervention of inflammatory disorders. Total triterpenes extracted from Euphorbium (TTE) and its major monomer euphol possess obvious anti-inflammatory activities, but their systematic mechanism regulating macrophage inflammation remains insufficiently clarified.
AIM OF THE STUDY
The present work was designed to verify the in vivo anti-inflammatory efficacy of TTE using a carrageenan-induced acute inflammatory model, and further clarify the anti-inflammatory mechanisms of TTE and its major active monomer euphol in LPS-triggered RAW264.7 macrophages and acute inflammatory animals models, focusing on the TLR4/MyD88/NF-κB signaling cascade and NLRP3-related signaling.
MATERIAL AND METHODS
In vivo, a carrageenan-induced acute rat paw edema model was established to preliminarily evaluate the anti-inflammatory effect of TTE and observe paw tissue histopathological damage. On the basis of confirmed efficacy of TTE, in vitro, RAW264.7 macrophages experiments were further conducted to exclude the molecular mechanism. Both TTE and its major active component euphol were applied in cell experiments. Cells were pretreated with TTE and euphol respectively followed by LPS stimulation. The secretion of multiple inflammatory mediators and cytokines was quantified via biochemical detection and ELISA assays. The protein and transcriptional profiles of core molecules associated with TLR4/MyD88/NF-κB pathway and NLRP3-related signaling were determined by Western blot and qRT-PCR, respectively. Furthermore, immunofluorescence staining was conducted to qualitatively observe the regulatory effects of TTE and euphol on LPS-induced NF-κB p65 nuclear translocation.
RESULTS
In vivo results showed that TTE obviously alleviated carrageenan-induced acute paw edema and reduced tissue inflammatory infiltration. Further in vitro cellular experiments demonstrated that both TTE and euphol markedly suppressed the overproduction of NO, PGE2, MCP-1 and core pro-inflammatory cytokines, downregulated the expression of iNOS and COX2 at both protein and mRNA levels. Mechanistically, both interventions coincided with decreased protein levels of TLR4, MyD88, NLRP3, ASC and caspase-1, alongside reduced phosphorylation of NF-κB p65 and IκBα. Correspondingly, the transcriptional levels of key genes involved in NF-κB and NLRP3-related signaling were also dramatically decreased. Consistent with the altered p65 phosphorylation status, immunofluorescence observations further verified that TTE and euphol correlated with weakened LPS-triggered NF-κB p65 nuclear accumulation, providing direct phenotypic evidence consistent with attenuated NF-κB signaling activation.
CONCLUSION
TTE exerts prominent in vivo anti-inflammatory effects in acute inflammatory injury. Both TTE and its active component euphol exert potent anti-inflammatory effects in vitro by dual regulation of the TLR4/MyD88/NF-κB inflammatory cascade and NLRP3-related signaling. These findings provide a basis for the verification of the traditional efficacies of Euphorbium.
Zhou Bai, Yan Liu, PanPan Yang et al.· Journal of Ethnopharmacology· 0 citations
Background Immunoglobulin A nephropathy (IgAN) is the most prevalent primary glomerulonephritis worldwide and a leading cause of end-stage renal disease, posing a significant threat to human health. Modified Huangqi Chifeng decoction (MHCD) has shown efficacy in ameliorating IgAN; however, its underlying regulatory mechanisms remain incompletely understood. Objective To evaluate the anti-inflammatory and anti-fibrotic effects of MHCD on IgAN and elucidate its molecular mechanisms through in vivo and in vitro experiments. Methods An IgAN rat model was established. The therapeutic effects of MHCD were assessed by measuring 24-hour urinary protein, Gd-IgA1, serum biochemistry, renal pathological damage, IgA deposition, inflammatory mediators, and fibrosis levels after 8 weeks of MHCD administration. ELISA was used to quantify inflammatory mediators; Western blot and immunohistochemistry were employed to analyze protein expression; PCR was performed to determine miR-146a levels. Subsequently, lipopolysaccharide-stimulated mesangial cells were used in vitro. Mesangial cell proliferation and the expression of TLR4/NF-κB signaling pathway components and downstream inflammatory factors was examined to verify the anti-inflammatory and anti-fibrotic mechanism of MHCD by overexpressing or silencing miR-146a. Results MHCD reduced urinary protein and serum Gd-IgA1 in IgAN rats, and alleviated renal IgA deposition, pathological injury, inflammation and fibrosis. Furthermore, MHCD inhibited renal expression of TLR4, MyD88, NF-κB p65 and NF-κB p-p65, and increased the level of miR-146a. Mechanistically, in vitro experiments demonstrated that MHCD exerted anti-inflammatory and anti-fibrotic effects by upregulating miR-146a and inhibiting the TLR4/NF-κB signaling pathway. Conclusion MHCD alleviates IgA nephropathy inflammation and renal fibrosis partially through upregulating miR-146a and subsequently suppressing the TLR4/NF-κB signaling pathway.
Yue Shi, Jing Liu, Sijia Ma et al.· Frontiers in Immunology· 0 citations