Dehydroandrographolide attenuates Toll-like receptor signaling by dual inhibition of MyD88- and TRIF-dependent pathways

Dehydroandrographolide attenuates Toll-like receptor signaling by dual inhibition of MyD88- and TRIF-dependent pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dehydroandrographolide attenuates Toll-like receptor signaling by dual inhibition of MyD88- and TRIF-dependent pathways Ye Eun Lee, Hanbin Ko, Dongwoo Lee, Younghyun Lee, Gyo Jeong Gu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8098180/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Background Toll-like receptors (TLRs) are key mediators of innate immune responses, and their dysregulation contributes to inflammatory diseases. Dehydroandrographolide (DAG), a diterpene lactone from Andrographis paniculata, is known for its anti-inflammatory activity, but its effects on individual branches of TLR signaling remain unclear. Methods RAW264.7 and 293T cells were used to evaluate the effects of DAG on MyD88- and TRIF-dependent signaling pathways. Luciferase reporter assays, Western blotting, RT-PCR, and nitrite assays were employed to assess NF-κB and IRF3 activation and inflammatory mediator expression. Statistical analysis was performed using one-way ANOVA. Results DAG significantly inhibited activation of NF-κB and IRF3 induced by TLR agonists (LPS, MALP-2, and Poly[ 1 ]) and by overexpression of MyD88- and TRIF-associated downstream molecules. Correspondingly, DAG suppressed iNOS, IFNβ, and IP-10 expression, indicating dual inhibition of both TLR signaling branches. Conclusions DAG exerts dual inhibitory effects on MyD88- and TRIF-dependent TLR signaling, attenuating inflammatory mediator expression. These findings suggest its potential as a therapeutic compound for inflammatory disorders. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Immunology Biological sciences/Molecular biology Toll-like receptor signaling Dehydroandrographolide MyD88-dependent pathway TRIF-dependent pathway Anti-inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Background Inflammation represents one of the body’s primary defense responses against microbial invasion and tissue injury [ 2 ]. This process involves intricate interactions among various cell types and signaling molecules, typically accompanied by vasodilation, increased leukocyte recruitment, and the release of pro-inflammatory mediators [ 3 ]. Such physiological changes play a crucial role in safeguarding damaged tissue and initiating its repair [ 4 ]. Based on duration and nature, inflammation can be classified into acute and chronic forms. Acute inflammation develops quickly in response to injury, infection, or tissue damage and is generally short-lived [ 5 ]. In contrast, chronic inflammation persists over extended periods, often resulting from continuous irritation or unresolved injury, and it can cause structural tissue alterations and damage. Persistent inflammatory activity is implicated in the onset and progression of many inflammatory disorders [ 6 ]. Toll-like receptors (TLRs) are a well-characterized subgroup of pattern recognition receptors (PRRs) that detect conserved molecular patterns from microbes and trigger immune responses [ 7 ]. They mediate the inflammatory reaction against pathogens including bacteria, viruses, and fungi, serving as a vital bridge between the innate and adaptive branches of immunity. TLRs also sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to initiate and fine-tune inflammatory cascades [ 8 ]. Ten types of TLRs have been identified in humans and thirteen in mice; they are located either on the plasma membrane or within endosomal compartments [ 9 ]. Membrane-localized TLRs generally recognize structural components of microbial surfaces, such as lipoproteins, lipopolysaccharides, and flagellins, whereas endosomal TLRs specialize in detecting microbial nucleic acids like double-stranded RNA, single-stranded RNA, or unmethylated CpG DNA [ 10 ]. Upon ligand recognition, TLRs transmit signals through two main routes: the MyD88- and the TRIF-dependent pathway. The MyD88-dependent route, used by most TLRs, primarily promotes the expression of inflammatory cytokines and chemokines. Meanwhile, the TRIF-dependent pathway is engaged by certain TLRs and induces production of interferons, while also regulating NF-κB via interaction with RIP1[ 11 ] . Dysregulation of TLR signaling has been linked to a variety of inflammatory and autoimmune disorders, as well as infectious diseases [ 12 ]. Thus, agents capable of modulating TLR pathways represent promising tools for controlling inflammation. Notably, medicinal plants—many of which possess both anti-cancer and anti-inflammatory activities—offer a valuable source of bioactive compounds that may support immune regulation and inflammation management [ 13 ]. Andrographis paniculata , belonging to the family Acanthaceae , has been traditionally utilized across China, India, and numerous Southeast Asian regions for centuries, primarily owing to its notable antipyretic and anti-inflammatory activities [ 14 ]. In traditional Chinese medicine, Andrographis paniculata has been prescribed for the management of persistent or recurrent infectious and inflammatory disorders, particularly those affecting the upper respiratory tract and causing intestinal diarrhea [ 15 – 17 ]. Among its bioactive constituents, dehydroandrographolide (DAG) has been identified as a compound with substantial immunomodulatory potential (Fig. 1 A) [ 1 ]. Despite these findings, the precise molecular interactions between dehydroandrographolide and TLRs in regulating immune responses have yet to be fully elucidated. This investigation, therefore, seeks to clarify the modulatory effects of dehydroandrographolide on TLR-mediated signaling, with specific emphasis on the MyD88- and TRIF-dependent pathways, aiming to advance understanding of its immune-regulating properties and explore its therapeutic prospects in inflammatory pathologies. 2. Methods 2.1. Aim, design, and setting of the study The aim of this study was to investigate the anti-inflammatory mechanisms of dehydroandrographolide (DAG), focusing on its regulatory effects on Toll-like receptor (TLR) signaling pathways. Specifically, we sought to determine whether DAG modulates both MyD88- and TRIF-dependent signaling cascades, which are two major branches of TLR-mediated immune responses. This study was designed as an in vitro experimental study using murine macrophage RAW264.7 cells and human embryonic kidney 293T cells, which are well-established models for analyzing TLR signaling and transcriptional activation. The experiments were conducted under controlled laboratory conditions at the Department of Biomedical Laboratory Science, Soonchunhyang University (Asan, Republic of Korea). 2.2. Reagents DAG was purchased from Cayman chemical and diluted in dimethylsulfoxide(DMSO). Lipopolysaccharide (LPS) was obtained from List Biological Laboratories (Ann Arbor, Michigan, USA; Cat. No.36841). Macrophage-activating lipopeptide-2 (MALP-2) was purchased from Alexis Biochemical (San Jose, CA, USA; Cat.No.421). Polyinosinic-polycytidylic acid (Poly[ 1 ]) was purchased from InvivoGen (San Diego, CA, USA; Cat.No. tlrl-pic-5). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise described. 2.3. Cell culture RAW264.7 cells (a murine monocytic cell line; ATCC TIB-71) and human embryonic kidney 293T cells(ATCC CRL-3216) were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C with 5% CO 2 atmosphere in a humidified incubator until confluence. 2.4. Cell viability test Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric assay. RAW 264.7 cells were treated with each compound for 4 h. Twenty microliters of the CellTiter 96 AQueous One Solution reagent (Promega, Madison, WI, USA) was added directly to the culture wells. The plate was then incubated for 4 h in a humidified, 5% CO 2 atmosphere, after which the absorbance at 490 nm was recorded using a 96-well plate reader. 2.5. Transfection and luciferase reporter gene assay Cells were seeded into 48-well plates at a density of 0.8 × 10 5 cells/ml and incubated at 37℃ in a 5% CO 2 /95% air environment. RAW264.7 and 293T cells were transfected with a luciferase plasmid and a HSP70-β‐galactosidase plasmid as an internal control using a p3000 and lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Cells were then treated with LPS, MALP-2, and Poly[I:C] for 8 h after being treated with DAG (20, 50µM) for 1 h. Luciferase and β‐galactosidase enzyme activities were determined using a Luciferase Assay System (Promega). Luciferase activity was normalized against β‐galactosidase activity. 2.6. Western blotting analysis Cells were seeded into 6-well plates at a density of 1.0 × 10 6 cells/ml and incubated at 37℃ in a 5% CO 2 /95% air environment for 48 h. RAW264.7 cells were pretreated with DAG (20, 50µM) for 1 h. They were then treated with agonists for 8 h. Total protein was extracted from cell lysates using RIPA lysis buffer. Cell lysates were subjected to 12% and 14% sodium dodecyl sulfate–polyacrylamide gel electrophoresis to separate proteins. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with PBS containing 0.1% Tween 20 and 5% nonfat dry milk for 24 h to prevent non-specific binding of antibodies. After the blocking step, membranes were incubated with specific primary antibodies and secondary antibodies conjugated with horseradish peroxidase (GE Healthcare, Chicago, IL, USA). Reactive bands were visualized using a Western Blot Detection System (iNtRON, Songnam, Korea). Membranes were stripped with 0.5 N NaOH for 20 min to reprobe with different antibodies. 2.7. Nitrite assay RAW 264.7 macrophages at a concentration of 0.8 × 10 5 cells/ml were seeded in 48-well plates and incubated for 24 h. Subsequently, they were treated with each compound in the presence or absence of LPS, MALP-2, or Poly[I:C] for 18 h. Samples (100 µl) of the culture medium were incubated with 150 µl Griess reagent (1% sulfanilamide and 0.1% naphthylethylene diamine in a 2.5% phosphoric acid solution) at room temperature for 5 min in a 96-well microplate. The absorbance at 570 nm was read using a plate reader, and the concentration of NO was then determined by the preparation of a standard calibration curve, using sodium nitrite as the standard. 2.8. Real-time RT-PCR analysis of IFNβ expression Total RNA was extracted using Ribospin™ (GeneAll, Seoul, Korea) according to the manufacturer's instructions. Total RNA (5 µg) was reverse-transcribed using a HyperScript™ for RT-PCR (GeneAll) and amplified with a Step One Plus Real-Time PCR System (Applied Biosystems) using a Power SYBR Green PCR Master kit (Applied Biosystems). Primers used to detect mouse IFNβ were as follows: forward primer 5′-TCCAAGAAAGGACGAACATTCG-3′, and reverse primer 5′-TGAGGACATCTCCCACGTCAA-3′. Primers for mouse β-actin (used as an internal control) were: forward primer 5′-TCATGAAGTGTGACGTTGACATCCGT-3′, and reverse primer 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′. The following PCR conditions were used: denaturation at 95°C for 10 min; and 40 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s. The specificity of PCR was assessed using a melting curve analysis. Fold induction of IFNβ expression was measured by real-time PCR in triplicate experiments relative to the vehicle control. 2.9. Data analysis Data were obtained from triplicate experiments. Values were expressed as the mean ± standard error of the mean (SEM). Differences in the data were evaluated using one-way ANOVA, with a P -value of less than 0.05 considered statistically significant. All analyses were conducted using the GraphPad Prism software version 10.3 (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Cytotoxicity of DAG The cytotoxic effect of DAG was evaluated in RAW 264.7 cells using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric assay. Cell viability was 91.4% at 50 µM DAG and decreased to 93.0% at 100 µM DAG (Fig. 1 B). Based on these results, a maximum concentration of 50 µM DAG was used in most subsequent experiments. 3.2. DAG suppresses NF-κB activation induced by TLR4 or TLR2 and TLR6 agonists NF-κB is a transcription factor that plays an important role in the human immune response [ 18 ], and activation of NF-κB through the MyD88-dependent pathways and TRIF-dependent pathways leads to inflammation by regulating the expression of several target genes. This study, therefore, examined whether LPS (TLR 4 agonist) or MALP-2 (TLR 2 and TLR 6 agonist) inhibits the activity of NF-κB using a luciferase reporter gene assay. The results indicate that DAG significantly inhibited NF-κB induced by LPS (Fig. 2 A) or MALP-2 (Fig. 2 B). This result indicates that DAG can inhibit the activity of NF-κB by modulating the signal transduction system through TLRs. 3.3. DAG suppresses iNOS expression induced by TLR4 or TLR2 and TLR6 agonists When TLRs recognize various agonists and send signals downstream, they induce the activation of NF-κB. Activated NF-κB induces the expression of inflammatory genes such as iNOS [ 19 ]. Therefore, this study investigated whether DAG could modulate iNOS expression induced by LPS or MALP-2. According to the iNOS-luciferase reporter gene assay (Fig. 3 A and B) and Western blot analysis (Fig. 3 C and D), DAG inhibited iNOS expression induced by LPS or MALP-2 in RAW 264.7 cells. Additionally, we were confirmed that the concentration of nitrite produced by iNOS decreased (Fig. 3 E and F). 3.4 DAG suppresses NF-κB activation induced by MyD88 downstream signaling components of TLRs MyD88-dependent pathway is the canonical adaptor of the inflammatory signaling pathway of Toll-like receptors [ 20 ]. From the above results, we found that DAG could inhibit the activity of NF-κB through the TLR signaling pathway. Therefore, we conducted experiments to determine whether DAG could also regulate the activation of NF-κB through the MyD88 signaling pathway. We transfected MyD88, IKKβ, and p65, which are downstream components of MyD88 dependent pathway, into 293T cells and performed a luciferase reporter gene assay. The results indicate that NF-κB induced by MyD88 (Fig. 4 A), IKKβ (Fig. 4 B), and p65 (Fig. 4 C) is significantly inhibited by DAG, suggesting that the molecular target of DAG is the MyD88-dependent downstream signaling components. 3.5. DAG suppresses IRF3 activation induced by TLR4 agonist IRF3 is activated exclusively through the TRIF signaling pathway. Therefore, we used LPS (TLR4 agonist) to activate TLR4, which signals through both the MyD88-dependent pathway and TRIF-dependent pathways. Therefore, it is employed to determine whether DAG can regulate the TRIF signaling pathway. Using a luciferase reporter gene assay with the IFNβ promoter domain containing the IRF3 binding site (IFNβ PRDIII-I), we confirmed that DAG significantly inhibited the activity of IRF3 induced by LPS (Fig. 5 A). These results are further confirmed by RT-PCR (Fig. 5 B). To further investigate the regulation of TRIF by DAG, we examined the expression of IP-10, so we confirmed by IP-10-luciferase reporter gene assay (Fig. 5 C) and Western blot (Fig. 5 D) that IP-10 induced by LPS is significantly inhibited by DAG. 3.6. DAG suppresses NF-κB activation and iNOS expression induced by TLR3 agonists TLR3 exclusively utilizes the TRIF pathway. Therefore, we used Poly[I:C] (a TLR3 agonist) to observe the regulation of NF-κB via the TRIF pathway. Our results showed that NF-κB induced by Poly[I:C] (Fig. 6 A) is significantly inhibited by DAG. This finding indicates that DAG can inhibit NF-kB activation through the TRIF-dependent pathway. Additionally, we confirmed that DAG inhibits Poly[I:C] induced iNOS expression using the iNOS-luciferase reporter gene assay (Fig. 6 B) and Western blot analysis (Fig. 6 C). Furthermore, a reduction in nitrite levels is observed in the nitrite assay (Fig. 6 D). 3.7. DAG suppresses IRF3 activation induced by TLR3 agonist We investigated whether DAG could inhibit the activation of IRF3 induced by the TLR3 agonist Poly[I:C]. The results showed that DAG significantly inhibited Poly[I:C] induced IRF3 activation, as shown by the luciferase reporter gene assay (Fig. 7 A). This inhibition is further confirmed by RT-PCR analysis (Fig. 7 B). Additionally, the IP-10 luciferase reporter gene assay (Fig. 7 C) and Western blot analysis (Fig. 7 D) also corroborated that DAG can inhibit IRF3 activation. The results indicate that DAG can regulate the TRIF signaling system. Therefore, we conducted experiments to identify molecular targets within the TRIF signaling system. TRIF, TBK1, and IRF3 are TRIF downstream components and are transfected into 293T cells to perform luciferase reporter gene assay. These findings indicate that IRF3, induced by TRIF (Fig. 8 A), TBK1 (Fig. 8 B), and IRF3 5D (Fig. 8 C), is significantly inhibited by DAG. These results indicate that the molecular target of DAG is above the TRIF signaling system. 4. Discussion This study demonstrates that DAG exerts broad inhibitory effects on Toll-like receptor (TLR)–mediated inflammatory responses by targeting both MyD88-dependent and TRIF-dependent pathways [ 21 ]. Using three distinct TLR agonists, LPS, MALP-2, and Poly[I:C], we observed that DAG attenuated downstream activation of key transcription factors, including NF-κB and IRF3, and consequently reduced the expression of inflammatory mediators such as iNOS and IP-10. The findings highlight a multifaceted role of DAG in modulating innate immune signaling. LPS, a well-characterized ligand for TLR4, activates both MyD88- and TRIF-mediated cascades, resulting in robust induction of proinflammatory cytokines. MALP-2, in contrast, signals primarily through TLR2 and TLR6, engaging the MyD88-dependent route [ 22 ]. Poly[I:C], as a synthetic analog of viral double-stranded RNA, activates TLR3 within endosomes and preferentially signals through TRIF [ 22 ]. Despite these differences in receptor specificity and signaling routes, DAG consistently suppressed downstream activation events, suggesting that its inhibitory effect occurs at a shared or converging point within these pathways. A notable aspect of this study is the concurrent suppression of NF-κB and IRF3 activation. NF-κB is a central regulator of inflammatory gene expression, including iNOS, which plays a critical role in nitric oxide production and inflammation resolution [ 23 ]. IRF3, on the other hand, is essential for antiviral defense, driving type I interferon production and chemokine expression such as IP-10 [ 24 , 25 ]. The simultaneous downregulation of these two transcription factors indicates that DAG’s action is not restricted to a single branch of the TLR signaling network but instead encompasses broader regulatory control. This observation expands upon previous findings that primarily focused on the TLR4–NF-κB axis, suggesting that DAG may influence common signaling intermediates shared by multiple TLRs rather than acting at the receptor level alone [ 26 , 27 ]. From a therapeutic perspective, these results suggest that DAG could serve as a promising candidate for treating diseases characterized by excessive or chronic inflammation. By targeting both MyD88- and TRIF-dependent pathways, DAG may offer an advantage over agents that selectively inhibit a single signaling route. Furthermore, the suppression of both proinflammatory and antiviral signaling suggests potential applicability in conditions where immune overactivation contributes to pathology, such as autoimmune disorders or viral-induced hyperinflammation. Notably, this dual regulatory property aligns with the traditional use of Andrographis paniculata extracts for managing systemic inflammatory and infectious conditions, thereby providing a molecular rationale that bridges empirical herbal efficacy with modern immunological mechanisms [ 27 ]. However, this study also raises important questions regarding the precise molecular target(s) of DAG within TLR signaling. Whether DAG interacts directly with adaptor proteins (e.g., MyD88 or TRIF), kinases, or transcriptional regulators remains to be elucidated.. Future work using phospho-proteomic or docking-based analyses could clarify whether DAG interferes with post-translational activation steps or adaptor assembly complexes. 5. Conclusion Our findings position DAG as a dual-pathway modulator of TLR signaling, capable of suppressing key inflammatory mediators through inhibition of both NF-κB and IRF3. This dual mechanism provides a strong rationale for further mechanistic and translational studies, with the ultimate goal of evaluating its therapeutic potential in inflammatory and immune-mediated diseases. our findings position DAG as a dual-pathway modulator of TLR signaling, capable of suppressing key inflammatory mediators through inhibition of both NF-κB and IRF3. This dual mechanism provides a strong rationale for further mechanistic and translational studies, with the ultimate goal of evaluating its therapeutic potential in inflammatory and immune-mediated diseases. Such investigations may also reveal whether DAG’s mode of action can be leveraged for multi-target modulation strategies that restore immune balance without complete suppression of innate defense responses. Abbreviations DAG Dehydroandrographolide TLR Toll-like receptor NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells IRF3 Interferon regulatory factor 3 iNOS Inducible nitric oxide synthase TRIF TIR-domain-containing adapter-inducing interferon-β MyD88 Myeloid differentiation primary response 88 Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests Funding Soonchunhyang University Research Fund financially supports this study. Author Contribution Y.E.L. and H.B.K. performed the experiments, curated the data, and conducted the formal analysis. D.W.L. contributed to the investigation. Y.H.L. provided advisory support. H.S.Y. and G.J.G. supervised the study, administered the project, secured funding, and were involved in writing the original draft as well as reviewing and editing the manuscript. 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Supplementary Files SupplementaryinformationDAGwesternblot.pdf Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 09 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviewers agreed at journal 06 Jan, 2026 Reviewers agreed at journal 19 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviewers agreed at journal 15 Dec, 2025 Reviewers agreed at journal 12 Dec, 2025 Reviewers invited by journal 12 Dec, 2025 Editor assigned by journal 12 Dec, 2025 Editor invited by journal 10 Dec, 2025 Submission checks completed at journal 28 Nov, 2025 First submitted to journal 28 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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18:15:07","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79132,"visible":true,"origin":"","legend":"","description":"","filename":"34bd2fa2049248178e8284266cba1df01structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/ac5d1593781847a8a5fd5961.xml"},{"id":98454914,"identity":"dad470bf-bed4-4f47-ae27-522604e2ec6e","added_by":"auto","created_at":"2025-12-17 18:15:07","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89137,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/e598760a0157f6a113982ddf.html"},{"id":98454891,"identity":"8be6e3bc-0f3d-403c-a0e2-f83f3922e598","added_by":"auto","created_at":"2025-12-17 18:15:06","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92481,"visible":true,"origin":"","legend":"\u003cp\u003eThe structure of dehydroandrographolide (DAG) (A) and Cell viability assay (B). RAW264.7 cells were treated with epoxomicin (20, 50, or 100 μM) for 4 h. The CellTiter 96 AQ\u003csub\u003eueous \u003c/sub\u003eOne Solution Reagent (20 ml/well) was added directly to culture wells. The plate was incubated at 37℃ for 4 h in a humidified, 5% CO\u003csub\u003e2 \u003c/sub\u003eatmosphere. The absorbance was recorded at 490 nm with a 96-well plate reader. Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/742f2be7763467a19c2eae3d.jpeg"},{"id":98623834,"identity":"91fa81eb-93f0-49e1-abf6-bd45c8bf94fe","added_by":"auto","created_at":"2025-12-19 17:07:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209040,"visible":true,"origin":"","legend":"\u003cp\u003eDAGsuppresses NF-κB activation induced by LPS and MALP-2. (A,B) RAW264.7 cells were transfected with NF-κB luciferase reporter plasmid, pre-treated with DAG (20 or 50 μM)for 1 h, and then treated with LPS or MALP-2 for an additional 8 h. Cell lysates were prepared and the luciferase enzyme activities were determined. Relative luciferase activity was normalized with b-gal activity. Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM. Statisticalsignificance was determined using a one-way ANOVA. *\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/dcfeab347818f192835493bb.jpeg"},{"id":98454896,"identity":"3712a370-ac25-4779-93b6-91498ad5886f","added_by":"auto","created_at":"2025-12-17 18:15:06","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":237221,"visible":true,"origin":"","legend":"\u003cp\u003eDAG inhibits iNOS expression induced by LPS and MALP-2\u003cstrong\u003e.\u003c/strong\u003e (A,B)RAW264.7 cells were transfected with iNOS luciferase reporter plasmid and pretreated with 20 or 50 μM DAG for 1h and then treated with LPS (A) or MALP-2 (B) for an additional 8 h. Cell lysates were prepared and luciferase enzyme activities were determined. Relative luciferase activity was normalized with b-gal activity. (C,D) RAW264.7 cells were pretreated with 20 or 50 mM DAG for 1 h and then further stimulated with LPS (C) or MALP-2 (10 ng/ml) (D) for an additional 8 h. Cell lysates were analyzed for iNOS and b-actin protein by immunoblots. (E,F) RAW 264.7 cells were pretreated with 20 or 50 mM DAG for 1 h and then treated with LPS (E), or MALP-2 (F), \u0026nbsp;for an additional 20 h. The amounts of nitrite in supernatant were measured using Griess reagent. Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM. Statistical significance was determined using a one-way ANOVA. *\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/0f86a735c9ce902cad6d86fa.jpeg"},{"id":98622844,"identity":"01ed3611-0840-4186-aba0-6c585805d4cb","added_by":"auto","created_at":"2025-12-19 17:02:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109147,"visible":true,"origin":"","legend":"\u003cp\u003eDAG suppresses NF-kB activation induced by downstream signaling components of MyD88-dependent pathway. (A-C) 293T cells were co-transfected with NF-kB luciferase reporter plasmid and the expression plasmid of MyD88 (A), IKKb (B), or p65 (C). Cells were further treated with DAG (20 or 50 μM) for 18 h. Relative luciferase activity was normalized with b-gal activity. Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM. Statistical significance was determined using a one-way ANOVA. *\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05; ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/01e63272099ad3ee52464a23.jpeg"},{"id":98622980,"identity":"c207a584-fe10-4104-bab9-5d45d5c7fe3e","added_by":"auto","created_at":"2025-12-19 17:03:52","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":198684,"visible":true,"origin":"","legend":"\u003cp\u003eEpoxomicin suppresses IRF3 activation induced by LPS. (A) RAW264.7 cells were transfected with IRF3 binding site (IFNb PRDIII-I) luciferase reporter plasmid, pre-treated with DAG (20 or 50 μM) for 1 h, and then treated with LPS \u0026nbsp;for an additional 8 h. Cell lysates were prepared and the luciferase and b-galactosidase enzyme activities were measured. luciferase activity was normalized with b-gal activity. (B) RAW264.7 cells were treated with DAG (20 or 50 μM) for 1 h and further stimulated with LPS for 18 h. Total RNAs were extracted and the levels of IFNb expression were determined by quantitative real-time RT-PCR analysis. IFNb expression was normalized with b-actin (internal control) expression. (C) RAW 264.7 cells were transfected with IP-10-luciferase reporter plasmid, pre-treated with DAG (20 or 50 μM) for 1 h, and then treated with LPS for an additional 8 h. Cell lysates were prepared and the luciferase and b-galactosidase enzyme activities were measured. luciferase activity was normalized with b-gal activity. (D) RAW 264.7 cells were pre-treated with DAG (20 or 50 μM) for 1 h and then treated with LPS for a further 8 h. Cell lysates were analyzed for IP-10 and β-actin protein by immunoblots. Veh, vehicle; DAG, dehydroandrographolide.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/5949a2d39c64c962a696fb8a.jpeg"},{"id":98454899,"identity":"eb9d2e52-5cc5-47a3-9420-2633e09f0c1b","added_by":"auto","created_at":"2025-12-17 18:15:07","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":202593,"visible":true,"origin":"","legend":"\u003cp\u003eDAG suppresses NF-κB activation induced by Poly[I:C]. (A) RAW264.7 cells were transfected with NF-κB luciferase reporter plasmid, pre-treated with DAG (20 or 50 μM) for 1 h, and then treated with Poly[I:C] for an additional 8 h. Cell lysates were prepared and the luciferase enzyme activities were determined. (B) RAW264.7 cells were transfected with iNOS luciferase reporter plasmid and pretreated with DAG (20 or 50 μM) for 1h and then treated with Poly[I:C] for an additional 8 h. Cell lysates were prepared and luciferase enzyme activities were determined. (C)RAW264.7 cells were pretreated with 20 or 50 mM DAG for 1 h and then further stimulated with Poly[I:C] for an additional 8 h. Cell lysates were analyzed for iNOS and b-actin protein by immunoblots. (D) RAW 264.7 cells were pretreated with 20 or 50 mM DAG for 1 h and then treated with Poly[I:C] for an additional 20 h. The amounts of nitrite in the supernatant were measured using Griess reagent. \u0026nbsp;Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM. Statisticalsignificance was determined using a one-way ANOVA. *\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/ecf238f09d3c44120373ae6a.jpeg"},{"id":98622899,"identity":"9f4cc931-425c-476e-9abb-9fef8e5ac043","added_by":"auto","created_at":"2025-12-19 17:03:27","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":181219,"visible":true,"origin":"","legend":"\u003cp\u003eDAG suppresses IRF3 activation induced by Poly[I:C].\u003cstrong\u003e \u003c/strong\u003e(A) RAW264.7 cells were transfected with IRF3 binding site (IFNbPRDIII-I) luciferase reporter plasmid, pre-treated with DAG (20 or 50 μM) for 1 h, and then treated with Poly[I:C] for an additional 8 h. Cell lysates were prepared and the luciferase and b-galactosidase enzyme activities were measured. (B) RAW264.7 cells were treated with DAG (20 or 50 μM) for 1 h and further stimulated with Poly[I:C] for 18 h. Total RNAs were extracted and the levels of IFNbexpression were determined by quantitative real-time RT-PCR analysis. IFNbexpression was normalized with b-actin (internal control) expression. (C) RAW 264.7 cells were transfected with IP-10-luciferase reporter plasmid, pre-treated with DAG (20 or 50 μM) for 1 h, and then treated with Poly[I:C] for an additional 8 h. Cell lysates were prepared and the luciferase and b-galactosidase enzyme activities were measured. (D) RAW 264.7 cells were pre-treated with DAG (20 or 50 μM) for 1 h and then treated with Poly[I:C] for a further 8 h. Cell lysates were analyzed for IP-10 and β-actin protein by immunoblots. Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM. Statistical significance was determined using a one-way ANOVA. *\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001. Veh, vehicle; DAG, dehydroandrographolide.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/b5fe552577ff6728cc559055.jpeg"},{"id":98623555,"identity":"53ba14f1-b640-4d39-8a1d-b33a5b404433","added_by":"auto","created_at":"2025-12-19 17:06:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":213170,"visible":true,"origin":"","legend":"\u003cp\u003eDAGsuppresses IRF3 activation induced by downstream signaling components of TRIF-dependent pathway. (A-C) 293T cells were co-transfected with IRF3 binding site (IFNbPRDIII-I)-luciferase reporter plasmid and the expression plasmid of TRIF (A), TBK1 (B), or IRF3 (C). Cells were further treated with DAG (20 or 50 μM) for 18 h. luciferase activity was normalized with b-gal activity. Results show representative results of 3 independent experiments. Values are expressed as the mean ± SEM. Statistical significance was determined using a one-way ANOVA. *\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05; **\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003ep \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/e668759fcfaaec2e09f44fbc.png"},{"id":98454903,"identity":"adac0d05-967c-4c90-a4e4-9d51003e9546","added_by":"auto","created_at":"2025-12-17 18:15:07","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":365329,"visible":true,"origin":"","legend":"\u003cp\u003eDehydroandrographolide (DAG) suppresses Toll-like receptor (TLR) signaling by inhibiting both MyD88- and TRIF-dependent pathways. This dual blockade attenuates downstream activation of NF-κB and IRF3, leading to reduced expression of pro-inflammatory mediators such as iNOS, COX-2, IFNβ, and IP-10. These findings highlight DAG as a potential anti-inflammatory agent derived from Andrographis paniculata.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/e689d8f5e1e5a89fcf562b75.png"},{"id":107351060,"identity":"44aaa50e-1641-4a93-8914-7ce030889bfa","added_by":"auto","created_at":"2026-04-20 16:08:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2092194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/f113b510-49bb-4268-8273-d7950a3ee41c.pdf"},{"id":98623285,"identity":"e16c84a9-2758-426d-9231-d9022d530a7f","added_by":"auto","created_at":"2025-12-19 17:05:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1703391,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationDAGwesternblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8098180/v1/3babdba81d5e302ba52f51ec.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dehydroandrographolide attenuates Toll-like receptor signaling by dual inhibition of MyD88- and TRIF-dependent pathways","fulltext":[{"header":"1. Background","content":"\u003cp\u003eInflammation represents one of the body\u0026rsquo;s primary defense responses against microbial invasion and tissue injury [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This process involves intricate interactions among various cell types and signaling molecules, typically accompanied by vasodilation, increased leukocyte recruitment, and the release of pro-inflammatory mediators [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Such physiological changes play a crucial role in safeguarding damaged tissue and initiating its repair [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Based on duration and nature, inflammation can be classified into acute and chronic forms. Acute inflammation develops quickly in response to injury, infection, or tissue damage and is generally short-lived [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In contrast, chronic inflammation persists over extended periods, often resulting from continuous irritation or unresolved injury, and it can cause structural tissue alterations and damage. Persistent inflammatory activity is implicated in the onset and progression of many inflammatory disorders [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eToll-like receptors (TLRs) are a well-characterized subgroup of pattern recognition receptors (PRRs) that detect conserved molecular patterns from microbes and trigger immune responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. They mediate the inflammatory reaction against pathogens including bacteria, viruses, and fungi, serving as a vital bridge between the innate and adaptive branches of immunity. TLRs also sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to initiate and fine-tune inflammatory cascades [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Ten types of TLRs have been identified in humans and thirteen in mice; they are located either on the plasma membrane or within endosomal compartments [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Membrane-localized TLRs generally recognize structural components of microbial surfaces, such as lipoproteins, lipopolysaccharides, and flagellins, whereas endosomal TLRs specialize in detecting microbial nucleic acids like double-stranded RNA, single-stranded RNA, or unmethylated CpG DNA [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Upon ligand recognition, TLRs transmit signals through two main routes: the MyD88- and the TRIF-dependent pathway. The MyD88-dependent route, used by most TLRs, primarily promotes the expression of inflammatory cytokines and chemokines. Meanwhile, the TRIF-dependent pathway is engaged by certain TLRs and induces production of interferons, while also regulating NF-κB via interaction with RIP1[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eDysregulation of TLR signaling has been linked to a variety of inflammatory and autoimmune disorders, as well as infectious diseases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, agents capable of modulating TLR pathways represent promising tools for controlling inflammation. Notably, medicinal plants\u0026mdash;many of which possess both anti-cancer and anti-inflammatory activities\u0026mdash;offer a valuable source of bioactive compounds that may support immune regulation and inflammation management [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eAndrographis paniculata\u003c/em\u003e, belonging to the family \u003cem\u003eAcanthaceae\u003c/em\u003e, has been traditionally utilized across China, India, and numerous Southeast Asian regions for centuries, primarily owing to its notable antipyretic and anti-inflammatory activities [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In traditional Chinese medicine, \u003cem\u003eAndrographis paniculata\u003c/em\u003e has been prescribed for the management of persistent or recurrent infectious and inflammatory disorders, particularly those affecting the upper respiratory tract and causing intestinal diarrhea [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among its bioactive constituents, dehydroandrographolide (DAG) has been identified as a compound with substantial immunomodulatory potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite these findings, the precise molecular interactions between dehydroandrographolide and TLRs in regulating immune responses have yet to be fully elucidated. This investigation, therefore, seeks to clarify the modulatory effects of dehydroandrographolide on TLR-mediated signaling, with specific emphasis on the MyD88- and TRIF-dependent pathways, aiming to advance understanding of its immune-regulating properties and explore its therapeutic prospects in inflammatory pathologies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Aim, design, and setting of the study\u003c/h2\u003e \u003cp\u003eThe aim of this study was to investigate the anti-inflammatory mechanisms of dehydroandrographolide (DAG), focusing on its regulatory effects on Toll-like receptor (TLR) signaling pathways. Specifically, we sought to determine whether DAG modulates both MyD88- and TRIF-dependent signaling cascades, which are two major branches of TLR-mediated immune responses.\u003c/p\u003e \u003cp\u003eThis study was designed as an \u003cem\u003ein vitro\u003c/em\u003e experimental study using murine macrophage RAW264.7 cells and human embryonic kidney 293T cells, which are well-established models for analyzing TLR signaling and transcriptional activation. The experiments were conducted under controlled laboratory conditions at the Department of Biomedical Laboratory Science, Soonchunhyang University (Asan, Republic of Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Reagents\u003c/h2\u003e \u003cp\u003eDAG was purchased from Cayman chemical and diluted in dimethylsulfoxide(DMSO). Lipopolysaccharide (LPS) was obtained from List Biological Laboratories (Ann Arbor, Michigan, USA; Cat. No.36841). Macrophage-activating lipopeptide-2 (MALP-2) was purchased from Alexis Biochemical (San Jose, CA, USA; Cat.No.421). Polyinosinic-polycytidylic acid (Poly[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]) was purchased from InvivoGen (San Diego, CA, USA; Cat.No. tlrl-pic-5). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell culture\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (a murine monocytic cell line; ATCC TIB-71) and human embryonic kidney 293T cells(ATCC CRL-3216) were cultured in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 \u0026micro;g/ml streptomycin at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere in a humidified incubator until confluence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell viability test\u003c/h2\u003e \u003cp\u003eCell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric assay. RAW 264.7 cells were treated with each compound for 4 h. Twenty microliters of the CellTiter 96 AQueous One Solution reagent (Promega, Madison, WI, USA) was added directly to the culture wells. The plate was then incubated for 4 h in a humidified, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, after which the absorbance at 490 nm was recorded using a 96-well plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Transfection and luciferase reporter gene assay\u003c/h2\u003e \u003cp\u003eCells were seeded into 48-well plates at a density of 0.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml and incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e/95% air environment. RAW264.7 and 293T cells were transfected with a luciferase plasmid and a HSP70-β‐galactosidase plasmid as an internal control using a p3000 and lipofectamine (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instruction. Cells were then treated with LPS, MALP-2, and Poly[I:C] for 8 h after being treated with DAG (20, 50\u0026micro;M) for 1 h. Luciferase and β‐galactosidase enzyme activities were determined using a Luciferase Assay System (Promega). Luciferase activity was normalized against β‐galactosidase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blotting analysis\u003c/h2\u003e \u003cp\u003eCells were seeded into 6-well plates at a density of 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/ml and incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e/95% air environment for 48 h. RAW264.7 cells were pretreated with DAG (20, 50\u0026micro;M) for 1 h. They were then treated with agonists for 8 h. Total protein was extracted from cell lysates using RIPA lysis buffer. Cell lysates were subjected to 12% and 14% sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis to separate proteins. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with PBS containing 0.1% Tween 20 and 5% nonfat dry milk for 24 h to prevent non-specific binding of antibodies. After the blocking step, membranes were incubated with specific primary antibodies and secondary antibodies conjugated with horseradish peroxidase (GE Healthcare, Chicago, IL, USA). Reactive bands were visualized using a Western Blot Detection System (iNtRON, Songnam, Korea). Membranes were stripped with 0.5 N NaOH for 20 min to reprobe with different antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Nitrite assay\u003c/h2\u003e \u003cp\u003eRAW 264.7 macrophages at a concentration of 0.8 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/ml were seeded in 48-well plates and incubated for 24 h. Subsequently, they were treated with each compound in the presence or absence of LPS, MALP-2, or Poly[I:C] for 18 h. Samples (100 \u0026micro;l) of the culture medium were incubated with 150 \u0026micro;l Griess reagent (1% sulfanilamide and 0.1% naphthylethylene diamine in a 2.5% phosphoric acid solution) at room temperature for 5 min in a 96-well microplate. The absorbance at 570 nm was read using a plate reader, and the concentration of NO was then determined by the preparation of a standard calibration curve, using sodium nitrite as the standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Real-time RT-PCR analysis of IFNβ expression\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using Ribospin\u0026trade; (GeneAll, Seoul, Korea) according to the manufacturer's instructions. Total RNA (5 \u0026micro;g) was reverse-transcribed using a HyperScript\u0026trade; for RT-PCR (GeneAll) and amplified with a Step One Plus Real-Time PCR System (Applied Biosystems) using a Power SYBR Green PCR Master kit (Applied Biosystems). Primers used to detect mouse IFNβ were as follows: forward primer 5\u0026prime;-TCCAAGAAAGGACGAACATTCG-3\u0026prime;, and reverse primer 5\u0026prime;-TGAGGACATCTCCCACGTCAA-3\u0026prime;. Primers for mouse β-actin (used as an internal control) were: forward primer 5\u0026prime;-TCATGAAGTGTGACGTTGACATCCGT-3\u0026prime;, and reverse primer 5\u0026prime;-CCTAGAAGCATTTGCGGTGCACGATG-3\u0026prime;. The following PCR conditions were used: denaturation at 95\u0026deg;C for 10 min; and 40 cycles of denaturation at 95\u0026deg;C for 15 s, annealing at 56\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 30 s. The specificity of PCR was assessed using a melting curve analysis. Fold induction of IFNβ expression was measured by real-time PCR in triplicate experiments relative to the vehicle control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Data analysis\u003c/h2\u003e \u003cp\u003eData were obtained from triplicate experiments. Values were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences in the data were evaluated using one-way ANOVA, with a \u003cem\u003eP\u003c/em\u003e-value of less than 0.05 considered statistically significant. All analyses were conducted using the GraphPad Prism software version 10.3 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cytotoxicity of DAG\u003c/h2\u003e \u003cp\u003eThe cytotoxic effect of DAG was evaluated in RAW 264.7 cells using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric assay. Cell viability was 91.4% at 50 \u0026micro;M DAG and decreased to 93.0% at 100 \u0026micro;M DAG (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on these results, a maximum concentration of 50 \u0026micro;M DAG was used in most subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. DAG suppresses NF-κB activation induced by TLR4 or TLR2 and TLR6 agonists\u003c/h2\u003e \u003cp\u003eNF-κB is a transcription factor that plays an important role in the human immune response [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and activation of NF-κB through the MyD88-dependent pathways and TRIF-dependent pathways leads to inflammation by regulating the expression of several target genes. This study, therefore, examined whether LPS (TLR 4 agonist) or MALP-2 (TLR 2 and TLR 6 agonist) inhibits the activity of NF-κB using a luciferase reporter gene assay. The results indicate that DAG significantly inhibited NF-κB induced by LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) or MALP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This result indicates that DAG can inhibit the activity of NF-κB by modulating the signal transduction system through TLRs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. DAG suppresses iNOS expression induced by TLR4 or TLR2 and TLR6 agonists\u003c/h2\u003e \u003cp\u003eWhen TLRs recognize various agonists and send signals downstream, they induce the activation of NF-κB. Activated NF-κB induces the expression of inflammatory genes such as iNOS [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, this study investigated whether DAG could modulate iNOS expression induced by LPS or MALP-2. According to the iNOS-luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B) and Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D), DAG inhibited iNOS expression induced by LPS or MALP-2 in RAW 264.7 cells. Additionally, we were confirmed that the concentration of nitrite produced by iNOS decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 DAG suppresses NF-κB activation induced by MyD88 downstream signaling components of TLRs\u003c/h2\u003e \u003cp\u003eMyD88-dependent pathway is the canonical adaptor of the inflammatory signaling pathway of Toll-like receptors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. From the above results, we found that DAG could inhibit the activity of NF-κB through the TLR signaling pathway. Therefore, we conducted experiments to determine whether DAG could also regulate the activation of NF-κB through the MyD88 signaling pathway. We transfected MyD88, IKKβ, and p65, which are downstream components of MyD88 dependent pathway, into 293T cells and performed a luciferase reporter gene assay. The results indicate that NF-κB induced by MyD88 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), IKKβ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), and p65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) is significantly inhibited by DAG, suggesting that the molecular target of DAG is the MyD88-dependent downstream signaling components.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. DAG suppresses IRF3 activation induced by TLR4 agonist\u003c/h2\u003e \u003cp\u003eIRF3 is activated exclusively through the TRIF signaling pathway. Therefore, we used LPS (TLR4 agonist) to activate TLR4, which signals through both the MyD88-dependent pathway and TRIF-dependent pathways. Therefore, it is employed to determine whether DAG can regulate the TRIF signaling pathway. Using a luciferase reporter gene assay with the IFNβ promoter domain containing the IRF3 binding site (IFNβ PRDIII-I), we confirmed that DAG significantly inhibited the activity of IRF3 induced by LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These results are further confirmed by RT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To further investigate the regulation of TRIF by DAG, we examined the expression of IP-10, so we confirmed by IP-10-luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) that IP-10 induced by LPS is significantly inhibited by DAG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. DAG suppresses NF-κB activation and iNOS expression induced by TLR3 agonists\u003c/h2\u003e \u003cp\u003eTLR3 exclusively utilizes the TRIF pathway. Therefore, we used Poly[I:C] (a TLR3 agonist) to observe the regulation of NF-κB via the TRIF pathway. Our results showed that NF-κB induced by Poly[I:C] (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) is significantly inhibited by DAG. This finding indicates that DAG can inhibit NF-kB activation through the TRIF-dependent pathway. Additionally, we confirmed that DAG inhibits Poly[I:C] induced iNOS expression using the iNOS-luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, a reduction in nitrite levels is observed in the nitrite assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7. DAG suppresses IRF3 activation induced by TLR3 agonist\u003c/h2\u003e \u003cp\u003eWe investigated whether DAG could inhibit the activation of IRF3 induced by the TLR3 agonist Poly[I:C]. The results showed that DAG significantly inhibited Poly[I:C] induced IRF3 activation, as shown by the luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). This inhibition is further confirmed by RT-PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, the IP-10 luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) also corroborated that DAG can inhibit IRF3 activation. The results indicate that DAG can regulate the TRIF signaling system. Therefore, we conducted experiments to identify molecular targets within the TRIF signaling system. TRIF, TBK1, and IRF3 are TRIF downstream components and are transfected into 293T cells to perform luciferase reporter gene assay. These findings indicate that IRF3, induced by TRIF (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA), TBK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), and IRF3 5D (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), is significantly inhibited by DAG. These results indicate that the molecular target of DAG is above the TRIF signaling system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrates that DAG exerts broad inhibitory effects on Toll-like receptor (TLR)\u0026ndash;mediated inflammatory responses by targeting both MyD88-dependent and TRIF-dependent pathways [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Using three distinct TLR agonists, LPS, MALP-2, and Poly[I:C], we observed that DAG attenuated downstream activation of key transcription factors, including NF-κB and IRF3, and consequently reduced the expression of inflammatory mediators such as iNOS and IP-10. The findings highlight a multifaceted role of DAG in modulating innate immune signaling. LPS, a well-characterized ligand for TLR4, activates both MyD88- and TRIF-mediated cascades, resulting in robust induction of proinflammatory cytokines. MALP-2, in contrast, signals primarily through TLR2 and TLR6, engaging the MyD88-dependent route [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Poly[I:C], as a synthetic analog of viral double-stranded RNA, activates TLR3 within endosomes and preferentially signals through TRIF [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Despite these differences in receptor specificity and signaling routes, DAG consistently suppressed downstream activation events, suggesting that its inhibitory effect occurs at a shared or converging point within these pathways.\u003c/p\u003e \u003cp\u003eA notable aspect of this study is the concurrent suppression of NF-κB and IRF3 activation. NF-κB is a central regulator of inflammatory gene expression, including iNOS, which plays a critical role in nitric oxide production and inflammation resolution [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. IRF3, on the other hand, is essential for antiviral defense, driving type I interferon production and chemokine expression such as IP-10 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The simultaneous downregulation of these two transcription factors indicates that DAG\u0026rsquo;s action is not restricted to a single branch of the TLR signaling network but instead encompasses broader regulatory control. This observation expands upon previous findings that primarily focused on the TLR4\u0026ndash;NF-κB axis, suggesting that DAG may influence common signaling intermediates shared by multiple TLRs rather than acting at the receptor level alone [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a therapeutic perspective, these results suggest that DAG could serve as a promising candidate for treating diseases characterized by excessive or chronic inflammation. By targeting both MyD88- and TRIF-dependent pathways, DAG may offer an advantage over agents that selectively inhibit a single signaling route. Furthermore, the suppression of both proinflammatory and antiviral signaling suggests potential applicability in conditions where immune overactivation contributes to pathology, such as autoimmune disorders or viral-induced hyperinflammation. Notably, this dual regulatory property aligns with the traditional use of \u003cem\u003eAndrographis paniculata\u003c/em\u003e extracts for managing systemic inflammatory and infectious conditions, thereby providing a molecular rationale that bridges empirical herbal efficacy with modern immunological mechanisms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, this study also raises important questions regarding the precise molecular target(s) of DAG within TLR signaling. Whether DAG interacts directly with adaptor proteins (e.g., MyD88 or TRIF), kinases, or transcriptional regulators remains to be elucidated.. Future work using phospho-proteomic or docking-based analyses could clarify whether DAG interferes with post-translational activation steps or adaptor assembly complexes.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur findings position DAG as a dual-pathway modulator of TLR signaling, capable of suppressing key inflammatory mediators through inhibition of both NF-κB and IRF3. This dual mechanism provides a strong rationale for further mechanistic and translational studies, with the ultimate goal of evaluating its therapeutic potential in inflammatory and immune-mediated diseases. our findings position DAG as a dual-pathway modulator of TLR signaling, capable of suppressing key inflammatory mediators through inhibition of both NF-κB and IRF3. This dual mechanism provides a strong rationale for further mechanistic and translational studies, with the ultimate goal of evaluating its therapeutic potential in inflammatory and immune-mediated diseases. Such investigations may also reveal whether DAG\u0026rsquo;s mode of action can be leveraged for multi-target modulation strategies that restore immune balance without complete suppression of innate defense responses.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDehydroandrographolide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTLR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eToll-like receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear factor kappa-light-chain-enhancer of activated B cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIRF3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon regulatory factor 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eiNOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInducible nitric oxide synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTRIF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTIR-domain-containing adapter-inducing interferon-β\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMyD88\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyeloid differentiation primary response 88\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eSoonchunhyang University Research Fund financially supports this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.E.L. and H.B.K. performed the experiments, curated the data, and conducted the formal analysis. D.W.L. contributed to the investigation. Y.H.L. provided advisory support. H.S.Y. and G.J.G. supervised the study, administered the project, secured funding, and were involved in writing the original draft as well as reviewing and editing the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu, Y. G. et al. Anti-inflammatory effect and pharmacokinetics of dehydroandrographolide, an active component of Andrographis paniculata, on Poly(I:C)-induced acute lung injury. \u003cem\u003eBiomed. Pharmacother\u003c/em\u003e. \u003cb\u003e174\u003c/b\u003e, 116456 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeghali, C. A. \u0026amp; Wright, T. M. Cytokines in acute and chronic inflammation. \u003cem\u003eFront. Biosci.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, d12\u0026ndash;26 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrero-Miliani, L., Nielsen, O. H., Andersen, P. S. \u0026amp; Girardin, S. E. Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. \u003cem\u003eClin. Exp. Immunol.\u003c/em\u003e \u003cb\u003e147\u003c/b\u003e (2), 227\u0026ndash;235 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, L. et al. Inflammatory responses and inflammation-associated diseases in organs. \u003cem\u003eOncotarget\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (6), 7204\u0026ndash;7218 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, R., Clermont, G., Vodovotz, Y. \u0026amp; Chow, C. C. The dynamics of acute inflammation. \u003cem\u003eJ. Theor. Biol.\u003c/em\u003e \u003cb\u003e230\u003c/b\u003e (2), 145\u0026ndash;155 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoilanen, E. Two faces of inflammation: an immunopharmacological view. \u003cem\u003eBasic. Clin. Pharmacol. Toxicol.\u003c/em\u003e \u003cb\u003e114\u003c/b\u003e (1), 2\u0026ndash;6 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkira, S. \u0026amp; Takeda, K. Toll-like receptor signalling. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e (7), 499\u0026ndash;511 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawai, T. \u0026amp; Akira, S. TLR signaling. \u003cem\u003eSemin Immunol.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e (1), 24\u0026ndash;32 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReuven, E. M., Fink, A. \u0026amp; Shai, Y. 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Sci.\u003c/em\u003e \u003cb\u003e1143\u003c/b\u003e, 21\u0026ndash;34 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrovska, B. B. Historical review of medicinal plants' usage. \u003cem\u003ePharmacogn Rev.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e (11), 1\u0026ndash;5 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, C., Shen, Y., Zhang, L., Wang, F. \u0026amp; Xiang, S. Pharmacological Effects and Pharmacokinetic Profiles of Dehydroandrographolide. \u003cem\u003eMediators Inflamm.\u003c/em\u003e \u003cb\u003e2025\u003c/b\u003e, 4123997 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin, H. et al. Dehydroandrographolide succinate attenuates dexamethasone-induced skeletal muscle atrophy by regulating Akt/GSK3beta and MuRF-1 pathways. \u003cem\u003eEur. J. Pharmacol.\u003c/em\u003e \u003cb\u003e990\u003c/b\u003e, 177265 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong, W. B. et al. 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NF-kappaB activation and IkappaB alpha dynamism involved in iNOS and chemokine induction in astroglial cells. \u003cem\u003eLife Sci.\u003c/em\u003e \u003cb\u003e68\u003c/b\u003e (15), 1695\u0026ndash;1701 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeguine, J. \u0026amp; Barton, G. M. MyD88: a central player in innate immune signaling. \u003cem\u003eF1000Prime Rep.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 97 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin, M. S. \u0026amp; Lee, J. O. Structures of TLR-ligand complexes. \u003cem\u003eCurr. Opin. Immunol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e (4), 414\u0026ndash;419 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWest, A. P., Koblansky, A. A. \u0026amp; Ghosh, S. Recognition and signaling by toll-like receptors. \u003cem\u003eAnnu. Rev. Cell. Dev. Biol.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e, 409\u0026ndash;437 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDowney, D. \u0026amp; Elborn, J. S. Nitric oxide, iNOS, and inflammation in cystic fibrosis. \u003cem\u003eJ. Pathol.\u003c/em\u003e \u003cb\u003e190\u003c/b\u003e (2), 115\u0026ndash;116 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColonna, M. TLR pathways and IFN-regulatory factors: to each its own. \u003cem\u003eEur. J. Immunol.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e (2), 306\u0026ndash;309 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKo, H. et al. Anti-inflammatory effects of Gingerenone A through modulation of toll-like receptor signaling pathways. \u003cem\u003eEur. J. Pharmacol.\u003c/em\u003e \u003cb\u003e983\u003c/b\u003e, 176997 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeng, Z. et al. Anti-Inflammatory Activity of Dehydroandrographolide by TLR4/NF-kappaB Signaling Pathway Inhibition in Bile Duct-Ligated Mice. \u003cem\u003eCell. Physiol. Biochem.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e (3), 1083\u0026ndash;1096 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao, Y., Yu, W. \u0026amp; Cai, H. Dehydroandrographolide facilitates M2 macrophage polarization by downregulating DUSP3 to inhibit sepsis-associated acute kidney injury. \u003cem\u003eImmun. Inflamm. Dis.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (4), e1249 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Toll-like receptor signaling, Dehydroandrographolide, MyD88-dependent pathway, TRIF-dependent pathway, Anti-inflammation","lastPublishedDoi":"10.21203/rs.3.rs-8098180/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8098180/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eToll-like receptors (TLRs) are key mediators of innate immune responses, and their dysregulation contributes to inflammatory diseases. Dehydroandrographolide (DAG), a diterpene lactone from Andrographis paniculata, is known for its anti-inflammatory activity, but its effects on individual branches of TLR signaling remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eRAW264.7 and 293T cells were used to evaluate the effects of DAG on MyD88- and TRIF-dependent signaling pathways. Luciferase reporter assays, Western blotting, RT-PCR, and nitrite assays were employed to assess NF-κB and IRF3 activation and inflammatory mediator expression. Statistical analysis was performed using one-way ANOVA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDAG significantly inhibited activation of NF-κB and IRF3 induced by TLR agonists (LPS, MALP-2, and Poly[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]) and by overexpression of MyD88- and TRIF-associated downstream molecules. Correspondingly, DAG suppressed iNOS, IFNβ, and IP-10 expression, indicating dual inhibition of both TLR signaling branches.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eDAG exerts dual inhibitory effects on MyD88- and TRIF-dependent TLR signaling, attenuating inflammatory mediator expression. These findings suggest its potential as a therapeutic compound for inflammatory disorders.\u003c/p\u003e","manuscriptTitle":"Dehydroandrographolide attenuates Toll-like receptor signaling by dual inhibition of MyD88- and TRIF-dependent pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 18:15:02","doi":"10.21203/rs.3.rs-8098180/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-09T13:32:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T17:07:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223453603858275556023199914600687427337","date":"2026-01-06T13:47:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33280507911618126216226113437802359699","date":"2025-12-19T12:03:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T23:06:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64313559136068884772395033173492655377","date":"2025-12-15T09:57:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62629622501378899999104452387832115284","date":"2025-12-12T23:02:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-12T11:37:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-12T11:16:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-10T13:46:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-28T07:24:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-28T07:16:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d785a72e-23e3-480b-af9b-5cc644946b91","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59778875,"name":"Biological sciences/Cell biology"},{"id":59778876,"name":"Health sciences/Diseases"},{"id":59778877,"name":"Biological sciences/Drug discovery"},{"id":59778878,"name":"Biological sciences/Immunology"},{"id":59778879,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-04-20T16:06:38+00:00","versionOfRecord":{"articleIdentity":"rs-8098180","link":"https://doi.org/10.1038/s41598-026-47514-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-16 15:57:48","publishedOnDateReadable":"April 16th, 2026"},"versionCreatedAt":"2025-12-17 18:15:02","video":"","vorDoi":"10.1038/s41598-026-47514-6","vorDoiUrl":"https://doi.org/10.1038/s41598-026-47514-6","workflowStages":[]},"version":"v1","identity":"rs-8098180","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8098180","identity":"rs-8098180","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}