Bufalin inhibits cytokine storm by regulating TLR4/TLR3 signaling pathway.

Bufalin inhibits cytokine storm by regulating TLR4/TLR3 signaling pathway. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 11 February 2025 V1 Latest version Share on Bufalin inhibits cytokine storm by regulating TLR4/TLR3 signaling pathway. Authors : Xixi Liu 0009-0002-4487-0174 , Chencheng Li , Jing Yang , Weiguang Zhang , Zhongxiao Hu , Xiaoli Zhang , Reaila Jianati , Fang Tian , Xingbin Dai , Zuqiong Xu , Biqing Chen 0000-0003-1443-1023 [email protected] , and Xuejun Zhu Authors Info & Affiliations https://doi.org/10.22541/au.173925755.56202203/v1 Published Immunity, Inflammation and Disease Version of record Peer review timeline 344 views 167 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Bufalin is one main component of the dried venom from Bufo gargarizans Cantor . Bufalin has anti-tumor, cardiotonic, anti-inflammatory and other physiological activities. However, in recent years, researchers have mainly paid attention to its anti-tumor effect and neglected its anti-inflammatory effect. We used lipopolysaccharide (TLR4 ligand) and poly inosinic acid (TLR3 ligand) to stimulate cultured macrophages to induce inflammatory condition, and found that bufalin could significantly reduce the production of pro-inflammatory factors (IL-6, TNF-α, IL-1β, IL-8, CXCL10, etc.). Transcriptome sequencing and molecular experiments showed that bufalin inhibited phosphorylation of IKBα and IRF3, and thus down-regulated Toll-like receptor pathway. Molecular docking predicted that one of the molecular targets of bufalin is MD2 coupled with lipopolysaccharide-activated TLR4. These findings not only support the pharmacological basis of using toad to treat inflammatory diseases in the Chinese medical history, but also provide a promising anti-inflammatory drug candidate for future clinical application. Bufalin inhibits cytokine storm by regulating TLR4/TLR3 signaling pathway. Xixi Liu a,b,1 , Chencheng Li a,c,1 ,Jing Yang a , Weiguang Zhang a , Zhongxiao Hu a , Xiaoli Zhang a , Reaila Jianati a ,Fang Tian d ,Xingbin Dai e ,Zuqiong Xu e , Biqing Chen d,* , Xuejun Zhu e,* 1. Affiliated Hospital of Nanjing University of Chinese Medicine, First Clinical Medical College of Nanjing University of Chinese Medicine, Nanjing 210029, Jiangsu, China 2. Department of General Medicine, The People’s Hospital of Lianxi District, Jiujiang City, Jiujiang 332005, Jiangxi, China 3. Wisdom Lake Academy of Pharmacy, Xi’ an Jiaotong-Liverpool University, Suzhou 215123, Jiangsu, China Central Laboratory, Affiliated Hospital of Nanjing University of Chinese Medicine/Jiangsu Province Hospital of Chinese Medicine, Nanjing 210029, Jiangsu, China Department of Hematology, Affiliated Hospital of Nanjing University of Chinese Medicine/Jiangsu Province Hospital of Chinese Medicine, Nanjing 210029, Jiangsu, China *Corresponding authors: [email protected] (ZhuX.J.); [email protected] (Chen B.Q.) 1 These authors contributed equally to this work. Abstract: Bufalin is one main component of the dried venom from Bufo gargarizans Cantor . Bufalin has anti-tumor, cardiotonic, anti-inflammatory and other physiological activities. However, in recent years, researchers have mainly paid attention to its anti-tumor effect and neglected its anti-inflammatory effect. We used lipopolysaccharide (TLR4 ligand) and poly inosinic acid (TLR3 ligand) to stimulate cultured macrophages to induce inflammatory condition, and found that bufalin could significantly reduce the production of pro-inflammatory factors (IL-6, TNF-α, IL-1β, IL-8, CXCL10, etc.). Transcriptome sequencing and molecular experiments showed that bufalin inhibited phosphorylation of IKBα and IRF3, and thus down-regulated Toll-like receptor pathway. Molecular docking predicted that one of the molecular targets of bufalin is MD2 coupled with lipopolysaccharide-activated TLR4. These findings not only support the pharmacological basis of using toad to treat inflammatory diseases in the Chinese medical history, but also provide a promising anti-inflammatory drug candidate for future clinical application. Keywords: Bufalin, TOLL-like receptors, inflammation, cytokine storm Introduction Inflammation is a defensive response of the immune system to harmful stimuli, which is helpful to remove invasive pathogens, promote tissue repair and maintain internal homeostasis [1, 2] . However, excessive or persistent inflammatory reaction can also cause acute injury or some chronic diseases. Cytokine storm is an uncontrolled systemic inflammatory reaction caused by excessive cytokines, which can lead to multiple organ failure and even death. Cytokine storms have been found in many infectious diseases, including severe acute respiratory syndrome and COVID-19 [4] . Immunotherapy such as chimeric antigen receptor T cell therapy can also lead to cytokine storm in clinical application. Cytokine storm is characterized by rapid proliferation and overactivation of T cells, macrophages and NK cells [5] . Monocytes/macrophages are the key mediators of cytokine storm [6, 7] . Excessive macrophage reaction could be harmful to the host. [10] . It is supposed that inflammation could be mitigated by inhibiting and reducing M1 macrophages and/or activating and increasing M2 macrophages [11] . Inflammation is related to the development and deterioration of most cancers [12] . Many anti-tumor targets are also anti-inflammatory targets, and many anti-tumor drugs have anti-inflammatory capability as well [13, 14] . As a widely used Chinese medicine famous for its anti-tumor effect [15] , the ethanol extract of the venom from Bufo gargarizans Cantor has also been used to treat various inflammatory diseases including tonsillitis and sore throat [17, 18] in the history. In the current decades, it has been used to treat hepatitis B and some purulent infections in clinic, and is believed to have anti-inflammatory and anti-microbial effects [20] . It mechanism has been revealed to reduce the infiltration of activated F4/80+ and/or CD68+ cells, significantly decreasing M1 macrophages and proinflammatory cytokine expression [16] . Bufalin is one of the main effective components of the venom from Bufo gargarizans Cantor , with relatively low side effects [19] . It has a variety of physiological effects including stimulating myocardial contraction and anti-tumor activity [16, 20] . Recent studies have found its inhibitory capability on the nuclear translocation of NF-κB in vitro , suggesting that bufalin may be used to treat inflammatory diseases [21] . This study evaluated the effect of bufalin on inflammatory cytokine storm at the cell level, proposed a possible molecular mechanism by analyzing transcriptome sequencing data and predicted a potential target of bufalin by molecular docking. Materials and methods: 2.1 Isolation and extraction of human peripheral blood mononuclear cell Peripheral blood samples were added with ficoll separation (Tianjin Haoyang Biological Products Technology Co. Ltd., China) in a ratio of 1:1 and centrifuged at 750 g for 20 min to purify peripheral blood mononuclear cells (PBMCs). Extracted PBMCs were then cultured in RPMI 1640 medium (Gibco Company, United States) containing 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co. Ltd., China) in a 5% CO 2 incubator at 37℃. PBMCs were stimulated by 500 ng/mL Lipopolysaccharide (LPS) (Sigma, Germany) or 50 ug/mL Poly inosinic acid (Poly I: C) (InvivoGen, United States) for 24 hours to simulate an inflammatory state. PBMCs were collected from six healthy volunteers, with the blood collection process approved by the Ethics Committee of Jiangsu Provincial Hospital of Traditional Chinese Medicine (Approval number 2022NL-124-02). 2.2 Cell culture Two monocyte cell lines, THP-1 and U937 (ATCC,USA), were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37℃ and 5% CO 2 . The cells were stimulated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (MCE, United States) for 24 hours to induce differentiation into M0 macrophages. Then PMA was discarded and replaced with 500 ng/mL LPS or 50 ug/mL Poly I:C for 24 hours to activate an inflammatory state. Bufalin (MCE) was co-cultured with the inflammation-activated macrophages for 24 hours as the experimental group. The blank group was only medium without drugs. 2.3 Detection of macrophage phenotype by flow cytometry The cultured cells were washed with PBS, digested and centrifuged with 5mM EDTA, with cell density adjusted to 1×10 6 cells /mL. The cells were incubated with fluorescent-labeled antibodies CD11b-APC (D12, BD Biosciences, United States) and CD86-PE (IT2.2, Biolegend, United States) independently at 4℃ for 30 min and then went through flow cytometry. 2.4 Detection of inflammatory cytokines by cytometric bead array (CBA) and enzyme-linked immunosorbent assay (ELISA) Cultured medium was collected and centrifuged at 1500 g for 3 minutes. The supernatant was collected for subsequent cytometric bead array with the Human Twelve Cytokines Detection Kit (Qingdao Riskell Biotechnology Co. Ltd., China) and ELISA (Ruixin Biotechnology Co. Ltd., China). CXCL10 and IFN-β were measured by ELISA, while all the other cytokines were measured by CBA. All steps in this experiment were carried out according to the manufacturer’s instruction. 2.5 Transcriptome sequencing 1×10 6 cells per sample were collected by centrifuge and lysed by Trizol. Total RNA was extracted and its quality was assessed by QUBIT and agarose gel electrophoresis. cDNA libraries are constructed by enriching for mRNA with Oligo(dT). The cDNA library was then sequenced on the Illumina platform to generate 6G data. The sequencing data were analyzed to quantify gene expressions. Gene set enrichment analysis (GSEA) and over representation analysis (ORT) were conducted by R package ClusterProfiler. 2.6 Real-time quantitative polymerase chain reaction (qPCR) 1×106 cells per sample were collected by centrifuge at 1500 g for 3 minutes. Total RNA was extracted by RNA extraction kit (Shanghai Feijie Biotechnology Co. Ltd., China) and reverse transcripted (Applied Biological Materials Inc, Canada) into cDNA according to the instructions, and qPCR (Applied Biological Materials Inc, Canada) was performed in the following procedures were set as 40 cycles of 95°C for 15 s, and 60°C for 1 min. 2.7 RNA interference Cultured monocytes were stimulated with 100ng/mL PMA for 24 hours and then 50ug/mL Poly I:C for 24 hours to simulate an inflammatory state. siRNAs targeting TLR4 and TRIF (synthesized by Shanghai Shenggong Bioengineering, China) were transfected respectively with Lipofectamine 2000 (Thermo Fisher Scientific, United States) for 24 hours, while negative control siRNA was used as the control. The culture medium was refreshed after 6 hours. 2.8 Western blot analysis Cultured cells were lysed by lysis buffer with protease and phosphatase inhibitors, and centrifuged at 14,000 g for 10 min to extract proteins. Proteins were quantified using the bicinchoninic acid assay kit and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 μg of each sample) and transferred onto polyvinylidene difluoride (PVDF) membranes for 90 minutes at 100 V. The membranes were incubated with 5% skim milk for 30 minutes for blocking, followed by incubation with primary antibodies against TLR3 (1:1000, Polyclonal, Affinity, United States), TLR4 (1:1000, 3G9A4, Proteintech, China), phospho-IκBα (1:1000, 14D4, Cell Signaling Technology, United States), IκBα (1:1000, L35A5, Cell Signaling Technology), GAPDH (1E6D9, 1:50000, Proteintech), IRF3 (1:5000, 1E6G8, Proteintech), and phospho-IRF3 (1:1000, Polyclonal, Cell Signaling Technology) at 4℃ overnight and secondary antibodies goat anti-rabbit IgG (1:5000, Polyclonal, Proteintech) and goat anti-mouse IgG (1:5000, Polyclonal, Proteintech) at room temperature for 1 hour. The blots were finally exposed using the ECL chemicalluminescence method. Images were analyzed by Image J software. 2.9 Molecular docking The structures of MD-2 protein (PDB ID：2E59) and bufalin were prepared by protein Preparation Guide and LigPrep module, respectively. Rigid body docking was first performed with default parameters using the molecular docking program Glide in SP mode(28,29) to screen the SPECS compound library . Ten conformations were generated and scored by glide score. The best scoring conformation is used for structural analysis, and the graph is generated by Pymol. 2.10 Statistical analysis Statistical analyses were performed by SPSS17.0. R Studio and GraphPad were adopted to draw figures. Student’s t test was used to compare means between two groups. P<0.05 was statistically significant. Results 3.1 Bufalin inhibits LPS-induced macrophage activation in vitro Under normal conditions, human monocyte THP-1 cells were round with smooth edges and no pseudopodia (Figures 1A and 1B, top). After stimulation with PMA and LPS for 24 hours, the cells displayed characteristics of activated macrophage, such as irregular morphology, and spindle-shaped pseudopodia (Figures 1A and 1B, center). Treatment with bufalin (8 nM) returned them to unactivated macrophage morphology, such as reducing the number of pseudopodia (Figures 1A and 1B, bottom). Flow cytometry analysis confirmed that THP-1 cells were activatedto M1 type macrophages (CD11b + CD86 + ) after PMA and LPS stimulation, while bufalin treatment led to a decrease in the percentage of M1 type macrophages (Figure 1C). 3.2 Bufalin inhibits cytokine storm in LPS-activated macrophages Activated macrophages secreted a large number of inflammatory cytokines (IL-6, IL-1β, IL-8, TNF-α, etc.).These cytokines could be significantly mitigated after bufalin treatment (Figure 2A). This anti-inflammatory effect of bufalin has been repeated in human monocytic cell line U937 and human primary PBMC (Figures 2B and 2C). 3.3 Bufalin mitigates cytokine storm in LPS-induced macrophages through TOLL-like receptor signaling pathway. To explore the molecular mechanism underlying the inflammation inhibitory effect of bufalin, gene expression profiles of the activated macrophages before and after bufalin treatment were compared. Over representation test (ORT) analysis revealed ten significantly enriched pathways (P<0.05) (Figure 3A). Gene set enrichment analysis (GSEA) also found ten pathways significantly changed (P value is not filtered) (Figure 3B). GSEA and ORT analyses overlapped on three pathways, which were all significantly down-regulated (Figure 3C). Among them, TOLL-like receptor pathway has been reported to be associated with inflammation [22] . Most genes in TOLL-like receptor pathway were significantly down-regulated (Figure 3D). 3.4 Bufalin abates cytokine storm in both LPS and Poly I: C activated macrophages through TLR3 pathway Further analysis of the RNAseq data revealed that bufalin significantly down-regulated TLR3-TRAF3-IKKε- pIRF3 pathway in LPS-activated macrophages (Figure 4A). Treatment of the Chinese Toad Venom, which is the raw extract where bufalin is one major component, resulted in a similar expression pattern, i.e., down-regulation of the TLR3 pathway in LPS-activated macrophages (Figure 4B). However, inhibition of the TLR3 pathway was not significant when the monocytes were not activated (Figure 4B). The significant reduction of TLR3, TRAF3 and IKKε were validated by qPCR (Figure 4C). and western blotting confirmed down-regulation of downstream phosphorylated IRF3 (Figures 4D). Since one important pathogenic ligand of TLR3 is viral double-stranded RNA (dsRNA), we then investigated whether activation of the TLR3 pathway by Poly I: C, a synthetic analog of dsRNA, could be impeded by bufalin [23, 24] . Poly I: C stimulated macrophages to produce a large number of cytokines (IL-1β, IL-8, TNF-α, CXCL10, IFN-β, etc.), while bufalin treatment could significantly mitigate them (Figure 5A). The results were replicated in human PBMC (Figure 5B). It was further confirmed that bufalin could down-regulate the TLR3 pathway, including TLR3, TRIF, and IKKε, in Poly I: C-activated macrophages as well (Figure 5C), and then decrease the phosphorylation of IRF3 (Figure 5D), which led to downstream decrease of cytokines such as CXCL10 (encoding IP-10). 3.5 TLR4 plays a necessary role in the effect of bufalin on inflammation-activated macrophages Since the origin of TLR3 pathway, TLR3, was down-regulated as well, it was supposed that there was at least a molecular target other than TLR3 in the TOLL-like receptor pathway. Thus, we predicted the targets of bufalin based on molecular docking by SuperPRED software. TLR4, in the LPS-activated state binding with myeloid differentiation protein 2 (MD2), hit with a high score as a potential predicted target of bufalin (Table 1). Molecular docking of bufalin and LPS-activated TLR4-MD2 complex displayed relatively high affinity with a docking pocket on MD2, composed of 133 CYS-136 VAL, 147 PHE-149 LEU-151 PHE-153 ILE, 44 ILE-46 ILE, and 61 LEU-63 ILE (Figure 6A). Since bufalin was predicted to bind LPS-activated TLR4 protein complex, we reexamined whether the downstream pathway of TLR4 was responsive to bufalin treatment. The results found that some cytokines including IL-6 and TNF-α were in the downstream of TLR4 pathway and significantly decreased(Figure 6B).Western Blot further confirmed inhibited phosphorylation by bufalin of IκBα, which was elevated in activated macrophages (Figure 6C).In order to find out whether TLR4 is the necessary node of Bufalin’s anti-inflammatory effect on monocyte-derived macrophages, we further interfered TLR4 with RNA. Bufalin can significantly down-regulate the TLR4/TLR3 pathway in the control group, but when TLR4 is knocked down, this inhibitory effect of Bufalin disappears (Figure 6D). This shows that TLR4 is an essential node in the molecular pathway of Bufalin inhibiting the hyper inflammation of monocyte-derived macrophages, and Bufalin can bind to TLR4-MD2 complex. We infer that Bufalin may indirectly inhibit the downstream pathways of TLR4 and TLR3 by inhibiting the binding of TLR4 to MD-2 (Figure 7). ­ Discussion In our previous studies, it was found that Chinese Toad extract could inhibit monocytic inflammation through TLR4 pathway [25] . This study found that bufalin, as one of the main components of Chinese Toad extract, could inhibit inflammatory reaction induced by bacterial membrane component LPS or virus RNA analog Poly I:C at cell level, and its mechanism may be through regulating TLR4/P-IκBα and TLR3/P-IRF3 pathways. Macrophages play an important role in the immune system. It is considered that the over-activation of natural immunity is one major factor for COVID-19 related cytokine storm [26] . Large-scale single cell RNA sequencing revealed that macrophages were the main source of inflammatory cytokines and the main host cells of SARS-CoV-2 virus in COVID-19 patients [27] . Therefore, discovering efficient inhibitor on activated macrophages could be helpful for many diseases involving over-inflammation. Current known macrophage inhibitors include BTK inhibitor GDC-0853 which prevents macrophage polarization by inhibiting TLR4/NF-κB signaling pathway [28] , dexamethasone which inhibits activity of M1 macrophage [29] , and cholesterol synthesis inhibitor statins which promote the formation of M2 macrophages [30, 31] . However, most treatments have obvious side effects [32] . As an active compound extracted from natural product, bufalin has shown a more prominent anti-inflammatory activity with few side effects, which means greater research and application prospect. Toll-like receptors constitute a key signal system that regulates macrophage function. Different toll-like receptors recognize distinct pathogen-related molecular patterns and trigger inflammation by producing inflammatory cytokines [22] .TLR4 recognizes bacterial LPS and produces pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [33] , while TLR3 recognizes viral double-stranded RNA and induces inflammatory chemokines/cytokines including CXCL10 and IFN-β [23, 34] . TLR3 helps to activate NFκB and IRF3 in macrophages [40] . By inhibiting TLR3/NLPR3/NFκB/IRF3 signaling pathway [41] , airway inflammation could be improved [42] . As bufalin can inhibit TLR3-IRF3 pathway, it suggests that bufalin may be used to relieve inflammation caused by virus infection. It is generally believed that as family members, TLR4 and TLR3 are parallel in functions. However, the current study found that TLR3 pathway was also affected when LPS stimulated TLR4. Other research has also reported indirect effect of TLR4 on TLR3 pathway [43, 44] . Tian et al. proved that LPS induces TLR3 expression through the TLR4-MyD88-IRAK-TRAF6-NF-κB signaling pathway in monocytes [43] , which was replicated in the current study. Furthermore, bufalin could inhibit the pro-inflammatory effect of LPS. When TLR4 was knocked down, the genes involved in TLR3 pathway were significantly down-regulated with downstream cytokines decreased. Moreover, after TLR4 knockdown, bufalin’s inhibitory effect on TLR3 pathway and cytokine level diminished. As suggested by molecular docking, bufalin might indirectly inhibit TLR3 pathway by acting on TLR4-MD2 complex. MD2 is a secreted glycoprotein which will bind the extracellular domain of TLR4 upon LPS stimulation, and triggers a series of downstream inflammatory reactions. Antibiotics which reduce its binding with LPS lead to down-regulation of inflammatory reaction [36] . Therefore, bufalin might competitively targeted TLR4-MD2 complex, which mitigate cytokine storm in macrophages by directly down-regulating TLR4-pIκBα pathway and indirectly down-regulating TLR3-pIRF3 pathway (Figure 7). However, M1 and M2 macrophages are not the only cell types involved in excessive release of cytokines. The effect of bufalin on other cytokine-releasing cells has not been touched in this study. Whether TLR4-MD2 complex is the direct target of bufalin needs further experiments. In addition, animal model of cytokine storm has not been established in this study, which is still far from clinical application. Besides, bufalin is a cardiac glycoside with low water solubility and slow clearance in vivo . The general therapeutic dose is about 60% of the toxic dose, and the therapeutic window is narrow. Therefore, it needs to be modified before clinical application. At present, there are some modified molecules, such as BF211, which need further experiments to investigate their influence on cytokine storm. To summary, this study shows that bufalin inhibits cytokine production in macrophages stimulated by bacteria and virus induced inflammation, and the potential underlying molecular mechanism is to regulate TLR4-pIκBα pathway directly and TLR3-pIRF3 pathway indirectly. This study provides a new insight for developing effective drugs to rapidly alleviate diseases involving macrophagic cytokine storm, such as COVID-19 [26, 27] , by inhibiting over-activated monocytes. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments: This work is supported by the National Natural Science Foundation of China (No.81673771), the Developing Program for High-level Academic Talent in Jiangsu Hospital of Chinese Medicine (No.y2021rc42), the Open Project of State Key Laboratory of Medical Immunology. (No.NKMI2021K18), the Science and Technology Development Program of Jiangsu Province-Clinical Frontier Technology (No. BE2016809), and the Postgraduate Research and Practice Innovation Program of Jiangsu Province (No. KYCX23-2122). Ethical approval The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the Helsinki Declaration (as revised in 2013). And written informed consent wasobtained from the patient donor in this study. This study was approved by the Ethical Review Committee Affiliated Hospital of Nanjing University of Chinese Medicine/Jiangsu Province Hospital of Chinese Medicine. Author Contributions CCL: Formal analysis, Writing - original draft. XXL: Validation, Formal analysis, Writing - original draft. JY: Methodology, Formal analysis. ZQX: Writing - original draft. WGZ: Methodology, Formal analysis. FT: Writing-review & editing. BQC: Project administration, Validation, Molecular analysis, Investigation, Writing -revised draft. XBD: Methodology, Resources. PJJ: Methodology, Resources. XJZ: Conceptualization, Methodology, Validation, Project administration, Formal analysis, Supervision, Resources, Investigation. Data Availability Statement The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request. Reference： 1.Sun SC. 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Conciliatory Anti-Allergic Decoction Attenuates Pyroptosis in RSV-Infected Asthmatic Mice and Lipopolysaccharide (LPS)-Induced 16HBE Cells by Inhibiting TLR3/NLRP3/NF-kappaB/IRF3 Signaling Pathway[J]. J Immunol Res. 2022; 2022:1800401.42. Liang Y, Liu X, Hu J, et al. The crude extract from the flowers of Trollius chinensis Bunge exerts anti-influenza virus effects through modulation of the TLR3 signaling pathway[J]. J Ethnopharmacol. 2023; 300:115-743.43. Tian X, Xu F, Lung WY, et al. Poly I:C enhances susceptibility to secondary pulmonary infections by gram-positive bacteria[J]. PLoS One. 2012; 7(9):e41879.44. Alexopoulou L, Holt AC, Medzhitov R, et al. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3[J]. Nature. 2001; 413(6857):732-738. Table 1：Prediction of bufalin molecular target Untitled Document Generated on Tue Feb 11 04:32:12 2025 by LaTeXML Fig.1. Bufalin inhibits PMA and LPS-induced macrophage activation. (A) Effects of Bufalin treatment on the morphology of macrophages under phase contrast microscope. (B) Under the white field microscope after Giemsa staining, the morphology of macrophages before and after Bufalin treatment was affected. (C) Flow cytometry showed the phenotype and activation level of macrophages. Control were THP-1 cells, Activates M0 were treated with PMA(100 ng/mL) for 24 hours, and then LPS (500 ng/mL) was added for 24 hours. Bufalin was treated with PMA+LPS and then treated with Bufalin 8nM for 24 hours. The data is expressed as the average of three replicates.*p < 0.05, **p < 0.01, *** P < 0.001. Fig. 2. Bufalin inhibits inflammatory factors secreted by LPS-induced activated macrophages. (A) the effect of bufalin on inflammatory factors secreted by monocytes/macrophages (THP-1) induced by PMA and LPS.(B) Effects of Bufalin on inflammatory factors secreted by monocytes/macrophages (U937) induced by PMA and LPS.(C) Effect of Bufalin on inflammatory factors secreted by peripheral blood mononuclear cells (PBMC) stimulated by LPS. A control is THP-1 cells, B control is U937 cells, C control is PBMC, Activates M0 were treated with PMA(100 ng/mL) for 24 hours, and then LPS (500 ng/mL) was added for 24 hours. Bufalin was treated with PMA+LPS and then treated with Bufalin 8nM for 24 hours. The data is expressed as the average of three independent replicates. *p < 0.05, **p < 0.01 and *** P < 0.001. Fig. 3. Bufalin down-regulates Toll-like receptor signaling pathway in activated macrophages. (A) signal pathways of genes with significant change times were analyzed by ORT enrichment( P < 0. 05), and the horizontal axis was the number of gene changes of activated monocytes/macrophages before and after Bufalin treatment. (B) Significantly changed genes enriched in 10 pathways by GSEA analysis ( P value is not filtered) . The horizontal axis is gene expression ratio of activated monocytes/macrophages before and after cinobufotalin treatment，taking as log10，color of the ridges indicate the significance level. (C) GSEA and ORT enrichment analysis show the enrichment trend of overlapping significant change signal pathways, with the vertical axis indicating the enrichment score and the horizontal axis indicating the accumulated gene number. (D) Heatmap shows the expression changes of TOLL pathway related genes in activated macrophages before and after Bufalin treatment. Control is an activated macrophage. Activated M0 were treated with PMA(100 ng/mL) for 24 hours and then LPS (500 ng/mL) was added for 24 hours. Bufalin was treated with PMA+LPS and then treated with Bufalin 8nM for 24 hours. Fig. 4. Bufalin down-regulates TLR4 and TLR3 signaling pathways in activated macrophages. (A) thermogram shows the expression changes of TLR3 pathway related genes of activated monocytes/macrophages before and after Bufalin treatment. (B) thermogram shows the mRNA expression changes of TLR3 pathway related genes of resting and activated monocytes/macrophages before and after cinobufagin treatment. (C) Bufalin on gene expression in TLR4/TLR3 pathway of activated macrophages. (D)Western blotting showed the effect of Bufalin on the expression of TLR3,IRF3, P-IRF3 proteins in activated macrophages, and the grey analysis of western blotting blot. Inactivated Macrophsges were THP-1 cells, and Activated M0/Activated Macrophsges were THP-1 cells, were treated with PMA(100 ng/mL) for 24 hours, and then LPS (500 ng/mL) was added for 24 hours. Bufalin 8nM is PMA+LPS, and then Bufalin 8nM is added for 24 hours. The data is expressed as the average of three independent replicates. *p < 0.05, **p < 0.01, *** P < 0.001. Fig. 5. Bufalin inhibits the inflammatory response induced by Poly I:C via the regulation of the TLR3/P-IRF3 signaling pathway. (A) The effects of Bufalin on inflammatory factors secreted by activated monocytes/macrophages (THP-1). (B) The effects of Bufalin on inflammatory factors secreted by peripheral blood mononuclear cells (PBMC) after stimulation. (C) The effects of Bufalin on gene expression in the TLR3 signaling pathway of macrophages after Poly I:C stimulation. (D) Western blotting showing the expression of TLR3, IRF3, and P-IRF3 in activated macrophages and Bufalin-treated cells, as well as the gray analysis of the Western blotting bands. A/C/D Control represents THP-1 cells, and B Control represents primary human PBMC cells. Activated M0 refers to the treatment of PMA (100 ng/mL) for 24 hours followed by the addition of Poly I:C (50 ng/mL) for 24 hours. Bufalin 8nM refers to the addition of Bufalin 8nM after PMA + Poly I:C treatment for 24 hours. The data is expressed as the average of three independent replicates. *p < 0.05, **p < 0.01, *** P < 0.001. Fig. 6. Bufalin indirectly downregulates the TLR3 pathway by affecting TLR4. (A) Bufalin binds to TLR4-MD2. (B) Bufalin on gene expression in TLR4 pathway of activated macrophages.(C) Western blotting showing the expression of TLR4, IκBα, and P-IκBα in activated macrophages and Bufalin-treated cells, as well as the gray analysis of the Western blotting bands. (D) Expression of related genes in the TLR3 pathway after TLR4 siRNA transfection and Bufalin treatment. Control siRNA/TLR4 siRNA was stimulated with PMA (100ng/ml) for 24 hours, followed by the addition of Poly I:C (50ug/ml) for 24 hours, and Control siRNA/TLR4 siRNA treatment for 6 hours followed by the addition of medium for 18 hours. Control siRNA +Bufalin/TLR4 siRNA+ Bufalin was stimulated with PMA (100ng/ml) for 24 hours, followed by the addition of Poly I:C (50ug/ml) for 24 hours, and Control siRNA/TLR4 siRNA treatment for 6 hours followed by the addition of Bufalin 8nM for 18 hours .The data is expressed as the average of three independent replicates, * means that compared with the Control siRNA group, *p < 0.05, **p < 0.01, *** P < 0.001. Fig. 7. Illustration showing the potential mechanism of bufalin on LPS/ Poly I:C activated macrophages. Information & Authors Information Version history V1 Version 1 11 February 2025 Peer review timeline Published Immunity, Inflammation and Disease Version of Record 4 Feb 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords animals cytokines inflammation Authors Affiliations Xixi Liu 0009-0002-4487-0174 Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Chencheng Li Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Jing Yang Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Weiguang Zhang Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Zhongxiao Hu Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Xiaoli Zhang Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Reaila Jianati Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine View all articles by this author Fang Tian Jiangsu Province Hospital of Chinese Medicine View all articles by this author Xingbin Dai Jiangsu Province Hospital of Chinese Medicine View all articles by this author Zuqiong Xu Jiangsu Province Hospital of Chinese Medicine View all articles by this author Biqing Chen 0000-0003-1443-1023 [email protected] Jiangsu Province Hospital of Chinese Medicine View all articles by this author Xuejun Zhu Jiangsu Province Hospital of Chinese Medicine View all articles by this author Metrics & Citations Metrics Article Usage 344 views 167 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Xixi Liu, Chencheng Li, Jing Yang, et al. 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