ETHNOPHARMACOLOGICAL RELEVANCE:In traditional Chinese medicine (TCM), colorectal cancer (CRC) is commonly associated with patterns such as damp-heat accumulation, heat toxin, and yin deficiency. Sanwu Huangqin Decoction (SWHQD) is a classical traditional Chinese medicine (TCM) formula composed of three medicinal herbs-Huangqin (Scutellaria baicalensis Georgi), Kushen (Sophora flavescens Ait.), and Dihuang (Rehmannia glutinosa Libosch.). It has been traditionally prescribed for clearing heat, eliminating dampness, and nourishing yin, particularly in the treatment of gastrointestinal disorders. Clinically, SWHQD has been used as an adjunctive intervention for CRC management. However, the precise pharmacological mechanisms underlying its anti-CRC activity remain incompletely characterized. AIM OF THE STUDY:This study sought to evaluate the anti-tumorigenic efficacy of SWHQD against CRC and to determine whether ferritinophagy-mediated ferroptosis contributes to its mechanism of action. MATERIALS AND METHODS:Human CRC cell lines (HCT116 and SW480) and a xenograft tumor model in BALB/c nude mice were used to evaluate the anti-tumor effects of SWHQD in vitro and in vivo. Ferroptosis-related indicators, including intracellular Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), were measured. The role of ferritinophagy was examined through analysis of the NCOA4/FTH1 pathway and NCOA4 gene silencing. RESULTS:SWHQD significantly suppressed CRC cell proliferation in vitro and inhibited tumor growth in vivo. Treatment induced hallmark features of ferroptosis, including elevated intracellular Fe2+, increased lipid ROS and MDA levels, and depletion of GSH, all of which were significantly abrogated by the ferroptosis inhibitors. Mechanistically, SWHQD upregulated nuclear receptor coactivator 4 (NCOA4), strengthened the interaction between NCOA4 and ferritin heavy chain 1 (FTH1), promoted autophagic degradation of FTH1, and consequently triggered iron release, thereby activating ferritinophagy-dependent ferroptosis. Silencing of NCOA4 markedly attenuated these biochemical and functional responses. CONCLUSIONS:SWHQD suppresses CRC through a mechanism involving the NCOA4/FTH1 pathway to induce ferritinophagy-dependent ferroptosis. These findings provide mechanistic evidence supporting the traditional application of SWHQD and highlight its potential as a complementary therapeutic strategy for CRC.

It has long been recognized that the intracellular replication of alphaviruses critically relies on several key host RNA-binding proteins (RBPs), including G3BP1/2 and FXR1/FXR2/FMR1. But how these RBPs modulate alphaviral replication, and whether it would be possible to target them for antiviral treatment, is less explored. Here, using Semliki Forest virus (SFV) as a model, we report that SFV non-structural protein 3 (nsP3) exploits G3BP to drive its condensation and transforms antiviral stress granules into proviral nsP3-G3BP co-condensates. The gel-like co-condensates enrich and protect viral genomic RNAs from host RNase degradation and promote viral translation and replication. nsP3-RBP co-condensation is widespread among alphaviruses, and condensate disruption is a plausible antiviral approach. Thus, these findings uncover a general anti-alphavirus strategy based on the conserved reliance of virus-host protein co-condensation.

Drug-induced liver injury (DILI), particularly from isoniazid (INH), is a major clinical concern. Ferroptosis is implicated in DILI, yet direct GPX4 stabilizers with defined binding sites remain undiscovered. While Schisandra chinensis lignans exhibit hepatoprotective potential, their mechanistic interplay with ferroptosis remains unexplored. Here, we delineate a novel molecular axis by which Schisandrin A (SinA) and Schisandrin B (SinB) mitigate INH-induced hepatotoxicity through ferroptosis suppression. We demonstrate that SinA/SinB significantly attenuates hepatic injury markers (ALT/AST), iron overload, lipid peroxidation, and glutathione depletion in vitro and in vivo. Strikingly, GPX4 knockout abolished their protective effects, underscoring GPX4 as the pivotal target. ITC and SPR revealed high-affinity binding of SinA/SinB to GPX4, while molecular docking identified K31 and K90 as critical residues for GPX4 interaction. Mutagenesis studies confirmed that K31/K90 substitutions abolished SinA/SinB’s efficacy, highlighting a structure-dependent mechanism. Compound binding reduced the interaction between GPX4 and TRIM25, inhibited the ubiquitination of GPX4, as evidenced by Co-IP and MD. This stabilizes GPX4, suppresses lipid peroxidation/iron accumulation, and rescues INH-induced ferroptosis in vitro and in vivo. These findings establish a novel mechanism of GPX4 regulation and provide a structural blueprint for anti-ferroptotic drug design.

Colorectal cancer (CRC) is one of the most common and deadly types of cancer globally. Ferroptosis, a type of regulated cell death that relies on iron, has become a promising target for treating CRC. Alkannin, a natural compound from Lithospermum erythrorhizon, exhibits anti-tumor activity, yet its mechanism in CRC is unclear. This study investigated alkannin's role in regulating ferroptosis via the Keap1/Nrf2/GPX4 axis. Using network pharmacology and experimental validation in HCT116 and SW480 cells and a xenograft mouse model, we found that alkannin markedly inhibited the viability, proliferation, and migratory capacity of CRC cells, demonstrating significant anti-tumor activity. Network pharmacology revealed a primary association between alkannin's therapeutic effects and the induction of ferroptosis, along with the regulation of oxidative stress pathways, with a notable focus on the Keap1/Nrf2 axis. Experimental evidence confirmed that alkannin induced ferroptosis, as reflected by increased intracellular Fe2+ levels and lipid peroxidation, along with reduced glutathione (GSH) content. These effects were reversed by ferroptosis inhibitors, which also attenuated alkannin-induced cytotoxicity. Mechanistically, alkannin enhanced Keap1 protein stability by suppressing its ubiquitination. It promoted the interaction between Keap1 and Nrf2, leading to decreased Nrf2 expression and inhibition of its nuclear translocation, thereby downregulating the expression of glutathione peroxidase 4 (GPX4), a key suppressor of ferroptosis. Genetic silencing of Keap1 significantly diminished alkannin-induced ferroptotic cell death. In vivo, alkannin effectively inhibited tumor growth in xenografted nude mice. Furthermore, it induced ferroptosis in tumor tissues, as evidenced by similar biochemical changes, which were counteracted by co-administration of a ferroptosis inhibitor. Consistently, alkannin upregulated Keap1 expression while reducing the protein levels of Nrf2 and GPX4 in tumor tissues. In conclusion, alkannin induces ferroptosis in CRC by stabilizing Keap1 to inhibit the Nrf2/GPX4 pathway, supporting its potential as a CRC therapeutic agent.

Many RNA-binding proteins (RBPs) bind poly(ADP-ribose) (PAR) despite lacking canonical PAR-binding domains, but the molecular basis and functional consequences of this interaction remain unclear. Because PAR can bind arginine-rich and hydrophobic regions that can also bind RNA, we hypothesized that PAR and RNA compete for shared sites on RBPs to regulate their function. Here, using proteomic and biochemical approaches, we identify a broad population of RBPs for which PAR and RNA have competitive interaction. This group is enriched for stress granule-associated proteins, including G3BP1/2. In G3BP1, PAR and RNA compete for binding a common site in an intrinsically disordered region (IDR3) that also undergoes covalent ADP-ribosylation. PAR binding disrupts the autoinhibitory IDR1-IDR3 interaction, shifting G3BP1 toward an open conformation known to promote condensation. PAR promotes early steps in condensation, after which it is replaced by RNA in a subsequent maturation phase. These studies show that PAR and RNA occupy distinct temporal and functional windows during condensate assembly, revealing mechanisms whereby stress granules shape the pool of translatable mRNAs.

Colorectal cancer (CRC), one of the leading causes of cancer-related mortality globally, urgently requires complementary and alternative therapies. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising anti-cancer strategy. Dendrobium officinale (D. officinale), a renowned traditional Chinese medicinal herb, is widely used in several Asian countries for its nutritional and therapeutic benefits. Although D. officinale has demonstrated anti-tumor effects, the molecular mechanisms underlying its action against CRC remain incompletely characterized. This study aimed to elucidate the role of D. officinale in suppressing CRC through the induction of ferroptosis and its regulatory effects on glutathione peroxidase 4 (GPX4), a key suppressor of ferroptosis. In vitro assays were conducted using HCT116 and SW480 CRC cell lines, and in vivo efficacy was evaluated in BALB/c nude mice bearing CRC xenografts. D. officinale significantly reduced CRC cell viability and proliferation in vitro and suppressed tumor growth in vivo. Induction of ferroptosis was evidenced by elevated levels of Fe2+, malondialdehyde (MDA), and lipid peroxidation, along with a depleted glutathione/oxidized glutathione disulfide (GSH/GSSG) ratio. Notably, these effects were reversed by ferroptosis inhibitors, including ferrostatin-1 (Fer-1) and deferoxamine. Consistently, D. officinale markedly downregulated GPX4 expression. Overexpression of GPX4 rescued D. officinale-induced ferroptosis, whereas GPX4 silencing exacerbated this effect. D. officinale suppresses CRC by triggering GPX4-dependent ferroptosis, providing a novel, naturally derived therapeutic approach. These findings bridge traditional medicine and modern oncology, establishing a foundation for developing targeted CRC treatments.

Myocardial infarction (MI) is the leading cause of cardiovascular disease-related death worldwide. Nonetheless, existing therapeutic approaches for MI are hampered by issues such as reliance on pharmacological agents and suboptimal patient adherence. Caffeic acid (CA) is a bioactive polyphenolic compound with important anti-inflammatory, anti-bacterial and anti-oxidant functions. Still, its specific role and mechanism in treating cardiovascular disease remain to be further studied. In recent years, a large number of studies have shown that the kelch-like ECH-associated protein 1/nuclear factor erythroid 2 related factor 2 (Keap1/Nrf2) pathway is a key factor in the occurrence and development of cardiovascular diseases. In this study, H2O2-induced oxidative stress model of H9c2 cells and left anterior descending branch (LAD) conjunctival induced acute myocardial infarction reperfusion (AMI/R) model were used to evaluate the protective effect of CA on the heart. The interaction between CA and Keap1 was analyzed by CA-labeled fluorescence probe, target fishing, isothermal titration calorimetry (ITC), protein crystallography and surface plasmon resonance (SPR). Our results suggested that CA binds Keap1 and degrades Keap1 in a p62-dependent manner, further promoting nuclear transcription of Nrf2 and thus effectively reducing oxidative stress. In addition, based on the three-dimensional eutectic structure, it was confirmed that CA directly targets Keap1 protein by interacting with residues M550 and N532, inducing conformation changes in Keap1 protein. We also found that the CA analog chlorogenic acid (GCA) can bind Keap1. In conclusion, this study elucidates a novel molecular mechanism and structural basis for the protective effects of CA against oxidative damage via the Keap1-Nrf2 pathway.

Radioresistance represents a substantial challenge in cancer treatment, particularly in esophageal squamous cell carcinoma (ESCC), where the underlying molecular mechanisms remain incompletely understood. Small nucleolar RNAs (snoRNAs), primarily located in the nucleolus, are noncoding RNAs whose roles in ESCC radiotherapy are unclear. In this study, an upregulated snoRNA, SNORA58 is identified in ESCC via a snoRNA PCR array. Furthermore, based on multicenter data, SNORA58 is established as a promising biomarker for predicting response to neoadjuvant chemoradiotherapy (nCRT). Patients with high SNORA58 expression levels presented a lower likelihood of achieving a complete response to nCRT and poorer clinical outcomes. Functionally, SNORA58 enhances cancer cell resistance to radiotherapy without affecting chemotherapeutic sensitivity. Mechanistically, SNORA58 stabilizes CTCF by inhibiting its ubiquitin-mediated degradation, leading to JNK1 downregulation and subsequent inactivation of the JNK signaling pathway; this disrupts intracellular iron homeostasis, thereby alleviating radiotherapy-induced ferroptosis. Notably, the administration of a JNK signaling activator significantly restored the radiosensitivity of high-SNORA58 ESCC cells both in vitro and in vivo. These findings elucidate the first demonstration of SNORA58 as a critical regulator of radioresistance in ESCC and reveal a novel link between snoRNAs and ferroptosis in this specific context, suggesting potential therapeutic strategies for managing ESCC.

Background Colorectal cancer (CRC) continues to represent a significant global public health challenge. Ferroptosis, a novel form of cell death dependent on iron and involving lipid peroxidation, has emerged as an effective strategy for treating various cancers with great potential for application. Purpose This study aimed to investigate the therapeutic potential of erianin, a novel dibenzyl compound isolated from the well-known herbal medicine Dendrobium chrysotoxum Lindl, in the treatment of CRC through induction of ferroptosis. Methods Human CRC HCT116 and SW480 cells were employed for in vitro investigations, while an AOM/DSS CRC animal model was established for in vivo experiments. Results The results demonstrated that erianin effectively inhibited the growth of CRC cells and suppressed tumorigenesis in the AOM/DSS CRC animal model. Erianin induced ferroptosis in CRC cells as evidenced by a significant increase in intracellular Fe2+ levels and lipid peroxides, along with a decrease in glutathione. Additionally, ferroptosis inhibitors reversed the cytotoxicity of erianin against CRC cells as well as its induction of ferroptosis. Notably, novel glutathione peroxidase 4 (GPX4), a core regulatory factor of ferroptosis, was found to be overexpressed in human primary colon adenocarcinoma tissues compared with normal tissues. However, erianin significantly reduced GPX4 expression by facilitating its ubiquitination and degradation. Furthermore, the overexpression of GPX4 mitigated erianin-induced ferroptotic cell death; conversely, the silencing of GPX4 amplified these effects. Conclusion Erianin demonstrates the potential to inhibit CRC by inducing ferroptosis through accelerating the ubiquitination and degradation of GPX4, indicating its promise as a therapeutic candidate against CRC.

The phase separation of biomolecules into so-called stress granules (SGs) allows the cell to tightly regulate translation activity in response to different stimuli, such as oxidative stress, starvation, or the recognition of non-cellular RNA. Recent reports suggest SGs induced during viral infection, may act as a crossroad between the cellular stress response and the activation of the innate immune response. Here, we aimed to dissect the role of SGs in the context of yellow fever virus (YFV) infection. We found that YFV infection resulted in translational shut-off from 24 hpi on followed by the formation of SGs at 48 hpi, a delay potentially associated with the ability of the YFV capsid to inhibit SG formation, through an interaction with the major SG scaffolding protein G3BP1. To elucidate the role of YFV-induced SGs during infection, we inhibited SG assembly using a small-molecule inhibitor and find that SG formation does not influence viral replication. Uncovering the first proteome of virus-induced SGs, our compositional analysis revealed a specific enrichment of proteins associated with mitochondrial processes in YFV-induced SGs. Indeed, we show that YFV infection results in mitochondrial damage and dysfunction. Together, we propose that YFV-induced SGs may be involved not only in the modulation of cellular homeostasis but also in influencing mitochondrial functions. Author Summary Viruses impose a major burden on the infected host – from structural rearrangements needed to assemble replication complexes, to exploiting cellular energy resources and genetic rewiring associated with antiviral responses. The assembly of membrane-less organelles such as stress granules (SGs) enable cells to rapidly tune cellular processes upon sensing of stresses such as viruses. Moreover, the cell’s innate immune response is proposed to be regulated by SGs and in turn many viruses disrupt or highjack their components. Yet, the molecular basis for SG functions during infection remain ambiguous. Here we investigated the interplay between yellow fever virus (YFV) infection and SGs. We demonstrate that infection with attenuated or pathogenic viruses result in the formation of SGs. Their compositional analysis reveal that they sequester mitochondrial proteins, correlating with altered mitochondrial functions during infection. This highlights a novel complex interplay between membrane-bound and membrane-less organelles which could present novel opportunities for antiviral therapies. ### Competing Interest Statement The authors have declared no competing interest.

Colorectal cancer (CRC), one of the primary causes of gastrointestinal malignancy-related mortality worldwide, encounters significant therapeutic challenges due to multidrug resistance. Multidrug resistance-associated protein 2 (MRP2), encoded by ABCC2, plays a crucial role in the development of drug resistance during clinical CRC treatment. This study elucidates a novel mechanism by which formononetin (FMNT), a bioactive isoflavone commonly found in food and medicinal-related plants, suppresses CRC via microRNA-490-3p (miR-490-3p)-mediated regulation of ABCC2 and synergizes with 5-fluorouracil (5-FU). HCT116 and SW480 cell lines as well as AOM/DSS-induced CRC models in wild-type and Mrp2-/- mice were employed. The results demonstrated that FMNT markedly suppresses CRC proliferation in vitro and tumorigenesis in vivo. Transcriptomic profiling identified ABCC2 as the top target associated with FMNT's efficacy. This finding was validated in clinical specimens, which revealed significant upregulation of MRP2 in human CRC tissues. FMNT markedly downregulated the MRP2 expression. Genetic ablation experiments further confirmed enhanced FMNT sensitivity in Mrp2-/- mice and MRP2 silencing CRC cells. Mechanistically, FMNT upregulated tumor-suppressive miR-490-3p, which directly targets the ABCC2 3'UTR, thereby establishing a regulatory axis corroborated by gain/loss-of-function experiments. Notably, overexpression of miR-490-3p augmented FMNT's antitumor effects, whereas inhibition of miR-490-3p diminished its efficacy. Therapeutic combination index analysis indicated robust synergy between FMNT and 5-FU, with combination therapy achieving superior tumor growth inhibition compared to monotherapies. Collectively, our findings uncover a novel miR-490-3p/ABCC2 regulatory mechanism underlying FMNT's antitumor activity and highlight its potential for chemosensitization through ABCC2 inhibition, providing a strong rationale for flavonoid-based adjuvant therapy in CRC management.

Stress granules (SGs) are dynamic RNA-protein assemblies that form in response to cellular stress and must be efficiently disassembled to restore normal cell function. Valosin-containing protein (VCP), an enzyme implicated in neurodegenerative diseases, is essential for SG disassembly, but whether and how this process is coordinated with SG assembly remains unclear. Here, we identify the VCP cofactor, Alveolar soft part sarcoma locus (ASPL) as a key regulator linking SG assembly and disassembly. ASPL promotes SG assembly by facilitating biomolecular condensation of Ras guanosine triphosphatase-activating protein-binding protein (G3BP) and stabilizing its interactions with other SG proteins. ASPL also facilitates phosphorylation and activation of VCP by UNC-51-like kinases 1 and 2 (ULK1/2), enabling G3BP extraction and efficient SG disassembly. Pathogenic VCP mutations that disrupt ASPL binding impair SG disassembly, a defect rescued by phosphomimetic mutations or ASPL depletion. Our findings suggest that disruptions in the ASPL-VCP interaction uncouple SG assembly and disassembly, representing a potential mechanism underlying VCP-associated neurodegenerative diseases.

The root of Aconitum carmichaelii Debx . (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg −1 ·d −1 ) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%–53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress and dissolve pre-existing stress granules. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Changji'an Formula (CJAF) is a Chinese herbal compound, which is effective against irritable bowel syndrome with predominant diarrhea (IBS-D) in clinic. However, the molecular mechanism has not been well defined. In the current study, the potential targets and signaling pathways of CJAF against IBS-D were predicted using network pharmacology analysis. The pharmacological mechanisms of CJAF against IBS-D and the potential mechanism were validated by using an IBS-D mouse model induced by enema with trinitrobenzene-sulfonic acid (TNBS) plus with restraint stress and further intervened with CJAF. A total of 232 active compounds of CJAF were obtained, a total of 397 potential targets for the active ingredients were retrieved and a total of 219 common targets were obtained as the potential targets of CJAF against IBS-D. GO and KEGG enrichment analyses showed that multiple targets were enriched and could be experimentally validated in a mouse model of IBS-D. The mechanisms were mainly converged on the immune and inflammatory pathways, especially the NF-κB, TNF and IL-17 signaling pathway, which were closely involved in the treatment of CJAF against IBS-D. Animal experiment showed that CJAF alleviated visceral hypersensitivity and diarrhea symptom of IBS-D. CJAF also restored the histological and ultrastructure damage of IBS-D. The result of Western blot showed that CJAF upregulated colonic tight junction proteins of ZO-1, Occludin and Claudin-1. Further results demonstrated that CJAF inhibited the protein expression of NF-κB/NLRP3 inflammasome pathway targets and downregulated proinflammatory mediators of IL-1β, IL-18, TNF-α. In conclusion, CJAF could effectively reduce inflammatory response and alleviate visceral hypersensitivity as well as diarrhea symptom of IBS-D by inhibiting the NF-κB/NLRP3 signaling pathway. This study not only reveals the mechanism of CJAF against IBS-D, but also provides a novel therapeutic strategy for IBS-D.

Melanoma is the most aggressive and difficult to treat of all skin cancers. Despite advances in the treatment of melanoma, the prognosis for melanoma patients remains poor, and the recurrence rate remains high. There is substantial evidence that Chinese herbals effectively prevent and treat melanoma. The bioactive ingredient Salvianolic acid B (SAB) found in Salvia miltiorrhiza, a well-known Chinese herbal with various biological functions, exhibits inhibitory activity against various cancers. A375 and mouse B16 cell lines were used to evaluate the main targets and mechanisms of SAB in inhibiting melanoma migration. Online bioinformatics analysis, Western blotting, immunofluorescence, molecular fishing, dot blot, and molecular docking assays were carried out to clarify the potential molecular mechanism. We found that SAB prevents the migration and invasion of melanoma cells by inhibiting the epithelial–mesenchymal transition (EMT) process of melanoma cells. As well as interacting directly with the N-terminal domain of β-actin, SAB enhanced its compactness and stability, thereby inhibiting the migration of cells. Taken together, SAB could significantly suppress the migration of melanoma cells via direct binding with β-actin, suggesting that SAB could be a helpful supplement that may enhance chemotherapeutic outcomes and benefit melanoma patients.

Aims: The Keap1-Nrf2 pathway is essential for antioxidant defense and addresses a variety of diseases related to oxidative stress damage. Caffeic acid (CA) is a biologically active natural compound with dihydroxy groups in positions 3 and 4, widely distributed in various fruits and other foods, with vigorous antioxidant activity. However, the molecular mechanism of actions of CA and its analogs (chlorogenic acid) against oxidative stress has not been well defined. The study investigated the molecular mechanism of CA could directly interact with Keap1 to activate Nrf2 to counteract ROS overproduction. Main methods: An H2O2-induced oxidative stress model in H9c2 cells was established. We clarified the molecular effets of CA binding the Keap1 by the method native mass spectrometry analysis, isothermal calorimetry (ITC), protein crystallography and SPR. Moreover, the complex crystal confirmed that the interaction between CA and Keap1 protein was disabled. Conclusions: Our findings suggested that CA and its analogs might be novel activators of the Keap1-Nrf2 signaling pathway and could induce conformational changes in Keap1 to promote p62-dependent autophagy and Nrf2 nuclear translocation to reduce oxidative stress effectively. More importantly, based on the three-dimensional cocrystal structure, CA was confirmed to directly target Keap1 by interacting with residues M550 and N532. Significance: The study provides new molecular mechanism and structure basis for the resistance of CA to oxidative damage through Keap1-Nrf2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE:Type 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic β-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. AIMS OF THE STUDY:This study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. MATERIALS AND METHODS:A mouse-derived pancreatic islet β-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2-/-) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. RESULTS:Network pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro, FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2-/- mice. CONCLUSIONS:This study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics.