Background: Non-small cell lung cancer (NSCLC) remains a significant health challenge, and the KRAS mutation plays a critical role in its development, especially KRASG12C. Rosmarinic acid (RA), a natural polyphenolic compound, has demonstrated robust anti-cancer activities. However, the precise molecular mechanisms underlying its anti-tumor effects in NSCLC remain poorly understood. Methods: We assessed RA's anti-tumor effects in vitro/in vivo NSCLC models. Activity-based protein profiling (ABPP) coupled with bioorthogonal click chemistry identified RA's direct molecular targets. Western blot and other techniques analyzed KRAS/AKT/ERK signaling, plus cell cycle/apoptosis molecule expression. The effect of RA on tumor suppression and the ability of tumor cells to evade macrophages in an NSCLC mouse model with KRASG12C mutation was evaluated. Results: RA significantly inhibited NSCLC cell proliferation and induced apoptosis by modulating the KRAS/AKT/ ERK signaling cascade. ABPP analysis revealed that RA directly binds to KRASG12C at the mutant cysteine-12 residue. Functional studies confirmed that RA-mediated cell cycle arrest and apoptosis depend on KRASG12C modulation. In KRASG12C-mutant mouse models, RA markedly suppressed tumor growth and reduced macrophage evasion by tumor cells. Conclusions: RA acts as a novel KRASG12C inhibitor that directly targets the mutant cysteine-12 residue, suppressing NSCLC progression through inhibition of the KRAS/AKT/ERK pathway and enhancement of anti-tumor immune activity. These findings highlight RA's therapeutic potential for KRASG12C-driven NSCLC and offer new insights for the development of targeted cancer therapies.

OBJECTIVES:To investigate the effect and mechanism of Anemoside B4 (AB4) on non-small cell lung cancer (NSCLC) metastasis. METHODS:In vitro, the MTT assay was used to evaluate the effect of AB4 on the viability of human NSCLC cell lines A549 and H1975. Cell scratch and Transwell assays were performed to assess the effect of AB4 on the migration and invasion of A549 and H1975 cells induced by transforming growth factor-β1 (TGF-β1). Western blotting and immunofluorescence were used to detect the expression of proteins related to epithelial-mesenchymal transition (EMT), the mitogen-activated protein kinase (MAPK) signaling pathway, and the oxidative stress signaling pathway. Invivo, a mouse model of melanoma lung metastasis was established by tail vein injection of B16-F10 cells to evaluate the effect of AB4 (20, 40 mg/kg) on melanoma lung metastasis. Blood routine parameters were measured, pathological changes in lung tissue were observed by hematoxylin and eosin staining, and the expression of EMT-, MAPK-, and oxidative stress signaling pathway-related proteins in lung tissue was analyzed by Western blotting. RESULTS:In vitro, the MTT assay showed that AB4 (5, 10, 20 μmol/L) had no cytotoxic effect. AB4 inhibited the migration and invasion of A549 and H1975 cells induced by TGF-β1; decreased the expression of N-cadherin, vimentin, Slug, and Snail while increasing E-cadherin expression in the EMT pathway; decreased Keap1 expression and increased Nrf2 expression in the oxidative stress pathway; and reduced the phosphorylation levels of JNK, ERK, and p38 in the MAPK pathway. Invivo, AB4 alleviated weight loss, inhibited melanoma lung metastasis, and the mechanism may be related to the inhibition of EMT, oxidative stress, and the MAPK signaling pathway in the lung tissue of model mice. CONCLUSIONS:AB4 inhibits tumor metastasis by modulating EMT mediated by the MAPK and Keap1/Nrf2 signaling pathways.

With non-small cell lung cancer (NSCLC) accounts for the majority of diagnoses, lung cancer continues to be the most common and deadly type of cancer worldwide. While treatment options have advanced, therapeutic efficacy remains constrained by drug resistance and side effects, highlighting the demand for novel therapeutic agents. Cauloside A, a natural compound isolated from Fructus Akebiae, has shown anti-tumor potential; however, its precise mechanism of action in NSCLC has remained unclear. This study demonstrates that Cauloside A inhibits tumor growth by interacting with TLR4 to initiate a novel pyroptotic pathway. In A549 and H1299 cell lines, Cauloside A dose-dependently reduced cell viability and induced pyroptosis, as evidenced by LDH release assays, transmission electron microscopy, and Annexin V/7-AAD staining. Mechanistic studies revealed that Cauloside A specifically activated GSDME cleavage via JNK phosphorylation and subsequent caspase-3 activation, independent of GSDMD. Importantly, cellular thermal shift assays, surface plasmon resonance, and molecular docking analyses identified TLR4 as a potential binding target of Cauloside A. In a C57BL/6 mouse syngeneic (allograft) model, Cauloside A treatment resulted in dose-dependent suppression of tumor growth, accompanied by reduced Ki67 and PCNA expression without apparent toxicity. Western blot analysis confirmed consistent activation of the TLR4-JNK-caspase-3-GSDME signaling axis in tumor tissues. These findings establish Cauloside A as a potential TLR4 modular that triggers GSDME-dependent pyroptosis, providing both mechanistic insights and a promising therapeutic strategy for NSCLC.

Migraine is a chronic neurological disorder. As a classic formula for promoting blood circulation and removing blood stasis, Xuefu Zhuyu Decoction (XFZYD) has shown definite clinical efficacy in the treatment of migraine with blood stasis syndrome; however, its biological mechanisms have not yet been fully elucidated and warrant further investigation. Therefore, in this study, we employed untargeted metabolomics, combined with transcriptomic sequencing, to identify endogenous differential metabolites in plasma and differentially expressed genes in brain tissue that were significantly regulated by XFZYD in migraine rats. We further explored the key targets and potential therapeutic mechanisms involved. Through integrated multi-omics analysis, the MAPK/ERK signaling pathway was ultimately identified as the key pathway regulated by XFZYD. Molecular biology experiments further confirmed that XFZYD modulated the expression of genes involved in inflammation, vascular function, and stress response within this pathway, acting on key genes such as COX-2, P-ERK1/2, Nr4a1, and Egr2, thereby intervening in two core pathological processes: vascular dysfunction and neurogenic inflammation. In summary, from both the transcriptomic and terminal metabolic levels, this study systematically elucidated the multidimensional mechanisms by which XFZYD treats migraine by reversing the "blood stasis" state and exerting its effect of "promoting blood circulation and removing blood stasis," thereby providing new research directions and a theoretical basis for its clinical application.

Anemoside B4 (AB4), a prominent triterpenoid saponin from Pulsatilla chinensis, has advanced to NMPA-approved clinical trials for ulcerative colitis. Despite its robust macroscopic efficacy across inflammatory, oncological, viral, and metabolic landscapes, its precise molecular wiring remains fragmented. Here, we distill AB4's pleiotropic actions into four core mechanistic axes: upstream pattern-recognition receptor (PRR) sensing, cell-fate trajectory control, immunometabolic coupling, and antiviral innate immune amplification. Critically, we identify a fundamental "context-dependency paradox"-exemplified by AB4's stringent suppression of the PI3K/Akt pathway in malignancies versus its forceful activation in diabetic skeletal muscle. We posit that this paradoxical bidirectional regulation is an artifact of the "resolution ceiling" imposed by traditional bulk-tissue analyses, which inherently mask cell-type-specific interactomes. To transcend this bottleneck, we propose a transformative "Omics-to-Atom" paradigm. By integrating single-cell/spatial transcriptomics, cell-type-specific chemoproteomics, cryo-electron microscopy (Cryo-EM), and CRISPR-based functional genomics, this framework dissects cellular heterogeneity to reveal dynamic, context-specific drug-protein assemblies. Ultimately, this paradigm not only clarifies AB4's mechanism for patient stratification and proactive safety prediction but also provides a scalable blueprint for modernizing natural product pharmacology into precision medicine.

ETHNOPHARMACOLOGICAL RELEVANCE:Myristica fragrans Houtt. (Nutmeg) is used in Traditional Chinese Medicine (TCM) to warm the middle energizer and resolve phlegm-dampness, which aligns with the TCM view of Acute Lung Injury (ALI) pathogenesis involving Lung and Spleen dysfunction. AIM OF THE STUDY:This study aimed to systematically decipher the protective effect and molecular mechanism of Myristica fragrans Houtt. (MF) against LPS-induced ALI using a multi-omics strategy. MATERIALS AND METHODS:The protective effect of MF was evaluated in an LPS-induced murine ALI model by assessing inflammatory cytokines and lung histopathology. MF's chemical profile and blood-absorbed components were identified by UHPLC-Q/TOF-MS/MS. Transcriptomics, WGCNA, network pharmacology, and molecular docking were integrated to predict core targets and pathways, which were further validated in LPS-stimulated RAW264.7 macrophages. RESULTS:MF dose-dependently alleviated ALI, reducing TNF-α and IL-6 levels and lung injury, with high-dose efficacy comparable to dexamethasone. Importantly, 3,4-Dimethoxycinnamic acid was first identified from MF and confirmed blood-absorbable. Twenty-four bioactive components (mainly phenolic acids and lignans) were identified in blood. Integrative analysis pinpointed TLR4 and NF-κB as core targets, enriched in TLR4/NF-κB signaling. Molecular docking confirmed their stable binding with key MF components. In vitro, MF suppressed inflammatory mediator release, downregulated iNOS/COX-2, and inhibited the TLR4/NF-κB pathway. CONCLUSIONS:MF protects against LPS-induced ALI by mitigating inflammation. Its bioactive components exert effects through multi-target inhibition of the TLR4/NF-κB pathway, providing a pharmacological basis for its potential use in ALI treatment.

Although injectable anemoside B4 (AB4) has therapeutic potential for atopic dermatitis (AD), its clinical use is restricted by safety and pharmacokinetic issues. Therefore, we designed 42 AB4 derivatives, established a preliminary SAR for anti-inflammatory and antiallergic activities, and selected three promising compounds for in vivo evaluation in an AD mouse model, leading to the identification of B4-39 as the lead compound. In a DNCB-induced AD mouse model, topical B4-39 (6.6 mg/kg) was more effective than dexamethasone and free from its side effects, while matching the efficacy of crisaborole at a much lower dose and providing better skin barrier repair. Mechanistically, B4-39 targets pyruvate carboxylase (PC)─a novel therapeutic target in AD─modulating the TCA cycle to suppress dendritic cell activation and concurrently inhibiting NF-κB and NLRP3 inflammasome signaling. Given its enhanced efficacy, steroid-sparing safety, novel PC-targeted action, and favorable topical delivery, B4-39 is a highly promising candidate for AD treatment.

Traditional Chinese medicine(TCM) capable of clearing heat and removing toxin is most commonly used in clinical practice and has the effect of removing fire-heat and toxin. Studies have shown that most of the Ilex plants have the effect of clearing heat and removing toxin, among which the varieties of I. cornuta, I. pubescens, I. rotunda, I. latifolia, and I. chinensis are most widely used. These plants generally contain triterpenoids and their glycosides, alkaloids, flavonoids, phenylpropanoids, and other chemical components, especially pentacyclic triterpenoids. According to their skeletons, pentacyclic triterpenoids can be divided into the oleanane type, the ursane type, the lupinane type, etc. Among them, ursane-type components are the most abundant, and 136 species have been found so far. These components have been proved to have pharmacological effects such as anti-inflammatory, anti-tumor, hypolipidemic, anti-thrombosis, cardiomyocyte-protective, antibacterial, and hepatoprotective effects. Therefore, this paper systematically reviews the domestic and foreign literature on Ilex plants with a focus on the research progress on pentacyclic triterpenoids and their pharmacological activities, aiming to provide reference for the development of TCM resources with the effect of clearing heat and removing toxin.

Neutropenia is a common complication in oncology patients receiving chemotherapy, and rapid regeneration of functional neutrophils is critical for effective management. Bletilla striata polysaccharide (BSP) has shown therapeutic potential, but its mechanisms and molecular targets remain unclear. Here, we demonstrate that BSP accelerates the recovery of white blood cells, particularly neutrophils, in a chemotherapy-induced neutropenia (CIN) mouse model with cyclophosphamide (CY). The regenerated neutrophils retained phagocytic activity against bacteria, and BSP treatment significantly reduced mortality in the endotoxin-induced mouse death model. Furthermore, BSP enhanced the repopulation of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and promoted cell-cycle entry, resulting in increased frequencies of long-term hematopoietic stem cells (LT-HSCs), multipotent progenitors 2 (MPP2), and MPP3/4 subsets. Both in vitro colony formation and in vivo competitive transplantation assays confirmed that BSP reshapes hematopoietic reconstitution and corrects aberrant myeloid differentiation. PCR array analysis of HSPCs indicated that this process is mediated by C/EBPε and its downstream genes (LTF, LCN2, and ELANE). Consistently, BSP failed to support myeloid reconstitution following C/EBPε knockdown in vitro. In a C/EBPε knockout mouse model, HSPCs repopulation and regeneration were impaired, and BSP failed to promote neutrophil recovery after CY challenge or the mobilization of MPP2 and MPP3/4 subsets. The regulatory effects of BSP on C/EBPε target genes were also abolished. In conclusion, our findings identify C/EBPε as a key mediator of BSP activity, driving HSPCs repopulation and restoring hematopoietic function. These results highlight BSP as a potential therapeutic strategy for chemotherapy-induced neutropenia.

Acute lung injury (ALI) carries high mortality with limited treatment options. Dihydrotanshinone (DHT), a bioactive component of Radix Salviae Miltiorrhizae, exhibits anti-inflammatory properties, though its molecular targets remain unclear. Given the critical role of NIMA-related kinase 7 (NEK7)-mediated NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation in inflammatory diseases, this study investigated DHT's anti-inflammatory effects through NEK7-NLRP3 regulation. In vitro, lipopolysaccharide (LPS) + ATP-stimulated macrophages were used to activate the NLRP3 inflammasome. In vivo, ALI was induced in BALB/c mice via intratracheal LPS instillation (4 mg/kg). NEK7 knockdown was achieved through intravenous and intranasal delivery of NEK7 siRNA. Protein expression of NEK7-NLRP3 components was analyzed in both models. Results indicated that LPS + ATP stimulation significantly upregulated NEK7-NLRP3 inflammasome components (NEK7, NLRP3, c-caspase-1, c-IL-1β) in macrophages and ALI tissues. DHT inhibited NEK7-NLRP3 complex formation, suppressing inflammasome assembly and activation. NEK7 knockdown eliminated IL-1β secretion in vitro and alleviated ALI in vivo. DHT treatment prevented pulmonary injury and reduced neutrophil infiltration. This study identifies DHT as a novel NEK7-NLRP3 inflammasome inhibitor, demonstrating its therapeutic potential for ALI treatment. These findings provide a new pharmacological approach targeting NEK7-mediated inflammasome activation in inflammatory lung diseases.

Six guaiane sesquiterpenoids were isolated from the Ainsliaea fragrans Champ., including four new compounds and two known compounds. Their structures were elucidated through comprehensive nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and electronic circular dichroism (ECD) analyses. Compared to other compounds, Compound 1, glucozaluzanin C (GC), which has an intact α, β-unsaturated ketone structure, demonstrated superior anti-inflammatory activity. In vitro and in vivo experimental studies had shown that GC had a significant effect in alleviating LPS-induced macrophage inflammation and acute liver injury. GC could target TLR4 and p65 proteins and significantly inhibit the activation and expression of NF-κB/MAPK pathways.

The pharmacological effects of ziyuglycoside II (ZYG II), a saponin derived from Sanguisorba officinalis L., were partially limited by low oral bioavailability (< 5 %) attributable to its poor water solubility, low membrane permeability and extensive first-pass metabolism. To enhance solubility, salt form was selected as ZYG II sodium salt (ZYG-II-Na) in this study, with systematic screening and characterization of its solid forms. To improve the bioavailability, the dry powder inhaler (DPI) of ZYG-II-Na was designed using the optimized solid form. Three crystalline forms (I, II, and III) and two amorphous forms (I and II) of ZYG-II-Na were identified and thoroughly characterized by structural analysis, stability assessment, powder evaluation, in vitro solubility studies, and in vivo pharmacokinetic assessments. Among these, amorphs I and II maintained supersaturation for longer period of time in purified water and Gamble's solution, respectively, indicating enhanced solubility stability. Crystal III exhibited remarkable humidity stability, whereas other solid forms required controlled humidity conditions for storage. The aerodynamic properties of ZYG-II-Na support its suitability for pulmonary delivery. In rats, the oral bioavailability of crystal I was only 3.53 %, whereas the bioavailability of its DPI administration increased to 8.54 %. Remarkably, amorph II further enhanced absorption, reaching an absolute bioavailability of 16.8 %, representing a 4.8-fold enhancement over oral administration. In conclusion, the cooperation of solid form optimization and inhalation delivery successfully overcomes the key barriers associated with poor bioavailability of saponin ZYG II.

Acute lung injury (ALI) is a severe disease caused by viral infection that triggers an uncontrolled inflammatory response. This study investigated the capacity of jasurolignoside (JO), a natural compound, to bind to Toll-like receptor 4 (TLR4) and treat ALI. The anti-inflammatory properties of JO were evaluated in vitro through Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, and co-immunoprecipitation. The investigation utilized a lipopolysaccharide (LPS)-induced ALI animal model to examine the therapeutic efficacy and mechanism of JO in vivo. JO attenuated inflammatory symptoms in infected cells and tissues by modulating the NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome and the nuclear factor κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. Molecular docking simulations revealed JO binding to TLR4 active sites, confirmed by cellular thermal shift assay. Surface plasmon resonance (SPR) demonstrated direct interaction between JO and TLR4 with a Kd value of 35.1 μmol·L-1. Moreover, JO inhibited tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 secretion and reduced leukocyte, neutrophil, lymphocyte, and macrophage infiltration in ALI-affected mice. JO also enhanced lung function and reduced ALI-related mortality. Immunohistochemical staining demonstrated JO's ability to suppress TLR4 expression in ALI-affected mouse lung tissue. This study establishes that JO can bind to TLR4 and effectively treat ALI, indicating its potential as a therapeutic agent for clinical applications.

RNA viruses have evolved various strategies to bypass the mitochondrial antiviral-signaling protein (MAVS) pathway, effectively sidestepping the host's innate immunity. Therefore, searching for MAVS signalosome regulators is crucial for the development of antiviral drugs. Here, we found RNA viral-drived MAVS lysosomal trafficking was inhibited by Anemoside B4 (B4), a triterpenoid saponin from the herbal medicine Pulsatilla chinensis (Bunge) Regel., has significant antiviral activity. We performed biotin-B4 probe tandem proteomic profiling and identified ubiquitin fold modifier 1 (UFM1), one of the newly discovered Ub-like proteins (UBLs), as a key target of B4. Using cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) analysis, and molecular docking analysis we showed that B4 directly bound to UFM1 through Lys34 and Ile57 sites and promoted UFMylation of target proteins. Furthermore, we found that MAVS UFMylation could promote polyubiquitination, K48- and K63-linked ubiquitination, yet preventing K27-linked ubiquitination. MAVS aggregation and innate immunity response were suppressed in virus infected UFM1 KO cells even in the presence of B4. Moreover, the decrease in K27-ubiquitin (Ub) binding on MAVS and lysosomal degradation induced by B4 was also significantly prevented by UFM1 deficiency. Importantly, to verify that MAVS UFMylation is the actual target for B4, cytoprotective and viral titer analysis were executed in cells lacking UFM1 challenged with Enterovirus 71 (EV71) and Influenza A virus (IAV). The findings uncover the new finding broad-spectrum antiviral mechanism of B4, suggesting that UFMylation of MAVS could be an advantageous approach for countering RNA viral infections.

Background and aim: Pulsatilla saponin (Ps) was isolated from Pulsatilla chinensis (Bunge) Regel, a traditional Chinese medicine, that has anti-proliferation, anti-inflammation, anti-tumor and immunomodulation activities. However, the anti-psoriasis activity of Ps and its underlying mechanisms have not been fully elucidated. This study aims to investigate the effect and potential mechanisms of Ps on psoriasis. Experimental procedure: Ps underwent quality control through HPLC and NMR analysis. Wound healing assay, MTT, clone assay, and EdU staining were used to detect HaCaT cells proliferation. Western blot and immunofluorescence were used to assess the expression of proteins. The th17 cells population was analyzed by flow cytometry. The levels of cytokines in the mice skin tissues were measured by RT-qPCR and ELISA. Results and conclusion: In vitro, Ps has an inhibition effect on the proliferation of M5-induced HaCaT cells. Ps inhibited proliferation by regulating NF-kappa B and JAK1/STAT3 pathways. Additionally, Ps decreased TNF-alpha, IL-1R, and IL-6 mRNA levels in M5-induced HaCaT cells. In vivo, Ps improved the pathological damage of Imiquimod (IMQ)-induced psoriasis BALB/c mice skin and reduced the Ki67 level in mice skin tissue. Further results showed that Ps decreased Th17 cells differentiation and IL-22, IL-17A, IL-6, IFN-gamma, TNF-alpha, and IL-1R secretion. Ps could ameliorate the psoriatic symptoms, decrease M5-induced HaCaT cell proliferation, and decrease the differentiation of Th17 cells in IMQ-induced psoriasis mice. Ps suppressed the release of inflammation cytokines by regulating NF-kappa B and JAK1/STAT3 pathways. Those results indicate that Ps has promising therapeutic potential for psoriasis treatment.

The gut-brain axis is dysregulated as a consequence of alterations in the gut microbiota. These alterations increase toxic microbial metabolites, endotoxemia, and the release of immune mediators and contribute to the development of depression. Cubebin is a dibenzyl butyrolactone lignan, and its stem is also known as Agaru in Tibetan areas, it is commonly used as a sedative and tranquilizing medicine. This study aimed to investigate the effects of cubebin on chronic stress-induced depression-like behavior in mice. Cubebin was observed to mitigate depressive-like behavior in chronic unpredictable mild stress (CUMS) mice, influence the restoration of their cerebral cortex and hippocampal tissue morphology, and enhance the abundance of relevant intestinal flora in depression model mice, particularly by decreasing the abundance of Clostridium, Dorea, and Ruminococcus. The final protein function expression was normalized by regulating depression-related metabolic pathways. Concomitantly, the concentrations of neurotransmitters serotonin (5-HT), norepinephrine (NE), and dopamine (DA) in the brains of mice in the model group were enhanced, and their depressive symptoms were mitigated. Our study findings suggest that cubebin may ameliorate CUMS-induced depression in mice by modulating the microbe-gut-brain axis, elucidating the key effect of gut metabolites on depressive symptoms.

BACKGROUND:Lung cancer persists as the foremost malignancy in terms of both incidence and mortality on a global scale. Paraptosis, a unique type of regulated cell death, differs from apoptosis in its reliance on protein synthesis and its morphological hallmarks, characterized by cytoplasmic vacuole formation and distension of the endoplasmic reticulum (ER) or mitochondrial swelling. Flemiphilippinin A (Flp-A), a bioactive compound isolated from Flemingia prostrata, exhibits potent antitumor activity against lung cancer. However, the precise molecular mechanism underlying Flp-A's anti-lung cancer effects-as well as its direct cellular targets-remain unclear, leaving a critical gap in current research. PURPOSE:To reveal the therapeutic role and molecular mechanism of Flp-A in lung carcinoma and, for the initial time, evaluate its effects on gefitinib resistance in lung carcinoma cells. METHODS:The efficacy and mechanism of Flp-A were probed through in vitro and in vivo experiments. In vitro assays included the MTT assay, plate cloning assay, EdU proliferation assay, cell morphology analysis, Western blotting, transcriptome sequencing, JC-1 staining, ROS detection, Seahorse energy metabolism analysis, immunofluorescence, molecular docking, and CETSA to evaluate the therapeutic effects and mechanistic action of Flp-A on lung carcinoma. In vivo experiments were conducted using a mouse xenograft model of lung cancer to assess the antitumor efficacy, systemic toxicity, and underlying mechanisms of Flp-A. RESULTS:This study illustrated the therapeutic potential of Flp-A against lung carcinoma through in vitro and in vivo experiments. Our findings revealed that Flp-A triggers paraptosis in lung The evidence points to the possibility that Flp-A can enhance the specificity of drug-resistant lung carcinoma cells to gefitinib. cells by targeting c-Myc and hyperactivating endoplasmic reticulum (ER) stress. Furthermore, excessive ER stress created a glutamine-deficient environment, which promoted CHOP accumulation in mitochondria. This led to mitochondrial membrane potential disruption, increased ROS production, and inhibition of mitochondrial oxidative phosphorylation. Additionally, we observed that low concentrations of Flp-A combined with gefitinib triggered excessive ER stress and activated paraptosis in gefitinib-resistant lung cancer cells. The evidence points to the possibility that Flp-A can enhance the sensitivity of drug-resistant lung carcinoma cells to gefitinib. CONCLUSION:This research demonstrates that Flp-A exerts potent anti-tumor effects against lung cancer by targeting c-Myc to induce excessive ER stress and mediating mitochondrial dysfunction through CHOP, ultimately triggering paraptosis. Furthermore, Flp-A increases sensitivity of lung carcinoma cells to gefitinib through paraptosis induction. Our work provides the first evidence for Flp-A's anti-lung cancer mechanism via the paraptosis pathway and confirms its safety profile. Additionally, we reveal Flp-A's capacity to bypass gefitinib resistance in lung carcinoma. These findings position Flp-A as a encouraging novel medical agent for lung cancer treatment, particularly for addressing drug resistance.

Background Acute gouty arthritis is a metabolic disorder caused by monosodium urate (MSU) accumulation, leading to NLRP3 inflammasome activation and joint inflammation. Anemoside B4 (B4), a pentacyclic triterpenoid saponin, exerts significant anti-inflammatory effects. However, the precise molecular mechanisms underlying its therapeutic action, particularly its targeting of key components in NLRP3 inflammasome activation, remain unclear. Purpose The aim of this study was to elucidate the therapeutic mechanisms and target of B4 in treating MSU-induced macrophage pyroptosis and acute gouty arthritis, focusing specifically, on its interaction with NEK7, a critical regulator of NLRP3 inflammasome activation. Methods Comprehensive in vitro and in vivo methods were employed to examine the effects and mechanisms of B4. In vitro analyses included Western blot, co-immunoprecipitation (Co-IP), and immunofluorescence assays to assess NLRP3 inflammasome components and NEK7-NLRP3 interactions. The binding of B4 to NEK7 was evaluated using molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS), NEK7 gene silencing, and site-specific amino acid mutation experiments. In vivo, MSU-induced acute gouty arthritis mouse models and NEK7 knockdown mouse models were used to demonstrate the therapeutic effects and specificity of B4. Results This study provides the first evidence that B4 significantly inhibits MSU-induced inflammation and pyroptosis in macrophages by directly targeting NEK7 and disrupting the NEK7-NLRP3 complex, thereby reducing NLRP3 inflammasome activation. Additionally, B4 effectively suppressed MSU-induced ROS production, mitochondrial damage, and NF-κB activation. In vivo, B4 alleviated symptoms of acute gouty arthritis, reduced NLRP3 expression, and demonstrated specificity for NEK7 in NEK7 knockdown mouse models. Conclusion This study highlights B4 as an effective inhibitor of NLRP3 inflammasome activation by directly targeting NEK7, thereby mitigating inflammation and pyroptosis in acute gouty arthritis. These findings position B4 as a prospective therapeutic candidate for the management of acute gouty arthritis, providing insights into its molecular targets and mechanisms.