Abstract Super-resolution fluorescence microscopy overcomes the optical diffraction limit and has significantly advanced our understanding of biological complexity within the framework of fluorescence labelling 1 . Fluorescence labelling underpins this capability, enabling high photon budgets, superior signal contrast, and tuneable photophysical properties essential for diverse super-resolution modalities 2,3 . In contrast, label-free Raman imaging offers intrinsic chemical specificity 4 , supporting applications ranging from biomolecular fingerprinting to cell metabolic mapping 5–8 and histopathological tissue characterization 9,10 . Despite its label-free advantage and chemical specificity, Raman imaging remains fundamentally limited in both spatial resolution and imaging contrast due to inherently low signal throughput and weak intrinsic Raman contrast 11–13 . Here, we introduce spatial-filtering nanoscopy (SFN), a physics-driven super-resolution strategy that achieves resolution enhancement through targeted signal purification instead of signal amplification, offering a conceptually distinct pathway beyond the diffraction limit. SFN synergistically integrates a sub-millimetre microsphere lens (SMML) with a standard confocal Raman microscope, harnessing two complementary physical effects: (i) the photonic redistribution effect (PRE), which narrows the lateral excitation profile, and (ii) the three-dimensional spatial filtering (3D-SFE), which effectively suppresses both lateral and axial background. We demonstrate SFN-enabled super-resolution Raman imaging of silicon nanostructures, intact cells, and tissue sections, achieving an effective lateral resolution of approximately 40 nm, and chemically resolving subcellular features including organelles and pseudopodia without exogenous labels. These findings establish SFN as a broadly generalizable hardware-based super-resolution strategy, readily deployable on standard confocal platforms and extensible across diverse optical imaging modalities.

Secondary bacterial infection is a major cause of severe progression and death in viral pneumonia. The excessive recruitment of neutrophils accompanied by the formation of neutrophil extracellular traps (NETs) plays an important role in excessive inflammation caused by viral-bacterial co-infection, but effective pharmacological interventions remain lacking. Our study revealed that quercitrin (QTN), a flavonoid commonly found in traditional Chinese medicine and daily diets, significantly reduced mortality, recruitment of neutrophils, the production of inflammatory cytokines, and viral and bacterial loads in H1N1 influenza and methicillin-resistant Staphylococcus aureus (MRSA) coinfection-induced pneumonia in mice. Mechanistically, QTN down-regulates the interaction between Histone H3 and myeloperoxidase (MPO), thereby mitigating excessive inflammation induced by NETs. Our study demonstrates that inhibiting NETosis with QTN is one of the effective strategies for co-infection treatment.

Ethnopharmacological relevanceGypsum Fibrosum (Shigao), a widely used mineral medicine in traditional Chinese medicine (TCM), is frequently incorporated into herbal formulations for the treatment of severe respiratory infections. However, its specific pharmacological role and the underlying mechanisms within complex formulas remain to be fully elucidated.Aim of the studyGypsum Fibrosum is abundant in metal ions with potential neuromodulatory properties. This study aimed to investigate the pharmacological effects and mechanisms of Gypsum Fibrosum within Xuanbai Chengqi Decoction (XBCQ) against severe viral pneumonia, with a focus on the TRPV1-mediated gut-brain-lung axis and pulmonary endothelial barrier protection.Materials and methodsA mouse model of H1N1-induced viral pneumonia was employed to evaluate the contribution of Gypsum Fibrosum to the therapeutic efficacy of XBCQ, with comparison to XBCQ lacking Gypsum Fibrosum and XBCQ substituted with Gypsum Ustum (calcined gypsum). Therapeutic effects were assessed by histopathological examination, cytokine measurement, and flow cytometry. Combined transcriptomic and single-cell RNA-sequencing analyses identified the β-adrenergic receptor (ADRB2) in pulmonary endothelial cells as a key target. The underlying neuroendocrine mechanism was further verified using RT-qPCR, immunofluorescence, western blotting, and intestinal TRPV1 nociceptor blockade with resiniferatoxin (RTX). Finally, the endothelial protective effects were validated in hypoxia-injured human umbilical vein endothelial cells (HUVECs) using an ADRB agonist.ResultsGypsum Fibrosum, but not Gypsum Ustum, contributed critically to the anti-inflammatory and lung-protective effects of XBCQ. Mechanistically, Gypsum Fibrosum suppressed the overexpression of intestinal TRPV1 nociceptors and their signaling transmission to the central nervous system (CNS), thereby attenuating hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis and excessive pulmonary sympathetic nerve activity. Such neuro-immune modulation restored ADRB2 receptor function and preserved pulmonary vascular endothelial integrity.ConclusionGypsum Fibrosum contributes to the therapeutic efficacy of XBCQ through a neuroregulatory mechanism centered on the TRPV1-mediated gut-brain-lung axis, thereby protecting the pulmonary endothelial barrier and enhancing XBCQ’s effectiveness against severe viral pneumonia.

BACKGROUND:α-Linolenic acid (ALA), an essential plant-derived polyunsaturated fatty acid with high safety, has been preliminarily shown to possess antiviral activity in vitro. However, the lack of in vivo activity evaluation and mechanism research has limited the understanding of its precise antiviral mechanism and therapeutic potential. PURPOSE:This study elucidates the mechanism by which ALA disrupts viral lipid envelopes, conferring broad-spectrum antiviral activity, and evaluates its feasibility as a natural botanical agent against respiratory enveloped viruses. METHODS:ALA (100 μM) was tested against H1N1, H3N2, and HCoV-229E in vitro. Its protective effect on the lungs were evaluated via intranasal and intraperitoneal administration in vivo. Raman spectroscopy, transmission electron microscopy, immunohistochemistry, and other techniques were employed to investigate the effects of ALA on viral envelopes, host membrane lipid signaling, pulmonary surfactant, lipoxygenases, and B-cell function. Additionally, a simulated mask application was tested to evaluate its practical antiviral potential. RESULTS:ALA exhibited differential effects on viral and host membranes. Transmission electron microscopy and Raman spectroscopy confirmed that it disrupts viral envelopes while restoring the lipid balance of host membranes. Intranasal administration reduced viral loads and tissue damage in H1N1-infected mice, enhanced pulmonary surfactant and lipoxygenase levels independently of B-cell activation. ALA also showed broad-spectrum activity against HCoV-229E and H3N2, and further simulated mask tests confirmed that ALA significantly impaired viral infectivity. CONCLUSION:This work establishes a unique dual mode of viral membrane destruction and host membrane protection for ALA. It innovatively reveals the lipid-targeted antiviral advantage of natural plant lipids, offering novel mechanistic insights and support for developing phytomedicine-based antiviral interventions.

Cryptococcal meningitis is a devastating fungal infection of the central nervous system that disproportionately affects immunocompromised individuals and carries high mortality. Amphotericin B (AmB), the clinical gold standard, exhibits potent fungicidal activity but is associated with substantial systemic toxicity. Nanoparticle-based delivery systems have been explored to enhance AmB therapeutic efficacy and mitigate systemic toxicity; however, inadequate blood-brain barrier (BBB) penetration and the absence of pathogen-specific targeting remain major obstacles. Herein, we developed a biomimetic targeted nanoplatform for intranasal delivery to address these challenges. The nanoplatform was constructed by coating self-assembled AmB and chlorin e6 (Ce6) hybrid nanoparticles with genetically engineered macrophage membranes overexpressing the β-glucan receptor Dectin-1. Following intranasal administration, the nanoplatform efficiently reached the meninges and specifically bound Cryptococcus neoformans via Dectin-1-mediated recognition, upon which ultrasound stimulation triggered reactive oxygen species (ROS) generation from Ce6 and rapid AmB release. In vitro studies confirmed the enhanced fungal targeting and antifungal efficacy of the engineered nanoplatform. In a murine model of cryptococcal meningitis, treatment with this nanoplatform significantly reduced brain fungal burden, alleviated histopathological damage, and prolonged survival compared with commercially available liposomal AmB. Collectively, these findings highlight the biomimetic targeted nanoplatform as a promising therapeutic strategy for fungal infections in the brain.

Co-infection with influenza A virus (IAV) and methicillin-resistant Staphylococcus aureus (MRSA) often causes severe pneumonia clinically; however, the role of innate immunity in this setting remains poorly understood. In our study, we established a murine co-infection model using low-lethality IAV and MRSA. Compared with single IAV or MRSA infection, co-infection with relatively low-lethality IAV and MRSA resulted in more severe pneumonia. Transcriptomic analysis indicated marked upregulation of genes involved in the pyroptotic signaling pathway. Consistently, flow cytometry and immunofluorescence analyses revealed caspase-1 activation and colocalization of gasdermin D (GSDMD) with macrophages in the lung. The RAW264.7 macrophage cell line was used for in vitro validation. Co-infection significantly enhanced the cleavage of caspase-1 and GSDMD in RAW264.7 cells. Furthermore, disulfiram, a pyroptosis inhibitor, was incorporated into the antiviral and antibacterial combination treatment. Although combined oseltamivir and linezolid treatment failed to fully alleviate lung injury, the inclusion of disulfiram, a GSDMD pore formation inhibitor, significantly ameliorated pneumonia symptoms and reduced inflammatory responses. Collectively, our findings highlight that macrophage pyroptosis contributes to the exacerbation of pneumonia induced by IAV and secondary MRSA co-infection. Inhibition of GSDMD-mediated pyroptosis may represent a viable therapeutic approach to alleviate disease severity and improve outcomes in lethal co-infection.

Antiviral agents are a cornerstone of therapeutic strategies against viral infections; however, the early-stage discovery and optimization of viral inhibitors remain time-consuming, resource-intensive, and mechanistically opaque. Here, we introduce a mechanism-guided strategy for rapid antiviral candidate identification and evaluation using confocal Raman microscopy (CRM) imaging across cellular and animal models. CRM enables label-free, quantitative, and spatially resolved assessment of drug efficacy at the single-cell level by correlating subcellular biochemical distributions, specifically of lipids, nucleic acids, and proteins, with virus-induced morphological alterations. To validate this approach, we evaluated α-linolenic acid (ALA) against influenza A virus (IAV) strain H1N1 PR8 in human non-small cell lung carcinoma A549 cells. CRM imaging revealed that ALA treatment restored lipid homeostasis, preserved nucleic acid integrity, and normalized protein expression profiles, changes those were spatially concordant with recovery of cellular architecture. These mechanistic insights directly informed the rational design of subsequent in vivo studies in murine models, markedly reducing reliance on empirical trial-and-error. Moreover, CRM was extended to ex vivo analysis of lung tissue, enabling high-resolution interrogation of alveolar architecture and surfactant layer composition in infected tissues. Collectively, our work establishes CRM as a rapid, mechanism-informed platform for prioritizing antiviral candidates, bridging molecular pharmacology and phenotypic response, and thereby accelerating the translation of promising leads into preclinical development.

Short-chain fatty acids (SCFAs) are widely recognized as important microbial metabolites of polysaccharides, playing crucial roles in modulating the intestinal immune system and protecting the intestinal barrier. This study investigated the effects of HCPM, a heteropolysaccharide from medicinal Houttuynia cordata, on the SCFAs production in mice infected with H1N1, and elucidated the potential mechanism underlying the therapy of H1N1-induced acute lung injury (ALI) through comparison with inactive Artemisia annua polysaccharide (AAP01-1). Our findings revealed that HCPM effectively alleviated ALI in H1N1-infected mice and specifically promoted acetate production in the feces rather than the serum acetate levels. In comparison, the inactive polysaccharide AAP01-1 had no significant impact on fecal acetate content. This evidence suggests that the therapeutic effects of HCPM could be mediated by intestinal acetate rather than serum acetate. The correlation analysis demonstrated that the abundance of Phocaeicola vulgatus specifically regulated by HCPM exhibited a strong correlation with the inflammatory indicators or the acetate levels in feces. In vitro, HCPM significantly enhanced acetate production by P. vulgatus. Acetate by enema restored the balance of Th17/Treg cells by activating GPR43 and inhibiting the JAK2/STAT3 pathway in the intestine. These findings suggest that acetate produced by the interaction between P. vulgatus and HCPM alleviates H1N1-induced ALI in mice through the GPR43/JAK2/STAT3 pathway in the intestine.

The coinfection of respiratory viruses and bacteria is a major cause of morbidity and mortality worldwide, despite the development of vaccines and powerful antibiotics. As a macromolecule that is difficult to absorb in the gastrointestinal tract, a homogeneous polysaccharide from Houttuynia cordata (HCPM) has been reported to exhibit anti-complement properties and alleviate influenza A virus (H1N1)-induced lung injury; however, the effects of HCPM without in vitro antiviral and antibacterial activities on more complicated pulmonary diseases resulting from viral-bacterial coinfection remains unclear. This study established a representative coinfection murine pneumonia model infected with H1N1 (0.2 LD50) and methicillin-resistant Staphylococcus aureus (MRSA, 107 CFU). HCPM significantly improved survival rate and weight loss, and ameliorated gut-lung damage and inflammatory cytokine production. Interestingly, the therapeutic effect of HCPM on intestinal damage preceded that in the lungs. Mechanistically, HCPM inhibited the overactivation of the intestinal complement (C3a and C5a) and suppressed the activation of the NLR family pyrin domain-containing 3 (NLRP3) pathway, which contributes to the regulation of the Treg/Th17 cell balance in the gut-lung axis. The results indicate the beneficial effects of an anti-complement polysaccharide against viral-bacterial coinfection pneumonia by modulating crosstalk between multiple immune regulatory networks.

Ethnopharmacological relevance: Ulcerative colitis (UC), a chronic inflammatory bowel disease, has become a significant public health challenge due to the limited effectiveness of available therapies. Huoxiang Zhengqi (HXZQ), a well-established traditional Chinese formula, shows potential in managing UC, as suggested by clinical and pharmacological studies. However, the active components and mechanisms responsible for its effects remain unclear. Aim of study: This study aimed to identify the bioactive components of HXZQ responsible for its therapeutic effects on UC and to elucidate their underlying mechanisms. Materials and methods: The effect of HXZQ against dextran sodium sulfate (DSS)-induced colitis was investigated. Ingredients in HXZQ were characterized and analyzed in colitic mice using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). In vitro, biological activity of compounds was assessed using lipopolysaccharide (LPS)-induced Ana-1 cells and bone marrow-derived macrophages (BMDMs), tumor necrosis factor-alpha (TNF-alpha)-induced Caco-2 cells, and isolated intestinal crypts from colitic mice. These results were confirmed in vivo. The targets of the components were identified through bioinformatics analysis and validated via molecular docking, enzyme inhibition assays, and in vivo experiments. Hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative real-time polymerase chain reaction (qPCR) were employed to confirm the pharmaceutical effects. Results: A clinical equivalent dose of HXZQ (2.5 mL/kg) effectively treated DSS-induced colitis. A total of 113 compounds were identified in HXZQ, with 35 compounds detected in colitic mice. Glycyrrhizic acid (GA) and patchouli alcohol (PA) emerged as key contributors to the anti-colitic effects of HXZQ. Further investigation revealed that HXZQ and its active components decreased the levels of pro-inflammatory cytokines TNF-alpha, interleukin-1(3 (IL-1(3), and interleukin-6 (IL-6) in colon, likely by inhibiting nuclear factor kappa-B (NF-kappa B) signaling pathway. This inhibition indirectly activated the intestinal farnesoid X receptor (FXR) signaling pathway, correcting bile acid imbalances caused by colitis. Additionally, these components significantly enhanced the expression of tight junction proteins ZO-1 and Occludin, as well as the adhesion protein E-cadherin, and reduced goblet cell loss, thereby repairing intestinal barrier injury. Mechanistically, GA and PA were found to inhibit 11(3-hydroxysteroid dehydrogenase 1 (11(3-HSD1) activity, leading to increased local active corticosterone levels in the intestine to exert anti-inflammatory effects. Notably, the inhibition of 11(3-HSD1 with the selective inhibitor BVT2733 (BVT) ameliorated colitis in mice. Conclusions: HXZQ exhibits therapeutic effects on UC, primarily through GA and PA inhibiting 11(3-HSD1. This suggests new natural therapy approaches for UC and positions 11(3-HSD1 as a potential target for colitis treatment.

ETHNOPHARMACOLOGICAL RELEVANCE:Bubali Cornu (BC) has a long history of use in traditional Chinese medicine for the treatment of various conditions, including high fever, hemoptysis, epistaxis, and infections. Despite its historical use, the active components responsible for its therapeutic effects, particularly in viral pneumonia, remain poorly understood. AIM OF THE STUDY:The objective of this study was to identify the active compounds in BC with antiviral activity against H1N1 and to evaluate their therapeutic potential in alleviating inflammation in H1N1-infected acute lung injury (ALI) mice. MATERIALS AND METHODS:The antiviral components of BC were isolated using silica gel column chromatography and high-performance liquid chromatography (HPLC) according to a bioactive guided separation strategy. Then, the protective effects of these components in H1N1-induced ALI mice were evaluated. To explore the potential mechanisms, network pharmacology (NP) analysis and qPCR were used to screen the related signaling pathways or proteins. Finally, the functions of the most effective compound were verified in H1N1-infected RAW264.7 macrophages, focusing on its regulatory effects on key proteins. RESULTS:The ethyl acetate fraction (EAF) of BC had a protective effect against ALI induced by H1N1 in mice. Subsequently, 13 compounds were isolated from EAF of BC, of which nicotinic acid (NA) showed the best antiviral activity in vitro. In vivo studies showed that NA treatment of ALI mice infected with H1N1 significantly reduced lung index and inflammation, and improved lung tissue morphology. Flow cytometry analysis revealed that NA reduced the over-recruitment of macrophages and neutrophils to alleviate the inflammatory storm. As verified by NP and qPCR, NA reduced the mRNA levels of NOS2 and NOX1 in the lungs of mice with viral pneumonia, but Western blot analysis only showed that NA inhibited the expression of NOS2. Finally, NA significantly inhibited the expression of NOS2 in RAW264.7 cells infected with H1N1, suggesting that NA might alleviate ALI induced by H1N1 by inhibiting NOS2 expression in pulmonary macrophages. CONCLUSION:The present study demonstrated that NA isolated from BC exhibited significant antiviral and anti-inflammatory properties in H1N1-induced ALI. NA might alleviate the progression of H1N1 pneumonia by inhibiting virus replication, reducing the over-recruitment of macrophages and neutrophils, and inhibiting the expression of NOS2. These findings provided valuable insights into the potential of BC in the treatment of viral infections, and highlighted the importance of exploring traditional animal-derived TCM in modern antiviral applications.

Background: Chronic infection with the hepatitis B virus (HBV) represents a significant global health concern. Baicalein, a naturally occurring flavone derived from the roots of Scutellaria baicalensis Georgi, has exhibited both anti-inflammatory and antiviral activities. S. baicalensis is extensively utilized in traditional Chinese medicine for the treatment of various liver disorders, including hepatitis. However, the specific anti-HBV effects of baicalein have not been fully elucidated. Purpose: This study aimed to investigate the inhibitory effects of baicalein on HBV and to elucidate its underlying mechanisms. Materials and Methods: The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were measured using enzyme-linked immunosorbent assay (ELISA) kits. Quantification of HBV DNA was performed using quantitative real-time polymerase chain reaction (qRT-PCR). Western blot analysis was conducted to evaluate proteins involved in autophagy, lysosomal acidification, and autophagy-related signaling pathways. Immunofluorescence microscopy was utilized to assess autophagic flux and lysosomal acidification. Results: Baicalein demonstrated significant inhibition of HBsAg, HBeAg, and HBV-DNA secretion in both in vivo and in vitro environments. Subsequent investigations revealed that baicalein disrupted the intracellular trafficking of the hepatitis B virus by inhibiting the CCDC88A-AKT-mTOR (Coiled coil domain containing protein 88A- protein kinase B-mammalian target of rapamycin) signaling pathway. Additionally, baicalein induced autophagy in HepG2 (Human hepatocellular carcinoma cell line 2) and HepG2.215 cell models. The anti- hepatitis B antigen effect of baicalein was partially attenuated when both early and late stages of autophagy were inhibited. A significant correlation was identified between the phosphorylation of AMPK alpha and the enhanced autophagy observed in baicalein-treated cells. Conclusions: This study elucidates a novel mechanism by which baicalein inhibits the hepatitis B virus (HBV). Specifically, baicalein exerts its antiviral effects by activating autophagy and suppressing the CCDC88A-AKTmTOR signaling pathway.

Bitter apricot kernel is a common traditional Chinese medicine used for lung diseases. Previous studies showed that Xuanbai-Chengqi decoction (XCD) containing bitter apricot kernel protected the alveolar and intestinal barriers in influenza-infected mice. However, the specific contribution of bitter apricot kernel and its active substances in viral pneumonia remain unclear. This study aimed to identify the main active ingredient in bitter apricot kernel and investigate its mechanism in protecting the alveolar epithelial barrier in viral pneumonia. Bitter apricot kernel was evaluated based on the efficacy differences between XCD and XCD without bitter apricot kernel. Amygdalin was identified through in vitro activity tests and verified in vivo. Immunohistochemistry, RT-qPCR, and WB were used to assess barrier protection and anti-inflammatory effects. The molecular mechanisms were explored using SPR/LC/MS and validated experimentally. Removing bitter apricot kernel significantly weakened XCD’s protective effect in influenza A virus-infected mice. Amygdalin showed anti-inflammatory, anti-hypoxia anti-influenza virus activities, and promoted endothelial cell migration in vitro. Amygdalin at 100 mg/kg effectively mitigated pulmonary injury and attenuated excessive inflammatory responses by regulating IL-6 and IL-10 in IAV-infected murine models. Oseltamivir is more effective than amygdalin in inhibiting the replication of influenza viruses and upregulating the expression level of IL-10. Amygdalin protected the alveolar barrier by restoring alveolar type II cells (AT2) and promoting alveolar regeneration, while upregulating surfactant protein A (SP-A) and aquaporin protein-5 (AQP5). Amygdalin bound selectively to vasoactive intestinal peptide receptor 1 (VIPR1) thereby upregulating cyclic adenosine monophosphate (cAMP) levels and the protein expression levels of Protein kinase A (PKA) and Phosphor-protein kinase A (p-PKA). Amygdalin is the key bioactive component of bitter apricot kernel, which exhibits protective effects in an IAV-induced pneumonia mouse model by activating the cAMP/PKA/p-PKA signaling cascade and recapitulating the biological effects of vasoactive intestinal peptide (VIP).

ETHNOPHARMACOLOGICAL RELEVANCE:The disruption of the pulmonary endothelial barrier is an important pathological feature of severe pneumonia. While our prior research has confirmed the therapeutic efficacy of the herbal formulation Xuanbai Chengqi Decoction (XBCQ) against severe pneumonia, the underlying molecular mechanisms remain elusive. AIM OF THE STUDY:This study was designed to elucidate the mechanism of XBCQ in preserving pulmonary endothelial integrity through sphingolipid metabolism modulation during severe influenza pneumonia. MATERIALS AND METHODS:This study employed an H1N1 influenza murine model (4 × LD50) to evaluate the therapeutic efficacy of XBCQ against viral pulmonary injury. Air-blood barrier protection was systematically assessed through quantitative analysis of surfactant protein expression and glycocalyx integrity in lung. Lipidomic perturbations and transcriptional profiles were characterized using high-resolution mass spectrometry and Illumina NovaSeq 6000 sequencing. The mechanism of XBCQ in regulating Smpd3 and downstream signaling was delineated through integrated in vivo and in vitro investigations. Furthermore, pulmonary endothelial preservation via nSMase2/S1PR2 modulation was validated using selective pharmacological inhibitors in both experimental systems. RESULTS:XBCQ treatment significantly ameliorated lung injury and preserved pulmonary endothelial integrity. Integrated transcriptomic and untargeted lipidomic analyses identified Smpd3 as the key gene responsible for orchestrating sphingolipid metabolic dysregulation in H1N1-infected mice. Administration of XBCQ rectified the sphingolipid imbalance, suppressed Smpd3 expression, and consequently modulated the S1PR2/eNOS signaling axis. Furthermore, pharmacological inhibition of nSMase2 and S1PR2 enhanced lung endothelial barrier integrity both in vivo and in vitro. Collectively, these findings substantiate the mechanism whereby XBCQ mitigates barrier dysfunction via the regulation of the Smpd3/S1PR2 pathway. CONCLUSIONS:This study demonstrates that XBCQ exerts its therapeutic effects by correcting sphingolipid metabolic dysregulation and regulating the S1PR2/eNOS signaling pathway. These results validate the potential therapeutic value of targeting Smpd3 and S1PR2 in the management of severe pneumonia.

Influenza virus causes millions of infections annually, with severe viral pneumonia accounting for a substantial proportion of associated mortality. Co-infections with other respiratory pathogens further worsen clinical outcomes, yet the underlying mechanisms remain poorly defined. Clinical observations have documented influenza–coronavirus co-infections, but their pathological interplay has not been elucidated.To address this gap, we established a murine model of sequential H1N1 (FM1 or PR8 strains) and human coronavirus 229E (HCoV-229E) infection, which recapitulated the fatal pneumonia observed in patients. Strikingly, co-infected mice exhibited accelerated mortality and exacerbated lung pathology despite reduced viral loads, revealing a paradoxical “low-virus, high-inflammation” state.Targeted transcriptional and functional analyses identified ZBP1-dependent necroptosis as the central driver of pathology. Lung tissues showed robust activation of the ZBP1–RIPK3–MLKL axis, which correlated with cytokine overproduction and histopathological damage. Notably, inhibition of RIPK3—rather than direct antiviral treatment—restored lung function and improved survival, highlighting the causal role of necroptosis independent of viral replication. Furthermore, this ZBP1-driven mechanism was conserved across diverse influenza strains, emphasizing its broad biological relevance.Collectively, our findings reveal a previously unrecognized immunopathological axis in which influenza-primed lungs undergo coronavirus-triggered necroptotic storm, directly linking viral co-infection to fatal pneumonia. These results identify ZBP1 and RIPK3 as promising therapeutic targets for decoupling inflammation from viral clearance in severe respiratory co-infections.

Qingfei Paidu decoction (QFPDD) has been extensively used in clinical treatments during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic. SARS-CoV-2 primarily invades host cells via its spike (S) protein binding to the angiotensin-converting enzyme 2 (ACE2) on the cell membrane, mediating viral-host membrane fusion. Blocking viral entry is a crucial step in preventing infection, with the interaction between the S receptor binding domain (S-RBD) and ACE2 being a key antiviral target. Given that SARS-CoV-2 predominantly affects the respiratory system and approximately 25% of patients suffering from corona virus disease 2019 (COVID-19) with gastrointestinal symptoms, we are committed to identifying more active ingredients in QFPDD that target the respiratory and gastrointestinal tracts of COVID-19 patients. Among medicinal plants, ephedra and liquorice derived from QFPDD, along with two other Chinese herbs, Platycodon grandiflorum and Radix Rhei Et Rhizome (rhubarb), have garnered our interest. These herbs have historically been used in traditional Chinese medicine (TCM) for treating infectious diseases with respiratory and digestive symptoms. Here, we established a library containing all components of the four individual herbs gathered from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and performed structure-based virtual screening to identify potential ACE2/S-RBD inhibitors. Subsequently, we selected 10 ingredients from the top 30 candidates and evaluated their activities using a pseudovirus neutralization assay. Delphinidin and deapio platycodin D (DPD) showed significant antiviral potential with half-maximal inhibitory concentration (IC 50 ) values of 45.35 µM and 1.38 µM, respectively. Furthermore, delphinidin also inhibited the 3-chymotrypsin-like protease (3CL pro ), indicating its dual-viral target inhibitory potential. Notably, DPD effectively suppressed HCoV-229E replication in BEL-7402 cells. This study not only provides a strategy for rapid identifying antiviral agents from TCM in anticipation of future pandemics but also offers theoretical and experimental evidence to support for the clinical use of QFPDD. Graphical Abstract

BackgroundThe protective role of gut microbiota and its metabolites against intestinal damage in sepsis patients remain unclear.MethodsFecal samples were acquired from patients categorized into sepsis and non-sepsis groups for analysis of microbial composition via 16S rRNA sequencing and untargeted metabolomics analysis. We assessed the impact of gut microbiota from sepsis patients on intestinal barriers in antibiotic-treated mice. Furthermore, We conducted spearman’s correlation analysis to examine the relationship between metabolites and the severity of sepsis. Additionally, we performed animal experiments to validate the functionality of identified metabolites.ResultsThe diversity of intestinal flora is decreased in patients with sepsis compared to the control group. Through fecal microbiota transplantation experiments, it was discovered that the gut microbiota derived from sepsis patients could induce intestinal damage in antibiotic-treated mice. Metabolomics analysis of the microbiota revealed a significant enrichment of the Valine, leucine, and isoleucine biosynthesis pathway. Further analysis showed a significant decrease in the abundance of L-valine in sepsis patients, which was negatively correlated with APACHE-II and SOFA scores. In sepsis mouse experiments, it was found that L-valine could alleviate sepsis-induced intestinal damage.ConclusionAlterations in microbial and metabolic features in the gut can affect the severity of sepsis. Furthermore, L-valine can protect against sepsis-induced intestinal injury.