Novel plant-derived exosome-like nanovesicles from pineapple: preparation, characterization, anti-inflammation effect via miR396-5p-Hrd1/NF-κB axis

Novel plant-derived exosome-like nanovesicles from pineapple: preparation, characterization, anti-inflammation effect via miR396-5p-Hrd1/NF-κB axis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel plant-derived exosome-like nanovesicles from pineapple: preparation, characterization, anti-inflammation effect via miR396-5p-Hrd1/NF-κB axis Weixi Xie, Zun Wang, Lang Deng, Siyuan Li, Yajing Wang, Wei Liu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9327044/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Plant-derived nanoparticles (PDNPs) have attracted considerable attention as promising bioactive nutraceutical agents. Pineapple ( Ananas comosus (Linn.) Merr. , Ac) is a functional fruit with notable anti-inflammatory and antioxidant effects, though its exact active components and mechanisms remain unclear. Our study shows that orally administered pineapple-derived nanoparticles (AcNPs) alleviate sepsis-induced lung and liver injury in mice. AcNPs preferentially accumulate in these organs, are mainly taken up by macrophages, and exert anti-inflammatory effects dependent on macrophage activity, as confirmed by clodronate liposome treatment. Mechanistic analyses integrating network pharmacology and RNA sequencing suggested that AcNPs primarily exert their effects via modulation of NF-κB–dependent inflammatory signaling. AcNPs suppressed NF-κB and inflammasome activation, reduced ROS-induced ER stress, inhibited M1 polarization, and promoted M2 polarization, thereby alleviating macrophage-mediated inflammation. Importantly, this inhibitory activity was attributed to RNA components within AcNPs rather than lipid or protein constituents. Deep RNA sequencing combined with miRNA mimic and inhibitor experiments identified miR396-5p as the principal bioactive molecule responsible for the suppression of macrophage inflammatory activation. Moreover, miR396-5p directly targets Hrd1, mediating the regulatory effect of AcNPs on NF-κB signaling. Collectively, these findings position AcNPs as a promising nanotherapeutic candidate with potential for the treatment of sepsis-induced acute lung/liver injury. Sepsis NF-κB Hrd1. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection( 1 ). Currently effective targeted therapies remain limited( 2 ). The pathogenesis of sepsis is primarily driven by excessive inflammatory responses and immune dysregulation( 3 , 4 ). During pathogenic infections, complex interactions between immune cells and inflammatory mediators form an intricate regulatory network( 5 ). Activation of immune cells initiates signaling cascades that release pro-inflammatory mediators, amplifying immune activation and leading to multi-organ dysfunction.( 5 , 6 ). The lungs and liver are particularly susceptible to early damage during sepsis( 7 ). Sepsis-induced acute lung injury or distress syndrome causes high mortality in critically ill patients( 8 ). Moreover, sepsis-related hepatic injury represents a strong independent predictor of mortality( 9 ). Early implementation of hepatoprotective strategies has been shown to improve survival outcomes( 10 ). Therefore, there is an urgent need to develop safer, more effective, and cost-efficient therapeutic strategies for sepsis. The pathogenesis of sepsis involves complex immune dysregulation and excessive inflammatory responses, in which the Hrd1 (HMG-CoA reductase degradation protein 1)-mediated NF-κB signaling pathway plays a crucial regulatory role( 11 – 13 ). Hrd1 is a key E3 ubiquitin ligase and a core component of the endoplasmic reticulum–associated degradation (ERAD) system( 14 ). In response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), Hrd1 promotes the ubiquitination and degradation of IκBα, thereby activating the NF-κB pathway and enhancing the transcription of pro-inflammatory mediators( 12 ). Besides activating immune cells, NF-κB regulates NLRP3 inflammasome assembly, promoting caspase-1 activation and IL-1β/IL-18 release, thereby amplifying inflammation and causing organ dysfunction( 15 – 17 ). Evidence indicates that excessive activation of the Hrd1/NF-κB axis and downstream inflammasomes is closely associated with disease severity and organ injury, especially in the lungs and liver. Inhibition of Hrd1 or NF-κB signaling has been shown to suppress inflammasome activation and reduce the production of pro-inflammatory mediators, thereby alleviating sepsis-induced acute lung and liver injury( 12 , 18 ). Collectively, these findings highlight the Hrd1/NF-κB/inflammasome axis as a key player in the pathophysiology of sepsis and a promising therapeutic target for mitigating multi-organ damage. Pineapple is a widely consumed tropical fruit with high nutritional value and notable medicinal properties, whose abundant dietary fiber and bioactive compounds confer potent natural pharmacological activities( 19 ). Previous studies have demonstrated that pineapple intake provides multiple health benefits, including anti-obesity, anti-inflammatory, and antioxidant effects( 20 – 23 ). However, the bioactive molecules and mechanisms remain unclear. Recently, plant-derived nanoparticles (PDNPs) have gained attention for their nucleic acids, lipids, and proteins that modulate human gene expression and aid disease treatment.( 24 ). For instance, ginger-derived extracellular vesicle-like nanoparticles have been shown to alleviate COVID-19-induced lung inflammation( 25 ), Rgl-exomiR-7972-mediated Rehmannia-derived EVs protect against LPS-induced lung injury and intestinal dysregulation( 26 ), and Prunus mume-derived EVLPs exert protective effects on ulcerative colitis via miR-159( 27 ). In the present study, we report for the first time that orally administered AcNPs preferentially accumulate in the lungs and liver and markedly mitigate sepsis-induced acute lung and liver injury. Mechanistically, AcNPs directly target Hrd1 via miR396-5p, thereby inhibiting the ubiquitination and degradation of IκB, which attenuates NF-κB signaling and inflammasome activation in macrophages, suppresses ROS-mediated endoplasmic reticulum stress, and modulates macrophage polarization. In summary, this study elucidates the active components and functional pathways of AcNPs in sepsis intervention, establishes a scientific foundation for their future clinical application, and highlights the translational potential of plant-derived PDNPs in disease treatment. 2. Materials and methods 2.1 AcNPs preparation Pineapple ( Ananas comosus ) were first washed with deionized water, peeled, and cut into small pieces to obtain Ananas comosus juice. The resulting juice was processed as follows: centrifuged at 3,000 × g for 20 min to remove dead cells, followed by centrifugation at 10,000 × g for 30 min to eliminate cell debris. The supernatant was then ultra-centrifuged at 120,000 × g for 60 min, and the pellets were resuspended in sterile PBS (Procell,China). The mixture was subsequently layered onto a sucrose gradient and ultra-centrifuged again at 120,000 × g for 90 min to purify AcNPs. Finally, AcNPs were diluted in sterile PBS and filtered through a 0.45 µm membrane. 2.2 AcNPs characterization To measure the particle size and concentration of AcNPs, the samples were diluted in PBS and analyzed using Nanoparticle Tracking Analysis (NTA) on a ZetaVIEW system (Particle Metrix, Germany). For morphological analysis, purified PM-EVLPs were deposited onto carbon-coated copper grids, stained with 1% phosphotungstic acid, air-dried, and subsequently observed under a HITACHI H-7650 transmission electron microscope. 2.3 Animal models To evaluate the therapeutic effects of AcNPs, 40 male C57BL/6J mice (6–8 weeks old) were randomly divided into five groups: CON (PBS,oral), CLP (PBS,oral), CLP + 20 mg/kg AcNPs (AcNPs, 20 mg/kg, oral), CLP + 80 mg/kg AcNPs (AcNPs, 80 mg/kg, oral), CLP + supernatant (supernatant, oral). The cecal ligation and puncture (CLP) was performed according to the following steps: mice were anesthetized with sodium pentobarbital, and a midline laparotomy was carried out to gently expose the cecum; According to the grouping, the cecum was ligated approximately 1 cm from the distal end with a 4 − 0 silk suture, taking care not to interrupt the blood supply of the ileocecal valve; The distal cecum was then punctured once or twice with a 22G or 21G needle, and a small amount of fecal content was gently extruded into the peritoneal cavity to induce polymicrobial infection; The cecum was then returned to the abdominal cavity, and the abdominal wall and skin were closed in layers; Control-operated mice underwent laparotomy with cecal exposure and repositioning without ligation or puncture. After 24 hours, mice were anesthetized with sodium pentobarbital and euthanized, and lung and liver tissues were collected for evaluation. To evaluate the toxicity of AcNPs in mice, 16 mice were randomly divided into two groups: CON (PBS, oral), AcNPs (AcNPs, 100 mg/kg, oral). After 24 hours, blood samples and the heart, liver, spleen, lungs, and kidneys were collected. To evaluate whether macrophages mediate the anti-inflammatory effects of AcNPs, 40 mice were randomly divided into five groups. Two groups received intraperitoneal injections of control liposomes, and three groups received intraperitoneal injections of clodronate liposomes. After 24 hours, the mice were treated as follows: ( 1 ) CON: oral PBS; ( 2 ) CLP + control liposomes: oral PBS; ( 3 ) CLP + AcNPs: oral AcNPs (80 mg/kg); ( 4 ) CLP + clodronate liposomes: oral PBS; ( 5 ) CLP + AcNPs + clodronate liposomes: oral AcNPs (80 mg/kg). 24 hours after these treatments, lung and liver injury were assessed. To evaluate the anti-inflammatory effect of miR396-5p, thirty-two mice were randomly divided into four groups: CON (PBS, oral), CLP (PBS, oral), CLP + 500 pmol miR396-5p (miR396-5p, 5 mg/kg, i.p. ), and CLP + 1000 pmol miR396-5p (miR396-5p, 20 mg/kg, i.p. ). Lung and liver injury levels were assessed 24 hours later. 2.4 Cell culture Primary peritoneal macrophages were isolated from 6-week-old male mice. Four days prior to harvest, the mice were intraperitoneally injected with 3 ml of 3% sodium thiosulfate (Sigma-Aldrich, USA). Cells were collected by peritoneal lavage with ice-cold RPMI-1640 medium (Gibco, USA). The lavage fluid was centrifuged at 1500 rpm for 10 min at 4°C, and the pellet was treated with red blood cell lysis buffer (Procell, China) for 10 min at 4°C. After a second centrifugation (1500 rpm, 10 min, 4°C), the remaining cells were resuspended and seeded into culture plates at a density of 1 × 10^6 cells per well. Following a 2-h adherence period, the cells were maintained at 37°C in a humidified 5% CO₂ incubator in complete medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Procell, China). RAW264.7 macrophages (Procell, China) were cultured under the same conditions (37°C, 5% CO₂) in complete medium supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Procell, China). 2.5 In vivo biodistribution assay For biodistribution studies, mice were administered AcNPs labeled with the near-infrared fluorescent dye DiR (Beyotime, China) via oral gavage. At designated time intervals (0, 6, 12, 24, and 48 h post-administration), animals were euthanized, and major organs were excised. The fluorescence intensity of DiR in the collected tissues was quantitatively assessed using a vivaCT80 small animal imaging system (SCANCO MEDICAL AG, Switzerland). 2.6 Cellular uptake of AcNPs AcNPs (30 µM) were labeled with the fluorescent dye DiO (Beyotime, China) and subsequently incubated with primary peritoneal macrophages for 0, 6, 12, and 24 h. The intracellular DiO fluorescence signal was then detected using a Cytation 5 imaging reader (BioTek, USA). 2.7 Histological analysis The heart, liver, spleen, lung, and kidney tissues were fixed in 4% paraformaldehyde solution (Servicebio, China), followed by paraffin embedding and sectioning. The sections were subsequently subjected to hematoxylin–eosin (H&E) staining for further evaluation. 2.8 ELISA assay Mice serum and bronchoalveolar lavage fluid (BALF) were subjected to enzyme-linked immunosorbent assay (ELISA) using commercial kits (Elabscience, China) to quantify the secretion levels of IL-6, TNF-α, and IL-1β. The absorbance of each well was subsequently measured and analyzed at 450 nm. 2.9 Wet/dry ratios Lung tissues were collected 24 hours after CLP model establishment, immediately weighed to determine the wet weight, and then dried in a 56°C oven for 60 hours until a constant dry weight was achieved. The ratio of wet weight to dry weight was calculated to assess pulmonary edema. 2.10 MPO assay The myeloperoxidase (MPO) assay was performed following the standardized protocols provided by Nanjing Jiancheng Bioengineering Institute (China). All procedures were carried out in strict accordance with the manufacturer’s instructions. The MPO activity was determined by measuring the absorbance at 460 nm, and the results were subsequently analyzed to assess neutrophil infiltration in the lung tissue. 2.11 MDA, SOD, GSH assay Commercial assay kits (Jiancheng Bioengineering Institute, China) were used to measure malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) levels according to the manufacturer’s instructions. Lung and liver tissues were homogenized, and the resulting homogenates were analyzed for MDA content, SOD activity, and GSH levels using the respective kits. 2.12 ALT, AST, LDH assay Commercial assay kits (Jiancheng Bioengineering Institute, China) were used to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels according to the manufacturer’s instructions. Liver was collected and analyzed for ALT, AST, and LDH activities using the respective kits. 2.13 RNA extraction and Quantitative real-time polymerase chain reaction Total RNA was extracted from lung tissues or cultured cells using TRIzol Reagent (Thermo Fisher Scientific, USA). cDNA was then synthesized with a Reverse Transcription Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was conducted to assess gene expression levels, employing SYBR Green chemistry (Promega, USA) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The amplification program included an initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds for combined annealing and extension. A melting curve analysis was performed at the end of the run from 60°C to 95°C to verify amplification specificity. The primer sequences used in this study are listed below. TNF-α: F-AGCCCCCAGTCTGTATCCTT, R-CTCCCTTTGCAGAACTCAGG ; IL-6: F-CTCCCTTTGCAGAACTCAGG, R-CTGTGAAGTCTCCTCTCCGG ; IL-1β: F-GGGCCTCAAAGGAAAGAATC, R-TACCAGTTGGGGAACTCTGC . 2.14 Western blot Protein lysates were prepared from tissues or cells using RIPA buffer (Solarbio, China), and the protein concentration was determined with a bicinchoninic acid (BCA) assay. Proteins were separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked for 1 hour in Tris-buffered saline containing 0.1% Tween 20 (TBST) supplemented with 5% (w/v) non-fat milk. Following blocking, membranes were incubated overnight at 4°C with the following primary antibodies: β-actin monoclonal antibody (1:5000, Proteintech, China), TNF-α monoclonal antibody (1:1000, Proteintech, China), IL-1β polyclonal antibody (1:1000, Proteintech, China), IL-6 monoclonal antibody (1:1000, Proteintech, China), NLRP3 polyclonal antibody (1:2000, Proteintech, China), ASC polyclonal antibody (1:1000, Proteintech, China), caspase-1 polyclonal antibody (1:1000, Proteintech, China), phospho-p65 polyclonal antibody (1:3000, Abcam, UK), p65 monoclonal antibody (1:1000, Abcam, UK), phospho-IκB polyclonal antibody (1:1000, CST, USA), IκB monoclonal antibody (1:1000, CST, USA), Pho-PERK monoclonal antibody (1:1000, Proteintech, China), PERK monoclonal antibody (1:1000, Proteintech, China), Pho-EIF2A polyclonal antibody (1:1000, Proteintech, China), EIF2A polyclonal antibody (1:1000, Proteintech, China), Hrd1 polyclonal antibody (1:1000, Proteintech, China), and ATF4 polyclonal antibody (1:1000, Proteintech, China). After primary antibody incubation, membranes were washed three times with TBST and then incubated at room temperature for 2 hours with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG monoclonal antibody (1:5000, Proteintech, China) or goat anti-mouse IgG monoclonal antibody (1:5000, Proteintech, China). Protein bands were visualized using Luminata™ Crescendo chemiluminescent substrate (Millipore, USA), and images were captured with a GeneGnome XRQ imaging system (Syngene, UK). 2.15 Flow cytometry M1 and M2 Macrophage Phenotyping: Primary peritoneal macrophages were collected after treatment and transferred to centrifuge tubes. Cells were resuspended in PBS and centrifuged at 1500 rpm for 15 minutes. The pellets were stained with APC-conjugated CD80 (1:500, Elabscience, China) or FITC-conjugated CD206 (1:500, Elabscience, China) for 15 minutes, and the fluorescence was measured using a BD LSRFortessa flow cytometer (BD, USA). Reactive Oxygen Species (ROS) Detection: Cells were washed three times with PBS and incubated with H2DCFDA working solution (Thermo Fisher Scientific, USA) in a 37°C, 5% CO₂ humidified incubator, protected from light, for 30 minutes. After three additional PBS washes, intracellular ROS levels were analyzed by flow cytometry (BD LSRFortessa, USA). Calcium Influx Measurement: Cells were incubated with Fluo-3 AM working solution (Solarbio, China) in HBSS (Solarbio, China) at 37°C for 20 minutes. Following this, the cells were washed with HEPES-buffered saline (Pricella, China) and further incubated in HBSS containing 1% fetal bovine serum (Gibco, USA) for 40 minutes. After a final wash, cells were resuspended in HEPES solution for analysis of intracellular calcium levels by flow cytometry (BD LSRFortessa, USA). 2.16 Network pharmacological screening of Pineapple-CLP-associated target pathways Potential phytochemical constituents of pineapple were systematically retrieved through data mining using the Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine (BATMAN-TCM, http://bionet.ncpsb.org.cn/batman-tcm/ ) in combination with an extensive literature survey, with search parameters defined as “Ingredient,” “Herb name,” or “Compound= Ananas comosus or Pineapple.” 2.17 Immunoprecipitation To examine the interaction between IκB and ubiquitinated proteins, cells were rinsed three times with PBS and lysed on ice for 1 hour using a lysis buffer supplemented with the protease inhibitor PMSF (Cat# P0100, Solarbio, China). The lysates were centrifuged at 12,000 rpm for 10 min at 4°C after removing debris. The supernatant was first incubated with Dynabeads™ Protein G (Cat# 10004D, Invitrogen, USA) for 3 h at 4°C to pre-clear nonspecific binding proteins. The magnetic beads were collected using DynaMag™-2 (Cat# 12321D, Invitrogen, USA), and the clarified supernatant was then incubated overnight at 4°C with an anti-IκB antibody. After incubation, fresh Dynabeads™ Protein G were added to capture the immune complexes, which were subsequently washed three times with cold lysis buffer. The immunoprecipitates were retrieved using DynaMag™-2 and subjected to Western blot analysis for detection of ubiquitinated IκB. 2.18 Statistical analysis All experimental results are presented as mean values ± standard deviation (SD). Statistical analyses were carried out using GraphPad Prism version 9.0 (GraphPad Software, USA). For datasets exhibiting normal distribution, differences among groups were evaluated by one-way analysis of variance (ANOVA). Statistical significance was defined as P < 0.05. 3. Results 3.1 Isolation and characterization of AcNPs Ultracentrifugation combined with sucrose density gradient centrifugation is currently regarded as the gold standard method for isolating and purifying exosome-like nanoparticles from plant sources( 28 ). In this study, AcNPs were extracted and purified from Pineapple pulp juice using sequential centrifugation with different relative centrifugal forces and sucrose density gradient solutions (Fig. 1 A). The morphology and particle size of AcNPs were then characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM images revealed that AcNPs appeared as spherical or ellipsoidal vesicles with a typical bilayer membrane structure, consistent with the general morphological features of exosome-like nanoparticles (Fig. 1 B). NTA analysis further indicated that the mean particle diameter of AcNPs was approximately 137.5 nm, with an average particle concentration of 3 × 10 6 particles/mL (Fig. 1 C). Lipidomic profiling demonstrated that AcNPs were enriched in hexosylceramide (HexCer, 16%), ceramide (Cer, 15.17%), phosphatidylethanolamine (PE, 14%), and triglycerides (TG, 13.83%), supporting the stability of the bilayer structure and providing part of the biochemical basis for their bioactivity (Fig. 1 D). In addition, untargeted metabolomic analysis showed that AcNPs were rich in terpenoids (30.77%), organic acids and derivatives (19.23%), phenolic acids and derivatives (17.31%), and steroids and steroid derivatives (8.65%), categories of metabolites that are closely associated with antioxidant activity, energy metabolism, inflammation regulation, and signal transduction, thereby highlighting the diverse biological and pharmacological potential of AcNPs (Fig. 1 E). Furthermore, proteomic comparison between pineapple proteins and AcNPs revealed 3253 overlapping proteins (Fig. 1 G). 3.2 Biodistribution and toxicity evaluation of AcNPs To evaluate the biodistribution and toxicity of AcNPs, DiR-labeled AcNPs were orally administered to healthy mice fasted for 24 hours. At designated time points post-administration (0, 6, 12, 24, and 48 hours), brain, kidney, heart, spleen, liver, lung, and gastrointestinal tissues were collected, and the distribution of AcNPs was monitored using the vivaCT80 small animal imaging system (Fig. 2 A). The results indicated that DiR-labeled AcNPs predominantly accumulated in the lungs and liver, reaching peak levels at 12 hours, with substantial signal remaining at 24 hours. Notably, most AcNPs were cleared from the body within 48 hours. Furthermore, administration of AcNPs at 100 mg/kg did not induce any pathological changes in the heart, liver, spleen, lung, or kidney (Fig. 2 B), and serum biochemical parameters including ALT, AST, CRE, and BUN showed no significant hepatotoxicity or nephrotoxicity (Fig. 2 C). These findings suggest that AcNPs preferentially accumulate in the lungs and liver, and preliminary evaluations indicate no obvious toxicity. 3.3 AcNPs attenuate CLP-induced lung and liver iinjury in mice Given the preferential accumulation of AcNPs in the lung and liver, as well as the compositional analysis indicating their anti-inflammatory and antioxidant potential, we employed a CLP model to induce pulmonary and hepatic injury in mice. Animals were treated with AcNPs at doses of 20 mg/kg, 80 mg/kg, or with the corresponding supernatant (Fig. 3 A). The results showed that AcNPs markedly reduced serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (Fig. 3 B). Histopathological examination of lung sections revealed a dose-dependent alleviation of CLP-induced inflammatory injury following AcNPs treatment (Fig. 3 C–D). Consistently, the lung wet-to-dry weight ratio demonstrated a reduction in pulmonary edema (Fig. 3 E). Moreover, AcNPs decreased MPO activity in lung homogenates (Fig. 3 F) and lowered inflammatory cytokine concentrations in both BALF and lung tissue (Fig. 3 G–J). In parallel, oxidative stress was mitigated, as evidenced by decreased MDA levels and elevated GSH and SOD levels in lung tissues (Fig. 3 J–L). Assessment of liver injury further confirmed the protective effects of AcNPs. AcNPs reversed CLP-induced elevations of ALT, AST, and ALP (Fig. 3 M–O), alleviated inflammatory histopathological damage in hepatic tissue (Fig. 3 P), and reduced TNF-α, IL-6, and IL-1β expression at both mRNA and protein levels in liver homogenates (Fig. 3 Q–U). Collectively, these findings demonstrate that AcNPs effectively attenuate CLP-induced lung and liver injury in mice. 3.4 Macrophages were central to AcNPs-induced attenuation of lung and liver injury. Macrophages play a pivotal regulatory role in both the initiation and resolution of inflammatory responses( 29 ). To further determine whether macrophages mediate the anti-inflammatory effects of AcNPs, mice were pretreated with either control liposomes or clodronate liposomes (Fig. 4 A). The results revealed that clodronate liposomes markedly induced macrophage depletion, eliminating approximately 87% of macrophages in the spleen (Fig. 4 D). Importantly, depletion of macrophages abolished the protective effects of AcNPs against CLP-induced lung and liver injury, as evidenced by exacerbated histopathological damage in lung and liver tissues, increased lung MPO activity, elevated lung W/D ratio, as well as aggravated hepatic injury markers (AST, ALT, and LDH). Moreover, the reduction in inflammatory cytokine levels in both lung and liver tissues induced by AcNPs was also significantly reversed (Fig. 4 B, C, E–L). Collectively, these findings demonstrate that the anti-inflammatory effects of AcNPs are mediated through a macrophage-dependent mechanism. 3.5 NF-κB signaling pathway mediates the effects of AcNPs on macrophages. Given that macrophages mediate the anti-inflammatory effects of AcNPs, we constructed a pharmacological target network between pineapple and sepsis, and identified 127 common targets using a Venn diagram (Fig. 5 A). Further protein–protein interaction (PPI) network analysis indicated that these targets were closely associated with NF-κB signaling, TNF signaling-mediated inflammation, and oxidative stress (Fig. 5 B-C). Subsequently, in an LPS-induced peritoneal macrophage model, RNA-seq was used to identify key signaling pathways modulated by AcNPs, and KEGG pathway enrichment analysis further revealed that NF-κB and TNF signaling mediated the anti-inflammatory effects of AcNPs (Fig. 5 D-E). These findings suggest that bioactive substance-rich AcNPs may exert their anti-inflammatory effects primarily through NF-κB signaling and its downstream inflammation-related pathways. 3.6 AcNPs inhibited LPS-induced NF-κB signaling pathway and inflammatory activation in macrophages. First, we evaluated the uptake of AcNPs by macrophages at the cellular level. DiO-labeled AcNPs were used to monitor macrophage internalization at 0, 3, 12, and 24 hours (Fig. 6 A-B). Subsequently, in LPS-induced inflammatory models using two types of macrophages (primary peritoneal macrophages and RAW264.7 cells), we observed a dose-dependent inhibition of NF-κB signaling upon AcNPs treatment (Fig. 6 C-D, G-H). The NLRP3 inflammasome, as a secondary signal downstream of NF-κB, was similarly suppressed by AcNPs (Fig. 6 E-F, I-J). Moreover, considering the critical role of macrophage M1/M2 polarization in inflammatory responses, we assessed the effect of AcNPs on macrophage phenotypes. The results indicated that AcNPs inhibited LPS-induced M1 polarization while promoting M2 polarization (Fig. 6 K). Collectively, these findings suggest that AcNPs suppress LPS-induced NF-κB signaling and inflammasome activation, inhibit M1 polarization, and facilitate M2 polarization in macrophages. 3.7 AcNPs attenuated LPS-induced oxidative stress and endoplasmic reticulum stress in macrophages. ROS-mediated ER stress represents a critical process in the inflammatory response( 30 ). To explore this, we assessed ROS production and ER stress in primary peritoneal macrophages following LPS stimulation. Using H2DCFDA, a fluorescent probe for reactive oxygen species, both flow cytometry and fluorescence microscopy revealed that AcNPs attenuated the LPS-induced elevation of ROS in a concentration-dependent manner (Fig. 7 A–C). Furthermore, targeted analysis of RNA-seq data indicated that AcNPs suppressed the expression of ER stress–related genes, a finding corroborated by western blot results showing downregulation of ER stress–associated proteins (Fig. 7 D–F). In addition, Fluo-3 AM, a probe commonly employed to assess Ca²⁺ influx during ER stress, demonstrated via flow cytometry and microscopy that AcNPs alleviated LPS-induced intracellular Ca²⁺ influx (Fig. 7 G–H). Collectively, these results suggest that AcNPs markedly suppress LPS-induced oxidative stress and ER stress in macrophages. 3.8 Identification of miR396-5p as a target of AcNPs and Hrd1 as its downstream effector. To identify the bioactive components responsible for the anti-inflammatory activity of AcNPs, we treated the nanoparticles with Proteinase K, RNase/DNase, and RNase/lipase to selectively inactivate proteins, nucleic acids, and lipids, respectively. The results indicated that the nucleic acid components within AcNPs are likely the key mediators of their anti-inflammatory effects (Fig. 8 A–B). Deep sequencing of miRNAs subsequently revealed six novel miRNAs highly enriched in AcNPs, including miR156b-3p, miR156b-5p, miR168a-5p, miR168b-5p, miR159-3p, and miR396-5p (Fig. 8 C). Synthetic mimics of these miRNAs were transfected into primary peritoneal macrophages, and both miR168a-5p and miR396-5p exhibited anti-inflammatory activity at a concentration of 60 µM, with miR396-5p showing a stronger effect (Fig. 8 D). By integrating RNA-seq data of AcNPs with psRobot-predicted targets, Hrd1 and Pstpip2 were identified as potential downstream targets of AcNP-derived miRNAs (Fig. 8 E–F). Hrd1 is an endoplasmic reticulum–associated E3 ubiquitin ligase that has been reported to regulate NF-κB signaling by promoting IκB ubiquitination. Treatment with miR396-5p mimics reduced macrophage activation upon LPS stimulation and suppressed Hrd1 expression, as further confirmed by luciferase reporter assays showing direct regulation of Hrd1 by miR396-5p (Fig. 8 G–I). Consistently, inhibition of miR396-5p abolished the effect of AcNPs on Hrd1 expression (Fig. 8 J–K). Moreover, we found that miR396-5p modulates NF-κB signaling through Hrd1-mediated ubiquitination of IκB (Fig. 8 L–M). Importantly, overexpression of Hrd1 reversed the anti-inflammatory effect of AcNPs, indicating that Hrd1 acts as a direct molecular target of AcNPs in regulating inflammation via the NF-κB pathway (Fig. 8 N–P). Collectively, these findings identify miR396-5p as a novel anti-inflammatory miRNA within AcNPs that directly targets Hrd1 and inhibits IκB ubiquitination. 3.9 miR396-5p mediates the in vitro anti-inflammatory activity of AcNPs. To further investigate whether miR396-5p mediates the regulatory effects of AcNPs on NF-κB signaling, inflammasome activation, and ERS, we examined the effects of miR396-5p mimic and inhibitor. The results showed that the miR396-5p mimic inhibited the activation of NF-κB and inflammasome signaling in a concentration-dependent manner, whereas the miR396-5p inhibitor reversed the inhibitory effects of AcNPs (Fig. 9 A–D). Moreover, similar results were observed in ROS-mediated ERS (Fig. 9 E–I). Collectively, these findings indicate that miR396-5p mediates the anti-inflammatory activity of AcNPs. 3.10 miR396-5p is a key target mediating the protective effects of AcNPs in vivo. Subsequently, we explored whether miR396-5p also exerts anti-inflammatory effects in CLP-induced septic mice (Fig. 10 A). The results showed that miR396-5p administration markedly reduced the serum levels of TNF-α, IL-6, and IL-1β in CLP mice (Fig. 10 B), alleviated pathological lung injury (Fig. 10 C–D), and decreased the concentrations of TNF-α, IL-6, and IL-1β as well as MPO activity (Fig. 10 E–F). In addition, miR396-5p treatment attenuated liver injury and inflammation in septic mice (Fig. 10 G–I). Furthermore, to determine the cellular target of miR396-5p, mice were treated with FITC-labeled miR396-5p followed by macrophage sorting using flow cytometry (Fig. 10 J). The results revealed a significant reduction in Hrd1 expression within pulmonary macrophages after miR396-5p treatment (Fig. 10 K–L). Collectively, these findings demonstrate that miR396-5p alleviates sepsis-induced lung and liver injury in vivo. 4. Discussions Sepsis, triggered by infection-induced systemic inflammatory response syndrome, can result in multiple organ dysfunction, with the liver and lungs being the most vulnerable targets( 31 ). During sepsis, the liver acts not only as a central hub of immune defense but also as a major site of inflammatory injury, manifesting as cholestasis, elevated transaminases, and dysregulation( 32 – 34 ). The lungs, on the other hand, undergo acute injury due to inflammatory cell infiltration, cytokine storms, and increased capillary permeability, ultimately progressing to ALI or ARDS( 35 , 36 ). These pathological changes markedly contribute to the high mortality associated with sepsis( 37 ). Although current clinical management primarily relies on anti-infective therapy, fluid resuscitation, and organ support, effective organ-specific interventions remain lacking. Recent evidence suggests that naturally derived nanoparticles, owing to their low toxicity, represent a promising therapeutic avenue( 38 , 39 ). Certain dietary-sourced NPs have demonstrated protective effects against acute hepatic and pulmonary injury. For instance, NPs from Platycodon grandiflorum alleviate LPS-induced ALI by modulating lipid metabolism and glycolysis( 40 ); Rehmanniae Radix exerts protective effects against ALI by targeting GPR161( 26 ); and pomegranate-derived NPs have been shown to attenuate hepatic injury in MASLD( 41 ). Notably, in our study, AcNPs were found to selectively target both the liver and lungs in mice, conferring multi-organ protection and enhancing systemic therapeutic efficacy without eliciting adverse reactions. In sepsis-induced acute lung and liver injury, although multiple cell types including neutrophils, epithelial cells, and endothelial cells play important roles, macrophages, as tissue-resident “sentinel” cells, are the first to recognize PAMPs and DAMPs( 3 , 42 , 43 ). They release large amounts of inflammatory cytokines and chemokines, orchestrating the recruitment of other immune cells and amplifying the inflammatory response, thereby exerting a central and initiating role( 5 , 44 ). In contrast, neutrophils, endothelial cells, epithelial cells, and lymphocytes mainly act downstream, contributing to barrier disruption, oxidative stress, and adaptive immune dysregulation( 45 , 46 ). While these cells serve as key executors of tissue injury, their activation largely depends on the early inflammatory signals provided by macrophages. Our study further demonstrated that clodronate liposomes treatment reversed the in vivo anti-inflammatory effects of AcNPs, indicating that macrophages are the primary target mediating the biological activity of AcNPs and highlighting the potential of AcNPs as a macrophage-centered therapeutic strategy. Therefore, we further explored the anti-inflammatory mechanism of AcNPs. Network pharmacology analysis based on pineapple and acute lung injury, together with KEGG enrichment analysis of RNA-seq data from LPS-treated macrophages, indicated that the NF-κB signaling pathway may serve as a key target of AcNPs. NF-κB is a classical inflammatory signaling pathway: LPS activates it through the TLR4 receptor, inducing TLR4 dimerization and recruitment of MyD88( 47 ). MyD88 subsequently recruits and activates IRAK, which forms a complex with TRAF6, leading to phosphorylation and ubiquitination of the downstream IκB complex( 48 ). This process releases NF-κB, allowing it to translocate into the nucleus and promote transcription of pro-inflammatory genes( 49 ). Within NF-κB signaling, Hrd1 binds to the deubiquitinating enzyme Usp15, rendering it inactive and unable to deubiquitinate IκBα, thereby causing hyperactivation of NF-κB( 12 ). Consistent with the RNA-seq and network pharmacology predictions, experimental results showed that AcNPs significantly inhibited the phosphorylation of p65 and IκB. Moreover, AcNPs treatment markedly suppressed NLRP3 inflammasome signaling, accompanied by inhibition of M1 macrophage polarization and promotion of M2 macrophage polarization. Oxidative stress–induced endoplasmic reticulum stress (ERS) represents a critical feature of inflammatory responses( 50 ). Excessive accumulation of intracellular reactive oxygen species (ROS) can disrupt protein folding and calcium homeostasis, activating ERS signaling pathways such as PERK, IRE1α, and ATF6, which in turn trigger downstream inflammatory gene expression( 51 ). In macrophages, ROS-mediated ERS is particularly pronounced, serving as a central node for the initiation, amplification, and maintenance of inflammation. Concurrently, ROS can promote phosphorylation and degradation of IκB, leading to nuclear translocation of NF-κB and activation of downstream inflammatory gene transcription. Activated NF-κB further enhances the production of proinflammatory mediators, including TNF-α, IL-1β, and IL-6, and promotes additional ROS generation, establishing a positive feedback loop that exacerbates ERS and oxidative stress, thereby amplifying the inflammatory response( 52 ). Previous studies further support this mechanism: dendrobine mitigates LPS-induced acute lung injury by modulating FAM134B-mediated ERS( 53 ); hyperglycemia impairs M2 polarization of hepatic macrophages via C/EBP homolog–mediated ERS, aggravating hepatic ischemia–reperfusion injury( 54 ); and exogenous hydrogen sulfide protects against rat liver injury by regulating ERS-induced macrophage autophagy( 55 ). Collectively, these studies suggest that the ROS–NF-κB–ERS network plays a pivotal role in controlling inflammation and represents a potential therapeutic target for macrophage-mediated inflammatory diseases. In the present study, we demonstrate for the first time that AcNPs significantly suppress ROS-mediated ERS, thereby alleviating LPS-induced macrophage inflammatory responses. Notably, AcNPs not only downregulate the expression of ERS-related genes and proteins but also inhibit NF-κB activation and promote the polarization of macrophages toward the anti-inflammatory M2 phenotype. These findings suggest that AcNPs may exert multi-level, multi-target regulation in the inflammatory microenvironment by modulating the ROS–NF-κB–ERS network. Although the precise upstream–downstream relationship between ERS and NF-κB remains to be fully elucidated, our data clearly indicate that AcNPs act synergistically to inhibit ROS, ERS, and NF-κB signaling, thereby exerting potent anti-inflammatory and tissue-protective effects. This provides a theoretical basis for developing macrophage-targeted nanotherapeutics and highlights the potential clinical value of AcNPs in multi-organ protection and inflammation modulation. To further identify the essential bioactive substances within AcNPs, we treated them with Proteinase K, RNase/DNase, and RNase/Lipids to selectively eliminate proteins, nucleic acids, and lipids, respectively, and then evaluated their impact on inflammatory responses. The results indicated that nucleic acids constitute the major basis of AcNPs’ biological activity. In recent years, plant-derived miRNAs have been recognized as a new category of dietary bioactive molecules capable of modulating endogenous mRNA expression( 56 ). Owing to their characteristic 2′-O-methyl modification at the terminal ends, plant miRNAs exhibit remarkable structural resilience, allowing them to withstand oxidative stress, corrosive conditions, and enzymatic degradation during gastrointestinal digestion. This stability under harsh environments underscores their potential as therapeutic molecules and dietary supplements. Supporting this concept, accumulating evidence has demonstrated their protective roles: miR159 carried by Prunus mume–derived EVLPs mitigates colitis symptoms, Teng et al( 27 ). reported that garlic-derived EVLPs enriched in miR-396e ameliorated nonalcoholic fatty liver disease, and Yu et al( 57 ). showed that fresh Rehmanniae Radix–derived EVLPs containing Rgl-exomiR-7972 alleviated LPS-induced acute lung injury( 26 ). In this study, systematic screening identified miR396-5p as a key mediator of the anti-inflammatory activity of AcNPs. Moreover, the overlap between RNA-seq results from AcNPs-treated macrophages and the predicted target genes of miR396-5p suggested that Hrd1 may serve as its direct downstream target. This conclusion was further validated by dual-luciferase reporter assays. Notably, only a limited number of miRNAs have been reported to directly target Hrd1, and these are mammalian-derived, including miR-513a-3p and miR-224. To our knowledge, this is the first demonstration that a plant-derived exogenous miRNA (miR396-5p) can directly target Hrd1, highlighting its potential as a novel therapeutic strategy for diseases in which Hrd1 plays a central pathogenic role. NPs derived from fruits, vegetables, and medicinal-food homologous plants exhibit remarkable advantages such as drug delivery vehicles. These NPs are broadly sourced, highly safe, and typically endowed with excellent biocompatibility and low immunogenicity, thereby minimizing the toxic side effects and immune rejection frequently encountered with synthetic nanomaterials. Their bilayer membrane structure and nanoscale size confer superior stability and permeability, enabling them to remain active for extended periods in circulation and to effectively traverse multiple biological barriers. Importantly, plant-derived NPs are intrinsically enriched with diverse bioactive molecules such as miRNAs, lipids, and polysaccharides, which not only serve as carriers but may also synergize exogenous therapeutics to potentiate overall therapeutic efficacy( 58 ). Unlike conventional synthetic vectors, these NPs possess innate tissue- and cell-targeting capacities—for instance, preferential accumulation in the gut or efficient uptake by macrophages—thereby facilitating precise drug delivery( 59 ). Moreover, their low production cost, straightforward preparation, and scalability highlight their sustainability and translational potential. Increasing evidence has demonstrated that loading small-molecule chemotherapeutics, proteins, or functional nucleic acids into plant-derived NPs markedly improves drug bioavailability, reduces off-target cytotoxicity, and enhances treatment outcomes. Collectively, these features underscore the broad promise of NPs from fruits, vegetables, and medicinal-food plants in shaping the next generation of drug delivery systems. 5. Conclusion In summary, our study is the first to demonstrate that orally administered AcNPs alleviate CLP-induced acute liver and lung injury in mice by targeting macrophages in these organs. Mechanistically, AcNPs deliver miR396-5p to suppress Hrd1, thereby reducing IκB ubiquitination and attenuating NF-κB–mediated inflammatory signaling as well as ROS-driven ERS. Overall, this work highlights plant-derived nanoparticles as a promising therapeutic strategy for sepsis and provides a potential platform for small nucleic acid drug delivery (Fig. 11 ). Abbreviations PDNPs, plant-derived nanoparticles; AcNPs, Ananas comosus (Linn.) Merr. , Ac-derived nanoparticles; Hrd1, HMG-CoA reductase degradation protein 1; ROS, reactive oxygen species; LPS, lipopolysaccharide; CLP, cecal ligation and puncture; PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; TNFα, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; IL-6, interleukin-6; NLRP3, NOD-like receptor family pyrin domain containing 3; EVLPs, extracellular vesicle-like particles; NTA, nanoparticle tracking analysis; BALF, bronchoalveolar lavage fluid; MPO, myeloperoxidase; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; ERS, endoplasmic reticulum stress; Hrd1, Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1. Declarations Data availability Data will be made available on request. Acknowledgements The authors thank the Medical Sci-Tech research Center of Ningxia Medical University (Medical Science Research Institution of Ningxia Hui Autonomous Region） for valuable help in our experiment. Author information Weixi Xie and Zun Wang have contributed equally to this work and share first authorship. Authors and Affiliations Xiangya Nursing School, Central South University, Changsha, Hunan, China Weixi Xie, Lang Deng, Siyuan Li, Yajing Wang, Wei Liu, Honghong Wang, Siyuan Tang. The School of Nursing, Ningxia Medical University, Yinchuan, Ningxia, China Zun Wang, Siyuan Tang. Contributions Honghong Wang, Siyuan Tang, Wei Liu: Conceptualization, Funding. Weixi Xie, Zun Wang: Writing original draft, Methodology, Investigation. Weixi Xie: Methodology, Investigation. Siyuan Li, Lang Deng, Yajing Wang: Investigation, Data curation, Supervision. All authors have read and agreed to the published version of the manuscript. Corresponding authors Correspondence to Wei Liu, Honghong Wang and Siyuan Tang. Ethics declarations Ethics approval These animals were procured from the Animal Center of Central South University. It has been approved by the Laboratory Animal Welfare and Ethics Committee of Animal Center, Central South University (Approval No.XMSB-2025-0595). Consent for publication Not applicable. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Fundigs This research was funded by the National Natural Science Foundation of China (grant numbers: 82170853); the Natural Science Foundation of Ningxia Hui Autonomous Region (grant number: 2024AAC02044); the National Natural Science Foundation of China (No. 82560441); the Natural Science Foundation of Hunan Province (No. 2024JJ6668); the Youth Science Foundation of Xiangya Hospital, Central South University (No. 2023Q13); and the Education and Teaching Reform Project of Ningxia Medical University (No. NYJY2025099). References Cecconi M, Evans L, Levy M, Rhodes A. Sepsis and septic shock. Lancet (London, England). 2018;392(10141):75-87. Giamarellos-Bourboulis EJ, Aschenbrenner AC, Bauer M, Bock C, Calandra T, Gat-Viks I, Kyriazopoulou E, Lupse M, Monneret G, Pickkers P, Schultze JL, van der Poll T, van de Veerdonk FL, Vlaar APJ, Weis S, Wiersinga WJ, Netea MG. The pathophysiology of sepsis and precision-medicine-based immunotherapy. Nature immunology. 2024;25(1):19-28. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9327044","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631099019,"identity":"d1bbdd3d-e90a-45ab-a59e-aa9e97c913bc","order_by":0,"name":"Weixi Xie","email":"","orcid":"","institution":"Xiangya Nursing School, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Weixi","middleName":"","lastName":"Xie","suffix":""},{"id":631099027,"identity":"34f2440e-1c6b-46e2-9162-0cf4303912a4","order_by":1,"name":"Zun Wang","email":"","orcid":"","institution":"The School of Nursing, Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zun","middleName":"","lastName":"Wang","suffix":""},{"id":631099034,"identity":"6beb04c7-98ca-433f-8341-1ddf49711e06","order_by":2,"name":"Lang Deng","email":"","orcid":"","institution":"Xiangya Nursing School, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lang","middleName":"","lastName":"Deng","suffix":""},{"id":631099041,"identity":"89763117-0753-454d-8238-c45a8f2b7bed","order_by":3,"name":"Siyuan Li","email":"","orcid":"","institution":"Xiangya Nursing School, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Siyuan","middleName":"","lastName":"Li","suffix":""},{"id":631099043,"identity":"fe3ad519-b2aa-4574-bf32-98579a48c000","order_by":4,"name":"Yajing Wang","email":"","orcid":"","institution":"Xiangya Nursing School, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yajing","middleName":"","lastName":"Wang","suffix":""},{"id":631099044,"identity":"4acd60c2-7413-425c-9f3e-2964be12c184","order_by":5,"name":"Wei Liu","email":"","orcid":"","institution":"Xiangya Nursing School, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Liu","suffix":""},{"id":631099046,"identity":"dc88535c-d207-4276-9dab-a1e2927d8df6","order_by":6,"name":"Honghong Wang","email":"","orcid":"","institution":"Xiangya Nursing School, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Honghong","middleName":"","lastName":"Wang","suffix":""},{"id":631099048,"identity":"82259529-bddc-4769-ae72-558fc2ef0927","order_by":7,"name":"Siyuan Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYPACGzl59gYwi7GBsGpmEJFmbNhzgDQthxMZbiQQqUV+Rv7BzwW/mBMYZz5+/JmHwUZ2wwHmZw/waTG4kcwsPbOPLY9dOs1Mmgfowg0H2MwN8GqRSGaQ5u3hKWacncPGzAN04YYDPGwS+B2WzPybt0ciseHmGWagw/4T1sJwI5lNmueHQWLDDR4GoMMOENZicOaxmTVvQwIwkNPMJOcYJBvPPMxmht9h7YmPb/P8+Q+MysOPP7ypsJPtO978DL/DBBKAcdEGt5QBGlH4AP8BIPGHkKpRMApGwSgY0QAAZdBGcnASiWsAAAAASUVORK5CYII=","orcid":"","institution":"The School of Nursing, Ningxia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Siyuan","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2026-04-05 14:58:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9327044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9327044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108254036,"identity":"f1fdab2c-f1d7-43db-9ba0-0edbe4b3a3d0","added_by":"auto","created_at":"2026-05-01 04:31:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExtraction and component characterization of AcNPs. \u003c/strong\u003e(A) Schematic diagram of the extraction process of AcNPs. (B) Transmission electron microscopy showing the structure of AcNPs (scale bar = 200 nm). (C) Particle size distribution of AcNPs measured by NTA. (D) Lipid analysis of AcNPs by lipidomics. (E) Untargeted metabolomic analysis of AcNPs. (F–G) Proteomic Venn diagram and volcano plot comparing pineapple and AcNPs.Data are presented as means ± standard deviation. (n=3, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/e2089a9c75fab4954fc1d5b3.png"},{"id":108491959,"identity":"199e56e4-c9cd-4356-ba06-3816ada0c15e","added_by":"auto","created_at":"2026-05-05 09:56:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":549406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiodistribution and in vivo toxicity of AcNPs. \u003c/strong\u003e(A) Biodistribution of 80 mg/kg DiR-labeled AcNPs at 0, 6, 12, 24, and 48 h post-oral administration in brain, kidney, heart, spleen, liver, lung, stomach, and small intestine. (B) H\u0026amp;E staining of heart, liver, spleen, lung, and kidney tissues 48 h after oral administration of 80 mg/kg AcNPs (scale bar = 100 μm). (C) Serum ALT, AST, CRE, and BUN levels indicating liver and kidney injury.Data are presented as means ± standard deviation. (n=6, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/6212f46b3f1af13bbb097453.png"},{"id":108491564,"identity":"cfceee36-f2cb-4fd8-b327-8acbc4d3c2cc","added_by":"auto","created_at":"2026-05-05 09:54:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":318707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcNPs alleviate CLP-induced lung and liver injury in mice.\u003c/strong\u003e (A) Schematic diagram of AcNPs treatment in CLP mice. (B) Serum levels of TNF-α, IL-6, and IL-1β detected by ELISA. (C–D) H\u0026amp;E staining showing lung injury and corresponding lung injury scores (scale bar = 100 μm). (E) Lung wet/dry weight ratio. (F) MPO activity in lung tissue. (G) mRNA levels of TNF-α, IL-6, and IL-1β in lung tissue detected by qPCR. (H–I) Protein expression of TNF-α, IL-6, and IL-1β in lung tissue determined by western blot and quantification. (J–L) MDA content, GSH-Px activity, and SOD activity in lung tissue. (M–O) AST, ALT, and LDH levels in liver tissue. (P) H\u0026amp;E staining showing liver injury (scale bar = 100 μm). (Q–S) mRNA levels of TNF-α, IL-6, and IL-1β in liver tissue detected by qPCR. (T–U) Protein expression of TNF-α, IL-6, and IL-1β in liver tissue determined by western blot and quantification.Data are presented as means ± standard deviation. (n=6, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/9c9faa26cfbd500797767c2f.png"},{"id":109067624,"identity":"331bfa12-146b-4059-a4df-853492ca2b26","added_by":"auto","created_at":"2026-05-12 09:58:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":366206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophages mediate the protective effects of AcNPs. \u003c/strong\u003e(A) Schematic diagram of AcNPs treatment in CLP mice with clodronate liposome administration. (B–C) H\u0026amp;E staining showing pathological changes in lung and liver tissues (scale bar = 100 μm). (D) Flow cytometry analysis showing depletion of lung macrophages by clodronate liposomes. (E) Lung injury score. (F) MPO content in lung tissue. (G) Lung wet/dry weight ratio. (H) mRNA levels of TNF-α, IL-6, and IL-1β in lung tissue detected by qPCR. (I–K) AST, ALT, and LDH levels in liver tissue. (L) mRNA levels of TNF-α, IL-6, and IL-1β in liver tissue detected by qPCR. Data are presented as means ± standard deviation. (n=6, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/0c1c5ce4b10e1fd8e83475f1.png"},{"id":108492541,"identity":"cd6c747e-a2be-4ad7-a0fb-d98e268a72df","added_by":"auto","created_at":"2026-05-05 09:58:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":355748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork pharmacology and RNA-seq reveal the targets and signaling pathways of AcNPs.\u003c/strong\u003e(A) Venn diagram showing the overlap between pineapple targets and acute lung injury–related targets. (B–C) PPI network analysis of the overlapping targets of pineapple and acute lung injury. (D) Heatmap showing gene expression changes regulated by AcNPs in RNA-seq. (E) KEGG enrichment analysis of signaling pathways identified by RNA-seq of AcNPs. Data are presented as means ± standard deviation. (n=3, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/48677fa6e16fa0614913ab62.png"},{"id":108491456,"identity":"23ad6faf-b06c-4b55-89f4-9303829a8042","added_by":"auto","created_at":"2026-05-05 09:53:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":255949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcNPs attenuate NF-κB and inflammasome signaling and modulate macrophage polarization\u003c/strong\u003e. (A–B) Distribution of DiO-labeled AcNPs in macrophages at 0, 3, 12, and 24 h. Scale bar = 50 μm. Red, DiO-AcNPs; blue, DAPI. (C–D) Western blot analysis of Pho-P65, P65, Pho-IκB, and IκB protein levels in primary peritoneal macrophages. (E–F) Western blot analysis of NLRP3, ASC, and cleaved caspase-1 protein levels in primary peritoneal macrophages. (G–H) Western blot analysis of Pho-P65, P65, Pho-IκB, and IκB protein levels in RAW264.7 cells. (I–J) Western blot analysis of NLRP3, ASC, and cleaved caspase-1 protein levels in RAW264.7 cells. (K) Flow cytometry analysis of APC-CD80-labeled M1 macrophages and FITC-CD206-labeled M2 macrophages. Data are presented as means ± standard deviation. (n=3, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/56103028ccab41a4b8bd4177.png"},{"id":108491428,"identity":"e733962b-caf7-4835-bb43-9ec2dc4cbb11","added_by":"auto","created_at":"2026-05-05 09:53:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":342428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAcNPs attenuate ROS-mediated ER stress in macrophages.\u003c/strong\u003e (A–B) Flow cytometry analysis of ROS labeled with H2DCFDA. (C) Fluorescence microscopy images of ROS labeled with H2DCFDA. Scale bar = 100 μm. (D) Heatmap of ER stress-related genes from RNA-seq data. (E–F) Western blot analysis of Pho-PERK, PERK, Pho-eIF2α, eIF2α, and ATF4 protein levels in primary peritoneal macrophages. (G) Flow cytometry analysis of intracellular calcium influx labeled with Fluo-3AM. (H) Fluorescence microscopy images of intracellular calcium influx labeled with Fluo-3AM. Scale bar = 100 μm. Data are presented as means ± standard deviation. (n=3, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/929961a3e454ded97f285292.png"},{"id":108254042,"identity":"4aa22859-f307-4e6e-a703-76f53a3ff629","added_by":"auto","created_at":"2026-05-01 04:31:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":237307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of miR396-5p–Hrd1 as the target of AcNPs.\u003c/strong\u003e (A) mRNA expression levels of TNF-α, IL-6, and IL-1β in primary peritoneal macrophages determined by qPCR. (B) mRNA expression levels of TNF-α, IL-6, and IL-1β in RAW264.7 cells determined by qPCR. (C) miRNA sequences highly expressed in AcNPs. (D) mRNA expression levels of TNF-α, IL-6, and IL-1β in primary peritoneal macrophages treated with miRNA mimic, determined by qPCR. (E) Venn diagram showing the overlap between downregulated genes identified by RNA-seq and predicted miRNA target genes. (F) Binding region between miR396-5p and Hrd1. (G–H) Western blot analysis of Hrd1 protein levels in primary peritoneal macrophages. (I) Dual-luciferase reporter assay demonstrating the interaction between miR396-5p and Hrd1. (J–K) Western blot analysis of Hrd1 protein levels in primary peritoneal macrophages. (L) Ubiquitination level of IκB determined by immunoprecipitation (IP) after miR396-5p treatment. (M) Ubiquitination level of IκB determined by IP after AcNPs treatment. (N–O) Western blot analysis of Hrd1 protein levels in primary peritoneal macrophages after Hrd1 overexpression. (P) mRNA expression levels of TNF-α, IL-6, and IL-1β in primary peritoneal macrophages after Hrd1 overexpression determined by qPCR. Data are presented as means ± standard deviation. (n=3, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/860f7416a11e260f0ab0349d.png"},{"id":108254045,"identity":"99f16d16-a264-478a-9dc6-cb55e6eeae40","added_by":"auto","created_at":"2026-05-01 04:31:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":261685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR396-5p mediates the anti-inflammatory activity of AcNPs in vitro.\u003c/strong\u003e (A–B) Western blot analysis of Pho-p65, p65, Pho-IκB, and IκB protein levels in primary peritoneal macrophages. (C–D) Western blot analysis of NLRP3, ASC, and cleaved caspase-1 protein levels in primary peritoneal macrophages. (E–F) Flow cytometric analysis of ROS generation labeled with H₂DCFDA. (G) Representative fluorescence microscopy images showing ROS labeled with H₂DCFDA. Scale bar = 100 μm. (H) Flow cytometric analysis of intracellular calcium influx labeled with Fluo-3 AM. (I) Representative fluorescence microscopy images showing intracellular calcium influx labeled with Fluo-3 AM. Scale bar = 100 μm. Data are presented as means ± standard deviation. (n=3, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/cca69c850026b54a2115e693.png"},{"id":108254046,"identity":"8dc0ed23-d9dc-408d-8080-576326ebbb53","added_by":"auto","created_at":"2026-05-01 04:31:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":307192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR396-5p alleviates sepsis-induced lung and liver injury in mice.\u003c/strong\u003e (A) Schematic diagram illustrates the treatment of CLP mice with miR396-5p. (B) Serum levels of TNF-α, IL-6, and IL-1β measured by ELISA. (C–D) H\u0026amp;E staining of lung tissue and corresponding lung injury scores (scale bar = 100 μm). (E) mRNA expression levels of TNF-α, IL-6, and IL-1β in lung tissue determined by qPCR. (F) Myeloperoxidase (MPO) activity in lung tissue. (G) H\u0026amp;E staining of liver tissue (scale bar = 100 μm). (H) AST, ALT, and LDH levels in liver tissue. (I) mRNA expression levels of TNF-α, IL-6, and IL-1β in liver tissue determined by qPCR. (J) Flow cytometric analysis of macrophages (Cy7-F4/80) internalizing FITC-labeled miR396-5p. (K–L) Western blot analysis of Hrd1 protein levels in primary peritoneal macrophages. Data are presented as means ± standard deviation. (n=6, \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01, and \u003cem\u003e***P\u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/804a2820c4c9b449b5ef2ea9.png"},{"id":108254043,"identity":"21c83d1d-fe93-499f-a8cc-5483f54b16e5","added_by":"auto","created_at":"2026-05-01 04:31:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":265427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the anti-inflammatory mechanism of AcNPs. \u003c/strong\u003eAcNPs deliver miR396-5p, which directly binds to Hrd1 and reduces the ubiquitination of IκB, thereby inhibiting the activation of the NF-κB signaling pathway and alleviating ROS-mediated ER stress (ERS). This process decreases M1-type macrophage polarization while promoting M2-type polarization, ultimately attenuating sepsis-induced lung and liver injury in mice.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/59c6d73d549608fa6cf735f1.png"},{"id":109070249,"identity":"9a0ad5ac-520b-4cd2-a227-b9d7b036a078","added_by":"auto","created_at":"2026-05-12 10:30:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3447111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9327044/v1/66b608fc-8e80-4ee9-8042-3ce341e555a0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eNovel plant-derived exosome-like nanovesicles from pineapple: preparation, characterization, anti-inflammation effect via miR396-5p-Hrd1/NF-κB axis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Currently effective targeted therapies remain limited(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The pathogenesis of sepsis is primarily driven by excessive inflammatory responses and immune dysregulation(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). During pathogenic infections, complex interactions between immune cells and inflammatory mediators form an intricate regulatory network(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Activation of immune cells initiates signaling cascades that release pro-inflammatory mediators, amplifying immune activation and leading to multi-organ dysfunction.(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The lungs and liver are particularly susceptible to early damage during sepsis(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Sepsis-induced acute lung injury or distress syndrome causes high mortality in critically ill patients(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Moreover, sepsis-related hepatic injury represents a strong independent predictor of mortality(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Early implementation of hepatoprotective strategies has been shown to improve survival outcomes(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, there is an urgent need to develop safer, more effective, and cost-efficient therapeutic strategies for sepsis.\u003c/p\u003e \u003cp\u003eThe pathogenesis of sepsis involves complex immune dysregulation and excessive inflammatory responses, in which the Hrd1 (HMG-CoA reductase degradation protein 1)-mediated NF-κB signaling pathway plays a crucial regulatory role(\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Hrd1 is a key E3 ubiquitin ligase and a core component of the endoplasmic reticulum\u0026ndash;associated degradation (ERAD) system(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In response to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), Hrd1 promotes the ubiquitination and degradation of IκBα, thereby activating the NF-κB pathway and enhancing the transcription of pro-inflammatory mediators(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Besides activating immune cells, NF-κB regulates NLRP3 inflammasome assembly, promoting caspase-1 activation and IL-1β/IL-18 release, thereby amplifying inflammation and causing organ dysfunction(\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Evidence indicates that excessive activation of the Hrd1/NF-κB axis and downstream inflammasomes is closely associated with disease severity and organ injury, especially in the lungs and liver. Inhibition of Hrd1 or NF-κB signaling has been shown to suppress inflammasome activation and reduce the production of pro-inflammatory mediators, thereby alleviating sepsis-induced acute lung and liver injury(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Collectively, these findings highlight the Hrd1/NF-κB/inflammasome axis as a key player in the pathophysiology of sepsis and a promising therapeutic target for mitigating multi-organ damage.\u003c/p\u003e \u003cp\u003ePineapple is a widely consumed tropical fruit with high nutritional value and notable medicinal properties, whose abundant dietary fiber and bioactive compounds confer potent natural pharmacological activities(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Previous studies have demonstrated that pineapple intake provides multiple health benefits, including anti-obesity, anti-inflammatory, and antioxidant effects(\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, the bioactive molecules and mechanisms remain unclear. Recently, plant-derived nanoparticles (PDNPs) have gained attention for their nucleic acids, lipids, and proteins that modulate human gene expression and aid disease treatment.(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). For instance, ginger-derived extracellular vesicle-like nanoparticles have been shown to alleviate COVID-19-induced lung inflammation(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), Rgl-exomiR-7972-mediated Rehmannia-derived EVs protect against LPS-induced lung injury and intestinal dysregulation(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), and Prunus mume-derived EVLPs exert protective effects on ulcerative colitis via miR-159(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In the present study, we report for the first time that orally administered AcNPs preferentially accumulate in the lungs and liver and markedly mitigate sepsis-induced acute lung and liver injury. Mechanistically, AcNPs directly target Hrd1 via miR396-5p, thereby inhibiting the ubiquitination and degradation of IκB, which attenuates NF-κB signaling and inflammasome activation in macrophages, suppresses ROS-mediated endoplasmic reticulum stress, and modulates macrophage polarization. In summary, this study elucidates the active components and functional pathways of AcNPs in sepsis intervention, establishes a scientific foundation for their future clinical application, and highlights the translational potential of plant-derived PDNPs in disease treatment.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 AcNPs preparation\u003c/h2\u003e \u003cp\u003ePineapple (\u003cem\u003eAnanas comosus\u003c/em\u003e) were first washed with deionized water, peeled, and cut into small pieces to obtain \u003cem\u003eAnanas comosus\u003c/em\u003e juice. The resulting juice was processed as follows: centrifuged at 3,000 \u0026times; g for 20 min to remove dead cells, followed by centrifugation at 10,000 \u0026times; g for 30 min to eliminate cell debris. The supernatant was then ultra-centrifuged at 120,000 \u0026times; g for 60 min, and the pellets were resuspended in sterile PBS (Procell,China). The mixture was subsequently layered onto a sucrose gradient and ultra-centrifuged again at 120,000 \u0026times; g for 90 min to purify AcNPs. Finally, AcNPs were diluted in sterile PBS and filtered through a 0.45 \u0026micro;m membrane.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 AcNPs characterization\u003c/h2\u003e \u003cp\u003eTo measure the particle size and concentration of AcNPs, the samples were diluted in PBS and analyzed using Nanoparticle Tracking Analysis (NTA) on a ZetaVIEW system (Particle Metrix, Germany). For morphological analysis, purified PM-EVLPs were deposited onto carbon-coated copper grids, stained with 1% phosphotungstic acid, air-dried, and subsequently observed under a HITACHI H-7650 transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animal models\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic effects of AcNPs, 40 male C57BL/6J mice (6\u0026ndash;8 weeks old) were randomly divided into five groups: CON (PBS,oral), CLP (PBS,oral), CLP\u0026thinsp;+\u0026thinsp;20 mg/kg AcNPs (AcNPs, 20 mg/kg, oral), CLP\u0026thinsp;+\u0026thinsp;80 mg/kg AcNPs (AcNPs, 80 mg/kg, oral), CLP\u0026thinsp;+\u0026thinsp;supernatant (supernatant, oral). The cecal ligation and puncture (CLP) was performed according to the following steps: mice were anesthetized with sodium pentobarbital, and a midline laparotomy was carried out to gently expose the cecum; According to the grouping, the cecum was ligated approximately 1 cm from the distal end with a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 silk suture, taking care not to interrupt the blood supply of the ileocecal valve; The distal cecum was then punctured once or twice with a 22G or 21G needle, and a small amount of fecal content was gently extruded into the peritoneal cavity to induce polymicrobial infection; The cecum was then returned to the abdominal cavity, and the abdominal wall and skin were closed in layers; Control-operated mice underwent laparotomy with cecal exposure and repositioning without ligation or puncture. After 24 hours, mice were anesthetized with sodium pentobarbital and euthanized, and lung and liver tissues were collected for evaluation.\u003c/p\u003e \u003cp\u003eTo evaluate the toxicity of AcNPs in mice, 16 mice were randomly divided into two groups: CON (PBS, oral), AcNPs (AcNPs, 100 mg/kg, oral). After 24 hours, blood samples and the heart, liver, spleen, lungs, and kidneys were collected.\u003c/p\u003e \u003cp\u003eTo evaluate whether macrophages mediate the anti-inflammatory effects of AcNPs, 40 mice were randomly divided into five groups. Two groups received intraperitoneal injections of control liposomes, and three groups received intraperitoneal injections of clodronate liposomes. After 24 hours, the mice were treated as follows: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) CON: oral PBS; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) CLP\u0026thinsp;+\u0026thinsp;control liposomes: oral PBS; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) CLP\u0026thinsp;+\u0026thinsp;AcNPs: oral AcNPs (80 mg/kg); (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) CLP\u0026thinsp;+\u0026thinsp;clodronate liposomes: oral PBS; (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) CLP\u0026thinsp;+\u0026thinsp;AcNPs\u0026thinsp;+\u0026thinsp;clodronate liposomes: oral AcNPs (80 mg/kg). 24 hours after these treatments, lung and liver injury were assessed.\u003c/p\u003e \u003cp\u003eTo evaluate the anti-inflammatory effect of miR396-5p, thirty-two mice were randomly divided into four groups: CON (PBS, oral), CLP (PBS, oral), CLP\u0026thinsp;+\u0026thinsp;500 pmol miR396-5p (miR396-5p, 5 mg/kg, \u003cem\u003ei.p.\u003c/em\u003e), and CLP\u0026thinsp;+\u0026thinsp;1000 pmol miR396-5p (miR396-5p, 20 mg/kg, \u003cem\u003ei.p.\u003c/em\u003e). Lung and liver injury levels were assessed 24 hours later.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell culture\u003c/h2\u003e \u003cp\u003ePrimary peritoneal macrophages were isolated from 6-week-old male mice. Four days prior to harvest, the mice were intraperitoneally injected with 3 ml of 3% sodium thiosulfate (Sigma-Aldrich, USA). Cells were collected by peritoneal lavage with ice-cold RPMI-1640 medium (Gibco, USA). The lavage fluid was centrifuged at 1500 rpm for 10 min at 4\u0026deg;C, and the pellet was treated with red blood cell lysis buffer (Procell, China) for 10 min at 4\u0026deg;C. After a second centrifugation (1500 rpm, 10 min, 4\u0026deg;C), the remaining cells were resuspended and seeded into culture plates at a density of 1 \u0026times; 10^6 cells per well. Following a 2-h adherence period, the cells were maintained at 37\u0026deg;C in a humidified 5% CO₂ incubator in complete medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Procell, China).\u003c/p\u003e \u003cp\u003eRAW264.7 macrophages (Procell, China) were cultured under the same conditions (37\u0026deg;C, 5% CO₂) in complete medium supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Procell, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 In vivo biodistribution assay\u003c/h2\u003e \u003cp\u003eFor biodistribution studies, mice were administered AcNPs labeled with the near-infrared fluorescent dye DiR (Beyotime, China) via oral gavage. At designated time intervals (0, 6, 12, 24, and 48 h post-administration), animals were euthanized, and major organs were excised. The fluorescence intensity of DiR in the collected tissues was quantitatively assessed using a vivaCT80 small animal imaging system (SCANCO MEDICAL AG, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cellular uptake of AcNPs\u003c/h2\u003e \u003cp\u003eAcNPs (30 \u0026micro;M) were labeled with the fluorescent dye DiO (Beyotime, China) and subsequently incubated with primary peritoneal macrophages for 0, 6, 12, and 24 h. The intracellular DiO fluorescence signal was then detected using a Cytation 5 imaging reader (BioTek, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological analysis\u003c/h2\u003e \u003cp\u003eThe heart, liver, spleen, lung, and kidney tissues were fixed in 4% paraformaldehyde solution (Servicebio, China), followed by paraffin embedding and sectioning. The sections were subsequently subjected to hematoxylin\u0026ndash;eosin (H\u0026amp;E) staining for further evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 ELISA assay\u003c/h2\u003e \u003cp\u003eMice serum and bronchoalveolar lavage fluid (BALF) were subjected to enzyme-linked immunosorbent assay (ELISA) using commercial kits (Elabscience, China) to quantify the secretion levels of IL-6, TNF-α, and IL-1β. The absorbance of each well was subsequently measured and analyzed at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Wet/dry ratios\u003c/h2\u003e \u003cp\u003eLung tissues were collected 24 hours after CLP model establishment, immediately weighed to determine the wet weight, and then dried in a 56\u0026deg;C oven for 60 hours until a constant dry weight was achieved. The ratio of wet weight to dry weight was calculated to assess pulmonary edema.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 MPO assay\u003c/h2\u003e \u003cp\u003eThe myeloperoxidase (MPO) assay was performed following the standardized protocols provided by Nanjing Jiancheng Bioengineering Institute (China). All procedures were carried out in strict accordance with the manufacturer\u0026rsquo;s instructions. The MPO activity was determined by measuring the absorbance at 460 nm, and the results were subsequently analyzed to assess neutrophil infiltration in the lung tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 MDA, SOD, GSH assay\u003c/h2\u003e \u003cp\u003eCommercial assay kits (Jiancheng Bioengineering Institute, China) were used to measure malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) levels according to the manufacturer\u0026rsquo;s instructions. Lung and liver tissues were homogenized, and the resulting homogenates were analyzed for MDA content, SOD activity, and GSH levels using the respective kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 ALT, AST, LDH assay\u003c/h2\u003e \u003cp\u003eCommercial assay kits (Jiancheng Bioengineering Institute, China) were used to determine alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels according to the manufacturer\u0026rsquo;s instructions. Liver was collected and analyzed for ALT, AST, and LDH activities using the respective kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 RNA extraction and Quantitative real-time polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from lung tissues or cultured cells using TRIzol Reagent (Thermo Fisher Scientific, USA). cDNA was then synthesized with a Reverse Transcription Kit (Thermo Fisher Scientific, USA) following the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR (qPCR) was conducted to assess gene expression levels, employing SYBR Green chemistry (Promega, USA) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The amplification program included an initial denaturation at 95\u0026deg;C for 2 minutes, followed by 40 cycles of 95\u0026deg;C for 3 seconds and 60\u0026deg;C for 30 seconds for combined annealing and extension. A melting curve analysis was performed at the end of the run from 60\u0026deg;C to 95\u0026deg;C to verify amplification specificity. The primer sequences used in this study are listed below. TNF-α: \u003cem\u003eF-AGCCCCCAGTCTGTATCCTT, R-CTCCCTTTGCAGAACTCAGG\u003c/em\u003e; IL-6: \u003cem\u003eF-CTCCCTTTGCAGAACTCAGG, R-CTGTGAAGTCTCCTCTCCGG\u003c/em\u003e; IL-1β: \u003cem\u003eF-GGGCCTCAAAGGAAAGAATC, R-TACCAGTTGGGGAACTCTGC\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Western blot\u003c/h2\u003e \u003cp\u003eProtein lysates were prepared from tissues or cells using RIPA buffer (Solarbio, China), and the protein concentration was determined with a bicinchoninic acid (BCA) assay. Proteins were separated by electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked for 1 hour in Tris-buffered saline containing 0.1% Tween 20 (TBST) supplemented with 5% (w/v) non-fat milk. Following blocking, membranes were incubated overnight at 4\u0026deg;C with the following primary antibodies: β-actin monoclonal antibody (1:5000, Proteintech, China), TNF-α monoclonal antibody (1:1000, Proteintech, China), IL-1β polyclonal antibody (1:1000, Proteintech, China), IL-6 monoclonal antibody (1:1000, Proteintech, China), NLRP3 polyclonal antibody (1:2000, Proteintech, China), ASC polyclonal antibody (1:1000, Proteintech, China), caspase-1 polyclonal antibody (1:1000, Proteintech, China), phospho-p65 polyclonal antibody (1:3000, Abcam, UK), p65 monoclonal antibody (1:1000, Abcam, UK), phospho-IκB polyclonal antibody (1:1000, CST, USA), IκB monoclonal antibody (1:1000, CST, USA), Pho-PERK monoclonal antibody (1:1000, Proteintech, China), PERK monoclonal antibody (1:1000, Proteintech, China), Pho-EIF2A polyclonal antibody (1:1000, Proteintech, China), EIF2A polyclonal antibody (1:1000, Proteintech, China), Hrd1 polyclonal antibody (1:1000, Proteintech, China), and ATF4 polyclonal antibody (1:1000, Proteintech, China). After primary antibody incubation, membranes were washed three times with TBST and then incubated at room temperature for 2 hours with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG monoclonal antibody (1:5000, Proteintech, China) or goat anti-mouse IgG monoclonal antibody (1:5000, Proteintech, China). Protein bands were visualized using Luminata\u0026trade; Crescendo chemiluminescent substrate (Millipore, USA), and images were captured with a GeneGnome XRQ imaging system (Syngene, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Flow cytometry\u003c/h2\u003e \u003cp\u003eM1 and M2 Macrophage Phenotyping: Primary peritoneal macrophages were collected after treatment and transferred to centrifuge tubes. Cells were resuspended in PBS and centrifuged at 1500 rpm for 15 minutes. The pellets were stained with APC-conjugated CD80 (1:500, Elabscience, China) or FITC-conjugated CD206 (1:500, Elabscience, China) for 15 minutes, and the fluorescence was measured using a BD LSRFortessa flow cytometer (BD, USA).\u003c/p\u003e \u003cp\u003eReactive Oxygen Species (ROS) Detection: Cells were washed three times with PBS and incubated with H2DCFDA working solution (Thermo Fisher Scientific, USA) in a 37\u0026deg;C, 5% CO₂ humidified incubator, protected from light, for 30 minutes. After three additional PBS washes, intracellular ROS levels were analyzed by flow cytometry (BD LSRFortessa, USA).\u003c/p\u003e \u003cp\u003eCalcium Influx Measurement: Cells were incubated with Fluo-3 AM working solution (Solarbio, China) in HBSS (Solarbio, China) at 37\u0026deg;C for 20 minutes. Following this, the cells were washed with HEPES-buffered saline (Pricella, China) and further incubated in HBSS containing 1% fetal bovine serum (Gibco, USA) for 40 minutes. After a final wash, cells were resuspended in HEPES solution for analysis of intracellular calcium levels by flow cytometry (BD LSRFortessa, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Network pharmacological screening of Pineapple-CLP-associated target pathways\u003c/h2\u003e \u003cp\u003ePotential phytochemical constituents of pineapple were systematically retrieved through data mining using the Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine (BATMAN-TCM, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bionet.ncpsb.org.cn/batman-tcm/\u003c/span\u003e\u003cspan address=\"http://bionet.ncpsb.org.cn/batman-tcm/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in combination with an extensive literature survey, with search parameters defined as \u0026ldquo;Ingredient,\u0026rdquo; \u0026ldquo;Herb name,\u0026rdquo; or \u0026ldquo;Compound= \u003cem\u003eAnanas comosus\u003c/em\u003e or Pineapple.\u0026rdquo;\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Immunoprecipitation\u003c/h2\u003e \u003cp\u003eTo examine the interaction between IκB and ubiquitinated proteins, cells were rinsed three times with PBS and lysed on ice for 1 hour using a lysis buffer supplemented with the protease inhibitor PMSF (Cat# P0100, Solarbio, China). The lysates were centrifuged at 12,000 rpm for 10 min at 4\u0026deg;C after removing debris. The supernatant was first incubated with Dynabeads\u0026trade; Protein G (Cat# 10004D, Invitrogen, USA) for 3 h at 4\u0026deg;C to pre-clear nonspecific binding proteins. The magnetic beads were collected using DynaMag\u0026trade;-2 (Cat# 12321D, Invitrogen, USA), and the clarified supernatant was then incubated overnight at 4\u0026deg;C with an anti-IκB antibody. After incubation, fresh Dynabeads\u0026trade; Protein G were added to capture the immune complexes, which were subsequently washed three times with cold lysis buffer. The immunoprecipitates were retrieved using DynaMag\u0026trade;-2 and subjected to Western blot analysis for detection of ubiquitinated IκB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental results are presented as mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were carried out using GraphPad Prism version 9.0 (GraphPad Software, USA). For datasets exhibiting normal distribution, differences among groups were evaluated by one-way analysis of variance (ANOVA). Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Isolation and characterization of AcNPs\u003c/h2\u003e \u003cp\u003eUltracentrifugation combined with sucrose density gradient centrifugation is currently regarded as the gold standard method for isolating and purifying exosome-like nanoparticles from plant sources(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In this study, AcNPs were extracted and purified from Pineapple pulp juice using sequential centrifugation with different relative centrifugal forces and sucrose density gradient solutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The morphology and particle size of AcNPs were then characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM images revealed that AcNPs appeared as spherical or ellipsoidal vesicles with a typical bilayer membrane structure, consistent with the general morphological features of exosome-like nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). NTA analysis further indicated that the mean particle diameter of AcNPs was approximately 137.5 nm, with an average particle concentration of 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e particles/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Lipidomic profiling demonstrated that AcNPs were enriched in hexosylceramide (HexCer, 16%), ceramide (Cer, 15.17%), phosphatidylethanolamine (PE, 14%), and triglycerides (TG, 13.83%), supporting the stability of the bilayer structure and providing part of the biochemical basis for their bioactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In addition, untargeted metabolomic analysis showed that AcNPs were rich in terpenoids (30.77%), organic acids and derivatives (19.23%), phenolic acids and derivatives (17.31%), and steroids and steroid derivatives (8.65%), categories of metabolites that are closely associated with antioxidant activity, energy metabolism, inflammation regulation, and signal transduction, thereby highlighting the diverse biological and pharmacological potential of AcNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Furthermore, proteomic comparison between pineapple proteins and AcNPs revealed 3253 overlapping proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biodistribution and toxicity evaluation of AcNPs\u003c/h2\u003e \u003cp\u003eTo evaluate the biodistribution and toxicity of AcNPs, DiR-labeled AcNPs were orally administered to healthy mice fasted for 24 hours. At designated time points post-administration (0, 6, 12, 24, and 48 hours), brain, kidney, heart, spleen, liver, lung, and gastrointestinal tissues were collected, and the distribution of AcNPs was monitored using the vivaCT80 small animal imaging system (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results indicated that DiR-labeled AcNPs predominantly accumulated in the lungs and liver, reaching peak levels at 12 hours, with substantial signal remaining at 24 hours. Notably, most AcNPs were cleared from the body within 48 hours. Furthermore, administration of AcNPs at 100 mg/kg did not induce any pathological changes in the heart, liver, spleen, lung, or kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and serum biochemical parameters including ALT, AST, CRE, and BUN showed no significant hepatotoxicity or nephrotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings suggest that AcNPs preferentially accumulate in the lungs and liver, and preliminary evaluations indicate no obvious toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 AcNPs attenuate CLP-induced lung and liver iinjury in mice\u003c/h2\u003e \u003cp\u003eGiven the preferential accumulation of AcNPs in the lung and liver, as well as the compositional analysis indicating their anti-inflammatory and antioxidant potential, we employed a CLP model to induce pulmonary and hepatic injury in mice. Animals were treated with AcNPs at doses of 20 mg/kg, 80 mg/kg, or with the corresponding supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results showed that AcNPs markedly reduced serum levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Histopathological examination of lung sections revealed a dose-dependent alleviation of CLP-induced inflammatory injury following AcNPs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;D). Consistently, the lung wet-to-dry weight ratio demonstrated a reduction in pulmonary edema (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Moreover, AcNPs decreased MPO activity in lung homogenates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and lowered inflammatory cytokine concentrations in both BALF and lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u0026ndash;J). In parallel, oxidative stress was mitigated, as evidenced by decreased MDA levels and elevated GSH and SOD levels in lung tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ\u0026ndash;L). Assessment of liver injury further confirmed the protective effects of AcNPs. AcNPs reversed CLP-induced elevations of ALT, AST, and ALP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM\u0026ndash;O), alleviated inflammatory histopathological damage in hepatic tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP), and reduced TNF-α, IL-6, and IL-1β expression at both mRNA and protein levels in liver homogenates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eQ\u0026ndash;U). Collectively, these findings demonstrate that AcNPs effectively attenuate CLP-induced lung and liver injury in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Macrophages were central to AcNPs-induced attenuation of lung and liver injury.\u003c/h2\u003e \u003cp\u003eMacrophages play a pivotal regulatory role in both the initiation and resolution of inflammatory responses(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). To further determine whether macrophages mediate the anti-inflammatory effects of AcNPs, mice were pretreated with either control liposomes or clodronate liposomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results revealed that clodronate liposomes markedly induced macrophage depletion, eliminating approximately 87% of macrophages in the spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Importantly, depletion of macrophages abolished the protective effects of AcNPs against CLP-induced lung and liver injury, as evidenced by exacerbated histopathological damage in lung and liver tissues, increased lung MPO activity, elevated lung W/D ratio, as well as aggravated hepatic injury markers (AST, ALT, and LDH). Moreover, the reduction in inflammatory cytokine levels in both lung and liver tissues induced by AcNPs was also significantly reversed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, E\u0026ndash;L). Collectively, these findings demonstrate that the anti-inflammatory effects of AcNPs are mediated through a macrophage-dependent mechanism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5 NF-κB signaling pathway mediates the effects of AcNPs on macrophages.\u003c/h2\u003e \u003cp\u003eGiven that macrophages mediate the anti-inflammatory effects of AcNPs, we constructed a pharmacological target network between pineapple and sepsis, and identified 127 common targets using a Venn diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Further protein\u0026ndash;protein interaction (PPI) network analysis indicated that these targets were closely associated with NF-κB signaling, TNF signaling-mediated inflammation, and oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Subsequently, in an LPS-induced peritoneal macrophage model, RNA-seq was used to identify key signaling pathways modulated by AcNPs, and KEGG pathway enrichment analysis further revealed that NF-κB and TNF signaling mediated the anti-inflammatory effects of AcNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). These findings suggest that bioactive substance-rich AcNPs may exert their anti-inflammatory effects primarily through NF-κB signaling and its downstream inflammation-related pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6 AcNPs inhibited LPS-induced NF-κB signaling pathway and inflammatory activation in macrophages.\u003c/h2\u003e \u003cp\u003eFirst, we evaluated the uptake of AcNPs by macrophages at the cellular level. DiO-labeled AcNPs were used to monitor macrophage internalization at 0, 3, 12, and 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Subsequently, in LPS-induced inflammatory models using two types of macrophages (primary peritoneal macrophages and RAW264.7 cells), we observed a dose-dependent inhibition of NF-κB signaling upon AcNPs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-D, G-H). The NLRP3 inflammasome, as a secondary signal downstream of NF-κB, was similarly suppressed by AcNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F, I-J). Moreover, considering the critical role of macrophage M1/M2 polarization in inflammatory responses, we assessed the effect of AcNPs on macrophage phenotypes. The results indicated that AcNPs inhibited LPS-induced M1 polarization while promoting M2 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Collectively, these findings suggest that AcNPs suppress LPS-induced NF-κB signaling and inflammasome activation, inhibit M1 polarization, and facilitate M2 polarization in macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.7 AcNPs attenuated LPS-induced oxidative stress and endoplasmic reticulum stress in macrophages.\u003c/h2\u003e \u003cp\u003eROS-mediated ER stress represents a critical process in the inflammatory response(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). To explore this, we assessed ROS production and ER stress in primary peritoneal macrophages following LPS stimulation. Using H2DCFDA, a fluorescent probe for reactive oxygen species, both flow cytometry and fluorescence microscopy revealed that AcNPs attenuated the LPS-induced elevation of ROS in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;C). Furthermore, targeted analysis of RNA-seq data indicated that AcNPs suppressed the expression of ER stress\u0026ndash;related genes, a finding corroborated by western blot results showing downregulation of ER stress\u0026ndash;associated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u0026ndash;F). In addition, Fluo-3 AM, a probe commonly employed to assess Ca\u0026sup2;⁺ influx during ER stress, demonstrated via flow cytometry and microscopy that AcNPs alleviated LPS-induced intracellular Ca\u0026sup2;⁺ influx (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG\u0026ndash;H). Collectively, these results suggest that AcNPs markedly suppress LPS-induced oxidative stress and ER stress in macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Identification of miR396-5p as a target of AcNPs and Hrd1 as its downstream effector.\u003c/h2\u003e \u003cp\u003eTo identify the bioactive components responsible for the anti-inflammatory activity of AcNPs, we treated the nanoparticles with Proteinase K, RNase/DNase, and RNase/lipase to selectively inactivate proteins, nucleic acids, and lipids, respectively. The results indicated that the nucleic acid components within AcNPs are likely the key mediators of their anti-inflammatory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;B). Deep sequencing of miRNAs subsequently revealed six novel miRNAs highly enriched in AcNPs, including miR156b-3p, miR156b-5p, miR168a-5p, miR168b-5p, miR159-3p, and miR396-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Synthetic mimics of these miRNAs were transfected into primary peritoneal macrophages, and both miR168a-5p and miR396-5p exhibited anti-inflammatory activity at a concentration of 60 \u0026micro;M, with miR396-5p showing a stronger effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). By integrating RNA-seq data of AcNPs with psRobot-predicted targets, Hrd1 and Pstpip2 were identified as potential downstream targets of AcNP-derived miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE\u0026ndash;F). Hrd1 is an endoplasmic reticulum\u0026ndash;associated E3 ubiquitin ligase that has been reported to regulate NF-κB signaling by promoting IκB ubiquitination. Treatment with miR396-5p mimics reduced macrophage activation upon LPS stimulation and suppressed Hrd1 expression, as further confirmed by luciferase reporter assays showing direct regulation of Hrd1 by miR396-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG\u0026ndash;I). Consistently, inhibition of miR396-5p abolished the effect of AcNPs on Hrd1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ\u0026ndash;K). Moreover, we found that miR396-5p modulates NF-κB signaling through Hrd1-mediated ubiquitination of IκB (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL\u0026ndash;M). Importantly, overexpression of Hrd1 reversed the anti-inflammatory effect of AcNPs, indicating that Hrd1 acts as a direct molecular target of AcNPs in regulating inflammation via the NF-κB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eN\u0026ndash;P). Collectively, these findings identify miR396-5p as a novel anti-inflammatory miRNA within AcNPs that directly targets Hrd1 and inhibits IκB ubiquitination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.9 miR396-5p mediates the in vitro anti-inflammatory activity of AcNPs.\u003c/h2\u003e \u003cp\u003eTo further investigate whether miR396-5p mediates the regulatory effects of AcNPs on NF-κB signaling, inflammasome activation, and ERS, we examined the effects of miR396-5p mimic and inhibitor. The results showed that the miR396-5p mimic inhibited the activation of NF-κB and inflammasome signaling in a concentration-dependent manner, whereas the miR396-5p inhibitor reversed the inhibitory effects of AcNPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;D). Moreover, similar results were observed in ROS-mediated ERS (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE\u0026ndash;I). Collectively, these findings indicate that miR396-5p mediates the anti-inflammatory activity of AcNPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.10 miR396-5p is a key target mediating the protective effects of AcNPs in vivo.\u003c/h2\u003e \u003cp\u003eSubsequently, we explored whether miR396-5p also exerts anti-inflammatory effects in CLP-induced septic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). The results showed that miR396-5p administration markedly reduced the serum levels of TNF-α, IL-6, and IL-1β in CLP mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB), alleviated pathological lung injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC\u0026ndash;D), and decreased the concentrations of TNF-α, IL-6, and IL-1β as well as MPO activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE\u0026ndash;F). In addition, miR396-5p treatment attenuated liver injury and inflammation in septic mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG\u0026ndash;I). Furthermore, to determine the cellular target of miR396-5p, mice were treated with FITC-labeled miR396-5p followed by macrophage sorting using flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eJ). The results revealed a significant reduction in Hrd1 expression within pulmonary macrophages after miR396-5p treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eK\u0026ndash;L). Collectively, these findings demonstrate that miR396-5p alleviates sepsis-induced lung and liver injury in vivo.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussions","content":"\u003cp\u003eSepsis, triggered by infection-induced systemic inflammatory response syndrome, can result in multiple organ dysfunction, with the liver and lungs being the most vulnerable targets(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). During sepsis, the liver acts not only as a central hub of immune defense but also as a major site of inflammatory injury, manifesting as cholestasis, elevated transaminases, and dysregulation(\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The lungs, on the other hand, undergo acute injury due to inflammatory cell infiltration, cytokine storms, and increased capillary permeability, ultimately progressing to ALI or ARDS(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). These pathological changes markedly contribute to the high mortality associated with sepsis(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Although current clinical management primarily relies on anti-infective therapy, fluid resuscitation, and organ support, effective organ-specific interventions remain lacking. Recent evidence suggests that naturally derived nanoparticles, owing to their low toxicity, represent a promising therapeutic avenue(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Certain dietary-sourced NPs have demonstrated protective effects against acute hepatic and pulmonary injury. For instance, NPs from Platycodon grandiflorum alleviate LPS-induced ALI by modulating lipid metabolism and glycolysis(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e); Rehmanniae Radix exerts protective effects against ALI by targeting GPR161(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e); and pomegranate-derived NPs have been shown to attenuate hepatic injury in MASLD(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Notably, in our study, AcNPs were found to selectively target both the liver and lungs in mice, conferring multi-organ protection and enhancing systemic therapeutic efficacy without eliciting adverse reactions.\u003c/p\u003e \u003cp\u003eIn sepsis-induced acute lung and liver injury, although multiple cell types including neutrophils, epithelial cells, and endothelial cells play important roles, macrophages, as tissue-resident \u0026ldquo;sentinel\u0026rdquo; cells, are the first to recognize PAMPs and DAMPs(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). They release large amounts of inflammatory cytokines and chemokines, orchestrating the recruitment of other immune cells and amplifying the inflammatory response, thereby exerting a central and initiating role(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). In contrast, neutrophils, endothelial cells, epithelial cells, and lymphocytes mainly act downstream, contributing to barrier disruption, oxidative stress, and adaptive immune dysregulation(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). While these cells serve as key executors of tissue injury, their activation largely depends on the early inflammatory signals provided by macrophages. Our study further demonstrated that clodronate liposomes treatment reversed the in vivo anti-inflammatory effects of AcNPs, indicating that macrophages are the primary target mediating the biological activity of AcNPs and highlighting the potential of AcNPs as a macrophage-centered therapeutic strategy.\u003c/p\u003e \u003cp\u003eTherefore, we further explored the anti-inflammatory mechanism of AcNPs. Network pharmacology analysis based on pineapple and acute lung injury, together with KEGG enrichment analysis of RNA-seq data from LPS-treated macrophages, indicated that the NF-κB signaling pathway may serve as a key target of AcNPs. NF-κB is a classical inflammatory signaling pathway: LPS activates it through the TLR4 receptor, inducing TLR4 dimerization and recruitment of MyD88(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). MyD88 subsequently recruits and activates IRAK, which forms a complex with TRAF6, leading to phosphorylation and ubiquitination of the downstream IκB complex(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This process releases NF-κB, allowing it to translocate into the nucleus and promote transcription of pro-inflammatory genes(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Within NF-κB signaling, Hrd1 binds to the deubiquitinating enzyme Usp15, rendering it inactive and unable to deubiquitinate IκBα, thereby causing hyperactivation of NF-κB(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Consistent with the RNA-seq and network pharmacology predictions, experimental results showed that AcNPs significantly inhibited the phosphorylation of p65 and IκB. Moreover, AcNPs treatment markedly suppressed NLRP3 inflammasome signaling, accompanied by inhibition of M1 macrophage polarization and promotion of M2 macrophage polarization.\u003c/p\u003e \u003cp\u003eOxidative stress\u0026ndash;induced endoplasmic reticulum stress (ERS) represents a critical feature of inflammatory responses(\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Excessive accumulation of intracellular reactive oxygen species (ROS) can disrupt protein folding and calcium homeostasis, activating ERS signaling pathways such as PERK, IRE1α, and ATF6, which in turn trigger downstream inflammatory gene expression(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). In macrophages, ROS-mediated ERS is particularly pronounced, serving as a central node for the initiation, amplification, and maintenance of inflammation. Concurrently, ROS can promote phosphorylation and degradation of IκB, leading to nuclear translocation of NF-κB and activation of downstream inflammatory gene transcription. Activated NF-κB further enhances the production of proinflammatory mediators, including TNF-α, IL-1β, and IL-6, and promotes additional ROS generation, establishing a positive feedback loop that exacerbates ERS and oxidative stress, thereby amplifying the inflammatory response(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Previous studies further support this mechanism: dendrobine mitigates LPS-induced acute lung injury by modulating FAM134B-mediated ERS(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e); hyperglycemia impairs M2 polarization of hepatic macrophages via C/EBP homolog\u0026ndash;mediated ERS, aggravating hepatic ischemia\u0026ndash;reperfusion injury(\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e); and exogenous hydrogen sulfide protects against rat liver injury by regulating ERS-induced macrophage autophagy(\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Collectively, these studies suggest that the ROS\u0026ndash;NF-κB\u0026ndash;ERS network plays a pivotal role in controlling inflammation and represents a potential therapeutic target for macrophage-mediated inflammatory diseases. In the present study, we demonstrate for the first time that AcNPs significantly suppress ROS-mediated ERS, thereby alleviating LPS-induced macrophage inflammatory responses. Notably, AcNPs not only downregulate the expression of ERS-related genes and proteins but also inhibit NF-κB activation and promote the polarization of macrophages toward the anti-inflammatory M2 phenotype. These findings suggest that AcNPs may exert multi-level, multi-target regulation in the inflammatory microenvironment by modulating the ROS\u0026ndash;NF-κB\u0026ndash;ERS network. Although the precise upstream\u0026ndash;downstream relationship between ERS and NF-κB remains to be fully elucidated, our data clearly indicate that AcNPs act synergistically to inhibit ROS, ERS, and NF-κB signaling, thereby exerting potent anti-inflammatory and tissue-protective effects. This provides a theoretical basis for developing macrophage-targeted nanotherapeutics and highlights the potential clinical value of AcNPs in multi-organ protection and inflammation modulation.\u003c/p\u003e \u003cp\u003eTo further identify the essential bioactive substances within AcNPs, we treated them with Proteinase K, RNase/DNase, and RNase/Lipids to selectively eliminate proteins, nucleic acids, and lipids, respectively, and then evaluated their impact on inflammatory responses. The results indicated that nucleic acids constitute the major basis of AcNPs\u0026rsquo; biological activity. In recent years, plant-derived miRNAs have been recognized as a new category of dietary bioactive molecules capable of modulating endogenous mRNA expression(\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Owing to their characteristic 2\u0026prime;-O-methyl modification at the terminal ends, plant miRNAs exhibit remarkable structural resilience, allowing them to withstand oxidative stress, corrosive conditions, and enzymatic degradation during gastrointestinal digestion. This stability under harsh environments underscores their potential as therapeutic molecules and dietary supplements. Supporting this concept, accumulating evidence has demonstrated their protective roles: miR159 carried by Prunus mume\u0026ndash;derived EVLPs mitigates colitis symptoms, Teng et al(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). reported that garlic-derived EVLPs enriched in miR-396e ameliorated nonalcoholic fatty liver disease, and Yu et al(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). showed that fresh Rehmanniae Radix\u0026ndash;derived EVLPs containing Rgl-exomiR-7972 alleviated LPS-induced acute lung injury(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In this study, systematic screening identified miR396-5p as a key mediator of the anti-inflammatory activity of AcNPs. Moreover, the overlap between RNA-seq results from AcNPs-treated macrophages and the predicted target genes of miR396-5p suggested that Hrd1 may serve as its direct downstream target. This conclusion was further validated by dual-luciferase reporter assays. Notably, only a limited number of miRNAs have been reported to directly target Hrd1, and these are mammalian-derived, including miR-513a-3p and miR-224. To our knowledge, this is the first demonstration that a plant-derived exogenous miRNA (miR396-5p) can directly target Hrd1, highlighting its potential as a novel therapeutic strategy for diseases in which Hrd1 plays a central pathogenic role.\u003c/p\u003e \u003cp\u003eNPs derived from fruits, vegetables, and medicinal-food homologous plants exhibit remarkable advantages such as drug delivery vehicles. These NPs are broadly sourced, highly safe, and typically endowed with excellent biocompatibility and low immunogenicity, thereby minimizing the toxic side effects and immune rejection frequently encountered with synthetic nanomaterials. Their bilayer membrane structure and nanoscale size confer superior stability and permeability, enabling them to remain active for extended periods in circulation and to effectively traverse multiple biological barriers. Importantly, plant-derived NPs are intrinsically enriched with diverse bioactive molecules such as miRNAs, lipids, and polysaccharides, which not only serve as carriers but may also synergize exogenous therapeutics to potentiate overall therapeutic efficacy(\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Unlike conventional synthetic vectors, these NPs possess innate tissue- and cell-targeting capacities\u0026mdash;for instance, preferential accumulation in the gut or efficient uptake by macrophages\u0026mdash;thereby facilitating precise drug delivery(\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Moreover, their low production cost, straightforward preparation, and scalability highlight their sustainability and translational potential. Increasing evidence has demonstrated that loading small-molecule chemotherapeutics, proteins, or functional nucleic acids into plant-derived NPs markedly improves drug bioavailability, reduces off-target cytotoxicity, and enhances treatment outcomes. Collectively, these features underscore the broad promise of NPs from fruits, vegetables, and medicinal-food plants in shaping the next generation of drug delivery systems.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our study is the first to demonstrate that orally administered AcNPs alleviate CLP-induced acute liver and lung injury in mice by targeting macrophages in these organs. Mechanistically, AcNPs deliver miR396-5p to suppress Hrd1, thereby reducing IκB ubiquitination and attenuating NF-κB\u0026ndash;mediated inflammatory signaling as well as ROS-driven ERS. Overall, this work highlights plant-derived nanoparticles as a promising therapeutic strategy for sepsis and provides a potential platform for small nucleic acid drug delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePDNPs, plant-derived nanoparticles; AcNPs, \u003cem\u003eAnanas comosus (Linn.) Merr.\u003c/em\u003e, Ac-derived nanoparticles; Hrd1, HMG-CoA reductase degradation protein 1; ROS, reactive oxygen species; LPS, lipopolysaccharide; CLP, cecal ligation and puncture; PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns; TNF\u0026alpha;, tumor necrosis factor alpha; IL-1\u0026beta;, interleukin-1 beta; IL-6, interleukin-6; NLRP3, NOD-like receptor family pyrin domain containing 3; EVLPs, extracellular vesicle-like particles; NTA, nanoparticle tracking analysis; BALF, bronchoalveolar lavage fluid; MPO, myeloperoxidase; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; ERS, endoplasmic reticulum stress; Hrd1, Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Medical Sci-Tech research Center of Ningxia Medical University (Medical Science Research Institution of Ningxia Hui Autonomous Region） for valuable help in our experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeixi Xie and Zun Wang have contributed equally to this work and share first authorship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiangya Nursing School, Central South University, Changsha, Hunan, China\u003c/p\u003e\n\u003cp\u003eWeixi Xie, Lang Deng, Siyuan Li, Yajing Wang, Wei Liu, Honghong Wang, Siyuan Tang.\u003c/p\u003e\n\u003cp\u003eThe School of Nursing, Ningxia Medical University, Yinchuan, Ningxia, China\u003c/p\u003e\n\u003cp\u003eZun Wang, Siyuan Tang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHonghong Wang, Siyuan Tang, Wei Liu: Conceptualization, Funding. Weixi Xie, Zun Wang: Writing original draft, Methodology, Investigation. Weixi Xie: Methodology, Investigation. Siyuan Li, Lang Deng, Yajing Wang: Investigation, Data curation, Supervision. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Wei Liu, Honghong Wang and Siyuan Tang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThese animals were procured from the Animal Center of Central South University. It has been approved by the Laboratory Animal Welfare and Ethics Committee of Animal Center, Central South University (Approval No.XMSB-2025-0595).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundigs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (grant numbers: 82170853); the Natural Science Foundation of Ningxia Hui Autonomous Region (grant number: 2024AAC02044); the National Natural Science Foundation of China (No. 82560441); the Natural Science Foundation of Hunan Province (No. 2024JJ6668); the Youth Science Foundation of Xiangya Hospital, Central South University (No. 2023Q13); and the Education and Teaching Reform Project of Ningxia Medical University (No. NYJY2025099).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCecconi M, Evans L, Levy M, Rhodes A. 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Cell host \u0026amp; microbe. 2018;24(5):637-652.e638.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sepsis, NF-κB, Hrd1.","lastPublishedDoi":"10.21203/rs.3.rs-9327044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9327044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlant-derived nanoparticles (PDNPs) have attracted considerable attention as promising bioactive nutraceutical agents. Pineapple (\u003cem\u003eAnanas comosus (Linn.) Merr.\u003c/em\u003e, Ac) is a functional fruit with notable anti-inflammatory and antioxidant effects, though its exact active components and mechanisms remain unclear. Our study shows that orally administered pineapple-derived nanoparticles (AcNPs) alleviate sepsis-induced lung and liver injury in mice. AcNPs preferentially accumulate in these organs, are mainly taken up by macrophages, and exert anti-inflammatory effects dependent on macrophage activity, as confirmed by clodronate liposome treatment. Mechanistic analyses integrating network pharmacology and RNA sequencing suggested that AcNPs primarily exert their effects via modulation of NF-κB\u0026ndash;dependent inflammatory signaling. AcNPs suppressed NF-κB and inflammasome activation, reduced ROS-induced ER stress, inhibited M1 polarization, and promoted M2 polarization, thereby alleviating macrophage-mediated inflammation. Importantly, this inhibitory activity was attributed to RNA components within AcNPs rather than lipid or protein constituents. Deep RNA sequencing combined with miRNA mimic and inhibitor experiments identified miR396-5p as the principal bioactive molecule responsible for the suppression of macrophage inflammatory activation. Moreover, miR396-5p directly targets Hrd1, mediating the regulatory effect of AcNPs on NF-κB signaling. Collectively, these findings position AcNPs as a promising nanotherapeutic candidate with potential for the treatment of sepsis-induced acute lung/liver injury.\u003c/p\u003e","manuscriptTitle":"Novel plant-derived exosome-like nanovesicles from pineapple: preparation, characterization, anti-inflammation effect via miR396-5p-Hrd1/NF-κB axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-01 04:31:39","doi":"10.21203/rs.3.rs-9327044/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-01T02:40:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T13:08:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-28T02:31:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100678766674300581010652038070523479771","date":"2026-04-22T04:02:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255354242094504284225966743581670807408","date":"2026-04-22T03:06:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285286534022495074160177503296397011114","date":"2026-04-22T03:04:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T02:49:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-06T05:45:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-06T05:44:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2026-04-05T14:43:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"339982cd-f3dd-44fd-8732-65d5c7ed4402","owner":[],"postedDate":"May 1st, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-01T02:40:34+00:00","index":16,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-01T04:31:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-01 04:31:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9327044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9327044","identity":"rs-9327044","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}