Adipogenic MSC-derived COL-3 Drives M2 Macrophage Polarization via PI3K-AKT to Alleviate Sepsis-induced Lung Injury

Adipogenic MSC-derived COL-3 Drives M2 Macrophage Polarization via PI3K-AKT to Alleviate Sepsis-induced Lung Injury | 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 Article Adipogenic MSC-derived COL-3 Drives M2 Macrophage Polarization via PI3K-AKT to Alleviate Sepsis-induced Lung Injury Qianmei Wang, Jiayao Zhang, Zelong Yang, Xianqi Wang, Dan Wu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8375617/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Sepsis-induced acute lung injury is a critical condition marked by uncontrolled inflammation and immune imbalance. Here, we investigated the role of extracellular matrix components derived from adipogenically differentiated adipose-derived stem cells in regulating macrophage polarization and lung injury. We found that type III collagen was significantly upregulated in the extracellular matrix after adipogenic induction and promoted macrophage polarization toward the anti-inflammatory M2 phenotype via the PI3K-AKT signaling pathway. In a mouse model of sepsis-induced lung injury, treatment with lyophilized extracellular matrix from adipogenic adipose-derived stem cells alleviated lung inflammation and injury, accompanied by increased infiltration of M2 macrophages. These results identify a novel mechanism by which stem cell-derived type III collagen modulates the immune microenvironment and suggest a new therapeutic approach for inflammatory lung diseases. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Immunology Biological sciences/Stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Sepsis is a systemic inflammatory syndrome caused by a dysregulated host response to infection, which can lead to multiple organ dysfunction, with the lungs being among the most frequently affected organs [ 1 ] . The mortality rate of sepsis-associated acute lung injury (ALI) can reach as high as 40%, significantly higher than that of ALI resulting from other causes [ 2 ] . Recent studies have shown that the polarization state of macrophages plays a critical regulatory role in the onset and progression of ALI [ 3 – 6 ] . However, the specific mechanisms underlying the dynamic imbalance between M1 and M2 macrophage subtypes remain unclear and warrant further investigation. Adipose-derived stem cells (ADSCs) are a type of adult stem cell with self-renewal capacity and multipotent differentiation potential [ 7 ] . In recent years, ADSCs have been widely used in basic and translational research on various inflammatory diseases and sepsis due to their remarkable immunomodulatory and tissue repair properties [ 8 – 10 ] . Previous studies have mainly focused on the effects of undifferentiated ADSCs in modulating inflammatory responses and promoting tissue repair, with evidence of protective effects observed in various animal models [ 11 – 12 ] . Notably, recent research has demonstrated that the functional state of stem cells can undergo dynamic changes during cell therapy in response to alterations in the microenvironment, with some cells further differentiating into terminal cell types [ 13 ] . As the terminally differentiated product of ADSCs, adipocytes more closely resemble mature adipose tissue in function and, theoretically, may be better suited to recapitulate the immunological microenvironment of adipose tissue and regulate immune responses. However, to date, the roles and mechanisms of adipogenically differentiated ADSCs in sepsis-associated acute lung injury remain poorly characterized [ 14 ] . Therefore, systematically elucidating the function and molecular mechanisms of adipogenically differentiated ADSCs in sepsis-induced lung injury is of great significance for advancing the theoretical basis and expanding the clinical application of stem cell-based therapies for inflammatory lung injury. After adipogenic induction, ADSCs secrete an extracellular matrix (ECM) with a composition and structure more similar to that of mature adipose tissue, with increased uniformity and controllability. Studies have shown that during adipogenic differentiation, the expression of key matrix proteins such as laminin and type III collagen (COL-3) is significantly upregulated. These changes not only reflect the dynamic regulatory capacity of ADSCs over ECM composition, but also suggest a potential role for the ECM in modulating the immune microenvironment. Given that macrophage polarization balance is a crucial component of the immune microenvironment in sepsis-induced ARDS, we hypothesize that adipogenically differentiated ADSCs, compared to undifferentiated ADSCs, are better able to recapitulate the unique immunomodulatory properties of adipose tissue, especially in regulating macrophage polarization. In this study, we explored the effects of ECM derived from adipogenically differentiated ADSCs on macrophage polarization. Through a series of phenotypic experiments, we observed a significant upregulation of type III collagen (COL-3) in the ECM, which was closely associated with the enrichment of M2 macrophages. This finding suggests that COL-3 may serve as a key active component in the regulation of macrophage polarization. Based on this, we further investigated the underlying mechanism and confirmed that COL-3 promotes M2 macrophage polarization via activation of the PI3K-AKT signaling pathway, thereby alleviating sepsis-induced lung injury. This study is expected to enrich the understanding of the immunomodulatory mechanisms of stem cell-derived ECM and provide new therapeutic targets for the treatment of inflammatory lung injury. RESULTS 1. Increased M2 Macrophage Population in the Late Phase of Sepsis-Induced Lung Injury in Mice To investigate the relationship between macrophage polarization status and the severity of sepsis-induced acute lung injury (ALI), a mouse model of septic lung injury was established using the cecal ligation and puncture (CLP) procedure (see Methods for details). After surgery, mice exhibited clear systemic inflammatory response symptoms, including lethargy, ruffled fur, reduced food intake, and purulent secretions at the canthi. Survival analysis revealed that the survival rate of mice in the CLP group decreased to 33.3% (3/9) at 24 hours post-operation, further dropped to 11.1% (1/9) at 36 hours, and reached 0% (0/9) at 48 hours. The survival rate in the control group also declined slightly over time, remaining at 66.7% (6/9) at 48 hours (Fig 1A). These results indicate that the model successfully induced severe sepsis. Additionally, the lung wet-to-dry weight ratio (W/D ratio) was significantly elevated in the CLP group, especially in mice with more severe symptoms, indicating substantial pulmonary edema (Fig 1B) and further validating the successful establishment of the model. At 24 hours post-surgery, samples were collected from the lungs, liver, and heart for hematoxylin and eosin (HE) staining (Fig 1C). The results revealed varying degrees of inflammatory cell infiltration in all three organs. The lungs exhibited marked interstitial edema and alveolar obstruction by exudates. Hepatic tissue showed hepatocyte swelling and narrowing of hepatic sinusoidal spaces, indicative of inflammatory edema. Myocardial tissue also exhibited edema, further confirming multi-organ inflammatory injury induced by sepsis. Subsequently, mice were categorized into mild, moderate, and severe lung injury groups based on the severity of their clinical symptoms prior to sample collection (Fig 1D). CD206 immunofluorescence staining was performed on lung tissues from each group to assess the distribution of M2 macrophages (Fig 1E). Quantitative analysis showed that the mild lung injury group had the highest proportion of CD206-positive areas (mean 2.86 ± 0.93%), followed by the moderate group (mean 1.79 ± 0.10%), and the severe group had the lowest proportion (mean 1.06 ± 0.47%). These results suggest that increased M2 macrophage abundance may be closely associated with better prognosis in sepsis-induced ALI and may play a critical role in the regulation of inflammation and tissue repair. 2. Adipogenic Differentiation of ADSCs Enhances M2 Macrophage Polarization via COL-3 To investigate whether adipogenic induction influences the regulatory capacity of adipose-derived stem cell (ADSC)-derived extracellular matrix (ECM) on macrophage polarization, ADSCs were first subjected to adipogenic differentiation and systematically characterized. As shown in Fig 2A, undifferentiated ADSCs displayed a typical spindle-shaped morphology with tightly aligned cells. In contrast, adipogenically induced ADSCs (AD-ADSCs) exhibited substantial lipid droplet accumulation, as evidenced by prominent red lipid deposition upon Oil Red O staining, confirming successful adipogenic differentiation. Both cell groups were then subjected to decellularization, and hematoxylin-eosin (HE) staining was used to preliminarily assess ECM retention (Fig 2B). The control group exhibited numerous residual nuclei, whereas nuclei were largely absent following decellularization, and the matrix structure was well preserved, indicating an effective decellularization process. To further evaluate the effects of ECM from different sources on macrophage polarization, the decellularized matrices were co-cultured with macrophages, and the proportion of M2 macrophages was assessed by CD206 immunofluorescence staining (Fig 2C). The results showed that the average proportion of CD206-positive area in the AD-ADSCs ECM group was 2.67 ± 0.48%, which was significantly higher than that in the ADSCs ECM group (1.33 ± 0.23%), indicating that ECM derived from adipogenically induced ADSCs has a greater capacity to promote M2 polarization. 3. Lyophilized ECM from Adipogenically Induced ADSCs Attenuates Sepsis-Induced Lung Injury and Promotes M2 Macrophage Polarization Given that ECM derived from adipogenically induced ADSCs (AD-ADSCs ECM) significantly promoted M2 macrophage polarization in vitro, we further evaluated its therapeutic effects in vivo. Lyophilized ECM powders were prepared according to the schematic workflow (Fig 3A), and their gross morphology was documented (Fig 3B), demonstrating the successful acquisition of homogeneous ECM lyophilized powder. Next, lyophilized ECM from undifferentiated ADSCs and from AD-ADSCs was respectively administered to mice with sepsis-induced acute lung injury established by cecal ligation and puncture (CLP) (Fig 3C). Mice were randomly divided into three groups: control (untreated), ADSCs ECM, and AD-ADSCs ECM. After two days of treatment, hematoxylin-eosin (HE) staining revealed that, compared to the control and ADSCs ECM groups, the AD-ADSCs ECM group exhibited reduced interstitial edema, more intact alveolar architecture, and markedly decreased inflammatory cell infiltration in lung tissue (Fig 3C). To further assess the immunomodulatory effects, CD206 immunofluorescence staining was performed to evaluate M2 macrophage infiltration in lung tissues from all three groups (Fig 3D). Quantitative analysis showed that the mean proportion of CD206-positive area in lung tissue was highest in the AD-ADSCs ECM group at 2 days (6.28 ± 0.76%), significantly greater than that in the 1-day treatment group (4.23 ± 0.45%) and the untreated group (0 days) (1.84 ± 0.94%). These results indicate that ECM derived from adipogenically induced ADSCs can also enhance M2 polarization in vivo. 4. Identify the key components in the ECM of adipogenically induced ADSCs (AD-ADSCs) that may mediate macrophage polarization, we first performed immunofluorescence staining for major collagens and adhesion molecules, assessing the expression of COL-1, COL-3, fibronectin, and laminin in ECM derived from undifferentiated ADSCs and AD-ADSCs (Fig 4A). Quantitative analysis revealed that the proportion of COL-3-positive area was significantly higher in the AD-ADSCs group (8.63 ± 1.18%) compared to the undifferentiated group (3.45 ± 1.95%, P < 0.05). There were no significant differences in the expression of COL-1, laminin, or fibronectin between the two groups (Fig 4B), suggesting that COL-3 may be the critical component of AD-ADSCs ECM responsible for regulating macrophage polarization. To verify the effect of COL-3 on inducing macrophage polarization toward the M2 phenotype, the four ECM components (COL-1, COL-3, fibronectin, and laminin) were individually coated onto culture plates, and RAW264.7 macrophages were co-cultured in vitro for 48 hours, followed by CD206 immunofluorescence staining (Fig 4C). Analysis of the CD206-positive area showed that the COL-3 group had the highest positive proportion (8.14 ± 1.09%), which was significantly greater than those of the COL-1 group (4.58 ± 1.19%), laminin group (6.09 ± 0.31%), and fibronectin group (2.67 ± 0.74%) (Fig 4D). To further explore the molecular mechanism of COL-3, macrophages treated with ADSCs ECM, AD-ADSCs ECM, or COL-3 were collected for assessment of CD206, AKT, and phosphorylated AKT (p-AKT) protein expression (Fig 4E). Western blot analysis demonstrated that both AKT and p-AKT expression were higher in macrophages treated with AD-ADSCs ECM and COL-3 compared to those treated with ADSCs ECM and the control group. Quantitative analysis showed that the AD-ADSCs ECM group had an AKT expression level of 1.43 ± 0.02 and p-AKT of 1.86 ± 0.05, both significantly higher than those in the ADSCs ECM group (AKT: 0.84 ± 0.06; p-AKT: 0.36 ± 0.02) and the control group (AKT: 0.80 ± 0.01; p-AKT: 0.70 ± 0.01) (Fig 4F). Collectively, these results demonstrate that the COL-3 content is markedly increased in the ECM of adipogenically induced ADSCs, and that COL-3 can enhance macrophage polarization toward the M2 phenotype by activating the PI3K-AKT signaling pathway, suggesting that COL-3 may be a key functional component in ECM-mediated immunomodulation. DISCUSSION In sepsis-associated acute respiratory distress syndrome (ARDS), aberrant macrophage polarization plays a pivotal role in disease progression [ 15 – 18 ] . Our study demonstrated that type III collagen (COL-3), a key extracellular matrix (ECM) component secreted by adipogenically differentiated adipose-derived stem cells (AD-ADSCs), is critical for promoting macrophage polarization toward the M2 phenotype, primarily via activation of the PI3K-AKT signaling pathway [ 19 – 22 ] . As one of the main components of ECM, collagen provides mechanical support for tissues and plays significant roles in regulating inflammation, angiogenesis, and tissue repair during injury [ 23 – 24 ] . COL-3, an important member of the collagen superfamily, is composed of three identical left-handed α1 chains wound into a stable right-handed triple helix structure [ 25 – 26 ] . This structure contains multiple integrin-binding sites that specifically interact with integrin receptors (such as α2β1 and α1β1) on macrophage surfaces, facilitating cell adhesion and migration, thus modulating the inflammatory microenvironment. The interaction between cells and ECM is crucial for macrophage polarization. During the early inflammatory phase, increased COL-3 expression forms a provisional matrix that directs inflammatory cells and fibroblasts to the injury site [ 27 ] . Subsequently, the N-terminal propeptide (CR) region of COL-3 binds to receptors on macrophage surfaces, activating the PI3K-AKT signaling pathway, thereby facilitating the shift from a pro-inflammatory M1 phenotype toward an anti-inflammatory and reparative M2 phenotype, as confirmed in our experiments . The Akt family comprises three serine/threonine protein kinases (Akt1, Akt2, Akt3), serving as critical effectors of the PI3K pathway. Upon PI3K-mediated conversion of PIP2 to PIP3, downstream proteins PDK1 and Akt are activated and bind to PIP3, leading to Akt phosphorylation. Extensive research has confirmed the essential role of the PI3K-AKT pathway in macrophage activation and gene expression regulation, with inhibition of this pathway significantly suppressing M2 macrophage polarization [28–30] . Lung macrophages include two primary subpopulations: alveolar macrophages (AM) and interstitial macrophages. AMs are further subdivided into resident and recruited AMs. Resident AMs typically exhibit an M2 phenotype, essential for maintaining pulmonary homeostasis, whereas recruited AMs commonly display a pro-inflammatory M1 phenotype in response to stimuli, exacerbating inflammation [31–34] . During the exudative phase of ALI/ARDS, resident AMs rapidly transition to the M1 phenotype upon infection, secreting numerous inflammatory cytokines such as IL-1β, IL-6, MCP-1, MIP-2, TNF-α, and ROS. This secretion triggers substantial neutrophil recruitment, leading to further lung tissue injury. Based on our findings, we speculate that AD-ADSC-derived COL-3 activates the PI3K-AKT signaling pathway in both resident and recruited AMs, facilitating their polarization from M1 to M2 phenotype. These polarized M2 macrophages subsequently express anti-inflammatory IL-10, limiting inflammation and enhancing the expression of tissue repair-associated proteins such as fibronectin-1, TGF-β-induced matrix-associated protein BIG-H3, and insulin-like growth factor 1, ultimately mitigating alveolar epithelial damage and restoring lung barrier function [ 35 – 37 ] . Distinct from traditional cytokine-mediated paracrine mechanisms of stem cells, the ECM component COL-3 offers enhanced stability, longevity, and reduced immunogenic and tumorigenic risks. Compared with other ECM components like COL-1, fibronectin, and laminin, COL-3 exhibits more specific and clearly defined roles in tissue remodeling and immune regulation, greatly enhancing its application potential. Moreover, recent advances have highlighted that mesenchymal stem cells (MSCs), including ADSCs, exert their immunomodulatory effects not only through cell–cell interactions, but also via paracrine mechanisms and ECM secretion, which can directly influence macrophage polarization [38–39] . For example, MSC-derived ECM and soluble factors have been shown to modulate macrophage phenotype and attenuate tissue inflammation in a variety of disease models, including sepsis-induced organ injury and osteoarthritis [38–39] . Clinically, the lyophilized nebulized formulation of COL-3 has significant advantages, including targeted delivery, extended shelf-life, and low production and storage costs, making it particularly suitable for high-risk sepsis-associated ARDS patients (such as those with APACHE II scores > 15), thus presenting a novel therapeutic strategy. However, our study has several limitations. First, the deeper molecular mechanisms by which COL-3 mediates macrophage polarization remain incompletely elucidated. Second, although the PI3K-AKT pathway activation is well-supported, further receptor-blocking or gene knockout studies are necessary to validate these findings comprehensively. Third, the degradation kinetics and stability of COL-3 in vivo remain unclear, potentially impacting clinical applicability. Future studies will focus on clarifying the precise binding mechanisms between COL-3 and integrins, identifying functional active peptide segments, and conducting extensive animal model validation to ensure safety and efficacy, laying a robust foundation for clinical translation. METHODS Adipose Tissue Collection and Processing Human subcutaneous adipose tissue was obtained from female patients aged 30–50 years who underwent elective liposuction at the Department of Plastic Surgery, the First Affiliated Hospital of the Air Force Medical University. All procedures were conducted in accordance with institutional ethical standards and the Declaration of Helsinki (1964) and its subsequent amendments or comparable ethical standards. The study protocol was approved by the Institutional Review Board of the hospital (approval no. KY20253363-1). Written informed consent was obtained from all donors prior to sample collection. The adipose tissue obtained by liposuction was immediately placed in a sterile transport container and delivered to the laboratory within 30 minutes. The tissue was washed three times with sterile PBS to remove residual blood, followed by centrifugation at 1000 rpm, with the upper layer of fat retained. Subsequently, a 0.1% Type I collagenase solution was added (tissue volume to enzyme solution volume ratio of 1:1), and the mixture was incubated at 37°C with constant agitation for 45 minutes. During the cell separation and purification stage, digestion was terminated by adding an equal volume of DMEM complete medium (containing 10% fetal bovine serum), and centrifugation at 1000 rpm was performed to discard the upper lipid layer and supernatant. The cells were then treated with red blood cell lysis buffer for 15 minutes, followed by centrifugation at 450g. The cell pellet was resuspended in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin), filtered through a 100 µm filter to remove tissue debris. Finally, the cells were seeded into 10 cm culture dishes and incubated at 37°C in a 5% CO₂ incubator. ADSCs Adipogenic Differentiation 1 mL of 0.1% gelatin was added to a six-well plate, mixed thoroughly, and allowed to stand for at least 30 minutes. After removing the excess liquid, cells were seeded into the plate. Cells to be induced were seeded at a density of 2×10⁴ cells/cm², with 2 mL of normal culture medium added per well, and cultured under standard conditions (37°C, 5% CO₂). Once the cells reached confluence, the medium was replaced with adipogenic induction solution A for 3 days, followed by solution B for 1 day. The two induction solutions were alternated, with daily observation of cell morphology. Oil Red O Staining After the induction period, the culture medium was discarded, and the cells were gently washed three times with PBS. Then, 4% paraformaldehyde was added for fixation at room temperature for 30 minutes, followed by PBS washing. The Oil Red O storage solution was mixed with distilled water (3:2), and the supernatant was collected after centrifugation to prepare the working staining solution. The working solution was added to each well, and staining was performed at room temperature for 30 minutes. After removing the staining solution, the cells were thoroughly washed with PBS. Finally, PBS was added, and the cells were observed under a microscope to assess lipid droplet staining. H&E Staining After fixation in 4% paraformaldehyde, tissue samples were processed for paraffin embedding and sectioned into 8 µm thick slices. The tissue sections were dewaxed and hydrated before staining with Hematoxylin and Eosin (H&E) to observe the tissue structure. Immunofluorescence Staining After dewaxing, the paraffin sections underwent antigen retrieval and were then blocked with 3% BSA at room temperature for 30 minutes. Following three washes with PBST, the primary antibody was applied according to the recommended concentration and incubated overnight at 4°C. After extensive washing with PBST, the corresponding fluorescence-labeled secondary antibody was added and incubated in the dark for 50 minutes. The sections were then washed with PBST, stained with DAPI for nuclear labeling, and observed under a fluorescence microscope after the application of anti-fluorescence quenching agent. Images were captured under the fluorescence microscope. Decellularization Procedure for ADSCs After ADSCs are cultured in dishes to the desired confluence, the culture medium is carefully aspirated, and the cells are gently rinsed three times with pre-chilled phosphate-buffered saline (PBS) to remove residual medium and non-adherent cells. Subsequently, a decellularization solution containing 0.5% Triton X-100 and 20 mM NH₄OH is added to completely cover the cell layer and incubated at room temperature for 3 minutes. Following decellularization, the dishes are rapidly washed three times with a large volume of PBS to thoroughly remove cellular debris and residual reagents. Next, a PBS solution containing 50 U/mL DNase I and 10 µg/mL RNase A is added, and the samples are incubated at 37°C for 30 minutes to digest any remaining nucleic acids. After treatment, the samples are washed several times with PBS until the wash solution is clear and free of bubbles. The resulting extracellular matrix can be stored in PBS at 4°C for short-term use or lyophilized for long-term preservation and subsequent experiments. Preparation of Lyophilized Extracellular Matrix (ECM) Powder ECM samples were prepared from both undifferentiated adipose-derived stem cells (ADSCs) and adipogenically induced ADSCs (AD-ADSCs) following decellularization. After thorough washing with phosphate-buffered saline (PBS), the ECM scaffolds were collected and subjected to lyophilization using a low-temperature sublimation method. Briefly, the decellularized ECM was first frozen at − 80°C overnight to ensure complete solidification. The frozen samples were then transferred to a vacuum freeze dryer, where primary drying was performed at a chamber pressure below 50 Pa and shelf temperature maintained between − 40°C and − 30°C. This process enabled sublimation of ice directly into vapor, minimizing thermal degradation of ECM components. Secondary drying was conducted gradually by increasing the shelf temperature to 0°C to remove residual bound water, yielding dry ECM powder. The lyophilized ECM was collected under sterile conditions, ground into fine powder using a sterile mortar and pestle, and stored at − 20°C until further use. Throughout the process, all procedures were performed under aseptic conditions to prevent contamination and preserve ECM bioactivity. Ethics approval and reporting All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Air Force Medical University (Xi’an, China) (approval no. IACUC-20241434). All animal procedures were carried out in accordance with relevant institutional guidelines and national regulations. This study is reported in accordance with the ARRIVE guidelines Sepsis Mouse Model (CLP) Male C57BL/6 mice (8–10 weeks old, 20–25 g) were purchased from the Animal Center of Air Force Medical University (Xi'an, China). Prior to modeling, mice were acclimated under standard laboratory conditions for 7 days to ensure consistency. The animals were randomly assigned to three groups: a sham-operated group (control), and two sepsis groups subjected to cecal ligation and puncture (CLP), including a mild CLP group (CLP-M) and a severe CLP group (CLP-S), based on the degree of ligation. The CLP procedure is a well-established murine model of sepsis. The surgical steps are as follows: Mice were anesthetized with isoflurane and secured in a supine position on the surgical platform. Abdominal hair was removed, and the skin was disinfected three times using povidone-iodine and ethanol. A 1 cm incision was made in the lower left abdomen to expose the peritoneal cavity. The cecum was carefully exteriorized. The cecum was ligated with suture material, then punctured twice using a 21G needle. A small amount of fecal material was gently extruded from the puncture site. The cecum was returned to the abdominal cavity, and the abdominal wall and skin were closed in two layers. Note: In the sham group, the cecum was exposed but neither ligated nor punctured; all other procedures were identical to the CLP group. Postoperative care included subcutaneous injection of 1 mL sterile saline for fluid resuscitation and provision of functional jelly for analgesia. The general behavior of mice was observed postoperatively, and a clinical disease score was recorded. Mice were euthanized at 6 h, 12 h, 24 h, and 48 h post-surgery for sample collection. Following anesthesia, blood was collected via retro-orbital puncture into EDTA-coated tubes and centrifuged to separate plasma. Liver, lung, kidney, and intestinal tissues were harvested for histological or immunofluorescent analysis. Euthanasia was performed by isoflurane overdose, with the isoflurane concentration adjusted to 5% or greater and exposure continued until at least 1 minute after breathing stopped. Death was confirmed by absence of respiration and heartbeat and loss of reflexes prior to tissue harvest. Aerosol Inhalation of Adipogenic ADSCs-derived ECM in C57BL/6J Mice Adipose-derived stem cells (ADSCs) were induced to undergo adipogenic differentiation for 14 days. After induction, the cells were decellularized, and the resulting extracellular matrix (ECM) was collected and lyophilized according to the methods described above. Before use, the lyophilized ECM powder was dissolved in sterile PBS and thoroughly mixed. Eight-week-old male C57BL/6J mice were randomly assigned to groups and placed in an exposure chamber. The mice received aerosolized ECM suspension via a nebulizer for 30 minutes daily for three consecutive days. Control mice received aerosolized PBS. Clinical Scoring of Disease Severity An experienced technician assessed the clinical severity of sepsis based on behavioral and physiological indicators following CLP surgery. The scoring system included five parameters: appearance (0–4 points), behavior at rest (0–3 points), response to stimulation (0–3 points), respiratory rate (0–3 points), and corneal secretions (0–5 points). The sum of individual scores was recorded as the final disease score at each observation time point. Statistical Analysis All experiments were performed at least in triplicate. Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, USA). Comparisons between two groups were performed using the unpaired Student’s t-test, and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Differences were considered statistically significant at P < 0.05. Declarations Acknowledgements Thank you to the staff of the Laboratory Animal Center of the Fourth Military Medical University for their dedicated care of the experimental animals. Author Contributions : Qianmei Wang: Conceptualization, Investigation, Data curation, Formal analysis, Animal experiments, Writing – original draft. Jiayao Zhang: Investigation, Methodology, Visualization, Writing – review & editing. Xianqi Wang: Investigation, Visualization. Zelong Yang: Investigation. Dan Wu: Investigation. Yanan Xu: Investigation. Peiwen Wang: Investigation. Heliang Fu: Investigation. Yuexiang Ma: Investigation. Qi Zhang: Investigation. Wen Yin: Conceptualization, Methodology, Supervision, Writing – review & editing. Junjie Li: Conceptualization, Supervision, Project administration, Funding acquisition, Writing – review & editing. Funding: This work was supported by the Natural Science Basic Research Program of Shaanxi Province (Grant Number: 2024JC-YBMS-735 and 2025JC-QYCX-083). Data Availability Statement: All data relevant to the study are included in the article. Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References Lelubre C, Vincent JL. Mechanisms and treatment of organ failure in sepsis. Nat Rev Nephrol. 2018;14(7):417-427. doi:10.1038/s41581-018-0005-7 Xie J, Wang H, Kang Y, et al. The Epidemiology of Sepsis in Chinese ICUs: A National Cross-Sectional Survey. Crit Care Med. 2020;48(3):e209-e218. doi:10.1097/CCM.0000000000004155 Wang Z, Wang Z. The role of macrophages polarization in sepsis-induced acute lung injury. Front Immunol. 2023;14:1209438. Published 2023 Aug 24. doi:10.3389/fimmu.2023.1209438 Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation [published correction appears in Nat Rev Immunol.2010 Jun;10(6):460]. Nat Rev Immunol. 2008;8(12):958-969. doi:10.1038/nri2448 Eming SA, Wynn TA, Martin P. Inflammation and metabolism in tissue repair and regeneration. Science. 2017;356(6342):1026-1030. doi:10.1126/science.aam7928 Hardbower DM, Asim M, Luis PB, et al. Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications. Proc Natl Acad Sci U S A. 2017;114(5):E751-E760. doi:10.1073/pnas.1614958114 Ren J, Lei G, Dong A, Cao S, Han X, Li H. Therapeutic potential of ADSC-derived exosomes in acute lung injury by regulating macrophage polarization through IRF7/NLRP3 signaling. Int Immunopharmacol. 2025;156:114658. doi:10.1016/j.intimp.2025.114658 Dong Z, Fu Y, Cai Z, Dai H, He Y. Recent advances in adipose-derived mesenchymal stem cell-derived exosomes for regulating macrophage polarization. Front Immunol. 2025;16:1525466. Published 2025 Feb 3. doi:10.3389/fimmu.2025.1525466 Liu C, Xiao K, Xie L. Advances in the Regulation of Macrophage Polarization by Mesenchymal Stem Cells and Implications for ALI/ARDS Treatment. Front Immunol. 2022;13:928134. Published 2022 Jul 8. doi:10.3389/fimmu.2022.928134 Chen Y, Yang L, Li X. Advances in Mesenchymal stem cells regulating macrophage polarization and treatment of sepsis-induced liver injury. Front Immunol. 2023;14:1238972. Published 2023 Oct 25. doi:10.3389/fimmu.2023.1238972 Cicuéndez M, Casarrubios L, Feito MJ, et al. Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence. Int J Mol Sci. 2021;22(8):3847. Published 2021 Apr 8. doi:10.3390/ijms22083847 Qian Y, Chen H, Pan T, et al. Autologous decellularized extracellular matrix promotes adipogenic differentiation of adipose derived stem cells in low serum culture system by regulating the ERK1/2-PPARγ pathway. Adipocyte. 2021;10(1):174-188. doi:10.1080/21623945.2021.1906509 Li J, Xu Y, Quan Y, et al. Type IV Collagen Promotes Adipogenic Differentiation of Adipose Stem Cells. Aesthetic Plast Surg. 2024;48(13):2536-2544. doi:10.1007/s00266-024-03890-w Shaik S, Martin EC, Hayes DJ, Gimble JM, Devireddy RV. Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq. Sci Rep. 2019;9(1):11800. Published 2019 Aug 13. doi:10.1038/s41598-019-48089-1 Morrell ED, Holton SE, Lawrance M, et al. The transcriptional and phenotypic characteristics that define alveolar macrophage subsets in acute hypoxemic respiratory failure. Nat Commun. 2023;14(1):7443. Published 2023 Nov 17. doi:10.1038/s41467-023-43223-0 Huang X, Xiu H, Zhang S, Zhang G. The Role of Macrophages in the Pathogenesis of ALI/ARDS. Mediators Inflamm. 2018;2018:1264913. Published 2018 May 13. doi:10.1155/2018/1264913 Cai Y, Shang L, Zhou F, et al. Macrophage pyroptosis and its crucial role in ALI/ARDS. Front Immunol. 2025;16:1530849. Published 2025 Feb 14. doi:10.3389/fimmu.2025.1530849 Li J, Ma W, Tang Z, et al. Macrophage‑driven pathogenesis in acute lung injury/acute respiratory disease syndrome: Harnessing natural products for therapeutic interventions (Review). Mol Med Rep. 2025;31(1):16. doi:10.3892/mmr.2024.13381 Guerau-de-Arellano M, Piedra-Quintero ZL, Tsichlis PN. Akt isoforms in the immune system. Front Immunol. 2022;13:990874. Published 2022 Aug 23. doi:10.3389/fimmu.2022.990874 Arranz A, Doxaki C, Vergadi E, et al. Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization. Proc Natl Acad Sci U S A. 2012;109(24):9517-9522. doi:10.1073/pnas.1119038109 Linton MF, Moslehi JJ, Babaev VR. Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis. Int J Mol Sci. 2019;20(11):2703. Published 2019 Jun 1. doi:10.3390/ijms20112703 Stewart DC, Brisson BK, Yen WK, et al. Type III Collagen Regulates Matrix Architecture and Mechanosensing during Wound Healing. J Invest Dermatol. 2025;145(4):919-938.e14. doi:10.1016/j.jid.2024.08.013 Browne S, Monaghan MG, Brauchle E, et al. Modulation of inflammation and angiogenesis and changes in ECM GAG-activity via dual delivery of nucleic acids. Biomaterials. 2015;69:133-147. doi:10.1016/j.biomaterials.2015.08.012 Singh D, Rai V, Agrawal DK. Regulation of Collagen I and Collagen III in Tissue Injury and Regeneration. Cardiol Cardiovasc Med. 2023;7(1):5-16. doi:10.26502/fccm.92920302 Kuivaniemi H, Tromp G. Type III collagen (COL3A1): Gene and protein structure, tissue distribution, and associated diseases. Gene. 2019;707:151-171. doi:10.1016/j.gene.2019.05.003 Parkin JD, San Antonio JD, Persikov AV, et al. The collαgen III fibril has a "flexi-rod" structure of flexible sequences interspersed with rigid bioactive domains including two with hemostatic roles. PLoS One. 2017;12(7):e0175582. Published 2017 Jul 13. doi:10.1371/journal.pone.0175582 Nakajima I, Yamaguchi T, Ozutsumi K, Aso H. Adipose tissue extracellular matrix: newly organized by adipocytes during differentiation. Differentiation. 1998;63(4):193-200. doi:10.1111/j.1432-0436.1998.00193.x Vergadi E, Ieronymaki E, Lyroni K, Vaporidi K, Tsatsanis C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J Immunol. 2017;198(3):1006-1014. doi:10.4049/jimmunol.1601515 Lu J, Xie L, Liu C, Zhang Q, Sun S. PTEN/PI3k/AKT Regulates Macrophage Polarization in Emphysematous mice. Scand J Immunol. 2017;85(6):395-405. doi:10.1111/sji.12545 Zhang LL, Zhang LF, Shi YB. Down-regulated paxillin suppresses cell proliferation and invasion by inhibiting M2 macrophage polarization in colon cancer. Biol Chem. 2018;399(11):1285-1295. doi:10.1515/hsz-2018-0002 Deng L, Ouyang B, Tang W, et al. Icariside II modulates pulmonary fibrosis via PI3K/Akt/β-catenin pathway inhibition of M2 macrophage program. Phytomedicine. 2024;130:155687. doi:10.1016/j.phymed.2024.155687 Shi T, Denney L, An H, Ho LP, Zheng Y. Alveolar and lung interstitial macrophages: Definitions, functions, and roles in lung fibrosis. J Leukoc Biol. 2021;110(1):107-114. doi:10.1002/JLB.3RU0720-418R Matsui T, Taniguchi S, Ishii M. Function of alveolar macrophages in lung cancer microenvironment. Inflamm Regen. 2024;44(1):23. Published 2024 May 8. doi:10.1186/s41232-024-00335-4 Ahmad S, Nasser W, Ahmad A. Epigenetic mechanisms of alveolar macrophage activation in chemical-induced acute lung injury. Front Immunol. 2024;15:1488913. Published 2024 Nov 8. doi:10.3389/fimmu.2024.1488913 Hou F, Wang H, Zheng K, et al. Distinct Transcriptional and Functional Differences of Lung Resident and Monocyte-Derived Alveolar Macrophages During the Recovery Period of Acute Lung Injury. Immune Netw. 2023;23(3):e24. Published 2023 Feb 23. doi:10.4110/in.2023.23.e24 Chen X, Tang J, Shuai W, Meng J, Feng J, Han Z. Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome. Inflamm Res. 2020;69(9):883-895. doi:10.1007/s00011-020-01378-2 Tu GW, Shi Y, Zheng YJ, et al. Glucocorticoid attenuates acute lung injury through induction of type 2 macrophage. J Transl Med. 2017;15(1):181. Published 2017 Aug 29. doi:10.1186/s12967-017-1284-7 Chen Y, Yang L, Li X. Advances in Mesenchymal stem cells regulating macrophage polarization and treatment of sepsis-induced liver injury. Front Immunol. 2023;14:1238972. Published 2023 Oct 25. doi:10.3389/fimmu.2023.1238972 Zhang X, Liu T, Ran C, et al. Immunoregulatory paracrine effect of mesenchymal stem cells and mechanism in the treatment of osteoarthritis. Front Cell Dev Biol. 2024;12:1411507. Published 2024 Jul 26. doi:10.3389/fcell.2024.1411507 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigure1.pdf Supplementary Figure 1. Uncropped Western blot images. (A) Uncropped CD206 Western blot membrane. The boxed region indicates the bands presented as CD206 in Figure 4E. (B) Uncropped AKT Western blot membrane. The boxed region indicates the bands presented as AKT in Figure 4E. (C) Uncropped phospho-AKT (p-AKT) Western blot membrane. The boxed region indicates the bands presented as p-AKT in Figure 4E. (D) Uncropped GAPDH Western blot membrane. The boxed region indicates the bands presented as GAPDH in Figure 4E. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 05 May, 2026 Reviews received at journal 02 May, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviews received at journal 04 Feb, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers invited by journal 29 Jan, 2026 Editor assigned by journal 29 Jan, 2026 Editor invited by journal 19 Jan, 2026 Submission checks completed at journal 14 Jan, 2026 First submitted to journal 14 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8375617","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":587626354,"identity":"fbb64f45-3e8d-4917-8d5d-2d006ffbfd9f","order_by":0,"name":"Qianmei Wang","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qianmei","middleName":"","lastName":"Wang","suffix":""},{"id":587626361,"identity":"2b150a74-0ae4-49cf-afb8-c452aab339ce","order_by":1,"name":"Jiayao Zhang","email":"","orcid":"","institution":"Department of Internal Medicine, The Fifth People’s Hospital of Jiangyou","correspondingAuthor":false,"prefix":"","firstName":"Jiayao","middleName":"","lastName":"Zhang","suffix":""},{"id":587626364,"identity":"1c267a3f-1843-45cb-a577-2efc903b9f1e","order_by":2,"name":"Zelong Yang","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zelong","middleName":"","lastName":"Yang","suffix":""},{"id":587626368,"identity":"25e5e76b-3c7b-4456-a754-025655867212","order_by":3,"name":"Xianqi Wang","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianqi","middleName":"","lastName":"Wang","suffix":""},{"id":587626369,"identity":"28a017b2-dcf8-4147-aaf1-b824291ae5d5","order_by":4,"name":"Dan Wu","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Wu","suffix":""},{"id":587626373,"identity":"3639aece-409b-4692-899a-042545b58411","order_by":5,"name":"Yanan Xu","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Xu","suffix":""},{"id":587626374,"identity":"5c29fc65-a3db-4ce2-a008-bc99224a00ec","order_by":6,"name":"Peiwen Wang","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peiwen","middleName":"","lastName":"Wang","suffix":""},{"id":587626375,"identity":"a3f32209-1d32-43d3-abfe-e84a3c00c825","order_by":7,"name":"Heliang Fu","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Heliang","middleName":"","lastName":"Fu","suffix":""},{"id":587626379,"identity":"ef629518-f010-4f93-9d3e-c563cf17f64e","order_by":8,"name":"Yuexiang Ma","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuexiang","middleName":"","lastName":"Ma","suffix":""},{"id":587626380,"identity":"3d9bbca6-4534-4a4e-9350-7dd9320aafbe","order_by":9,"name":"Qi Zhang","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Zhang","suffix":""},{"id":587626381,"identity":"68657d51-3c95-4923-b512-6c5f5ddd31c1","order_by":10,"name":"Wen Yin","email":"","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Yin","suffix":""},{"id":587626382,"identity":"e776420f-a115-4b33-b30a-bb8a45ef604b","order_by":11,"name":"Junjie Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDCCA2DSgpmxvbHxQUKFhJw8EVoYGxgYJJgZew4fNvhwxsLYsIFILUCUliY5s60iEWovbsB3vPn5g497JNiZG3IMpHnnSSQwNjA/fHQDjxbJM8cMG2c8Azqs4YyBMe82iTx2BjZj4xw8Wgxu5DA28xwAamnsMUgGailmbOBhkyZOSzOPwWHeORKJDQeI1tLGltg4s4EILSC/zJwB0tLDfJjhwzEJY8NmAn4BhtiDDx8O2CQbzn/Y/iOhpk5Onr354WN8WmAgGRGDzEQoBwE7wulkFIyCUTAKRiwAAL0WT3/9OzoOAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Emergency, Xijing Hospital, Fourth Military Medical University","correspondingAuthor":true,"prefix":"","firstName":"Junjie","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-12-16 11:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8375617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8375617/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103056329,"identity":"2eea4253-f959-469e-8305-9b405ee49183","added_by":"auto","created_at":"2026-02-20 09:06:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2535557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSeptic lung injury severity is associated with systemic inflammation and M2 macrophage polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Kaplan–Meier survival curve of mice within 48 hours after CLP surgery(n = 10)\u003c/p\u003e\n\u003cp\u003e(B) Lung wet-to-dry weight ratio (W/D) in mice stratified by lung injury severity. (C) Representative H\u0026amp;E-stained histological sections of lung, liver, and heart tissues at 24 hours post-CLP\u003c/p\u003e\n\u003cp\u003e(D) Lung histopathology of mice grouped into mild, moderate, and severe lung injury categories\u003c/p\u003e\n\u003cp\u003e(E) Immunofluorescence staining for CD206 (green, M2 macrophage marker) and DAPI (blue, nuclear counterstain) in lung tissues.Quantitative analysis of positive area fraction (%) for CD206 staining is shown on the right\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8375617/v1/19ba8e715a454fcb35c23f03.png"},{"id":102321778,"identity":"67bae3d3-d991-42af-b9fb-d6f2a9cece15","added_by":"auto","created_at":"2026-02-10 13:52:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1052735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipogenic differentiation of ADSCs enhances their ECM-mediated promotion of M2 macrophage polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative morphological images of ADSCs before and after adipogenic induction. Undifferentiated ADSCs exhibit spindle-shaped morphology, while adipogenically differentiated ADSCs (AD-ADSCs) show abundant lipid droplets, confirmed by Oil Red O staining\u003c/p\u003e\n\u003cp\u003e(B) HE staining of ECM scaffolds before and after decellularization\u003c/p\u003e\n\u003cp\u003e(C) Immunofluorescence staining of macrophages cultured on ECMs derived from ADSCs or AD-ADSCs. M2 macrophages were identified by CD206 (green), and nuclei were counterstained with DAPI (blue)\u003c/p\u003e\n\u003cp\u003e(D) Quantitative analysis of positive area fraction (%) for CD206 staining\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8375617/v1/efdb01171ef7fa3780307824.png"},{"id":102321772,"identity":"84b3893e-82ba-4208-a1b3-22daf2af2158","added_by":"auto","created_at":"2026-02-10 13:52:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2259198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLyophilized ECM from adipogenically induced ADSCs (AD-ADSCs) alleviates sepsis-induced lung injury and enhances M2 macrophage polarization in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of the preparation process for lyophilized ECM powder derived from AD-ADSCs\u003c/p\u003e\n\u003cp\u003e(B) Representative macroscopic image of the lyophilized ECM powder\u003c/p\u003e\n\u003cp\u003e(C) H\u0026amp;E staining of lung tissues from CLP-induced septic mice treated with PBS (control), lyophilized ECM from undifferentiated ADSCs (ADSCs ECM), or from adipogenically induced ADSCs (AD-ADSCs ECM)\u003c/p\u003e\n\u003cp\u003eCD206 immunofluorescence staining of lung sections from each treatment group.Quantitative analysis of positive area fraction (%) for CD206 staining is shown on the right\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8375617/v1/935f31b415c241605d7f3fa1.png"},{"id":102321774,"identity":"f1057cc7-7e62-41b3-9731-c146a76bd4bb","added_by":"auto","created_at":"2026-02-10 13:52:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1035233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCOL-3 is enriched in AD-ADSC-derived ECM and promotes M2 macrophage polarization via the PI3K-AKT signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e（A）Immunofluorescence staining of ECM proteins (COL-1, COL-3, fibronectin, and laminin) in ECM derived from undifferentiated ADSCs and adipogenically differentiated ADSCs (AD-ADSCs)\u003c/p\u003e\n\u003cp\u003e（B）Quantification of the positive area fraction for COL-1, COL-3, fibronectin, and laminin in ECM\u003c/p\u003e\n\u003cp\u003e（C）Immunofluorescence staining for CD206 in RAW264.7 macrophages cultured on substrates coated with individual ECM proteins (COL-1, COL-3, fibronectin, and laminin)\u003c/p\u003e\n\u003cp\u003e（D）Quantification of CD206-positive area in RAW264.7 macrophages\u003c/p\u003e\n\u003cp\u003e（E）Western blot analysis of PI3K, AKT, and phosphorylated AKT (p-AKT) expression in macrophages treated with ECM derived from ADSCs, AD-ADSCs, or purified COL-3 protein. (Original blots are presented in Supplementary Figure 1.)\u003c/p\u003e\n\u003cp\u003e（F）Densitometric quantification of relative protein expression levels from Western blot data\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8375617/v1/8f798633799989f963dddf17.png"},{"id":103056785,"identity":"8e2f4e30-58f7-4b83-9bee-be68700673cd","added_by":"auto","created_at":"2026-02-20 09:24:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8739484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8375617/v1/8d7b646d-97ca-4f9d-8265-3c04c683fe03.pdf"},{"id":102321776,"identity":"d2a232f3-c38c-4683-a848-49be0c1f57a2","added_by":"auto","created_at":"2026-02-10 13:52:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5676744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Uncropped Western blot images.\u003c/strong\u003e\u003cbr\u003e\n(A) Uncropped CD206 Western blot membrane. The boxed region indicates the bands presented as CD206 in Figure 4E.\u003cbr\u003e\n(B) Uncropped AKT Western blot membrane. The boxed region indicates the bands presented as AKT in Figure 4E.\u003cbr\u003e\n(C) Uncropped phospho-AKT (p-AKT) Western blot membrane. The boxed region indicates the bands presented as p-AKT in Figure 4E.\u003cbr\u003e\n(D) Uncropped GAPDH Western blot membrane. The boxed region indicates the bands presented as GAPDH in Figure 4E.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8375617/v1/bcd39bb32eb7a79e6224f4e9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adipogenic MSC-derived COL-3 Drives M2 Macrophage Polarization via PI3K-AKT to Alleviate Sepsis-induced Lung Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is a systemic inflammatory syndrome caused by a dysregulated host response to infection, which can lead to multiple organ dysfunction, with the lungs being among the most frequently affected organs \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. The mortality rate of sepsis-associated acute lung injury (ALI) can reach as high as 40%, significantly higher than that of ALI resulting from other causes \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Recent studies have shown that the polarization state of macrophages plays a critical regulatory role in the onset and progression of ALI \u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, the specific mechanisms underlying the dynamic imbalance between M1 and M2 macrophage subtypes remain unclear and warrant further investigation.\u003c/p\u003e \u003cp\u003eAdipose-derived stem cells (ADSCs) are a type of adult stem cell with self-renewal capacity and multipotent differentiation potential \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In recent years, ADSCs have been widely used in basic and translational research on various inflammatory diseases and sepsis due to their remarkable immunomodulatory and tissue repair properties \u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Previous studies have mainly focused on the effects of undifferentiated ADSCs in modulating inflammatory responses and promoting tissue repair, with evidence of protective effects observed in various animal models \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Notably, recent research has demonstrated that the functional state of stem cells can undergo dynamic changes during cell therapy in response to alterations in the microenvironment, with some cells further differentiating into terminal cell types \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. As the terminally differentiated product of ADSCs, adipocytes more closely resemble mature adipose tissue in function and, theoretically, may be better suited to recapitulate the immunological microenvironment of adipose tissue and regulate immune responses. However, to date, the roles and mechanisms of adipogenically differentiated ADSCs in sepsis-associated acute lung injury remain poorly characterized \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Therefore, systematically elucidating the function and molecular mechanisms of adipogenically differentiated ADSCs in sepsis-induced lung injury is of great significance for advancing the theoretical basis and expanding the clinical application of stem cell-based therapies for inflammatory lung injury.\u003c/p\u003e \u003cp\u003eAfter adipogenic induction, ADSCs secrete an extracellular matrix (ECM) with a composition and structure more similar to that of mature adipose tissue, with increased uniformity and controllability. Studies have shown that during adipogenic differentiation, the expression of key matrix proteins such as laminin and type III collagen (COL-3) is significantly upregulated. These changes not only reflect the dynamic regulatory capacity of ADSCs over ECM composition, but also suggest a potential role for the ECM in modulating the immune microenvironment. Given that macrophage polarization balance is a crucial component of the immune microenvironment in sepsis-induced ARDS, we hypothesize that adipogenically differentiated ADSCs, compared to undifferentiated ADSCs, are better able to recapitulate the unique immunomodulatory properties of adipose tissue, especially in regulating macrophage polarization.\u003c/p\u003e \u003cp\u003eIn this study, we explored the effects of ECM derived from adipogenically differentiated ADSCs on macrophage polarization. Through a series of phenotypic experiments, we observed a significant upregulation of type III collagen (COL-3) in the ECM, which was closely associated with the enrichment of M2 macrophages. This finding suggests that COL-3 may serve as a key active component in the regulation of macrophage polarization. Based on this, we further investigated the underlying mechanism and confirmed that COL-3 promotes M2 macrophage polarization via activation of the PI3K-AKT signaling pathway, thereby alleviating sepsis-induced lung injury. This study is expected to enrich the understanding of the immunomodulatory mechanisms of stem cell-derived ECM and provide new therapeutic targets for the treatment of inflammatory lung injury.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e1. Increased M2 Macrophage Population in the Late Phase of Sepsis-Induced Lung Injury in Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the relationship between macrophage polarization status and the severity of sepsis-induced acute lung injury (ALI), a mouse model of septic lung injury was established using the cecal ligation and puncture (CLP) procedure (see Methods for details). After surgery, mice exhibited clear systemic inflammatory response symptoms, including lethargy, ruffled fur, reduced food intake, and purulent secretions at the canthi.\u003c/p\u003e\n\u003cp\u003eSurvival analysis revealed that the survival rate of mice in the CLP group decreased to 33.3% (3/9) at 24 hours post-operation, further dropped to 11.1% (1/9) at 36 hours, and reached 0% (0/9) at 48 hours. The survival rate in the control group also declined slightly over time, remaining at 66.7% (6/9) at 48 hours (Fig 1A). These results indicate that the model successfully induced severe sepsis. Additionally, the lung wet-to-dry weight ratio (W/D ratio) was significantly elevated in the CLP group, especially in mice with more severe symptoms, indicating substantial pulmonary edema (Fig 1B) and further validating the successful establishment of the model.\u003c/p\u003e\n\u003cp\u003eAt 24 hours post-surgery, samples were collected from the lungs, liver, and heart for hematoxylin and eosin (HE) staining (Fig 1C). The results revealed varying degrees of inflammatory cell infiltration in all three organs. The lungs exhibited marked interstitial edema and alveolar obstruction by exudates. Hepatic tissue showed hepatocyte swelling and narrowing of hepatic sinusoidal spaces, indicative of inflammatory edema. Myocardial tissue also exhibited edema, further confirming multi-organ inflammatory injury induced by sepsis.\u003c/p\u003e\n\u003cp\u003eSubsequently, mice were categorized into mild, moderate, and severe lung injury groups based on the severity of their clinical symptoms prior to sample collection (Fig 1D). CD206 immunofluorescence staining was performed on lung tissues from each group to assess the distribution of M2 macrophages (Fig 1E). Quantitative analysis showed that the mild lung injury group had the highest proportion of CD206-positive areas (mean 2.86 \u0026plusmn; 0.93%), followed by the moderate group (mean 1.79 \u0026plusmn; 0.10%), and the severe group had the lowest proportion (mean 1.06 \u0026plusmn; 0.47%).\u003c/p\u003e\n\u003cp\u003eThese results suggest that increased M2 macrophage abundance may be closely associated with better prognosis in sepsis-induced ALI and may play a critical role in the regulation of inflammation and tissue repair.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Adipogenic Differentiation of ADSCs Enhances M2 Macrophage Polarization via COL-3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether adipogenic induction influences the regulatory capacity of adipose-derived stem cell (ADSC)-derived extracellular matrix (ECM) on macrophage polarization, ADSCs were first subjected to adipogenic differentiation and systematically characterized.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig 2A, undifferentiated ADSCs displayed a typical spindle-shaped morphology with tightly aligned cells. In contrast, adipogenically induced ADSCs (AD-ADSCs) exhibited substantial lipid droplet accumulation, as evidenced by prominent red lipid deposition upon Oil Red O staining, confirming successful adipogenic differentiation.\u003c/p\u003e\n\u003cp\u003eBoth cell groups were then subjected to decellularization, and hematoxylin-eosin (HE) staining was used to preliminarily assess ECM retention (Fig 2B). The control group exhibited numerous residual nuclei, whereas nuclei were largely absent following decellularization, and the matrix structure was well preserved, indicating an effective decellularization process.\u003c/p\u003e\n\u003cp\u003eTo further evaluate the effects of ECM from different sources on macrophage polarization, the decellularized matrices were co-cultured with macrophages, and the proportion of M2 macrophages was assessed by CD206 immunofluorescence staining (Fig 2C). The results showed that the average proportion of CD206-positive area in the AD-ADSCs ECM group was 2.67 \u0026plusmn; 0.48%, which was significantly higher than that in the ADSCs ECM group (1.33 \u0026plusmn; 0.23%), indicating that ECM derived from adipogenically induced ADSCs has a greater capacity to promote M2 polarization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Lyophilized ECM from Adipogenically Induced ADSCs Attenuates Sepsis-Induced Lung Injury and Promotes M2 Macrophage Polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that ECM derived from adipogenically induced ADSCs (AD-ADSCs ECM) significantly promoted M2 macrophage polarization in vitro, we further evaluated its therapeutic effects in vivo. Lyophilized ECM powders were prepared according to the schematic workflow (Fig 3A), and their gross morphology was documented (Fig 3B), demonstrating the successful acquisition of homogeneous ECM lyophilized powder.\u003c/p\u003e\n\u003cp\u003eNext, lyophilized ECM from undifferentiated ADSCs and from AD-ADSCs was respectively administered to mice with sepsis-induced acute lung injury established by cecal ligation and puncture (CLP) (Fig 3C). Mice were randomly divided into three groups: control (untreated), ADSCs ECM, and AD-ADSCs ECM. After two days of treatment, hematoxylin-eosin (HE) staining revealed that, compared to the control and ADSCs ECM groups, the AD-ADSCs ECM group exhibited reduced interstitial edema, more intact alveolar architecture, and markedly decreased inflammatory cell infiltration in lung tissue (Fig 3C).\u003c/p\u003e\n\u003cp\u003eTo further assess the immunomodulatory effects, CD206 immunofluorescence staining was performed to evaluate M2 macrophage infiltration in lung tissues from all three groups (Fig 3D). Quantitative analysis showed that the mean proportion of CD206-positive area in lung tissue was highest in the AD-ADSCs ECM group at 2 days (6.28 \u0026plusmn; 0.76%), significantly greater than that in the 1-day treatment group (4.23 \u0026plusmn; 0.45%) and the untreated group (0 days) (1.84 \u0026plusmn; 0.94%). These results indicate that ECM derived from adipogenically induced ADSCs can also enhance M2 polarization in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Identify the key components in the ECM of adipogenically induced ADSCs\u003c/strong\u003e (AD-ADSCs) that may mediate macrophage polarization, we first performed immunofluorescence staining for major collagens and adhesion molecules, assessing the expression of COL-1, COL-3, fibronectin, and laminin in ECM derived from undifferentiated ADSCs and AD-ADSCs (Fig 4A). Quantitative analysis revealed that the proportion of COL-3-positive area was significantly higher in the AD-ADSCs group (8.63 \u0026plusmn; 1.18%) compared to the undifferentiated group (3.45 \u0026plusmn; 1.95%, P \u0026lt; 0.05). There were no significant differences in the expression of COL-1, laminin, or fibronectin between the two groups (Fig 4B), suggesting that COL-3 may be the critical component of AD-ADSCs ECM responsible for regulating macrophage polarization.\u003c/p\u003e\n\u003cp\u003eTo verify the effect of COL-3 on inducing macrophage polarization toward the M2 phenotype, the four ECM components (COL-1, COL-3, fibronectin, and laminin) were individually coated onto culture plates, and RAW264.7 macrophages were co-cultured in vitro for 48 hours, followed by CD206 immunofluorescence staining (Fig 4C). Analysis of the CD206-positive area showed that the COL-3 group had the highest positive proportion (8.14 \u0026plusmn; 1.09%), which was significantly greater than those of the COL-1 group (4.58 \u0026plusmn; 1.19%), laminin group (6.09 \u0026plusmn; 0.31%), and fibronectin group (2.67 \u0026plusmn; 0.74%) (Fig 4D).\u003c/p\u003e\n\u003cp\u003eTo further explore the molecular mechanism of COL-3, macrophages treated with ADSCs ECM, AD-ADSCs ECM, or COL-3 were collected for assessment of CD206, AKT, and phosphorylated AKT (p-AKT) protein expression (Fig 4E). Western blot analysis demonstrated that both AKT and p-AKT expression were higher in macrophages treated with AD-ADSCs ECM and COL-3 compared to those treated with ADSCs ECM and the control group. Quantitative analysis showed that the AD-ADSCs ECM group had an AKT expression level of 1.43 \u0026plusmn; 0.02 and p-AKT of 1.86 \u0026plusmn; 0.05, both significantly higher than those in the ADSCs ECM group (AKT: 0.84 \u0026plusmn; 0.06; p-AKT: 0.36 \u0026plusmn; 0.02) and the control group (AKT: 0.80 \u0026plusmn; 0.01; p-AKT: 0.70 \u0026plusmn; 0.01) (Fig 4F).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that the COL-3 content is markedly increased in the ECM of adipogenically induced ADSCs, and that COL-3 can enhance macrophage polarization toward the M2 phenotype by activating the PI3K-AKT signaling pathway, suggesting that COL-3 may be a key functional component in ECM-mediated immunomodulation.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn sepsis-associated acute respiratory distress syndrome (ARDS), aberrant macrophage polarization plays a pivotal role in disease progression \u003csup\u003e[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Our study demonstrated that type III collagen (COL-3), a key extracellular matrix (ECM) component secreted by adipogenically differentiated adipose-derived stem cells (AD-ADSCs), is critical for promoting macrophage polarization toward the M2 phenotype, primarily via activation of the PI3K-AKT signaling pathway \u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs one of the main components of ECM, collagen provides mechanical support for tissues and plays significant roles in regulating inflammation, angiogenesis, and tissue repair during injury \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. COL-3, an important member of the collagen superfamily, is composed of three identical left-handed α1 chains wound into a stable right-handed triple helix structure \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. This structure contains multiple integrin-binding sites that specifically interact with integrin receptors (such as α2β1 and α1β1) on macrophage surfaces, facilitating cell adhesion and migration, thus modulating the inflammatory microenvironment. The interaction between cells and ECM is crucial for macrophage polarization. During the early inflammatory phase, increased COL-3 expression forms a provisional matrix that directs inflammatory cells and fibroblasts to the injury site \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Subsequently, the N-terminal propeptide (CR) region of COL-3 binds to receptors on macrophage surfaces, activating the PI3K-AKT signaling pathway, thereby facilitating the shift from a pro-inflammatory M1 phenotype toward an anti-inflammatory and reparative M2 phenotype, as confirmed in our experiments .\u003c/p\u003e \u003cp\u003eThe Akt family comprises three serine/threonine protein kinases (Akt1, Akt2, Akt3), serving as critical effectors of the PI3K pathway. Upon PI3K-mediated conversion of PIP2 to PIP3, downstream proteins PDK1 and Akt are activated and bind to PIP3, leading to Akt phosphorylation. Extensive research has confirmed the essential role of the PI3K-AKT pathway in macrophage activation and gene expression regulation, with inhibition of this pathway significantly suppressing M2 macrophage polarization \u003csup\u003e[28\u0026ndash;30]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLung macrophages include two primary subpopulations: alveolar macrophages (AM) and interstitial macrophages. AMs are further subdivided into resident and recruited AMs. Resident AMs typically exhibit an M2 phenotype, essential for maintaining pulmonary homeostasis, whereas recruited AMs commonly display a pro-inflammatory M1 phenotype in response to stimuli, exacerbating inflammation\u003csup\u003e[31\u0026ndash;34]\u003c/sup\u003e. During the exudative phase of ALI/ARDS, resident AMs rapidly transition to the M1 phenotype upon infection, secreting numerous inflammatory cytokines such as IL-1β, IL-6, MCP-1, MIP-2, TNF-α, and ROS. This secretion triggers substantial neutrophil recruitment, leading to further lung tissue injury. Based on our findings, we speculate that AD-ADSC-derived COL-3 activates the PI3K-AKT signaling pathway in both resident and recruited AMs, facilitating their polarization from M1 to M2 phenotype. These polarized M2 macrophages subsequently express anti-inflammatory IL-10, limiting inflammation and enhancing the expression of tissue repair-associated proteins such as fibronectin-1, TGF-β-induced matrix-associated protein BIG-H3, and insulin-like growth factor 1, ultimately mitigating alveolar epithelial damage and restoring lung barrier function \u003csup\u003e[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDistinct from traditional cytokine-mediated paracrine mechanisms of stem cells, the ECM component COL-3 offers enhanced stability, longevity, and reduced immunogenic and tumorigenic risks. Compared with other ECM components like COL-1, fibronectin, and laminin, COL-3 exhibits more specific and clearly defined roles in tissue remodeling and immune regulation, greatly enhancing its application potential. Moreover, recent advances have highlighted that mesenchymal stem cells (MSCs), including ADSCs, exert their immunomodulatory effects not only through cell\u0026ndash;cell interactions, but also via paracrine mechanisms and ECM secretion, which can directly influence macrophage polarization\u003csup\u003e[38\u0026ndash;39]\u003c/sup\u003e. For example, MSC-derived ECM and soluble factors have been shown to modulate macrophage phenotype and attenuate tissue inflammation in a variety of disease models, including sepsis-induced organ injury and osteoarthritis\u003csup\u003e[38\u0026ndash;39]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eClinically, the lyophilized nebulized formulation of COL-3 has significant advantages, including targeted delivery, extended shelf-life, and low production and storage costs, making it particularly suitable for high-risk sepsis-associated ARDS patients (such as those with APACHE II scores\u0026thinsp;\u0026gt;\u0026thinsp;15), thus presenting a novel therapeutic strategy.\u003c/p\u003e \u003cp\u003eHowever, our study has several limitations. First, the deeper molecular mechanisms by which COL-3 mediates macrophage polarization remain incompletely elucidated. Second, although the PI3K-AKT pathway activation is well-supported, further receptor-blocking or gene knockout studies are necessary to validate these findings comprehensively. Third, the degradation kinetics and stability of COL-3 in vivo remain unclear, potentially impacting clinical applicability. Future studies will focus on clarifying the precise binding mechanisms between COL-3 and integrins, identifying functional active peptide segments, and conducting extensive animal model validation to ensure safety and efficacy, laying a robust foundation for clinical translation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAdipose Tissue Collection and Processing\u003c/h2\u003e \u003cp\u003eHuman subcutaneous adipose tissue was obtained from female patients aged 30\u0026ndash;50 years who underwent elective liposuction at the Department of Plastic Surgery, the First Affiliated Hospital of the Air Force Medical University. All procedures were conducted in accordance with institutional ethical standards and the Declaration of Helsinki (1964) and its subsequent amendments or comparable ethical standards. The study protocol was approved by the Institutional Review Board of the hospital (approval no. KY20253363-1). Written informed consent was obtained from all donors prior to sample collection.\u003c/p\u003e \u003cp\u003eThe adipose tissue obtained by liposuction was immediately placed in a sterile transport container and delivered to the laboratory within 30 minutes. The tissue was washed three times with sterile PBS to remove residual blood, followed by centrifugation at 1000 rpm, with the upper layer of fat retained. Subsequently, a 0.1% Type I collagenase solution was added (tissue volume to enzyme solution volume ratio of 1:1), and the mixture was incubated at 37\u0026deg;C with constant agitation for 45 minutes. During the cell separation and purification stage, digestion was terminated by adding an equal volume of DMEM complete medium (containing 10% fetal bovine serum), and centrifugation at 1000 rpm was performed to discard the upper lipid layer and supernatant. The cells were then treated with red blood cell lysis buffer for 15 minutes, followed by centrifugation at 450g. The cell pellet was resuspended in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin), filtered through a 100 \u0026micro;m filter to remove tissue debris. Finally, the cells were seeded into 10 cm culture dishes and incubated at 37\u0026deg;C in a 5% CO₂ incubator.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eADSCs Adipogenic Differentiation\u003c/h3\u003e\n\u003cp\u003e1 mL of 0.1% gelatin was added to a six-well plate, mixed thoroughly, and allowed to stand for at least 30 minutes. After removing the excess liquid, cells were seeded into the plate. Cells to be induced were seeded at a density of 2\u0026times;10⁴ cells/cm\u0026sup2;, with 2 mL of normal culture medium added per well, and cultured under standard conditions (37\u0026deg;C, 5% CO₂). Once the cells reached confluence, the medium was replaced with adipogenic induction solution A for 3 days, followed by solution B for 1 day. The two induction solutions were alternated, with daily observation of cell morphology.\u003c/p\u003e\n\u003ch3\u003eOil Red O Staining\u003c/h3\u003e\n\u003cp\u003eAfter the induction period, the culture medium was discarded, and the cells were gently washed three times with PBS. Then, 4% paraformaldehyde was added for fixation at room temperature for 30 minutes, followed by PBS washing. The Oil Red O storage solution was mixed with distilled water (3:2), and the supernatant was collected after centrifugation to prepare the working staining solution. The working solution was added to each well, and staining was performed at room temperature for 30 minutes. After removing the staining solution, the cells were thoroughly washed with PBS. Finally, PBS was added, and the cells were observed under a microscope to assess lipid droplet staining.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E Staining\u003c/h2\u003e \u003cp\u003eAfter fixation in 4% paraformaldehyde, tissue samples were processed for paraffin embedding and sectioned into 8 \u0026micro;m thick slices. The tissue sections were dewaxed and hydrated before staining with Hematoxylin and Eosin (H\u0026amp;E) to observe the tissue structure.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eAfter dewaxing, the paraffin sections underwent antigen retrieval and were then blocked with 3% BSA at room temperature for 30 minutes. Following three washes with PBST, the primary antibody was applied according to the recommended concentration and incubated overnight at 4\u0026deg;C. After extensive washing with PBST, the corresponding fluorescence-labeled secondary antibody was added and incubated in the dark for 50 minutes. The sections were then washed with PBST, stained with DAPI for nuclear labeling, and observed under a fluorescence microscope after the application of anti-fluorescence quenching agent. Images were captured under the fluorescence microscope.\u003c/p\u003e\n\u003ch3\u003eDecellularization Procedure for ADSCs\u003c/h3\u003e\n\u003cp\u003eAfter ADSCs are cultured in dishes to the desired confluence, the culture medium is carefully aspirated, and the cells are gently rinsed three times with pre-chilled phosphate-buffered saline (PBS) to remove residual medium and non-adherent cells. Subsequently, a decellularization solution containing 0.5% Triton X-100 and 20 mM NH₄OH is added to completely cover the cell layer and incubated at room temperature for 3 minutes. Following decellularization, the dishes are rapidly washed three times with a large volume of PBS to thoroughly remove cellular debris and residual reagents. Next, a PBS solution containing 50 U/mL DNase I and 10 \u0026micro;g/mL RNase A is added, and the samples are incubated at 37\u0026deg;C for 30 minutes to digest any remaining nucleic acids. After treatment, the samples are washed several times with PBS until the wash solution is clear and free of bubbles. The resulting extracellular matrix can be stored in PBS at 4\u0026deg;C for short-term use or lyophilized for long-term preservation and subsequent experiments.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Lyophilized Extracellular Matrix (ECM) Powder\u003c/h2\u003e \u003cp\u003eECM samples were prepared from both undifferentiated adipose-derived stem cells (ADSCs) and adipogenically induced ADSCs (AD-ADSCs) following decellularization. After thorough washing with phosphate-buffered saline (PBS), the ECM scaffolds were collected and subjected to lyophilization using a low-temperature sublimation method.\u003c/p\u003e \u003cp\u003eBriefly, the decellularized ECM was first frozen at \u0026minus;\u0026thinsp;80\u0026deg;C overnight to ensure complete solidification. The frozen samples were then transferred to a vacuum freeze dryer, where primary drying was performed at a chamber pressure below 50 Pa and shelf temperature maintained between \u0026minus;\u0026thinsp;40\u0026deg;C and \u0026minus;\u0026thinsp;30\u0026deg;C. This process enabled sublimation of ice directly into vapor, minimizing thermal degradation of ECM components. Secondary drying was conducted gradually by increasing the shelf temperature to 0\u0026deg;C to remove residual bound water, yielding dry ECM powder.\u003c/p\u003e \u003cp\u003eThe lyophilized ECM was collected under sterile conditions, ground into fine powder using a sterile mortar and pestle, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further use. Throughout the process, all procedures were performed under aseptic conditions to prevent contamination and preserve ECM bioactivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval and reporting\u003c/h2\u003e \u003cp\u003e All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Air Force Medical University (Xi\u0026rsquo;an, China) (approval no. IACUC-20241434). All animal procedures were carried out in accordance with relevant institutional guidelines and national regulations. This study is reported in accordance with the ARRIVE guidelines\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSepsis Mouse Model (CLP)\u003c/h2\u003e \u003cp\u003eMale C57BL/6 mice (8\u0026ndash;10 weeks old, 20\u0026ndash;25 g) were purchased from the Animal Center of Air Force Medical University (Xi'an, China). Prior to modeling, mice were acclimated under standard laboratory conditions for 7 days to ensure consistency. The animals were randomly assigned to three groups: a sham-operated group (control), and two sepsis groups subjected to cecal ligation and puncture (CLP), including a mild CLP group (CLP-M) and a severe CLP group (CLP-S), based on the degree of ligation. The CLP procedure is a well-established murine model of sepsis. The surgical steps are as follows:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMice were anesthetized with isoflurane and secured in a supine position on the surgical platform.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAbdominal hair was removed, and the skin was disinfected three times using povidone-iodine and ethanol.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eA 1 cm incision was made in the lower left abdomen to expose the peritoneal cavity. The cecum was carefully exteriorized.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe cecum was ligated with suture material, then punctured twice using a 21G needle. A small amount of fecal material was gently extruded from the puncture site.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe cecum was returned to the abdominal cavity, and the abdominal wall and skin were closed in two layers. \u003cem\u003eNote: In the sham group, the cecum was exposed but neither ligated nor punctured; all other procedures were identical to the CLP group.\u003c/em\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePostoperative care included subcutaneous injection of 1 mL sterile saline for fluid resuscitation and provision of functional jelly for analgesia.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe general behavior of mice was observed postoperatively, and a clinical disease score was recorded.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMice were euthanized at 6 h, 12 h, 24 h, and 48 h post-surgery for sample collection. Following anesthesia, blood was collected via retro-orbital puncture into EDTA-coated tubes and centrifuged to separate plasma. Liver, lung, kidney, and intestinal tissues were harvested for histological or immunofluorescent analysis.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eEuthanasia was performed by isoflurane overdose, with the isoflurane concentration adjusted to 5% or greater and exposure continued until at least 1 minute after breathing stopped. Death was confirmed by absence of respiration and heartbeat and loss of reflexes prior to tissue harvest.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAerosol Inhalation of Adipogenic ADSCs-derived ECM in C57BL/6J Mice\u003c/h2\u003e \u003cp\u003eAdipose-derived stem cells (ADSCs) were induced to undergo adipogenic differentiation for 14 days. After induction, the cells were decellularized, and the resulting extracellular matrix (ECM) was collected and lyophilized according to the methods described above. Before use, the lyophilized ECM powder was dissolved in sterile PBS and thoroughly mixed. Eight-week-old male C57BL/6J mice were randomly assigned to groups and placed in an exposure chamber. The mice received aerosolized ECM suspension via a nebulizer for 30 minutes daily for three consecutive days. Control mice received aerosolized PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eClinical Scoring of Disease Severity\u003c/h2\u003e \u003cp\u003eAn experienced technician assessed the clinical severity of sepsis based on behavioral and physiological indicators following CLP surgery. The scoring system included five parameters: appearance (0\u0026ndash;4 points), behavior at rest (0\u0026ndash;3 points), response to stimulation (0\u0026ndash;3 points), respiratory rate (0\u0026ndash;3 points), and corneal secretions (0\u0026ndash;5 points). The sum of individual scores was recorded as the final disease score at each observation time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least in triplicate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 8.0 (GraphPad Software, USA). Comparisons between two groups were performed using the unpaired Student\u0026rsquo;s t-test, and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThank you to the staff of the Laboratory Animal Center of the Fourth Military Medical University for their dedicated care of the experimental animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions :\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQianmei Wang: Conceptualization, Investigation, Data curation, Formal analysis, Animal experiments, Writing \u0026ndash; original draft.\u003c/p\u003e\n\u003cp\u003eJiayao Zhang: Investigation, Methodology, Visualization, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eXianqi Wang: Investigation, Visualization.\u003c/p\u003e\n\u003cp\u003eZelong Yang: Investigation.\u003c/p\u003e\n\u003cp\u003eDan Wu: Investigation.\u003c/p\u003e\n\u003cp\u003eYanan Xu: Investigation.\u003c/p\u003e\n\u003cp\u003ePeiwen Wang: Investigation.\u003c/p\u003e\n\u003cp\u003eHeliang Fu: Investigation.\u003c/p\u003e\n\u003cp\u003eYuexiang Ma: Investigation.\u003c/p\u003e\n\u003cp\u003eQi Zhang: Investigation.\u003c/p\u003e\n\u003cp\u003eWen Yin: Conceptualization, Methodology, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eJunjie Li: Conceptualization, Supervision, Project administration, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Basic Research Program of Shaanxi Province (Grant Number: 2024JC-YBMS-735 and 2025JC-QYCX-083).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data relevant to the study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLelubre C, Vincent JL. Mechanisms and treatment of organ failure in sepsis. Nat Rev Nephrol. 2018;14(7):417-427. doi:10.1038/s41581-018-0005-7\u003c/li\u003e\n\u003cli\u003eXie J, Wang H, Kang Y, et al. The Epidemiology of Sepsis in Chinese ICUs: A National Cross-Sectional Survey. Crit Care Med. 2020;48(3):e209-e218. doi:10.1097/CCM.0000000000004155\u003c/li\u003e\n\u003cli\u003eWang Z, Wang Z. The role of macrophages polarization in sepsis-induced acute lung injury. Front Immunol. 2023;14:1209438. Published 2023 Aug 24. doi:10.3389/fimmu.2023.1209438\u003c/li\u003e\n\u003cli\u003eMosser DM, Edwards JP. Exploring the full spectrum of macrophage activation [published correction appears in Nat Rev Immunol.2010 Jun;10(6):460]. Nat Rev Immunol. 2008;8(12):958-969. doi:10.1038/nri2448\u003c/li\u003e\n\u003cli\u003eEming SA, Wynn TA, Martin P. Inflammation and metabolism in tissue repair and regeneration. Science. 2017;356(6342):1026-1030. doi:10.1126/science.aam7928\u003c/li\u003e\n\u003cli\u003eHardbower DM, Asim M, Luis PB, et al. Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications. Proc Natl Acad Sci U S A. 2017;114(5):E751-E760. doi:10.1073/pnas.1614958114\u003c/li\u003e\n\u003cli\u003eRen J, Lei G, Dong A, Cao S, Han X, Li H. Therapeutic potential of ADSC-derived exosomes in acute lung injury by regulating macrophage polarization through IRF7/NLRP3 signaling. Int Immunopharmacol. 2025;156:114658. doi:10.1016/j.intimp.2025.114658\u003c/li\u003e\n\u003cli\u003eDong Z, Fu Y, Cai Z, Dai H, He Y. Recent advances in adipose-derived mesenchymal stem cell-derived exosomes for regulating macrophage polarization. Front Immunol. 2025;16:1525466. Published 2025 Feb 3. doi:10.3389/fimmu.2025.1525466\u003c/li\u003e\n\u003cli\u003eLiu C, Xiao K, Xie L. Advances in the Regulation of Macrophage Polarization by Mesenchymal Stem Cells and Implications for ALI/ARDS Treatment. Front Immunol. 2022;13:928134. Published 2022 Jul 8. doi:10.3389/fimmu.2022.928134\u003c/li\u003e\n\u003cli\u003eChen Y, Yang L, Li X. Advances in Mesenchymal stem cells regulating macrophage polarization and treatment of sepsis-induced liver injury. Front Immunol. 2023;14:1238972. Published 2023 Oct 25. doi:10.3389/fimmu.2023.1238972\u003c/li\u003e\n\u003cli\u003eCicu\u0026eacute;ndez M, Casarrubios L, Feito MJ, et al. Effects of Human and Porcine Adipose Extracellular Matrices Decellularized by Enzymatic or Chemical Methods on Macrophage Polarization and Immunocompetence. Int J Mol Sci. 2021;22(8):3847. Published 2021 Apr 8. doi:10.3390/ijms22083847\u003c/li\u003e\n\u003cli\u003eQian Y, Chen H, Pan T, et al. Autologous decellularized extracellular matrix promotes adipogenic differentiation of adipose derived stem cells in low serum culture system by regulating the ERK1/2-PPAR\u0026gamma; pathway. Adipocyte. 2021;10(1):174-188. doi:10.1080/21623945.2021.1906509\u003c/li\u003e\n\u003cli\u003eLi J, Xu Y, Quan Y, et al. Type IV Collagen Promotes Adipogenic Differentiation of Adipose Stem Cells. Aesthetic Plast Surg. 2024;48(13):2536-2544. doi:10.1007/s00266-024-03890-w\u003c/li\u003e\n\u003cli\u003eShaik S, Martin EC, Hayes DJ, Gimble JM, Devireddy RV. Transcriptomic Profiling of Adipose Derived Stem Cells Undergoing Osteogenesis by RNA-Seq. Sci Rep. 2019;9(1):11800. Published 2019 Aug 13. doi:10.1038/s41598-019-48089-1\u003c/li\u003e\n\u003cli\u003eMorrell ED, Holton SE, Lawrance M, et al. The transcriptional and phenotypic characteristics that define alveolar macrophage subsets in acute hypoxemic respiratory failure. Nat Commun. 2023;14(1):7443. Published 2023 Nov 17. doi:10.1038/s41467-023-43223-0\u003c/li\u003e\n\u003cli\u003eHuang X, Xiu H, Zhang S, Zhang G. The Role of Macrophages in the Pathogenesis of ALI/ARDS. Mediators Inflamm. 2018;2018:1264913. Published 2018 May 13. doi:10.1155/2018/1264913\u003c/li\u003e\n\u003cli\u003eCai Y, Shang L, Zhou F, et al. Macrophage pyroptosis and its crucial role in ALI/ARDS. Front Immunol. 2025;16:1530849. Published 2025 Feb 14. doi:10.3389/fimmu.2025.1530849\u003c/li\u003e\n\u003cli\u003eLi J, Ma W, Tang Z, et al. Macrophage‑driven pathogenesis in acute lung injury/acute respiratory disease syndrome: Harnessing natural products for therapeutic interventions (Review). Mol Med Rep. 2025;31(1):16. doi:10.3892/mmr.2024.13381\u003c/li\u003e\n\u003cli\u003eGuerau-de-Arellano M, Piedra-Quintero ZL, Tsichlis PN. Akt isoforms in the immune system. Front Immunol. 2022;13:990874. Published 2022 Aug 23. doi:10.3389/fimmu.2022.990874\u003c/li\u003e\n\u003cli\u003eArranz A, Doxaki C, Vergadi E, et al. Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization. Proc Natl Acad Sci U S A. 2012;109(24):9517-9522. doi:10.1073/pnas.1119038109\u003c/li\u003e\n\u003cli\u003eLinton MF, Moslehi JJ, Babaev VR. Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis. Int J Mol Sci. 2019;20(11):2703. Published 2019 Jun 1. doi:10.3390/ijms20112703\u003c/li\u003e\n\u003cli\u003eStewart DC, Brisson BK, Yen WK, et al. Type III Collagen Regulates Matrix Architecture and Mechanosensing during Wound Healing. J Invest Dermatol. 2025;145(4):919-938.e14. doi:10.1016/j.jid.2024.08.013\u003c/li\u003e\n\u003cli\u003eBrowne S, Monaghan MG, Brauchle E, et al. Modulation of inflammation and angiogenesis and changes in ECM GAG-activity via dual delivery of nucleic acids. Biomaterials. 2015;69:133-147. doi:10.1016/j.biomaterials.2015.08.012\u003c/li\u003e\n\u003cli\u003eSingh D, Rai V, Agrawal DK. Regulation of Collagen I and Collagen III in Tissue Injury and Regeneration. Cardiol Cardiovasc Med. 2023;7(1):5-16. doi:10.26502/fccm.92920302\u003c/li\u003e\n\u003cli\u003eKuivaniemi H, Tromp G. Type III collagen (COL3A1): Gene and protein structure, tissue distribution, and associated diseases. Gene. 2019;707:151-171. doi:10.1016/j.gene.2019.05.003\u003c/li\u003e\n\u003cli\u003eParkin JD, San Antonio JD, Persikov AV, et al. The coll\u0026alpha;gen III fibril has a \u0026quot;flexi-rod\u0026quot; structure of flexible sequences interspersed with rigid bioactive domains including two with hemostatic roles. PLoS One. 2017;12(7):e0175582. Published 2017 Jul 13. doi:10.1371/journal.pone.0175582\u003c/li\u003e\n\u003cli\u003eNakajima I, Yamaguchi T, Ozutsumi K, Aso H. Adipose tissue extracellular matrix: newly organized by adipocytes during differentiation. Differentiation. 1998;63(4):193-200. doi:10.1111/j.1432-0436.1998.00193.x\u003c/li\u003e\n\u003cli\u003eVergadi E, Ieronymaki E, Lyroni K, Vaporidi K, Tsatsanis C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J Immunol. 2017;198(3):1006-1014. doi:10.4049/jimmunol.1601515\u003c/li\u003e\n\u003cli\u003eLu J, Xie L, Liu C, Zhang Q, Sun S. PTEN/PI3k/AKT Regulates Macrophage Polarization in Emphysematous mice. Scand J Immunol. 2017;85(6):395-405. doi:10.1111/sji.12545\u003c/li\u003e\n\u003cli\u003eZhang LL, Zhang LF, Shi YB. Down-regulated paxillin suppresses cell proliferation and invasion by inhibiting M2 macrophage polarization in colon cancer. Biol Chem. 2018;399(11):1285-1295. doi:10.1515/hsz-2018-0002\u003c/li\u003e\n\u003cli\u003eDeng L, Ouyang B, Tang W, et al. Icariside II modulates pulmonary fibrosis via PI3K/Akt/\u0026beta;-catenin pathway inhibition of M2 macrophage program. Phytomedicine. 2024;130:155687. doi:10.1016/j.phymed.2024.155687\u003c/li\u003e\n\u003cli\u003eShi T, Denney L, An H, Ho LP, Zheng Y. Alveolar and lung interstitial macrophages: Definitions, functions, and roles in lung fibrosis. J Leukoc Biol. 2021;110(1):107-114. doi:10.1002/JLB.3RU0720-418R\u003c/li\u003e\n\u003cli\u003eMatsui T, Taniguchi S, Ishii M. Function of alveolar macrophages in lung cancer microenvironment. Inflamm Regen. 2024;44(1):23. Published 2024 May 8. doi:10.1186/s41232-024-00335-4\u003c/li\u003e\n\u003cli\u003eAhmad S, Nasser W, Ahmad A. Epigenetic mechanisms of alveolar macrophage activation in chemical-induced acute lung injury. Front Immunol. 2024;15:1488913. Published 2024 Nov 8. doi:10.3389/fimmu.2024.1488913\u003c/li\u003e\n\u003cli\u003eHou F, Wang H, Zheng K, et al. Distinct Transcriptional and Functional Differences of Lung Resident and Monocyte-Derived Alveolar Macrophages During the Recovery Period of Acute Lung Injury. Immune Netw. 2023;23(3):e24. Published 2023 Feb 23. doi:10.4110/in.2023.23.e24\u003c/li\u003e\n\u003cli\u003eChen X, Tang J, Shuai W, Meng J, Feng J, Han Z. Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome. Inflamm Res. 2020;69(9):883-895. doi:10.1007/s00011-020-01378-2\u003c/li\u003e\n\u003cli\u003eTu GW, Shi Y, Zheng YJ, et al. Glucocorticoid attenuates acute lung injury through induction of type 2 macrophage. J Transl Med. 2017;15(1):181. Published 2017 Aug 29. doi:10.1186/s12967-017-1284-7\u003c/li\u003e\n\u003cli\u003eChen Y, Yang L, Li X. Advances in Mesenchymal stem cells regulating macrophage polarization and treatment of sepsis-induced liver injury. Front Immunol. 2023;14:1238972. Published 2023 Oct 25. doi:10.3389/fimmu.2023.1238972\u003c/li\u003e\n\u003cli\u003eZhang X, Liu T, Ran C, et al. Immunoregulatory paracrine effect of mesenchymal stem cells and mechanism in the treatment of osteoarthritis. Front Cell Dev Biol. 2024;12:1411507. Published 2024 Jul 26. doi:10.3389/fcell.2024.1411507\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8375617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8375617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepsis-induced acute lung injury is a critical condition marked by uncontrolled inflammation and immune imbalance. Here, we investigated the role of extracellular matrix components derived from adipogenically differentiated adipose-derived stem cells in regulating macrophage polarization and lung injury. We found that type III collagen was significantly upregulated in the extracellular matrix after adipogenic induction and promoted macrophage polarization toward the anti-inflammatory M2 phenotype via the PI3K-AKT signaling pathway. In a mouse model of sepsis-induced lung injury, treatment with lyophilized extracellular matrix from adipogenic adipose-derived stem cells alleviated lung inflammation and injury, accompanied by increased infiltration of M2 macrophages. These results identify a novel mechanism by which stem cell-derived type III collagen modulates the immune microenvironment and suggest a new therapeutic approach for inflammatory lung diseases.\u003c/p\u003e","manuscriptTitle":"Adipogenic MSC-derived COL-3 Drives M2 Macrophage Polarization via PI3K-AKT to Alleviate Sepsis-induced Lung Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 13:51:49","doi":"10.21203/rs.3.rs-8375617/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-05T07:28:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:13:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282538168248086893811155637567930908267","date":"2026-04-22T13:31:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T08:07:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246181092026605082879268330832720713342","date":"2026-01-29T06:30:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-29T06:08:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-29T06:03:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-19T13:14:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-15T02:34:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-15T02:29:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c90b0f32-e05d-4bb3-b070-c76696eaa04e","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-05T07:28:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:13:34+00:00","index":115,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62521260,"name":"Biological sciences/Cell biology"},{"id":62521261,"name":"Health sciences/Diseases"},{"id":62521262,"name":"Biological sciences/Immunology"},{"id":62521263,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-05-05T07:40:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 13:51:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8375617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8375617","identity":"rs-8375617","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}