Club cell secretory protein 16 up-regulates cell proliferation, inhibits inflammation and pyroptosis against particular matter 2.5 -induced epithelium damage in asthmatic mice

Club cell secretory protein 16 up-regulates cell proliferation, inhibits inflammation and pyroptosis against particular matter 2.5 -induced epithelium damage in asthmatic mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Club cell secretory protein 16 up-regulates cell proliferation, inhibits inflammation and pyroptosis against particular matter 2.5 -induced epithelium damage in asthmatic mice Jinle Lin, Xiaowen Chen, Yuehua Chen, Xiaobing Zeng, Jie Yao, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4651501/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction : Club cell secretory protein (CC16) is reported to have multiple protective functions in airway diseases, including anti-inflammatory, immunomodulatory and antioxidant. This study aims to determine whether CC16 can repair lung injury caused by particular matter 2.5(PM 2.5 ) exposure in asthmatic mice. Methods In the ovalbumin (OVA)-induced asthma murine study, 6-week-old male C57BL/6J mice were primary exposed to PM 2.5 for 24 hours and following treated with CC16, Artery blood gas, lung function,histopathology and immunohistochemical staining were detected. The BEAS-2B cell line was primary exposed to PM 2.5 for 24 hours and then treated with CC16 subsequently, fluorescence and electron microscopy, protein and RNA of inflammation and pyroptosis, and RNA Sequencing were detected. Results In the OVA-induced asthmatic mice after exposure of PM 2.5 treatment with CC16 ameliorated PM 2.5 -induced lung tissue damage, respiratory acidosis and restore the increased airway resistance after PM 2.5 -exposed group, accompanied with the inhibition in the protein of inflammation and pyroptosis.Moreover, CC16 increased cell proliferation, ameliorated pyroptotic cell death induced by PM 2.5 and inhibited the expression on the protein and RNA of inflammation and pyroptosis. Transcriptome analysis revealed that CC16 down-regulate genes associated with inflammatory adhesion, while up-regulating proliferation genes,like E-Twenty-Six-1. Conclusions CC16 could repair airway epithelium PM 2.5 -induced damage in asthma mice by up-regulating cell proliferation,inhibiting pyroptosis and imflammation, which it will been used as a novel therapeutic agent to alleviate the health risks of PM 2.5 exposure in future. CC16 inflammation pyroptosis PM2.5 asthma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 INTRODUCTION Particulate matter 2.5(PM 2.5 ) refers to a mix of tiny solid and liquid particles of < 2.5 µm in diameter suspended in the air. 1 – 2 High concentrations of PM 2.5 increase the morbidity and severity of asthma. The disease is often accompanied by damage to the airway epithelium cells (AECs), the critical frontline of the respiratory tract for defence and homeostasis. It is hypothesized that epithelial dysfunction is the principal mechanism of asthma pathogenesis. 3 PM 2.5 aggravates asthmatic inflammation and results in signal-related pyroptosis. 4 – 5 Activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3) inflammasome leads to the release of cysteine-containing aspartate specific protease 1(Caspase 1)-dependent, proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 as well as gasdermin D-related swelling and membrane rupture of cells, followed by pyroptotic death. 4 Elevated concentrations of NLRP-3 and IL-1β have been observed in the sputum of non-smokers with asthma. 6 In addition, PM 2.5 orchestrates lung inflammation and enhances the expression of NLRP 3. The use of NLRP 3 inhibitor MCC950, caspase 1 inhibitor Ac-YVAD-CHO and IL-1β neutralizing antibodies had effectively decreased the asthmatic inflammation. 7 Club cell secretory protein (CC16) belongs to the secretoglobin superfamily and predominantly secreted by AECs, with anti-inflammatory and immunoregulatory functions. 8 – 9 Our previous clinical study found the abrupt elevation of serum CC16 in critically ill patients indicates a potential association with secondary acute respiratory distress syndrome (ARDS) and its subsequent return to normal levels signifies a favorable prognosis. 10 Further research has highlighted the CC16 inhibits toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) inflammatory pathways in vitro. 11 Another research on LPS and bacterial-induced lung injury in mice elucidated that extracellular vesicles-CC16 could be a potential therapeutic agent for acute lung injury(ALI) by inhibiting the inflammatory and DNA damage responses by reducing NF-κB signaling. 12 Moreover, epidemiological studies have shown decreased serum CC16 levels in patients with asthma. 13 The CC16 A38G polymorphism may contribute to the development of late-onset asthma. 14 However, how CC16 repairs the damaged epithelium in the airways of asthmatic has not been elucidated. CC16 had repair properties in a AECs model of short-term high concentration exposure to PM 2.5 . Consequently, further examination of the repair mechanisms and effects of CC16 in the model following short-term exposure to PM 2.5 is warranted. In this study, the repair mechanisms of CC16 in PM 2.5 -induced lung injury on asthma model mice and human bronchial epithelial (BEAS-2B) cell line were investigated. METHODS The following experimental methods are a brief description. For details, see the Appendix S1. The work has been reported in line with the ARRIVE guidelines 2.0. Collection of PM 2.5 and sample preparation PM 2.5 samples were obtained from the rooftops of the platform at the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences (100 m near the motorway) in August 2020, using high-volume air samplers (Thermo Fischer Scientific, Waltham, USA). Pieces of quartz fibre filter membrane (20×25-cm 2 ) loaded with PM 2.5 were washed, filtered, centrifuged and freeze-dried to produce PM 2.5 freeze-dried powder.In the experiment, the powder was dissolved in normal saline or PBS. Mice model study Male C57BL/6J mice were randomly divided into six groups (n = 20 per group) and suffered different treatments.The Research Animal Care Committee of Southern Medical University approved the study protocols. All experiments involving animals complied with the ARRIVE guidelines. 15 Mice in the six groups were treated with normal saline(Control group), OVA(Asthma group), OVA + PM 2.5 (24 h)(PM 2.5 (24 h) group), OVA + PM 2.5 (48h)(PM 2.5 (48 h) group), OVA + PM 2.5 +CC16(24 h)(PM 2.5 +CC16 (24 h) group), OVA + PM 2.5 +CC16 (48 h)(PM 2.5 +CC16 (48 h) group). Mice were sensitized with OVA to induce asthma model as following: on the 1st, 7th, 14th day of the experiment, mice were sensitized with OVA (1 mg OVA and 100 mg Al(OH)3; Sigma, St. Louis, USA) or normal saline via intraperitoneal injection, followed by inhalation exposure to 1% OVA or saline aerosol via compression atomizers (402AI, JS, CN). On days 21–27 to promote challenge, 30 min/day. During the challenge of asthma model, the mice were inhalation exposure to PM 2.5 ( 2 mg / kg ) for 2 hour on the 28th day. The PM 2.5 +CC16 (24 h) group were exposed to 2 mg/kg CC16 (USCN Sciences Co., Wuhan, CN) for 2 hour on the 29th day while the PM 2.5 +CC16 (48 h) group were treated with the same treatment on the 29th and 30th day. At the end of the last treatment,all the mice were tested lung function, anesthetized and sacrificed to collect arterial blood and lung tissues for further research(Fig. 1 A). Artery blood gas Four mice were anesthetized with pentobarbital (40 mg/kg) and the partial pressure of oxygen (PaO 2 ), carbon dioxide (PCO 2 ), hydrogen potential (pH), and anion gap (AG) in abdominal aortic blood were analyzed using a radiometer (ABL825, Radiometer). Lung function Five anesthetized mice were assessed for their airway responsiveness to different doses of methacholine by using the FinePointe RC (Buxco Research Systems, DSI, MN, USA). For each dose, lung resistance was measured and computed over 3 min. The respiratory data were acquired and presented with the Fine Pointe station. Histopathology and immunohistochemical staining For scoring analysis, the sections were stained with hematoxylin–eosin(HE), using the Smith Lung Injury Scoring System to assess injury severity. The sections were used the primary antibodies of TLR 4 (1:100, Abcam, UK), Anti-Phospho-NF-κB (1:150, Abcam), NLRP 3 (1:200, Abcam), Caspase 1 (1:500, Bioss, CN), IL-1β (1:100, Abcam), and Gasdermin D (1:150, Abcam) tand performed the immunohistochemical(IHC) staining. IHC images were randomly scanned by a light scanning microscope at ×200 magnification and Image J to quantify the high positive percentage contribution. BEAS-2B cell sourcing, exposure to PM 2.5 , and treatment of CC16 BEAS-2B cells were incubated with PM 2.5 (100 and 200 µg/mL) for 24 h. In the subgroup with PM 2.5 (100 µg/mL), BEAS-2B cells were exposed to 0.25, 0.5, and 1 µg/mL CC16 (R&D Systems, Catalog #: 4218-UT,Minneapolis, MN, USA) for 24 h after PM 2.5 exposure. The protocol for generating cell models is illustrated in Fig. 1 B. Cell Counting Kit-8 assay The CCK-8 solution (Abcam, USA) was added to the culture medium and incubated for 2 h after PM 2.5 or CC16 administration following the manufacturer’s instructions. The viability of living cells in each group was tested using a microplate reader at a 450-nm optical density. Light and fluorescence microscopy Cell viability was assessed by light-sheet microscopy. The types of cell death were assessed using the Annexin V-FITC/PI Fluorescence Microscopy Kit (BD Biosciences, San Diego, USA) and Hoechst 33342 (BD Biosciences) following the manufacturer’s instructions.The slice was observed under a confocal fluorescence microscope (Nikon, Tokyo, Japan) at 405, 488, and 640 nm excitation wavelength. Scanning electron microscopy The sample was tsprayed with an ion sputter, and observed and photographed with an electron microscope (Zeiss, MERLIN, Germany). Western blotting The proteins of lung tissue samples and cell lysates were run on SDS-PAGE. Western blot was performed using primary antibodies against TLR 4, NF-κB, polyclonalanti-NF-κB, Caspase 3, NLRP 3, Caspase 1, IL-1β, Gasdermin D, high mobility group box 1 (HMGB1), E-Twenty-Six-1(ETS1) and β-actin. We used Image J to quantify the WB strip gray value. Real-time PCR Real-time PCR was conducted using the QuantiFast SYBR Green PCR kit (Invitrogen, Carlsbad, USA) on the Roche Light Cycler 480II system. For details, see the Appendix S1.The primer list is as follows in Table 1 Database construction, analysis, and validation based on transcriptome data BEAS-2B cells treated with either CC16 or PM 2.5 were subjected to RNA extraction using TRIzol's protocol. cDNA was compounded following the Total RNA seq (H/M/R) Library Prep Kit instructions. The mRNA seq library underwent 100 bp double ended-sequencing using HiSeq2000. The Fatsqc tool is utilized for quality control of mRNA sequencing data. The gene expression level is then calculated using Cufflinks, with default parameters being employed. Finally, Gene Ontology analysis is conducted using DAVID. The Western Blot technique is utilized to verify the protein expression of genes that have undergone screening. Statistical analysis Data are presented as the mean ± standard deviation. Statistical analysis were performed with SPSS 14.0 statistical software (SPSS Inc., Chicago, IL, USA). The Student's t -test was performed to analyze the difference between the two groups. One-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test was performed for comparisons among multiple experimental groups. P < 0.05 was considered as significance. Ethics/Ethical Approval . The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The mouse procedures outlined in this study received approval from the Research Ethics Committee of Southern Medical University (Approval No. BYL20231202). All procedures were conducted following the Guide for the Care and Use of Laboratory Animals. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. RESULTS Lung tissue damage, respiratory acidosis, and increased airway resistance in asthmatic mice due to PM 2.5 − exposure Arterial blood gas analysis showed that exposure to PM 2.5 for 24 h induced a decline in PO 2 and exposure for 48 h caused a decrease in pH and elevation in PCO 2 compared with the control group mice (Fig. 2 A). The results of the pulmonary function evaluation indicated a significant increase in airway resistance variables, specific airwayresistance (sRaw) and peak inspiratory flow(PIF), among mice exposed to PM 2.5 (Fig. 2 B), suggesting an increase in airway resistance, airway hyperresponsiveness, and respiratory distress. Exposure to PM 2.5 can exacerbate airway inflammation in asthmatic mice(Fig. 2 C- 2 D). Immunohistochemical and western blot in PM 2.5 exposed asthmatic mice Immunohistochemical staining indicate that exposure to PM 2.5 has the potential to induce the expression of TLR4, NLRP3, Caspase-1, Gasdermin D, and IL-1β in mouse lung tissue (Fig. 3 A). Additionally, Western blot results suggest that inflammatory pathways, including TLR4, Anti-Phospho NF-κB, Caspase-3, and NF-κB, as well as pyrolytic channels such as NLRP3, Caspase-1, Gasdermin D, and IL-1β, exhibit increased expression levels following PM 2.5 exposure when compared to pre-exposure levels (Fig. 3 B). Besides, the above protein levels still increased after 48 hours of exposure to PM 2.5 . The administration of CC16 relived acute lung injury, respiratory acidosis, and airway distress in asthmatic mice due to PM 2.5 − exposure The arterial blood gas analysis indicates that the group of CC16 treatment exhibited a significant improvement in blood gas parameters compared to the two other asthma mice model groups exposed to PM 2.5 (Fig. 4 A), manifesting as an increase in pH value, an increase in PaO 2 and a decrease in PaCO 2 . However, the changes in AG were less pronounced. The results of the pulmonary function evaluation indicate that PM 2.5 -induced abnormal sRaw and PIF can be ameliorated by CC16, as depicted in the accompanying figure (Fig. 4 B). Specifically, mice treated with CC16 for 24 hours exhibited restoration of sRaw and PIF to normal levels, in contrast to those exposed to PM 2.5 . Furthermore, a 48-h treatment with CC16 resulted in a complete normalization of sRaw.The results of HE staining indicate that exposure to PM 2.5 induces lung injury characterized by the inflammatory response, bleeding, and necrosis, which can be ameliorated by CC16 treatment, leading to a reduction in the Smith score of lung injury(Fig. 4 C- 4 D). CC16 resulted in the suppression of increased levels of inflammation and pyroptosis proteins in asthmatic mice exposed to PM 2.5 Both IHC and Western blot analyses indicate that nebulization therapy with CC16 suppresses the expression of inflammatory and pyrolytic signaling proteins in lung tissue. In mice treated with CC16 nebulization, the levels of proteins TLR4, Caspase-1, and IL-1β were lower and the expression levels of NF-κ B. NLRP3, Caspase-1, Gasdermin D, and IL-1 β was inhibited (Fig. 5 A-B). PM 2.5 caused pyroptosis and increased activation of inflammatory in BEAS-2B cells To investigate effects of PM 2.5 , We used PM 2.5 to stimulate BEAS-2B cells and. CCK-8 assays and observed changes in cell morphology and cell viability. Annexin V-FITC/PI/Hochest33342 staining analysis and Confocal microscopy inspection suggested that BEAS-2B cells died (Fig. 6 A-B). Moreover, scanning electron microscope verified that PM 2.5 compromised the structural integrity of the cellular membrane, such as cell membrane perforation, membrane surface, and irregular shape (Fig. 6 C). Based on the results of previous animal experiments, we focus on some cell pyroptosis and inflammatory proteins. The protein blotting analysis indicates that exposure to PM 2.5 and LPS led to the observation of phosphorylated NF-κB in the inflammatory signal channel within the cell signal, as compared to the control group. Additionally, the expression of caspase-3 cleavers, and HMGB1 was found to be higher, while the expression of Caspase-1 shear, Gasdermin D shear, and IL-1βshear in the pyrolytic signaling pathway was significantly increased compared to the control group (Fig. 7 A-D). Elevated levels of RNA transcription were observed for Caspase-1, Gasdermin D, and IL-1 β in the PM 2.5 and LPS exposure groups when compared to the control group (Fig. 7 E). These result showed PM 2.5 caused pyroptosis and increased activation of inflammatory in BEAS-2B cell. The administration of CC16 ameliorated in PM 2.5 -induced pyroptosis and inflammation in BEAS-2B cells Changes in cell morphology and cell viability were observed after treating the cells with different concentrations of CC16 in PM 2.5 -contaminated BEAS-2B cells to investigate the function of CC16 in pyroptosis and inflammation.Based on optical microscopy and PI staining, the results showed that CC16 treatment prevented the damage to cell viability caused by PM 2.5 , the levels of cell viability were higher than those in the PM 2.5 group. Besides, it was observed that the proliferation of BEAS-2B cells exposed to PM 2.5 was dependent on both the dosage and duration of CC16 treatment(Fig. 8 A-B). Further studies found that treatment with CC16 effectively decreased pyroptosis induced by PM 2.5 . Through the utilization of Confocal microscopy in conjunction with fluorescence staining, CC16 facilitated the repair of the cell membrane. Ultimately, our findings were substantiated by scanning electron microscopy, that the surviving cells exhibited a smooth and unblemished cell membrane devoid of any surface protrusions indicative of pyroptosis, and their morphology remained intact without any discernible deformities (Fig. 8 C). Western blot analysis indicates that the expression of phosphorylated NF-κB, P38, Caspase-3, Caspase-1, Gasdmin D, HMGB1, and IL-1β was significantly reduced in the CC16 treatment group compared to the PM 2.5 exposure group (Fig. 9 A-D). The RT-PCR analysis indicates that the transcription levels of Caspase-1, Gasdmin D and IL-1β were lower in the CC16 treatment group compared to the PM 2.5 exposure group treated with high-pressure sterilization(Fig. 9 E).These results demonstrated that CC16 effectively effectively suppresses the upregulation of inflammation and pyroptosis signaling pathways induced by exposure to PM 2.5 . CC16 regulated genes expression profiles in PM 2.5 -induced alveolar epithelial cells We employed transcriptome analysis to investigate the regulatory influence of on gene in control, LPS, PM 2.5 , and PM 2.5+ CC16 groups. Principal component analysis (PCA) was used to assess the quantitative repeatability of differentially expressed genes and to determine the statistical consistency of the quantitative results of cell samples(Fig. 10 A). PM 2.5 induced 1100 up-regulated differentially expressed genes and 1180 downregulated genes. 37 differentially expressed genes are commonly up-regulated by CC16, while 20 are down-regulated (Fig. 10 B-C). After CC16 treatment, the expression of 10 genes that were originally upregulated in the PM 2.5 group decreased, while the expression of 21 genes that were originally downregulated in the PM 2.5 group increased(Table S2 ). Differentially expressed genes will be screened and subjected to Gene Ontology (GO) enrichment analysis using a database. The functional classification and pathways of significantly enriched differentially expressed proteins (P < 0.05) will be visually presented through bubble plots. The identified pathways primarily comprised were: collagen-containing extracellular matrix, response to oxygen levels, regulation of lipid metabolic process, response to hypoxia, response to decreased oxygen levels, response to the drug, lysosomal membrane, lytic vacuole membrane, vacuolar membrane, response to interleukin-1, intracellular lipid transport, collagen trimerblood microparticle, regulation of intracellular lipid transport, regulation of intracellular sterol transport, regulation of intracellular cholesterol transport, regulation of receptor catabolic, process low-density lipoprotein particle receptor catabolic process, estrous cycle(Fig. 10 D). However, upon CC16 intervention, only ETS1,ZBTB38, PSMD2, EIF3A, FuUS, and C15orf52 were observed to be down-regulated (Fig. 10 E). ETS1 was chosen and subsequently validated through Western Blot analysis, confirming that the expression of ETS1 was indeed up-regulated by CC16 (Fig. 10 F). DISCUSSION The risk of asthma exacerbation increases because of exposure to a high concentration of PM 2.5 . 16 However, previous studies did not clearly state the relationship between PM 2.5 and the mechanism of acute asthmatic attacks. Our results suggested that PM 2.5 caused respiratory acidosis, increased airway resistance, lung tissue damage, inflammation, and airway epithelium dysfunction while upregulating inflammation and pyroptosis proteins. However, CC16 ameliorated these pathological damage through downregulating inflammation and pyroptosis proteins. The final key result of this study is that CC16 acts mechanistically to offer protection against lung damage caused by PM 2.5 . Hence, there is potential interest in using CC16-based therapeutics for preventing asthma exacerbation. PM 2.5 particles are small and can reach the distal end of the airway with respiration, causing respiratory system damage. 17 More and more research evidence suggests that PM 2.5 can cause various cell death, including autophagy, necrosis, apoptosis, pyroptosis and ferroptosis. 18 – 19 We in vivo and vitro data showed pyroptosis proteins NLRP3, Caspase-1, Gasdermin D, and IL-1β in PM 2.5 -induced asthma. This type of pyroptosis is detected through the classical pathway through inflammasomes, recruiting and activating caspase-1, which cleaves and activates IL-18 and IL-1βfor inflammatory factors, cleave the N-terminal sequence of GSDMD, causing it to bind to the membrane and produce membrane pores, leading to cell pyroptosis. 20 – 21 Similarly, Wang et al. and Huang et al.found that PM 2.5 -induced lung toxicity via suppression of NLRP3 inflammasome-mediated pyroptosis. 22 – 23 Besides, Xiong et al. reported that deleting the pyroptosis-associated protein NLRP3 in macrophages might minimize pyroptosis and PM 2.5 -induced lung toxicity. 24 CC16 is so richly secreted by bronchial club cells and other epithelial cells that it is one of the most abundant proteins in respiratory secretions. 25 The relationship of CC16 and asthma is revealed. A plot study found circulating CC16 deficits were associated with a 20% increased odds of asthma. Furthermore, there appeared to be a dose effect, as more frequent symptoms were more strongly associated with low CC16(40% increased odds) than less frequent symptoms. 26 Another research found low CC16 mRNA expression levels in bronchial epithelial cells were associated with asthma severity. 27 Recently, several studies have been performed using CC16, and most of the investigations have focused on regulating inflammatory pathways in immune cells. 28 Pang et al. observed that recombinant CC16 inhibits tumor necrosis factor-α (TNF-α), IL-6, and IL-8 production in LPS-treated RAW264.7 cells. 29 Moreover, CC16 has been reported to inhibit NF-κB nuclear translocation and matrix metalloprotein 9 production in LPS-stimulated rat tracheal epithelial cells. 30 In sepsis-induced acute lung injury, Janicova et al. noted that endogenous CC16 modulates early macrophage-driven inflammation as an intrinsic anti-inflammatory signal. In study on particle-induced inflammation, Cui et al. revealed that rCC16 significantly lowered the IL-1β, TNF-α, and IL-6 protein and mRNA levels in THP-1 macrophages. Moreover, they showed that CC16 attenuated the increase in pro-IL-1β, NLRP3, and caspase 1 levels induced by exposure to silica particles. 31 In our study, CC16 repairs airway epithelial proteins by targeting signal proteins related to pyroptosis and inflammation, promoting cell proliferation. This dual role and mechanism of action suggest further exploration of CC16 in airway epithelial repair. Our results from previous experiments demonstrated that rCC16 inhibited LPS-stimulated apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ERK1/2 pathway and inhibited the release of inflammatory factors by inactivating the TLR-4/NF-κB signaling pathway. 11 In this study, we revealed that PM 2.5 -induced pyroptosis comprises the inactivation of the NLRP3/caspase 1 signaling pathway and up-regulated expression of inflammatory mediator via the inactivation of the TLR4/NF-κB/MAPK/caspase 3 signaling pathway. However, they were both simultaneously inhibited by rCC16. Especially, CC16 appears to be highly specific of HMGB1. Our cell and animal models demonstrated that CC16 could inhibit the PM 2.5 -induced release of HMGB1. Liu et al. documented that CC16 attenuates house dust mite-mediated airway inflammation and damage via suppression of airway epithelial cell apoptosis in an HMGB1-dependent manner. 32 Furthermore, Huang et al. identified that HMGB1 could promote the dysfunction of the epithelial barrier in synergy with IL-1β. 33 Nevertheless, the underlying mechanism of how CC16 inhibits HMGB1 and IL-1β needs further investigation. There is evidence suggesting that the binding capacities of CC16 are attributed to a modifying change between an activator and a receptor component protein inside and outside the cells. This protein is a dimer with two reverse polypeptide chains, thus forming a hydrophobic pocket for binding small lipophilic molecules. 34 Johnson identified that VLA-4, created by a highly conserved sequence of amino acids (leucine-valine-aspartic acid), has vital mechanistic implications as a novel receptor. VLA-4 could impact neutrophil recruitment and leukocyte adhesion during infection by binding to integrin α4β1. 35 These findings emphasize the underlying mechanism of CC16 as an intrinsic anti-inflammatory signal in the case of lung injury induced by PM 2.5 , sepsis, and toxicity. According to the transcriptome analysis, CC16 has been observed to impede alveolar epithelial cells pyroptosis after PM 2.5 exposure. This effect could be attributed to the upregulation of genes associated with cellular proliferation and repair, and down-regulation of genes linked to adhesion and lipid metabolism. Among the up-regulated genes, ETS1 is closely related to the AKT/mTOR proliferation pathway and governs the downstream NF- κB. 36 However, there are several limitations in our study. First, apparent conceptual improvements are needed, especially regarding the experimental setup of the animal model. To determine whether CC16 moderates anaphylactic inflammation in the asthma airways or influences PM 2.5 -mediated inflammation, it would be beneficial to use four control groups: Control + OVA,Control + OVA + CC16, Control + PM 2.5 , and Control + PM 2.5 +CC16. Dr. Wang Aili, a member of our research team, augmented the control group in accordance with the experimental design and subsequently verified that CC16 exhibits reparative properties in response to injuries induced by both PM 2.5 and OVA. The forthcoming publication of her article is anticipated.. It would be appropriate to design a new research study specifically focused on investigating the relationship between asthma and PM 2.5 . An additional animal experiment will be conducted by adding the above group. However, too few examples support the significant difference in sRAW between asthma and the negative control. Overall, data from HE, arterial blood gas, and IL 1β in BAL support the successful construction of the asthma model. CONCLUSIONS In conclusion, CC16 can mitigate PM 2.5− induced airway epithelial damage by regulating inflammatory and pyroptosis signaling pathways.These findings suggest that CC16 shows promise as a therapeutic intervention for alleviating the health risks of PM 2.5 exposure in the asthmatic patients. Declarations Author Contributions. Jinle Lin, Xiaowen Chen, Yuehua Chen, Xiaobing Zeng participated in the question conception, data analysis, and manuscript draft preparation. Jian Wu was the general director of the project, responsible for the project funding, designing, writing, and checking. Jie Yao, Fang Wang, and Yuyang Miao performed the cellular and animal experiments.Shaohua Luo, Lei Jiang, Wenxue Hu, Xiaolong Liu, Jing Zhang, Zhongpeng Li, and Siping Zhou abstracted the data collection and conducted formal analysis. Qingli Dou, and Wenwu Zhang provided critical appraisals of the study and assisted with the development of the study question. All authors have read and approved the manuscript. Funding .This work was supported through funding from Jian Wu (National Natural Science Fund of China, Grant No. 81970012; Key-Area Research and Development Program of Guangdong Province, Grant No. 2019B020227006; GuangDong Basic and Applied Basic Research Foundation, Grant No. KS0120220270), Jinle Lin (National Natural Science Fund of China, Grant No.82302462; Guang Dong Basic and Applied Basic Research Foundation, Grant No. 2022A1515111206, Guangdong Provincial Medical Science and Technology Research Fund project, Grant No.20221115145253272, Shenzhen Key Medical Discipline Construction Fund, SZXK047; High-level medical team of Shenzhen "Three Famous Medical and Health Project"; Key Laboratory of Emergency and Trauma (Hainan Medical University), Ministry of Education, Grant No. KLET-201902); Science and Technology Planning Project of Shenzen Municipality Grant No. YJ20230807140904010. All funder contributed equally to this investigation. Data Availability .The data used during the current study are available from the corresponding author on reasonable request. Conflict of Interest. The authors declare that they have no conflict of interests. Consent for publication. All authors confirm their consent for publication. Ethics approval and consent to participate. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The mouse procedures outlined in this study received approval from the Research Ethics Committee of Southern Medical University (Title: A study on the mechanism by which exosome-loaded CC16 mediates the regulation of alveolar epithelial cell pyroptosis in the treatment of ARDS through LVD in conjunction with ETS-1, Approval No. BYL20231202, Date of approval December 4, 2023). All procedures were conducted following the Guide for the Care and Use of Laboratory Animals. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The patient(s) or their guardian(s)/legally authorized representative(s)/next of kin provided written informed consent for participation in the study and/or the use of samples. Acknowledgement. We thank all investigators for their excellent assistance in this research. Pulmonary function test was undertaken by Xiu Yu and Hongchang Chen (Institute of Respiratory Diseases, Shenzhen People's Hospital). We wish to thank Xinhui Bi (Institute of Geochemistry, Chinese Academy of Sciences) and Qi Fan and Shenxun Zhou (Sun Yat-sen University) for collecting and Xinzhi Bi (Guangzhou Institute of Geographical Chemistry) for analyzing PM 2.5 , as well as Jiajie Shan and Jian Wang (School of Medicine, Southern China University of Technology) for their technical assistance. 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Part Fibre Toxicol 2020; 17: Additional Declarations No competing interests reported. Supplementary Files Supplyment.docx SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. Cite Share Download PDF Status: Posted Version 1 posted 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-4651501","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":325172253,"identity":"719cd71b-d3ab-4a60-9087-ac7e2126c1ad","order_by":0,"name":"Jinle Lin","email":"","orcid":"","institution":"People’s Hospital of Shenzhen Baoan District, The Second Affiliated Hospital of Shenzhen University","correspondingAuthor":false,"prefix":"","firstName":"Jinle","middleName":"","lastName":"Lin","suffix":""},{"id":325172254,"identity":"9abe18d0-3f37-4dd8-bd70-004c06823473","order_by":1,"name":"Xiaowen 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Academy of Medical Sciences, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhongpeng","middleName":"","lastName":"Li","suffix":""},{"id":325172283,"identity":"a079ce78-ed62-4b1e-9346-b5c8251c89a5","order_by":15,"name":"Siping Zhou","email":"","orcid":"","institution":"Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siping","middleName":"","lastName":"Zhou","suffix":""},{"id":325172285,"identity":"438b4aca-aaf1-484c-90ff-831363f15eb7","order_by":16,"name":"Jian Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYLACCQMGBn4om7GBaC2SIKUHiNYCAgYHiNUiPyPHTMKiwC7P+PzhZ9IfGGxkNxxgfvYAr+E3gFokDJKLzW6kmUkcYEgz3nCAzdwArxaJ3G1ALcyJ227wsAG1HE7ccADIwO8wsJb6xM39Z0Ba/hPWwnADrAVoOEMOSMsBwloMzrz/bCFhcDxxxo00Y4szBsnGMw+zmeF3WHta4m2JP9WJ/f2HH96oqLCT7Tve/Ay/wwQSGJgRKkBBxYxXPRDwH2Bg/EBI0SgYBaNgFIxsAABixEgkhg/V4QAAAABJRU5ErkJggg==","orcid":"","institution":"Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-06-28 02:17:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4651501/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4651501/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61016646,"identity":"59f77b47-75fd-4f3c-bf09-459d2ea14811","added_by":"auto","created_at":"2024-07-24 15:27:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":677764,"visible":true,"origin":"","legend":"\u003cp\u003eThe asthma mice model was induced via the intraperitoneal injection and atomization of OVA until Day 27. PM\u003csub\u003e2.5\u003c/sub\u003e (2 mg/kg) was administrated to the mice in the PM\u003csub\u003e2.5\u003c/sub\u003e group on Day 28. CC16 (2 mg/kg) was administered to the mice in the PM\u003csub\u003e2.5\u003c/sub\u003e+CC16 group on Day 28 after exposure to PM\u003csub\u003e2.5\u003c/sub\u003e. BEAS-2B cells were incubated with (100 μg/mL, and 200 μg/mL) PM\u003csub\u003e2.5\u003c/sub\u003e with 24 h and 48 h, CC16 (0.25 μg/mL, 0.5 μg/mL, and 1 μg/mL) was administered to the cell medium after the cells were exposed to 100 μg/mL PM\u003csub\u003e2.5\u003c/sub\u003e for 24 h.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/d7edaefa111d44fe5a91ef2f.png"},{"id":61016635,"identity":"8a2754ba-ed6a-4deb-a23e-f257ac60479d","added_by":"auto","created_at":"2024-07-24 15:27:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1229793,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of OVA induction and exposure to PM\u003csub\u003e 2.5\u003c/sub\u003e on mice. \u003cstrong\u003ea\u003c/strong\u003e blood gas ,\u003cstrong\u003eb \u003c/strong\u003eThe lung function,\u003cstrong\u003ec\u003c/strong\u003e HE staining of lung tissue, The red arrow denotes the injury of ciliated cells in the trachea, the blue arrow denotes inflammatory reaction, hemorrhage, and necrosis.\u003cstrong\u003ed\u003c/strong\u003e Injury score in Lung tissue.(n = 4 in each group) \u003cem\u003ep\u003c/em\u003e ＜ 0.05(*).\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/c6265436683f534773195760.png"},{"id":61016636,"identity":"b7964ec0-7c11-4e38-8e19-8f87c901b04c","added_by":"auto","created_at":"2024-07-24 15:27:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1565249,"visible":true,"origin":"","legend":"\u003cp\u003eThe lung injury were induced by OVA and exposed to PM\u003csub\u003e2.5\u003c/sub\u003e.\u003cstrong\u003ea \u003c/strong\u003eImmunohistochemical staining(n = 10 in each group). \u003cstrong\u003eb \u003c/strong\u003eWestern blot of lung tissue(n = 3 in each group). Full-length blots/gels are presented in Supplementary Figure 3\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/2fcc118bf9733e9e3dab8973.png"},{"id":61016640,"identity":"d4a8b02f-7028-49f3-ba69-902d514afe82","added_by":"auto","created_at":"2024-07-24 15:27:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":883600,"visible":true,"origin":"","legend":"\u003cp\u003eThe evaluation of CC16 on mice after exposure to PM\u003csub\u003e2.5\u003c/sub\u003e. \u003cstrong\u003ea \u003c/strong\u003eblood gas, \u003cstrong\u003eb\u003c/strong\u003e lung function, \u003cstrong\u003ec\u003c/strong\u003e HE staining of lung tissue, The red arrow denotes inflammatory reaction, the blue arrow denotes repariment of lung tissue.\u003cstrong\u003ed\u003c/strong\u003e Injury score in Lung tissue.(n = 4 in each group). \u003cem\u003ep\u003c/em\u003e ＜ 0.05(*),\u003cem\u003ep\u003c/em\u003e ＜ 0.01(**).\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/cfe7bdd965e3aa516892955f.png"},{"id":61016641,"identity":"9a13a907-ddf7-4bb6-82c4-3163dfb94ed7","added_by":"auto","created_at":"2024-07-24 15:27:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1671134,"visible":true,"origin":"","legend":"\u003cp\u003eIHC and Western blot were used to measure the protein expression levels of CC16-treated mice after exposure to PM\u003csub\u003e2.5\u003c/sub\u003e.\u003cstrong\u003e a\u003c/strong\u003e Immunohistochemical staining (n = 10 in each group). \u003cstrong\u003eb\u003c/strong\u003e Western blot of lung tissue (n = 3 in each group). Full-length blots/gels are presented in Supplementary Figure 5.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/da35f4940b1a3f3a8aa5a77b.png"},{"id":61016638,"identity":"c241fee0-1eb0-409f-97c3-b511d588f0f3","added_by":"auto","created_at":"2024-07-24 15:27:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":848105,"visible":true,"origin":"","legend":"\u003cp\u003eLight microscopy, CCK-8 analysis, confocal microscopy, and electron microscopy of the alterations in BEAS-2B cells exposed to PM\u003csub\u003e2.5\u003c/sub\u003e. \u003cstrong\u003ea\u003c/strong\u003e Light microscopy. \u003cstrong\u003eb\u003c/strong\u003e Cell viability was inferred from CCK-8 analysis. \u003cstrong\u003ec\u003c/strong\u003e Confocal microscopy and electron microscopy.*\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 vs. control group. \u003cem\u003ep\u003c/em\u003e ＜ 0.05(*), \u003cem\u003ep\u003c/em\u003e ＜ 0.01(#).\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/3848e177762b9ef1d0ec3b4a.png"},{"id":61016644,"identity":"e79bf926-46b6-4d74-917c-d731026a3c2e","added_by":"auto","created_at":"2024-07-24 15:27:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":823036,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot and RNA analysis of inflammatory and pyroptosis signaling pathways. \u003cstrong\u003ea-d\u003c/strong\u003e Western blot. \u003cstrong\u003ee \u003c/strong\u003eRNA analysis.\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05(*) vs. control group. Full-length blots/gels are presented in Supplementary Figure 7\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/413d0d79dd59175280d0ca17.png"},{"id":61017383,"identity":"66f71c4e-9d7f-4785-bad9-d4accd85dcd4","added_by":"auto","created_at":"2024-07-24 15:35:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1348828,"visible":true,"origin":"","legend":"\u003cp\u003eLight microscopy, CCK-8 analysis,confocal microscopy, and electron microscopy to determine the viability of BEAS-2B cells treated with CC16. \u003cstrong\u003ea\u003c/strong\u003eLight microscopy. \u003cstrong\u003eb\u003c/strong\u003e Cell viability was inferred from CCK-8 analysis. \u003cstrong\u003ec\u003c/strong\u003e Confocal microscopy and electron microscopy.\u003cem\u003ep\u003c/em\u003e ＜ 0.05(*), \u003cem\u003ep\u003c/em\u003e ＜0.01(**) vs. control group.\u003c/p\u003e","description":"","filename":"Picture8.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/305c83c343a349509fd55867.png"},{"id":61016645,"identity":"db1ac766-5eb1-4774-b508-f0df605a017a","added_by":"auto","created_at":"2024-07-24 15:27:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":667696,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot and PCR analysis of inflammatory and pyroptosis signaling pathways after CC16 treatment. \u003cstrong\u003ea-b\u003c/strong\u003e Western blot. \u003cstrong\u003ec \u003c/strong\u003eRNA analysis.\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05(*) vs. control group. Full-length blots/gels are presented in Supplementary Figure 9\u003c/p\u003e","description":"","filename":"Picture9.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/3d2309f6d30b118ad8333063.png"},{"id":61018222,"identity":"8d0e2dac-c3fc-48bf-a8c4-7a5abee4019a","added_by":"auto","created_at":"2024-07-24 15:43:01","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":513431,"visible":true,"origin":"","legend":"\u003cp\u003eThe intervention of CC16 on AEC Pyroptosis following exposure to PM\u003csub\u003e2.5\u003c/sub\u003e utilizing transcriptome analysis.\u003cstrong\u003ea\u003c/strong\u003e Principal Component Analysis (PCA). \u003cstrong\u003eb \u003c/strong\u003etwo volcano plots display differential genes between the PM\u003csub\u003e2.5\u003c/sub\u003e and control groups.\u003cstrong\u003ec\u003c/strong\u003e two volcano plots display differential genes between the CC16 group and the PM\u003csub\u003e2.5\u003c/sub\u003e group. \u003cstrong\u003ed \u003c/strong\u003eGO analysis investigate the pathophysiological process involved in the intervention of CC16. \u003cstrong\u003ee \u003c/strong\u003eHeat map of differential gene expression induced by CC16 intervention.\u003cstrong\u003ef \u003c/strong\u003eWestern blot validation was performed on ETS1 of PM\u003csub\u003e2.5\u003c/sub\u003e exposed-AEC pyroptosis induced by CC16 intervention. Full-length blots/gels are presented in Supplementary Figure 10.\u003c/p\u003e","description":"","filename":"Picture10.png","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/4dc3a70cb677de07f140ca63.png"},{"id":61916873,"identity":"d4c9fae7-6da0-404e-8e1b-016bb246c887","added_by":"auto","created_at":"2024-08-07 05:07:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12629692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/53337128-3e34-4a18-b5ff-909325444d09.pdf"},{"id":61017384,"identity":"ba8b1a65-3448-4eef-b102-b060c9a9a553","added_by":"auto","created_at":"2024-07-24 15:35:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUPPORTING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional supporting information can be found online in the Supporting Information section at the end of this article.\u003c/p\u003e","description":"","filename":"Supplyment.docx","url":"https://assets-eu.researchsquare.com/files/rs-4651501/v1/9ecbdd14fb5e64b2eb2d5073.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Club cell secretory protein 16 up-regulates cell proliferation, inhibits inflammation and pyroptosis against particular matter 2.5 -induced epithelium damage in asthmatic mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eParticulate matter 2.5(PM\u003csub\u003e2.5\u003c/sub\u003e) refers to a mix of tiny solid and liquid particles of \u0026lt;\u0026thinsp;2.5 \u0026micro;m in diameter suspended in the air.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e High concentrations of PM\u003csub\u003e2.5\u003c/sub\u003e increase the morbidity and severity of asthma. The disease is often accompanied by damage to the airway epithelium cells (AECs), the critical frontline of the respiratory tract for defence and homeostasis. It is hypothesized that epithelial dysfunction is the principal mechanism of asthma pathogenesis.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePM\u003csub\u003e2.5\u003c/sub\u003e aggravates asthmatic inflammation and results in signal-related pyroptosis.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3) inflammasome leads to the release of cysteine-containing aspartate specific protease 1(Caspase 1)-dependent, proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 as well as gasdermin D-related swelling and membrane rupture of cells, followed by pyroptotic death.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Elevated concentrations of NLRP-3 and IL-1β have been observed in the sputum of non-smokers with asthma.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e In addition, PM\u003csub\u003e2.5\u003c/sub\u003e orchestrates lung inflammation and enhances the expression of NLRP 3. The use of NLRP 3 inhibitor MCC950, caspase 1 inhibitor Ac-YVAD-CHO and IL-1β neutralizing antibodies had effectively decreased the asthmatic inflammation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eClub cell secretory protein (CC16) belongs to the secretoglobin superfamily and predominantly secreted by AECs, with anti-inflammatory and immunoregulatory functions.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Our previous clinical study found the abrupt elevation of serum CC16 in critically ill patients indicates a potential association with secondary acute respiratory distress syndrome (ARDS) and its subsequent return to normal levels signifies a favorable prognosis.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Further research has highlighted the CC16 inhibits toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) inflammatory pathways in vitro.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Another research on LPS and bacterial-induced lung injury in mice elucidated that extracellular vesicles-CC16 could be a potential therapeutic agent for acute lung injury(ALI) by inhibiting the inflammatory and DNA damage responses by reducing NF-κB signaling.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Moreover, epidemiological studies have shown decreased serum CC16 levels in patients with asthma.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The CC16 A38G polymorphism may contribute to the development of late-onset asthma.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e However, how CC16 repairs the damaged epithelium in the airways of asthmatic has not been elucidated. CC16 had repair properties in a AECs model of short-term high concentration exposure to PM\u003csub\u003e2.5\u003c/sub\u003e. Consequently, further examination of the repair mechanisms and effects of CC16 in the model following short-term exposure to PM\u003csub\u003e2.5\u003c/sub\u003e is warranted.\u003c/p\u003e \u003cp\u003eIn this study, the repair mechanisms of CC16 in PM\u003csub\u003e2.5\u003c/sub\u003e-induced lung injury on asthma model mice and human bronchial epithelial (BEAS-2B) cell line were investigated.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eThe following experimental methods are a brief description. For details, see the Appendix S1. The work has been reported in line with the ARRIVE guidelines 2.0.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection of PM\u003csub\u003e2.5\u003c/sub\u003e and sample preparation\u003c/h2\u003e \u003cp\u003ePM\u003csub\u003e2.5\u003c/sub\u003e samples were obtained from the rooftops of the platform at the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences (100 m near the motorway) in August 2020, using high-volume air samplers (Thermo Fischer Scientific, Waltham, USA). Pieces of quartz fibre filter membrane (20\u0026times;25-cm\u003csup\u003e2\u003c/sup\u003e) loaded with PM\u003csub\u003e2.5\u003c/sub\u003e were washed, filtered, centrifuged and freeze-dried to produce PM\u003csub\u003e2.5\u003c/sub\u003e freeze-dried powder.In the experiment, the powder was dissolved in normal saline or PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMice model study\u003c/h2\u003e \u003cp\u003e Male C57BL/6J mice were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;20 per group) and suffered different treatments.The Research Animal Care Committee of Southern Medical University approved the study protocols. All experiments involving animals complied with the ARRIVE guidelines.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Mice in the six groups were treated with normal saline(Control group), OVA(Asthma group), OVA\u0026thinsp;+\u0026thinsp;PM\u003csub\u003e2.5\u003c/sub\u003e(24 h)(PM\u003csub\u003e2.5\u003c/sub\u003e (24 h) group), OVA\u0026thinsp;+\u0026thinsp;PM\u003csub\u003e2.5\u003c/sub\u003e(48h)(PM\u003csub\u003e2.5\u003c/sub\u003e (48 h) group), OVA\u0026thinsp;+\u0026thinsp;PM\u003csub\u003e2.5\u003c/sub\u003e+CC16(24 h)(PM\u003csub\u003e2.5\u003c/sub\u003e+CC16 (24 h) group), OVA\u0026thinsp;+\u0026thinsp;PM\u003csub\u003e2.5\u003c/sub\u003e+CC16 (48 h)(PM\u003csub\u003e2.5\u003c/sub\u003e+CC16 (48 h) group). Mice were sensitized with OVA to induce asthma model as following: on the 1st, 7th, 14th day of the experiment, mice were sensitized with OVA (1 mg OVA and 100 mg Al(OH)3; Sigma, St. Louis, USA) or normal saline via intraperitoneal injection, followed by inhalation exposure to 1% OVA or saline aerosol via compression atomizers (402AI, JS, CN). On days 21\u0026ndash;27 to promote challenge, 30 min/day. During the challenge of asthma model, the mice were inhalation exposure to PM\u003csub\u003e2.5\u003c/sub\u003e( 2 mg / kg ) for 2 hour on the 28th day. The PM\u003csub\u003e2.5\u003c/sub\u003e+CC16 (24 h) group were exposed to 2 mg/kg CC16 (USCN Sciences Co., Wuhan, CN) for 2 hour on the 29th day while the PM\u003csub\u003e2.5\u003c/sub\u003e+CC16 (48 h) group were treated with the same treatment on the 29th and 30th day. At the end of the last treatment,all the mice were tested lung function, anesthetized and sacrificed to collect arterial blood and lung tissues for further research(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eArtery blood gas\u003c/h2\u003e \u003cp\u003eFour mice were anesthetized with pentobarbital (40 mg/kg) and the partial pressure of oxygen (PaO\u003csub\u003e2\u003c/sub\u003e), carbon dioxide (PCO\u003csub\u003e2\u003c/sub\u003e), hydrogen potential (pH), and anion gap (AG) in abdominal aortic blood were analyzed using a radiometer (ABL825, Radiometer).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eLung function\u003c/h2\u003e \u003cp\u003eFive anesthetized mice were assessed for their airway responsiveness to different doses of methacholine by using the FinePointe RC (Buxco Research Systems, DSI, MN, USA). For each dose, lung resistance was measured and computed over 3 min. The respiratory data were acquired and presented with the Fine Pointe station.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology and immunohistochemical staining\u003c/h2\u003e \u003cp\u003eFor scoring analysis, the sections were stained with hematoxylin\u0026ndash;eosin(HE), using the Smith Lung Injury Scoring System to assess injury severity. The sections were used the primary antibodies of TLR 4 (1:100, Abcam, UK), Anti-Phospho-NF-κB (1:150, Abcam), NLRP 3 (1:200, Abcam), Caspase 1 (1:500, Bioss, CN), IL-1β (1:100, Abcam), and Gasdermin D (1:150, Abcam) tand performed the immunohistochemical(IHC) staining. IHC images were randomly scanned by a light scanning microscope at \u0026times;200 magnification and Image J to quantify the high positive percentage contribution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBEAS-2B cell sourcing, exposure to PM\u003csub\u003e2.5\u003c/sub\u003e, and treatment of CC16\u003c/h2\u003e \u003cp\u003eBEAS-2B cells were incubated with PM\u003csub\u003e2.5\u003c/sub\u003e (100 and 200 \u0026micro;g/mL) for 24 h. In the subgroup with PM\u003csub\u003e2.5\u003c/sub\u003e (100 \u0026micro;g/mL), BEAS-2B cells were exposed to 0.25, 0.5, and 1 \u0026micro;g/mL CC16 (R\u0026amp;D Systems, Catalog #: 4218-UT,Minneapolis, MN, USA) for 24 h after PM\u003csub\u003e2.5\u003c/sub\u003e exposure. The protocol for generating cell models is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell Counting Kit-8 assay\u003c/h2\u003e \u003cp\u003eThe CCK-8 solution (Abcam, USA) was added to the culture medium and incubated for 2 h after PM\u003csub\u003e2.5\u003c/sub\u003e or CC16 administration following the manufacturer\u0026rsquo;s instructions. The viability of living cells in each group was tested using a microplate reader at a 450-nm optical density.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLight and fluorescence microscopy\u003c/h2\u003e \u003cp\u003eCell viability was assessed by light-sheet microscopy. The types of cell death were assessed using the Annexin V-FITC/PI Fluorescence Microscopy Kit (BD Biosciences, San Diego, USA) and Hoechst 33342 (BD Biosciences) following the manufacturer\u0026rsquo;s instructions.The slice was observed under a confocal fluorescence microscope (Nikon, Tokyo, Japan) at 405, 488, and 640 nm excitation wavelength.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy\u003c/h2\u003e \u003cp\u003eThe sample was tsprayed with an ion sputter, and observed and photographed with an electron microscope (Zeiss, MERLIN, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe proteins of lung tissue samples and cell lysates were run on SDS-PAGE. Western blot was performed using primary antibodies against TLR 4, NF-κB, polyclonalanti-NF-κB, Caspase 3, NLRP 3, Caspase 1, IL-1β, Gasdermin D, high mobility group box 1 (HMGB1), E-Twenty-Six-1(ETS1) and β-actin. We used Image J to quantify the WB strip gray value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReal-time PCR\u003c/h2\u003e \u003cp\u003eReal-time PCR was conducted using the QuantiFast SYBR Green PCR kit (Invitrogen, Carlsbad, USA) on the Roche Light Cycler 480II system. For details, see the Appendix S1.The primer list is as follows in Table\u0026nbsp;1\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDatabase construction, analysis, and validation based on transcriptome data\u003c/h2\u003e \u003cp\u003eBEAS-2B cells treated with either CC16 or PM\u003csub\u003e2.5\u003c/sub\u003e were subjected to RNA extraction using TRIzol's protocol. cDNA was compounded following the Total RNA seq (H/M/R) Library Prep Kit instructions. The mRNA seq library underwent 100 bp double ended-sequencing using HiSeq2000. The Fatsqc tool is utilized for quality control of mRNA sequencing data. The gene expression level is then calculated using Cufflinks, with default parameters being employed. Finally, Gene Ontology analysis is conducted using DAVID. The Western Blot technique is utilized to verify the protein expression of genes that have undergone screening.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis were performed with SPSS 14.0 statistical software (SPSS Inc., Chicago, IL, USA). The Student's \u003cem\u003et\u003c/em\u003e-test was performed to analyze the difference between the two groups. One-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s post-hoc test was performed for comparisons among multiple experimental groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significance.\u003c/p\u003e \u003cp\u003e\u003cb\u003eEthics/Ethical Approval\u003c/b\u003e. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The mouse procedures outlined in this study received approval from the Research Ethics Committee of Southern Medical University (Approval No. BYL20231202). All procedures were conducted following the Guide for the Care and Use of Laboratory Animals. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eLung tissue damage, respiratory acidosis, and increased airway resistance in asthmatic mice due to PM\u003c/b\u003e \u003csub\u003e \u003cb\u003e2.5\u003c/b\u003e \u0026minus;\u003c/sub\u003e \u003cb\u003eexposure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eArterial blood gas analysis showed that exposure to PM\u003csub\u003e2.5\u003c/sub\u003e for 24 h induced a decline in PO\u003csub\u003e2\u003c/sub\u003e and exposure for 48 h caused a decrease in pH and elevation in PCO\u003csub\u003e2\u003c/sub\u003e compared with the control group mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results of the pulmonary function evaluation indicated a significant increase in airway resistance variables, specific airwayresistance (sRaw) and peak inspiratory flow(PIF), among mice exposed to PM\u003csub\u003e2.5\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting an increase in airway resistance, airway hyperresponsiveness, and respiratory distress. Exposure to PM\u003csub\u003e2.5\u003c/sub\u003e can exacerbate airway inflammation in asthmatic mice(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical and western blot in PM\u003csub\u003e2.5\u003c/sub\u003e exposed asthmatic mice\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining indicate that exposure to PM\u003csub\u003e2.5\u003c/sub\u003e has the potential to induce the expression of TLR4, NLRP3, Caspase-1, Gasdermin D, and IL-1β in mouse lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, Western blot results suggest that inflammatory pathways, including TLR4, Anti-Phospho NF-κB, Caspase-3, and NF-κB, as well as pyrolytic channels such as NLRP3, Caspase-1, Gasdermin D, and IL-1β, exhibit increased expression levels following PM\u003csub\u003e2.5\u003c/sub\u003e exposure when compared to pre-exposure levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Besides, the above protein levels still increased after 48 hours of exposure to PM\u003csub\u003e2.5\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe administration of CC16 relived acute lung injury, respiratory acidosis, and airway distress in asthmatic mice due to PM\u003c/b\u003e \u003csub\u003e \u003cb\u003e2.5\u003c/b\u003e \u0026minus;\u003c/sub\u003e \u003cb\u003eexposure\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe arterial blood gas analysis indicates that the group of CC16 treatment exhibited a significant improvement in blood gas parameters compared to the two other asthma mice model groups exposed to PM\u003csub\u003e2.5\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), manifesting as an increase in pH value, an increase in PaO\u003csub\u003e2\u003c/sub\u003e and a decrease in PaCO\u003csub\u003e2\u003c/sub\u003e. However, the changes in AG were less pronounced. The results of the pulmonary function evaluation indicate that PM\u003csub\u003e2.5\u003c/sub\u003e-induced abnormal sRaw and PIF can be ameliorated by CC16, as depicted in the accompanying figure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Specifically, mice treated with CC16 for 24 hours exhibited restoration of sRaw and PIF to normal levels, in contrast to those exposed to PM\u003csub\u003e2.5\u003c/sub\u003e. Furthermore, a 48-h treatment with CC16 resulted in a complete normalization of sRaw.The results of HE staining indicate that exposure to PM\u003csub\u003e2.5\u003c/sub\u003e induces lung injury characterized by the inflammatory response, bleeding, and necrosis, which can be ameliorated by CC16 treatment, leading to a reduction in the Smith score of lung injury(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCC16 resulted in the suppression of increased levels of inflammation and pyroptosis proteins in asthmatic mice exposed to PM\u003c/b\u003e \u003csub\u003e \u003cb\u003e2.5\u003c/b\u003e \u003c/sub\u003e \u003c/p\u003e \u003cp\u003eBoth IHC and Western blot analyses indicate that nebulization therapy with CC16 suppresses the expression of inflammatory and pyrolytic signaling proteins in lung tissue. In mice treated with CC16 nebulization, the levels of proteins TLR4, Caspase-1, and IL-1β were lower and the expression levels of NF-κ B. NLRP3, Caspase-1, Gasdermin D, and IL-1 β was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePM\u003csub\u003e2.5\u003c/sub\u003e caused pyroptosis and increased activation of inflammatory in BEAS-2B cells\u003c/h2\u003e \u003cp\u003eTo investigate effects of PM\u003csub\u003e2.5\u003c/sub\u003e, We used PM\u003csub\u003e2.5\u003c/sub\u003e to stimulate BEAS-2B cells and. CCK-8 assays and observed changes in cell morphology and cell viability. Annexin V-FITC/PI/Hochest33342 staining analysis and Confocal microscopy inspection suggested that BEAS-2B cells died (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Moreover, scanning electron microscope verified that PM\u003csub\u003e2.5\u003c/sub\u003e compromised the structural integrity of the cellular membrane, such as cell membrane perforation, membrane surface, and irregular shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Based on the results of previous animal experiments, we focus on some cell pyroptosis and inflammatory proteins. The protein blotting analysis indicates that exposure to PM\u003csub\u003e2.5\u003c/sub\u003e and LPS led to the observation of phosphorylated NF-κB in the inflammatory signal channel within the cell signal, as compared to the control group. Additionally, the expression of caspase-3 cleavers, and HMGB1 was found to be higher, while the expression of Caspase-1 shear, Gasdermin D shear, and IL-1βshear in the pyrolytic signaling pathway was significantly increased compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). Elevated levels of RNA transcription were observed for Caspase-1, Gasdermin D, and IL-1 β in the PM\u003csub\u003e2.5\u003c/sub\u003e and LPS exposure groups when compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These result showed PM\u003csub\u003e2.5\u003c/sub\u003e caused pyroptosis and increased activation of inflammatory in BEAS-2B cell.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThe administration of CC16 ameliorated in PM\u003csub\u003e2.5\u003c/sub\u003e-induced pyroptosis and inflammation in BEAS-2B cells\u003c/h2\u003e \u003cp\u003eChanges in cell morphology and cell viability were observed after treating the cells with different concentrations of CC16 in PM\u003csub\u003e2.5\u003c/sub\u003e-contaminated BEAS-2B cells to investigate the function of CC16 in pyroptosis and inflammation.Based on optical microscopy and PI staining, the results showed that CC16 treatment prevented the damage to cell viability caused by PM\u003csub\u003e2.5\u003c/sub\u003e, the levels of cell viability were higher than those in the PM\u003csub\u003e2.5\u003c/sub\u003e group. Besides, it was observed that the proliferation of BEAS-2B cells exposed to PM\u003csub\u003e2.5\u003c/sub\u003e was dependent on both the dosage and duration of CC16 treatment(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). Further studies found that treatment with CC16 effectively decreased pyroptosis induced by PM\u003csub\u003e2.5\u003c/sub\u003e. Through the utilization of Confocal microscopy in conjunction with fluorescence staining, CC16 facilitated the repair of the cell membrane. Ultimately, our findings were substantiated by scanning electron microscopy, that the surviving cells exhibited a smooth and unblemished cell membrane devoid of any surface protrusions indicative of pyroptosis, and their morphology remained intact without any discernible deformities (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Western blot analysis indicates that the expression of phosphorylated NF-κB, P38, Caspase-3, Caspase-1, Gasdmin D, HMGB1, and IL-1β was significantly reduced in the CC16 treatment group compared to the PM\u003csub\u003e2.5\u003c/sub\u003e exposure group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-D). The RT-PCR analysis indicates that the transcription levels of Caspase-1, Gasdmin D and IL-1β were lower in the CC16 treatment group compared to the PM\u003csub\u003e2.5\u003c/sub\u003e exposure group treated with high-pressure sterilization(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).These results demonstrated that CC16 effectively effectively suppresses the upregulation of inflammation and pyroptosis signaling pathways induced by exposure to PM\u003csub\u003e2.5\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCC16 regulated genes expression profiles in PM\u003csub\u003e2.5\u003c/sub\u003e-induced alveolar epithelial cells\u003c/h2\u003e \u003cp\u003eWe employed transcriptome analysis to investigate the regulatory influence of on gene in control, LPS, PM\u003csub\u003e2.5\u003c/sub\u003e, and PM\u003csub\u003e2.5+\u003c/sub\u003eCC16 groups. Principal component analysis (PCA) was used to assess the quantitative repeatability of differentially expressed genes and to determine the statistical consistency of the quantitative results of cell samples(Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). PM\u003csub\u003e2.5\u003c/sub\u003e induced 1100 up-regulated differentially expressed genes and 1180 downregulated genes. 37 differentially expressed genes are commonly up-regulated by CC16, while 20 are down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB-C). After CC16 treatment, the expression of 10 genes that were originally upregulated in the PM\u003csub\u003e2.5\u003c/sub\u003e group decreased, while the expression of 21 genes that were originally downregulated in the PM\u003csub\u003e2.5\u003c/sub\u003e group increased(Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Differentially expressed genes will be screened and subjected to Gene Ontology (GO) enrichment analysis using a database. The functional classification and pathways of significantly enriched differentially expressed proteins (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) will be visually presented through bubble plots. The identified pathways primarily comprised were: collagen-containing extracellular matrix, response to oxygen levels, regulation of lipid metabolic process, response to hypoxia, response to decreased oxygen levels, response to the drug, lysosomal membrane, lytic vacuole membrane, vacuolar membrane, response to interleukin-1, intracellular lipid transport, collagen trimerblood microparticle, regulation of intracellular lipid transport, regulation of intracellular sterol transport, regulation of intracellular cholesterol transport, regulation of receptor catabolic, process low-density lipoprotein particle receptor catabolic process, estrous cycle(Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD). However, upon CC16 intervention, only ETS1,ZBTB38, PSMD2, EIF3A, FuUS, and C15orf52 were observed to be down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE). ETS1 was chosen and subsequently validated through Western Blot analysis, confirming that the expression of ETS1 was indeed up-regulated by CC16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe risk of asthma exacerbation increases because of exposure to a high concentration of PM\u003csub\u003e2.5\u003c/sub\u003e.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e However, previous studies did not clearly state the relationship between PM\u003csub\u003e2.5\u003c/sub\u003e and the mechanism of acute asthmatic attacks. Our results suggested that PM\u003csub\u003e2.5\u003c/sub\u003e caused respiratory acidosis, increased airway resistance, lung tissue damage, inflammation, and airway epithelium dysfunction while upregulating inflammation and pyroptosis proteins. However, CC16 ameliorated these pathological damage through downregulating inflammation and pyroptosis proteins. The final key result of this study is that CC16 acts mechanistically to offer protection against lung damage caused by PM\u003csub\u003e2.5\u003c/sub\u003e. Hence, there is potential interest in using CC16-based therapeutics for preventing asthma exacerbation.\u003c/p\u003e \u003cp\u003ePM\u003csub\u003e2.5\u003c/sub\u003e particles are small and can reach the distal end of the airway with respiration, causing respiratory system damage.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e More and more research evidence suggests that PM\u003csub\u003e2.5\u003c/sub\u003e can cause various cell death, including autophagy, necrosis, apoptosis, pyroptosis and ferroptosis.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e We in vivo and vitro data showed pyroptosis proteins NLRP3, Caspase-1, Gasdermin D, and IL-1β in PM\u003csub\u003e2.5\u003c/sub\u003e-induced asthma. This type of pyroptosis is detected through the classical pathway through inflammasomes, recruiting and activating caspase-1, which cleaves and activates IL-18 and IL-1βfor inflammatory factors, cleave the N-terminal sequence of GSDMD, causing it to bind to the membrane and produce membrane pores, leading to cell pyroptosis.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Similarly, Wang et al. and Huang et al.found that PM\u003csub\u003e2.5\u003c/sub\u003e-induced lung toxicity via suppression of NLRP3 inflammasome-mediated pyroptosis.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Besides, Xiong et al. reported that deleting the pyroptosis-associated protein NLRP3 in macrophages might minimize pyroptosis and PM\u003csub\u003e2.5\u003c/sub\u003e-induced lung toxicity.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCC16 is so richly secreted by bronchial club cells and other epithelial cells that it is one of the most abundant proteins in respiratory secretions.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The relationship of CC16 and asthma is revealed. A plot study found circulating CC16 deficits were associated with a 20% increased odds of asthma. Furthermore, there appeared to be a dose effect, as more frequent symptoms were more strongly associated with low CC16(40% increased odds) than less frequent symptoms.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Another research found low CC16 mRNA expression levels in bronchial epithelial cells were associated with asthma severity.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Recently, several studies have been performed using CC16, and most of the investigations have focused on regulating inflammatory pathways in immune cells.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Pang et al. observed that recombinant CC16 inhibits tumor necrosis factor-α (TNF-α), IL-6, and IL-8 production in LPS-treated RAW264.7 cells.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Moreover, CC16 has been reported to inhibit NF-κB nuclear translocation and matrix metalloprotein 9 production in LPS-stimulated rat tracheal epithelial cells.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In sepsis-induced acute lung injury, Janicova et al. noted that endogenous CC16 modulates early macrophage-driven inflammation as an intrinsic anti-inflammatory signal. In study on particle-induced inflammation, Cui et al. revealed that rCC16 significantly lowered the IL-1β, TNF-α, and IL-6 protein and mRNA levels in THP-1 macrophages. Moreover, they showed that CC16 attenuated the increase in pro-IL-1β, NLRP3, and caspase 1 levels induced by exposure to silica particles.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In our study, CC16 repairs airway epithelial proteins by targeting signal proteins related to pyroptosis and inflammation, promoting cell proliferation. This dual role and mechanism of action suggest further exploration of CC16 in airway epithelial repair.\u003c/p\u003e \u003cp\u003eOur results from previous experiments demonstrated that rCC16 inhibited LPS-stimulated apoptosis by activating the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ERK1/2 pathway and inhibited the release of inflammatory factors by inactivating the TLR-4/NF-κB signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e In this study, we revealed that PM\u003csub\u003e2.5\u003c/sub\u003e-induced pyroptosis comprises the inactivation of the NLRP3/caspase 1 signaling pathway and up-regulated expression of inflammatory mediator via the inactivation of the TLR4/NF-κB/MAPK/caspase 3 signaling pathway. However, they were both simultaneously inhibited by rCC16. Especially, CC16 appears to be highly specific of HMGB1. Our cell and animal models demonstrated that CC16 could inhibit the PM\u003csub\u003e2.5\u003c/sub\u003e-induced release of HMGB1. Liu et al. documented that CC16 attenuates house dust mite-mediated airway inflammation and damage via suppression of airway epithelial cell apoptosis in an HMGB1-dependent manner.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Furthermore, Huang et al. identified that HMGB1 could promote the dysfunction of the epithelial barrier in synergy with IL-1β.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Nevertheless, the underlying mechanism of how CC16 inhibits HMGB1 and IL-1β needs further investigation.\u003c/p\u003e \u003cp\u003eThere is evidence suggesting that the binding capacities of CC16 are attributed to a modifying change between an activator and a receptor component protein inside and outside the cells. This protein is a dimer with two reverse polypeptide chains, thus forming a hydrophobic pocket for binding small lipophilic molecules.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Johnson identified that VLA-4, created by a highly conserved sequence of amino acids (leucine-valine-aspartic acid), has vital mechanistic implications as a novel receptor. VLA-4 could impact neutrophil recruitment and leukocyte adhesion during infection by binding to integrin α4β1.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e These findings emphasize the underlying mechanism of CC16 as an intrinsic anti-inflammatory signal in the case of lung injury induced by PM\u003csub\u003e2.5\u003c/sub\u003e, sepsis, and toxicity.\u003c/p\u003e \u003cp\u003eAccording to the transcriptome analysis, CC16 has been observed to impede alveolar epithelial cells pyroptosis after PM\u003csub\u003e2.5\u003c/sub\u003e exposure. This effect could be attributed to the upregulation of genes associated with cellular proliferation and repair, and down-regulation of genes linked to adhesion and lipid metabolism. Among the up-regulated genes, ETS1 is closely related to the AKT/mTOR proliferation pathway and governs the downstream NF- κB.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHowever, there are several limitations in our study. First, apparent conceptual improvements are needed, especially regarding the experimental setup of the animal model. To determine whether CC16 moderates anaphylactic inflammation in the asthma airways or influences PM\u003csub\u003e2.5\u003c/sub\u003e-mediated inflammation, it would be beneficial to use four control groups: Control\u0026thinsp;+\u0026thinsp;OVA,Control\u0026thinsp;+\u0026thinsp;OVA\u0026thinsp;+\u0026thinsp;CC16, Control\u0026thinsp;+\u0026thinsp;PM\u003csub\u003e2.5\u003c/sub\u003e, and Control\u0026thinsp;+\u0026thinsp;PM\u003csub\u003e2.5\u003c/sub\u003e+CC16. Dr. Wang Aili, a member of our research team, augmented the control group in accordance with the experimental design and subsequently verified that CC16 exhibits reparative properties in response to injuries induced by both PM\u003csub\u003e2.5\u003c/sub\u003e and OVA. The forthcoming publication of her article is anticipated.. It would be appropriate to design a new research study specifically focused on investigating the relationship between asthma and PM\u003csub\u003e2.5\u003c/sub\u003e. An additional animal experiment will be conducted by adding the above group. However, too few examples support the significant difference in sRAW between asthma and the negative control. Overall, data from HE, arterial blood gas, and IL 1β in BAL support the successful construction of the asthma model.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, CC16 can mitigate PM\u003csub\u003e2.5\u0026minus;\u003c/sub\u003einduced airway epithelial damage by regulating inflammatory and pyroptosis signaling pathways.These findings suggest that CC16 shows promise as a therapeutic intervention for alleviating the health risks of PM\u003csub\u003e2.5\u003c/sub\u003e exposure in the asthmatic patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions.\u003c/strong\u003eJinle Lin,\u0026nbsp;Xiaowen Chen, Yuehua Chen,\u0026nbsp;Xiaobing Zeng\u0026nbsp;participated in the question conception, data analysis, and manuscript draft preparation.\u0026nbsp;Jian Wu\u0026nbsp;was the general director of the project, responsible for the project funding, designing, writing, and checking.\u0026nbsp;Jie Yao,\u0026nbsp;Fang Wang, and\u0026nbsp;Yuyang Miao\u0026nbsp;performed the cellular and animal experiments.Shaohua Luo,\u0026nbsp;Lei Jiang,\u0026nbsp;Wenxue Hu,\u0026nbsp;Xiaolong Liu,\u0026nbsp;Jing Zhang,\u0026nbsp;Zhongpeng Li, and\u0026nbsp;Siping Zhou\u0026nbsp;abstracted the data collection and conducted formal analysis.\u0026nbsp;Qingli Dou, and\u0026nbsp;Wenwu Zhang\u0026nbsp;provided critical appraisals of the study and assisted with the development of the study question. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e.This work was supported through funding from Jian Wu (National Natural Science Fund of China,\u0026nbsp;Grant No. 81970012; Key-Area Research and Development Program of Guangdong Province,\u0026nbsp;Grant No. 2019B020227006; GuangDong\u0026nbsp;Basic\u0026nbsp;and\u0026nbsp;Applied\u0026nbsp;Basic\u0026nbsp;Research\u0026nbsp;Foundation,\u0026nbsp;Grant No. KS0120220270),\u0026nbsp;Jinle Lin (National Natural Science Fund of China,\u0026nbsp;Grant No.82302462;\u0026nbsp;Guang\u0026nbsp;Dong Basic and Applied Basic Research Foundation,\u0026nbsp;Grant No. 2022A1515111206,\u0026nbsp;Guangdong Provincial Medical Science and Technology Research Fund project,\u0026nbsp;Grant No.20221115145253272,\u0026nbsp;Shenzhen Key Medical Discipline Construction Fund,\u0026nbsp;SZXK047; High-level medical team of Shenzhen \u0026quot;Three Famous Medical and Health Project\u0026quot;;\u0026nbsp;Key Laboratory of Emergency and Trauma (Hainan Medical University),\u0026nbsp;Ministry of Education,\u0026nbsp;Grant No. KLET-201902); Science and Technology Planning Project of Shenzen Municipality Grant No. YJ20230807140904010. All funder contributed equally to this investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e.The data used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epublication.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors confirm their consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The mouse procedures outlined in this study received approval from the Research Ethics Committee of Southern Medical University (Title: A study on the mechanism by which exosome-loaded CC16 mediates the regulation of alveolar epithelial cell pyroptosis in the treatment of ARDS through LVD in conjunction with ETS-1,\u0026nbsp;Approval No. BYL20231202, Date of approval December 4, 2023). All procedures were conducted following the Guide for the Care and Use of Laboratory Animals. The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The patient(s) or their guardian(s)/legally authorized representative(s)/next of kin provided written informed consent for participation in the study and/or the use of samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all investigators for their excellent assistance in this research. Pulmonary function test was undertaken by Xiu Yu and Hongchang Chen (Institute of Respiratory Diseases,\u0026nbsp;Shenzhen People\u0026apos;s Hospital). We wish to thank Xinhui Bi (Institute of Geochemistry,\u0026nbsp;Chinese Academy of Sciences) and Qi Fan and Shenxun Zhou (Sun Yat-sen University) for collecting and Xinzhi Bi (Guangzhou Institute of Geographical Chemistry) for analyzing PM\u003csub\u003e2.5\u003c/sub\u003e, as well as Jiajie Shan and Jian Wang (School of Medicine, Southern China University of Technology) for their technical assistance. We thanks to the language-editor of Abdelouahab Bellou. The authors declare that artificial intelligence is not used in this study \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang Y, Duan X and Wang L. Spatial-Temporal Evolution of PM(2.5) Concentration and its Socioeconomic Influence Factors in Chinese Cities in 2014⁻2017. Int J Environ Res Public Health 2019; 16.\u003c/li\u003e\n\u003cli\u003eZhang L, Wilson JP and MacDonald B, et al. The changing PM2.5 dynamics of global megacities based on long-term remotely sensed observations. Environ. Int. 2020; 142: 105862.\u003c/li\u003e\n\u003cli\u003eWei H, Yuan W and Yu H, et al. Cytotoxicity induced by fine particulate matter (PM(2.5)) via mitochondria-mediated apoptosis pathway in rat alveolar macrophages. Environ Sci Pollut Res Int 2021; 28: 25819-25829.\u003c/li\u003e\n\u003cli\u003eFrey A, Lunding LP and Ehlers JC, et al. More Than Just a Barrier: The Immune Functions of the Airway Epithelium in Asthma Pathogenesis. 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Med. 2021; 27: 11.\u003c/li\u003e\n\u003cli\u003eHuang W, Zhao H and Dong H, et al. High-mobility group box 1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway. Int. J. Mol. Med. 2016; 37: 1189-1198.\u003c/li\u003e\n\u003cli\u003eLakind JS, Holgate ST and Ownby DR, et al. A critical review of the use of Clara cell secretory protein (CC16) as a biomarker of acute or chronic pulmonary effects. Biomarkers 2007; 12: 445-467.\u003c/li\u003e\n\u003cli\u003eJohnson M, Younis US and Menghani SV, et al. CC16 Binding to \u0026alpha;(4)\u0026beta;(1) Integrin Protects against Mycoplasma pneumoniae Infection. Am J Respir Crit Care Med 2021; 203: 1410-1418.\u003c/li\u003e\n\u003cli\u003eChen YC, Chuang TY and Liu CW, et al. Particulate matters increase epithelial-mesenchymal transition and lung fibrosis through the ETS-1/NF-\u0026kappa;B-dependent pathway in lung epithelial cells. Part Fibre Toxicol 2020; 17: \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CC16, inflammation, pyroptosis, PM2.5, asthma","lastPublishedDoi":"10.21203/rs.3.rs-4651501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4651501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e \u003cp\u003e: Club cell secretory protein (CC16) is reported to have multiple protective functions in airway diseases, including anti-inflammatory, immunomodulatory and antioxidant. This study aims to determine whether CC16 can repair lung injury caused by particular matter 2.5(PM\u003csub\u003e2.5\u003c/sub\u003e) exposure in asthmatic mice.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn the ovalbumin (OVA)-induced asthma murine study, 6-week-old male C57BL/6J mice were primary exposed to PM\u003csub\u003e2.5\u003c/sub\u003e for 24 hours and following treated with CC16, Artery blood gas, lung function,histopathology and immunohistochemical staining were detected. The BEAS-2B cell line was primary exposed to PM\u003csub\u003e2.5\u003c/sub\u003e for 24 hours and then treated with CC16 subsequently, fluorescence and electron microscopy, protein and RNA of inflammation and pyroptosis, and RNA Sequencing were detected.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the OVA-induced asthmatic mice after exposure of PM\u003csub\u003e2.5\u003c/sub\u003e treatment with CC16 ameliorated PM\u003csub\u003e2.5\u003c/sub\u003e-induced lung tissue damage, respiratory acidosis and restore the increased airway resistance after PM\u003csub\u003e2.5\u003c/sub\u003e-exposed group, accompanied with the inhibition in the protein of inflammation and pyroptosis.Moreover, CC16 increased cell proliferation, ameliorated pyroptotic cell death induced by PM\u003csub\u003e2.5\u003c/sub\u003e and inhibited the expression on the protein and RNA of inflammation and pyroptosis. Transcriptome analysis revealed that CC16 down-regulate genes associated with inflammatory adhesion, while up-regulating proliferation genes,like E-Twenty-Six-1.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCC16 could repair airway epithelium PM\u003csub\u003e2.5\u003c/sub\u003e-induced damage in asthma mice by up-regulating cell proliferation,inhibiting pyroptosis and imflammation, which it will been used as a novel therapeutic agent to alleviate the health risks of PM\u003csub\u003e2.5\u003c/sub\u003e exposure in future.\u003c/p\u003e","manuscriptTitle":"Club cell secretory protein 16 up-regulates cell proliferation, inhibits inflammation and pyroptosis against particular matter 2.5 -induced epithelium damage in asthmatic mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 15:26:55","doi":"10.21203/rs.3.rs-4651501/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9160ac24-d9d2-47ea-9d2c-5912d558bad1","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-07T04:59:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-24 15:26:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4651501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4651501","identity":"rs-4651501","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}