Oligosaccharides from Polygonatum cyrtonema Hua ameliorate colitis-induced lung injury via inhibiting inflammation and oxidative stress

Oligosaccharides from Polygonatum cyrtonema Hua ameliorate colitis-induced lung injury via inhibiting inflammation and oxidative stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Oligosaccharides from Polygonatum cyrtonema Hua ameliorate colitis-induced lung injury via inhibiting inflammation and oxidative stress Jin Xu, Chuankang Tang, Qianyu He, Yu Lu, Pei Luo, Jianbo Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6166677/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 Lung disease is one of the parenteral manifestations of ulcerative colitis (UC), and still requires attention to the impact on the lungs while treating UC. Oligosaccharides from Polygonatum cyrtonema Hua (PFOS) has the therapeutic potential for lung injury associated with enteritis.To investigate the mechanism of PFOS in treating colitis-induced lung injury through NF-kB/Nrf2 signaling pathway, this study constructed in vitro and in vivo injury models using DSS and LPS. PFOS was used for the drug treatment, and Nrf2 inhibitor ML385 was used to interfere with Nrf2/HO-1 signaling pathway in the cells. In vivo results showed that PFOS reduces lung tissue damage associated with enteritis by protecting the epithelial barrier and regulating the Nrf2/NF-κB signaling pathways. PFOS upregulates antioxidant proteins (Nrf2, HO-1, NQO-1) and suppresses pro-inflammatory markers (p65, p-p65, IKK-β), indicating its antioxidant and anti-inflammatory effects. In vitro results showed that PFOS suppressed epithelial barrier damages, inflammation and oxidative stress in lung epithelial cells by inhibiting the NF-kB pathway and upregulating the Nrf2 signaling pathway. Additionally, PFOS intervention ameliorated the DSS-induced amino acid and lipid metabolism disorder.In conclusion,the DSS induced enteritis associated lung injury model, PFOS inhibited epithelial barrier damages, inflammation and oxidative stress of lung suppressed NF-kB and up regulated Nrf2 signaling pathway. Additionally, PFOS’s impact on intestinal metabolites may contribute to further protect lung tissue. Biological sciences/Drug discovery Biological sciences/Molecular biology PFOS colitis-induced lung injury NF-kB/IKK-β Nrf2/HO-1/NQO-1 mucosal barrier intestinal metabolites Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction The "Lung-Intestinal Axis" links the lungs and intestines through a shared developmental origin, common mucosal immune system, similar immunomodulatory mechanisms, and a shared molecular foundation [ 1 – 2 ]. Mechanisms like inflammatory mediator circulation, gut microbiota dysbiosis, and oxidative stress play crucial roles in this axis, linking lung and intestinal health[ 3 ]. These mechanisms have implications for understanding diseases that affect both the lungs and the intestines, such as those triggered by infections or chronic inflammation[ 4 ]. The genus Polygonatum has long been recognized for its medicinal and nutritional value, being widely utilized as a traditional tonic food and medicinal ingredient across various countries, including China, Pakistan, Iran, Japan, and others, despite regional cultural differences [ 5 ]. The primary active compounds identified in Polygonatum species include steroidal saponins, flavonoids, polysaccharides, and alkaloids [ 6 ]. In our recent work, we successfully isolated and characterized oligosaccharides from Polygonatum cyrtonema Hua (PFOS), and explored several of their biological activities[ 7 , 8 ]. Notably, PFOS were shown to mitigate intestinal damage induced by dextran sodium sulfate (DSS), with mechanisms involving anti-inflammatory and antioxidant effects [ 8 ]. Furthermore, PFOS significantly reduced mortality in mice with lipopolysaccharide (LPS)-induced peritonitis and ameliorated the associated acute lung injury [ 7 ]. In clinical work, the interplay between gut and lung health has garnered increasing attention, particularly in conditions where symptoms manifest across both systems. For example, an analysis of clinical characteristics in 140 patients with SARS-CoV-2 infection in Wuhan revealed that 39.6% of patients exhibited gastrointestinal symptoms, underscoring the gut-lung axis [ 9 ]. Similarly, data from the RHINE study indicated that individuals with inflammatory bowel disease (IBD), particularly females with ulcerative colitis (UC), exhibited a higher prevalence of asthma and respiratory symptoms [ 10 ]. Moreover, researchers have demonstrated that DSS-induced colitis in genetically susceptible models is sufficient to induce lung inflammation and tissue damage, highlighting the potential for developing therapeutic strategies aimed at reducing comorbidities in IBD patients[ 11 ].Given our previous findings on the protective effects of PFOS in both gut and lung injuries, we sought to further investigate its potential effects on lung injury associated with enteritis. Researches indicated that nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in inhibiting ferroptosis, thereby alleviating intestinal ischemia/reperfusion-induced acute lung injury[ 12 , 13 ]. Additionally, cigarette smoke has been found to promote the production of endogenous reactive oxygen species (ROS) in patients with chronic obstructive pulmonary disease (COPD), potentially exacerbating intestinal disorders[ 3 ]. These findings suggest that Nrf2 may have a pivotal role in diseases involving gut-lung crosstalk. Nrf2 is now widely recognized to regulate over 200 genes encoding enzymes involved in antioxidant defense mechanisms, including glutathione peroxidase (GPx), glutathione (GSH), and heme oxygenase-1 (HO-1) synthesis[ 14 ]. However, its role in innate immunity and the regulation of immune-related gene expression remains less well understood. Emerging evidence indicates that activation of the Nrf2/HO-1 signaling pathway exerts significant anti-inflammatory effects[ 15 ]. Notably, sinomenine has been shown to modulate macrophage polarization toward the M2 phenotype in an Nrf2-dependent manner, while inhibiting IκBα phosphorylation and nuclear translocation of NF-κB, thus identifying the upstream target of sinomenine in modulating NF-κB activity[ 16 ]. Therefore, basing on the findings from previous studies, we established a mouse model of DSS-induced enteritis-associated lung injury to investigate the effects of PFOS on lung pathology in this context. The study employed various biotechnological approaches to assess the efficacy and underlying mechanisms of PFOS, with a particular focus on oxidative stress and inflammatory responses. Additionally, we developed cellular models using lung epithelial cells to further validate the efficacy and elucidate the mechanisms of action of PFOS in this system. 2. Materials and methods 2.1 Materials The preparation of PFOS was provided as the previous report[17]. Dextran sulfate sodium (DSS) was obtained from Yuanye Biotechnology Co. Ltd. (S14049, molecular weight: 50,000, Shanghai, China). CAT assay kits, SOD assay kits, MDA assay kits and GPX2 assay kits were purchased from JSBOSSEN Biotechnology Company (Nanjing, China) or from Jiancheng Biotechnology Company (Nanjing, China). Human IL-1β, IL-6, TNF-α, IL-10 ELISA kits were purchased from JSBOSSEN. The primary antibodies against ZO-1,Claudin-1, p65,p-p65, IKK-β, Nrf2,HO-1,NQO-1, Keap-1were from Santa Cruz or Proteintech. Nrf2 inhibitor ML385 were purchased from MCE. 2.2 Animals and treatment Ethical statement. The animal procedures in this study followed the“Principles of Laboratory Animal Care” published by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” developed by the Institute of Laboratory Animal Resources and published by the National Institutes of Health in 2011 (8th edition). All experimental procedures were conducted in strict accordance with the guidelines approved by the Animal Ethics Committee of Southwest Medical University (20240229-01). All the reported methods are in accordance with the ARRIVE guidelines. Male C57BL/6 mice (6–8 weeks old) which were obtained from Chengdu Yaokang Biotechnology Co. Ltd were used for all the studies. All mice were maintained in the room with a 12 h/12 h light/ dark cycle, constant temperature (20–25◦C) and allowed access to water and food adlibitum. After an acclimatization period of one week, the mice were grouped according to weight. Mice were divided into 4 groups, Group 1 (normal control): no DSS exposure and no treatment; Group 2 (negative control): DSS exposure with the same volume PBS treatment; Group 3–4: DSS exposure and 0.5 and 2kg/day PFOS treatment. The mice were orally gavaged with the same volume of PBS or PFOS for two weeks. And then, acute colitis was induced by administering 3% DSS salt to the mice for 7 days. In brief, DSS was dissolved in drinking water to a concentration of 3%, and the mice were allowed to drink freely. The DSS solution was replaced daily. 2.3 Histological analysis Lung tissues were fixed in 4% neutral formaldehyde for 24 h, embedded in paraffin and cut into 2 um sections. Sections dewaxed to water were stained with hematoxylin and eosin (H&E) followed by microscopic observations. The Smith score was used to evaluate lung injury[18]. Briefly, the pathological injury score of lung tissue needs to evaluate the degree of hemorrhage and edema in 6 different fields of the same section: no injury, 0 score; Damage area less than 25%, 1 score; The damage area is 25%~50%, 2 points; The damage area is 50%~75%, 3 points; More than 75% of the damage. Four points. The average total score of each group was divided into lung injury scores. 2.4 Immunohistochemical analysis After the lung sections were repaired in citrate buffer at high temperature, H2O2 was added to block the removal of endogenous enzymes. After the first antibody 4 spent the night, the second antibody was added and incubated for 45min. Diluent cell signaling11724S with DAB(kit, sigalstainR DAB Diluent cell signaling11724S); hematoxylin staining.Pictures were obtained by the light microscope (Olympus, Japan). 2.5 Immunofluorescence The lung sections were fixed with paraformaldehyde for 10min, the EDTA was used for antigen repair, BSA was added, and the serum was blocked for 30min.The first antibody was incubated at 4°C overnight, and the second antibody was incubated at room temperature for 50min. DAPI dye solution was added to lung sections and incubated at room temperature for 10min away from light. The slices are slightly dried and sealed with an anti-fluorescence attenuation sealant. The images were observed and collected under a fluorescence microscope. According to the results of immunofluorescence experiments, the nuclei stained by DAPI were blue under ultraviolet excitation, and the positive expression was luciferin labeled green. 2.6 Cell culture and cell viability assay Human normal lung epithelial cell A549(purchased from Guangzhou Rongman Biotechnology Co., LTD.) requires a complete culture medium consisting of 1% double antibody, 90%DMEM high sugar medium and 10%FBS, cultured in a 5%CO2 37℃ incubator.The optimal concentration of PFOS in LPS-induced A549 pneumonia was determined by CCK-8 method. In brief, logarithmic cells were collected and 96-well plates were inoculated at a cell density of 1,000 to 10,000 cells/well. After 24 hours of incubation, seven concentrations of PFOS were administered at 0.3125uM, 0.625uM, 1.5uM, 5uM, 10uM, 15uM and 20uM, respectively. Additionally, 10ug/ml LPS was administered for 24h. After 24h, take out the 96-well plate and add 10ul CCK-8 solution to each hole. Incubation was continued in the cell incubator, and the OD value of each well at 450nm was measured by enzyme-labeler after 1h and 2h incubation, respectively. According to OD value, the optimal concentration of oligosaccharides was selected to continue the sequence experiment. 2.7 Cell experimental A549 cells (2×10 5 cells/ml) were seeded in 6-well plates, and then the cells were cultured at 37.0 ◦C with 5 % CO2 for 24 h. Cells were treated with PFOS (5uM) for 12h, and intervened with ML385 (10uM, a specific inhibitor of Nrf2) and/or 10ug/ml LPS for 24 h. Cell suspension was collected and then stored at −80℃ for detecting antioxidant activity. Cell samples were collected for protein tests. 2.8 Western blot analysis 1 ml RIPA pyrolysis liquid containing 10ul PMSA protease inhibitor was added into 100 mg lung tissue or cells extract, and ground in ice until the tissue was fully lysed. The lysed sample was centrifuged at 4 ℃, 12,000 rpm/min, 20 min and collected the supernatant. Add 25ul loading buffer (SDS) to the supernatant, 816 metal bath was heated at 95 ℃ and boiled for 8 min. An equal amount of protein (45 μg) was loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes(PVDF). Block the membrane with 5% skim milk powder solution for 1 h and then incubated overnight at 4 ◦C with specific primary antibodies. The horseradish peroxidase (HRP)-coupled secondary antibody was then added and incubated for 1.5 h at room temperature. Bands were then visualized through chemiluminescence by using Omni-ECL™Femto Light Chemiluminescenc Kit. Protein expression was then quantified with Image J software. 2.9 Enzyme-linked immunosorbent assay The levels of inflammatory cytokines in the supernatants of A549 cells subjected to LPS and the levels of oxidative stress related indicators in the cell extract were quantified using the human ELISA kits (JSBOSSEN, Nanjing, China) as per the manufacturer’s instructions. The level of MPO in lung tissue homogenate were quantified using the mouse ELISA kits (Jiancheng, Nanjing, China). 2.10 Detection of oxidative stress related indicators activity in lung tissue The activity levels of SOD, GPX2, CAT, MDA in lung tissues were detected using biochemical detection kits according to the instructions of manufacturer, respectively. 2.11 Quantitative real-time PCR Total RNA of lung tissues was extracted by using TRIZOL reagent (Invitrogen, USA). cDNA synthesis was completed according to the PrimeScriptTM RT Reagent Kit (Takara, Japan)with gDNA Eraser (Perfect Real Time) Kit (Takara, Japan). Real-time PCR was performed using SYBR ® RT-qPCR kit (Takara, Japan) in strict accor[1]dance with the instructions. Reaction conditions: pre-denaturation (95 ℃, 2 min); Denaturation (95 ℃, 5 s); Annealing (55 ℃~ 60 ℃, 10 s); Amplification program (55 cycles); Extend (55 ℃ to 95◦℃) and analyze the melting point curve. All the primers used in this study are listed inSupplementary Table S1. 2.12 Non-targeted metabolomics analysis of cecal metabolites Weigh 50mg of each specimen into a 2 mL centrifuge tube and add a 6 mm diameter grinding bead. A 400μL mixture of methanol and water was prepared according to the 4:1 volume ratio, and 0.02 mg/mL of L-2-chlorophenylalanine was added to the mixture to prepare the extraction solution, and then added to the centrifuge tube. The sample solution was ground in a frozen tissue grinder for 6 min (-10℃, 50 Hz), and then extracted by ultrasound at low temperature for 30 min (5℃, 40 kHz). The sample was placed at -20℃ for 30 min, centrifuged for 15 min (4℃, 13000 g), and the supernatant was transferred to the injection vial with internal intubation for machine analysis. The samples were analyzed by LC-MS/MS using Thermo Field ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system. Chromatographic conditions were set: 3ul samples were separated by HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm) and then entered mass spectrometry for detection. Mobile phase A consists of 95% water +5% acetonitrile (containing 0.1% formic acid) and mobile phase B consists of 47.5% acetonitrile +47.5% isopropyl alcohol +5% water (containing 0.1% formic acid). The flow rate was 0.40 mL/min and the column temperature was 40℃. Set mass spectrum conditions: The sample quality spectrum signal is collected in positive and negative ion scanning mode, and the quality scanning range is 70-1050m/z. The flow rate of sheath gas is 50psi, the flow rate of auxiliary gas is 13psi, the heating temperature of auxiliary gas is 425℃, the positive mode ion spray voltage is set to 3500 V, the negative mode ion spray voltage is set to -3500 V, the ion transport tube temperature is 325℃, and the normalized collision energy is 20-40-60 V. The primary mass spectrometry resolution was 60000, the secondary mass spectrometry resolution was 7500, and the data were collected by DDA mode. After the completion of the computer, the LC-MS raw data was imported into the metabolomics processing software for analysis. 2.13 Statistical analysis All experiments were repeated for three or more times, and the data of each group were expressed as mean± SD or mean±SEM. SPSS21.0 was used for paired t test and one-way statistical analysis of variance. #p < 0.05, ##p < 0.01, ###p 0.05, indicating no statistical significance in pairwise comparison. 3. Results 3.1 PFOS reduced lung damage associated with DSS-induced enteritis Intestinal barrier dysfunction is a known mechanism contributing to acute lung injury (ALI) in various extrapulmonary diseases[19,20]. Given that our previous research demonstrated the protective effects of PFOS on the intestinal barrier, this study aimed to evaluate its effectiveness in mitigating lung injury associated with enteritis in a DSS-induced mouse model. First, gross examination of lung specimens revealed notable differences among the experimental groups. As shown in Fig. 1A, lungs from the control group exhibited a glossy, uniform pink appearance and elasticity. In contrast, lungs from the DSS model group appeared dark red, dull, and swollen. However, pre-administration of two doses of PFOS in the DSS-induced colitis model led to a marked improvement in lung appearance, with lungs exhibiting a pink color with scattered dark red hemorrhagic spots but retaining relative glossiness. Histopathological analysis using HE staining further confirmed that PFOS administration significantly reduced lung damage. Lungs from the colitis group showed extensive inflammatory cell infiltration, whereas PFOS-treated mice displayed a notable decrease in the histopathological scores, indicating reduced lung injury compared to the DSS model group (Fig. 1B). Additionally, immunofluorescence analysis of mucosal epithelial barrier-associated proteins, ZO-1 and Claudin-1, revealed that PFOS pre-administration reduced the accumulation of ZO-1 and Claudin-1 droplets and partially restored the original ring-like structure of the cell membrane (Fig. 1C-D). Previous studies have shown that liquid-liquid phase separation of ZO proteins drives the polymerization of tight junction proteins into a continuous belt[21]. Therefore, we postulated that PFOS may enhance the formation of tight epithelial barrier junctions by promoting ZO-1 phase separation, thereby mitigating lung injury associated with enteritis. 3.2 PFOS treatment reduced inflammatory response in lung tissues of colitis mice We further investigated the effect of PFOS on lung macrophages by analyzing the surface-specific antigen F4/80 through immunohistochemical staining. As shown in Fig. 2A, the positive rate of macrophages in the alveolar septum and peribronchus was significantly higher in the DSS group compared to the normal control group. However, PFOS administration markedly inhibited macrophage recruitment. Additionally, the accumulation of neutrophils, closely linked to increased myeloperoxidase (MPO) activity, was reflected by a dramatic elevation in MPO levels in the lung tissues of colitis mice. PFOS treatment significantly reduced these elevated MPO levels (Fig. 2B). Furthermore, mRNA expression analysis of inflammatory cytokines revealed that oral administration of DSS significantly increased IL-1β, IL-6, and TNF-α levels in lung tissues compared to the control group. Consistent with its inhibitory effect on inflammatory cell infiltration, PFOS treatment (2 mg/kg) suppressed the expression of these proinflammatory factors (Fig. 2C-F). Collectively, these findings indicate that PFOS treatment effectively alleviates lung inflammation in colitis-induced mice. 3.3 PFOS treatment reduced the oxidative stress in lung tissues of colitis mice Several studies have reported that the active ingredients of natural medicines exert protective effects against acute lung injury via the Nrf2 signaling pathway[22]. Similarly, our previous studies also confirmed the antioxidant effects of PFOS[8]. In this study, we investigated whether PFOS could inhibit colitis-induced oxidative stress in the lungs. Malondialdehyde (MDA) is a key indicator of oxidative stress and the body's antioxidant capacity. As shown in Fig. 3A, MDA levels were significantly elevated in the lung tissues of colitis mice, while PFOS treatment (2 mg/kg) substantially reduced these levels. Additionally, we found that PFOS treatment at both 0.5 mg/kg and 2mg/kg significantly restored the activity of key antioxidant enzymes, including SOD, CAT, and GPX2. PFOS also promoted the mRNA expression of these antioxidant enzymes (Fig. 3B-G). Collectively, these data indicate that PFOS attenuates colitis-induced lung injury through its antioxidant activity. 3.4 PFOS administration suppressed NF-kB signaling pathway and up regulated Nrf2 signaling pathway to reduce inflammation and oxidative stress The expression of various inflammatory cytokines is regulated by the NF-κB pathway, while Nrf2 serves as a key transcription factor mediating antioxidant signaling. Therefore, we further investigated whether PFOS could modulate the NF-κB/Nrf2 signaling pathways to reduce inflammatory responses and oxidative stress in lung tissues. As shown in Fig. 4A, oral administration of DSS in mice activated the expression of NF-κB protein in lung tissues, leading to inflammation. However, PFOS treatment significantly inhibited NF-κB expression, and phosphorylation of p65 was markedly reduced. Further analysis revealed that PFOS exerted its anti-inflammatory effects by decreasing the protein expression of IKKβ following administration of PFOS at 5 mg/kg (Fig.4A), a result confirmed by immunohistochemical staining (Fig.4B). Additionally, immunohistochemistry detected IL-1β, a downstream pro-inflammatory factor of the NF-κB pathway, and PFOS treatment significantly inhibited IL-1β expression, consistent with its anti-inflammatory effects (Fig. 4C). We also explored whether the antioxidant activity of PFOS was mediated through the regulation of the Nrf2 signaling pathway. DSS treatment significantly suppressed the expression of Nrf2, HO-1, and NQO-1 proteins in the lung tissues of colitis-induced mice (Fig.4D). However, PFOS treatment restored the expression of these Nrf2 pathway proteins. Interestingly, no statistically significant changes in Keap-1 expression were observed across all groups. Immunohistochemical staining further confirmed the expression of Nrf2 and HO-1 proteins in the lung tissues (Fig.4E-F).Taken together, these results demonstrate that PFOS alleviates lung injury by inhibiting the NF-κB signaling pathway and upregulating the Nrf2 signaling pathway. 3.5 PFOS inhibited LPS-induced epithelial barrier damages, inflammation and oxidative stress in lung epithelial cells LPS is known to damage the epithelial barrier by inducing the rapid redistribution and reducing the expression of ZO-1 in tight junctions[23]. Additionally, LPS is commonly used to establish in vitro models of inflammation and oxidative stress[24]. In this study, we developed an in vitro model using LPS-induced lung epithelial cells (A549) to verify the efficacy and mechanisms of PFOS in mitigating epithelial barrier damage, inflammation, and oxidative stress in the lungs(Fig. 5A). The CCK8 assay was used to assess cell viability after PFOS treatment. The results showed that PFOS at a concentration of 10 mM displayed a downward trend in viability, with 20 mM demonstrating significant toxicity compared to the control group (Fig. 5B). Based on these findings, we selected 5 mM PFOS for subsequent experiments. Since Nrf2 is known to negatively regulate the NF-κB pathway[25], we included an Nrf2 inhibitor (ML385) to preliminarily explore the crosstalk between Nrf2 and NF-κB signaling. Consistent with previous research, LPS exposure significantly reduced the expression of the tight junction proteins ZO-1 and Claudin-1 in A549 cells (Fig. 5C). However, PFOS treatment partially restored the expression of these proteins, while the protective effect was significantly inhibited by the Nrf2 inhibitor (Fig. 5C). As shown in Fig. 5D, LPS exposure also significantly increased the production of myeloperoxidase (MPO), a marker of neutrophil activation, as well as the proinflammatory cytokines TNF-α, IL-1β, and IL-6. PFOS treatment in LPS-stimulated A549 cells markedly reduced the levels of these cytokines and MPO. Given that the NF-κB pathway is a critical regulator of inflammation, we investigated the changes in NF-κB signaling through Western blot analysis. PFOS treatment significantly suppressed the expression of p65, p-p65, and IKK-β in LPS-stimulated A549 cells (Fig. 5E). Although the addition of the Nrf2 inhibitor resulted in a downward trend in NF-κB-related protein expression, the changes were not statistically significant (Fig. 5E). These results suggest that the anti-inflammatory effects of PFOS are closely related to the inhibition of NF-κB pathway activation, with potential signaling crosstalk between Nrf2 and NF-κB, though this crosstalk may not involve direct negative regulation of NF-κB by Nrf2. To further explore the antioxidant effects of PFOS, we measured the activity of several antioxidant enzymes in cell extracts. LPS exposure led to a decrease in the activities of SOD, GPX2, and CAT, while increasing malondialdehyde (MDA) levels in the cell supernatant (Fig. 5F). PFOS treatment significantly reduced MDA expression and enhanced the activity of SOD, GPX2, and CAT, though these effects were almost completely blocked by the Nrf2 inhibitor ML385 (Fig. 5F). Subsequent Western blot analysis showed that the antioxidant effect of PFOS was mediated by activation of the Nrf2 signaling pathway. As shown in Fig. 5G, LPS significantly inhibited the protein expression of Nrf2, HO-1, and NQO-1. In contrast, PFOS treatment restored the expression of these proteins to levels close to those of the normal control, and the use of ML385 confirmed that the antioxidant effects of PFOS depend on the Nrf2 pathway. Collectively, these results demonstrate that PFOS alleviates LPS-induced epithelial barrier damage, inflammation, and oxidative stress in A549 cells by inhibiting the NF-κB pathway and upregulating the Nrf2 signaling pathway. 3.6 Intestinal metabolites affected PFOS exhibits potential correlations to the lung inflammation In previous studies, oligosaccharides were found to partially restore imbalances in the intestinal flora in a DSS-induced enteritis mouse model[6]. In the present study, non-targeted fecal metabolomics was employed to investigate the effects of PFOS pre-intervention on intestinal metabolites in mice, aiming to determine whether the pulmonary protective effects of PFOS are related to changes in intestinal metabolites. First, principal component analysis (PCA) revealed varying degrees of inter-group differences among stool samples across different experimental groups. Notably, the distribution pattern of intestinal metabolites after PFOS intervention was more similar to that of the normal control group than the model group (Fig. 6A-B). Further pairwise comparisons were conducted using orthogonal partial least squares discriminant analysis (OPLS-DA). Permutation tests were performed, and differential metabolites were identified using selection criteria (p1.0). These metabolites were visualized in S-plots and volcano plots, highlighting those with significant differences (Fig. 7A-E). Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted for the significantly altered metabolites. As illustrated in Fig. 7F, compared with the normal control group, the metabolites in the model group were significantly enriched in pathways related to amino acid metabolism (five pathways) and lipid metabolism (three pathways). These results indicate that DSS intervention induced significant disturbances in amino acid and lipid metabolism in mice with enteritis. However, PFOS intervention ameliorated the DSS-induced disruptions in these metabolic processes (Fig. 8A-E). It is worth noting that PFOS intervention also resulted in the enrichment of differentially expressed metabolites in three key signaling pathways: cAMP, PPAR, and TRP, all of which are known to be involved in the regulation of NF-κB and Nrf2 signaling pathways[26-28] (Fig. 8F). Cluster analysis of the significantly altered metabolites across all groups was performed, and a heatmap was generated to visually represent the expression levels of these compounds in each group. As shown in Fig. 9, the metabolic profile of the DSS model group was distinctly separated from that of the normal control group and the PFOS pre-intervention group. Furthermore, the metabolic profiles of the normal control and PFOS pre-intervention groups were similar, consistent with previous findings.In conclusion, PFOS intervention mitigates DSS-induced metabolic disorders in mice and may play a role in regulating inflammation and oxidative stress through alterations in key metabolites. 4. Discussion UC is a systemic inflammatory condition known to affect various organs, including the lungs, with nearly half of UC patients exhibiting low-grade respiratory tract inflammation[29,30]. The gut-lung crosstalk is thought to involve microbial products, metabolites, mucosal barrier dysfunction, and lymphocyte homing[31,32]. In this study, a DSS-induced enteritis mouse model was used to assess lung tissue damage, confirming lung injury consistent with previous research[33].PFOS, previously shown to protect against intestinal and pulmonary inflammation[7,8], was administered as a pre-intervention. Results demonstrated that PFOS, particularly at higher doses, reduced lung injury scores in the mice (Figure 1B). DSS-induced disruption of tight junction proteins ZO-1 and Claudin-1, critical for epithelial barrier integrity[34,35], was mitigated by PFOS, which restored the membrane structure of these proteins and reduced droplet-like accumulations observed in DSS-treated mice (Figure 1C-D). Moreover, PFOS increased the expression of ZO-1 and Claudin-1 in cell experiments compared to the LPS model group (Figure 5C), suggesting that it may enhance tight junction formation by regulating protein phase separation and expression. This, in turn, could restore epithelial barrier function and alleviate DSS-induced lung injury. Inflammation plays a critical role in compromising epithelial barriers in both the lungs and intestines. When these barriers are damaged, bacterial toxins like lipopolysaccharide (LPS) can enter the systemic circulation, intensifying immune responses in distant tissues such as the lungs[24]. This study focused on the effects of PFOS on lung inflammation associated with enteritis. In the DSS-induced colitis model, F4/80 immunohistochemical staining, a marker for macrophage infiltration, and myeloperoxidase (MPO), a neutrophil infiltration marker, were significantly elevated in the lung tissues of the model group. However, PFOS (2 mg/kg) treatment notably reduced the infiltration of both macrophages and neutrophils (Figure 2A-B). Additionally, PFOS significantly suppressed mRNA transcription of pro-inflammatory cytokines in lung tissues of mice and reduced the expression of pro-inflammatory proteins in human lung epithelial cell line A549 (Figure 2C-F). Given that NF-κB is a key transcription factor responsible for activating inflammatory mediators[36], we further explored the involvement of the NF-κB signaling pathway in the mechanism of PFOS. NF-κB activation typically depends on IκB kinase (IKK) activity[37]. Our findings showed that PFOS markedly decreased the expression of IKK, p65, and phosphorylated p65 (p-p65), thereby inhibiting NF-κB signaling activation in both the lung tissue of mice with enteritis-associated lung injury and LPS-induced lung epithelial cell injury (Figure 4A and Figure5E).Here, PFOS attenuates lung inflammation by downregulating the NF-κB/IKK signaling pathway, providing a protective effect against pneumonia in mice with colitis. From a molecular pathology perspective, the accumulation of excessive reactive oxygen species (ROS) in lung tissue is often associated with the activation of pro-inflammatory signaling pathways[38]. In this study, both in vivo and in vitro experiments demonstrated that PFOS treatment significantly reduced the activity of the lipid peroxidation marker malondialdehyde (MDA). Additionally, PFOS intervention upregulated the transcription and activity of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase 2 (GPX2) (Figure 3 and Figure5F).Nuclear factor erythroid 2-related factor 2 (Nrf2), a central transcription factor involved in the regulation of the body’s antioxidant defense mechanisms, controls the expression of these antioxidant enzymes[39]. We further investigated the effect of PFOS on the Nrf2 signaling pathway. Our results confirmed that PFOS significantly increased the protein expression of Nrf2 and its downstream targets, heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO-1). Notably, PFOS-induced activation of Nrf2 was independent of Kelch-like ECH-associated protein 1 (Keap1), which traditionally regulates Nrf2. Previous studies have demonstrated that Nrf2 activation can occur independently of Keap1, with protein kinases playing a crucial role in modulating Nrf2 activity[40].These findings suggest that PFOS exerts its protective effects, in part, by activating the Nrf2 signaling pathway (Figure 4D and Figure5G). Furthermore, evidence indicates crosstalk between the Nrf2 and NF-κB pathways. The Nrf2 pathway can suppress NF-κB activation and inhibit NF-κB-mediated transcription, while NF-κB signaling can downregulate Nrf2 activity[41]. To explore this interaction further, we used the Nrf2 inhibitor ML385 to investigate whether PFOS affects both pathways. The results confirmed that PFOS-mediated lung protection is closely related to Nrf2 activation, and consistent with previous findings, Nrf2 negatively regulated NF-κB signaling (Figure5). Finally, it is important to note that intestinal metabolites play a significant role in modulating immune responses in lung diseases[42]. In this study, non-targeted fecal metabolomics analysis revealed substantial changes in the metabolites of DSS-induced enteritis mice. KEGG pathway enrichment analysis showed that these altered metabolites were mainly involved in amino acid metabolism, lipid metabolism, and mineral absorption, indicating that DSS-induced enteritis caused disruptions in amino acid and lipid metabolism (Figure7F). After PFOS pre-intervention, these metabolic imbalances were ameliorated. Of particular interest (Figure8F), several of the significantly altered metabolites were enriched in pathways related to cAMP, peroxisome proliferator-activated receptor (PPAR), and transient receptor potential (TRP), all of which are known to regulate NF-κB and Nrf2 signaling[26-28]. Taken together, these findings suggest that PFOS not only affects intestinal metabolites but also modulates key signaling pathways, supporting the hypothesis that PFOS may function as a next-generation prebiotic. This is in line with previous research indicating that PFOS positively impacts the gut microbiota[6], further highlighting its potential therapeutic role. 5. Conclusions In conclusion, this study shows that PFOS reduces lung tissue damage associated with enteritis by protecting the epithelial barrier and regulating the Nrf2/NF-κB signaling pathways (Fig.10). PFOS upregulates antioxidant proteins (Nrf2, HO-1, NQO-1) and suppresses pro-inflammatory markers (p65, p-p65, IKK-β), indicating its antioxidant and anti-inflammatory effects. Additionally, PFOS’s impact on intestinal metabolites may contribute to its regulation of inflammation and oxidative stress, further protecting lung tissue. These findings suggest PFOS as a potential therapeutic agent for lung injury linked to enteritis. Abbreviations The following abbreviations are used in this manuscript: UC, ulcerative colitis; PFOS, Oligosaccharides from Polygonatum cyrtonema Hua; DSS, dextran sodium sulfate; LPS, lipopolysaccharide; IBD, inflammatory bowel disease; Nrf2, nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; COPD, chronic obstructive pulmonary disease; GPx, glutathione peroxidase; GSH, glutathione; HO-1, heme oxygenase-1; H&E, hematoxylin and eosin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride membranes; HRP. horseradish peroxidase; ALI ,acute lung injury; MPO, myeloperoxidase; MDA, Malondialdehyde; PCA, principal component analysis; OPLS-DA, orthogonal partial least squares discriminant analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; IKK, IκB kinase, p-p65, phosphorylated p65; NQO-1, NAD(P)H quinone dehydrogenase 1; PPAR, peroxisome proliferator-activated receptor; TRP, transient receptor potential. Declarations CRediT authorship contribution statement Jin Xu: Investigation, Data curation, Visualization, Writing original draft. Chuangkang Tang: Investigation, Visualization, Writing original draft. Yuqian He: Investigation, Data curation. Yu Lu: Investigation,Visualization. Pei Luo: Supervision, Writing - review & editing. Jianbo Wu: Funding acqui[1]sition, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing finan[1]cial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the Sichuan Province Science and Technology Agency Grant (2024NSFSC0591)，and Luzhou-Southwest Medical University University's scientific research project (2018-ZRQN-159). Compliance with Ethics Requirements All Institutional and National Guidelines for the care and use of animals were followed. 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The role of Nrf2 in the pathogenesis and treatment of ulcerative colitis.Front Immunol. Jun 5 :141200111. (2023). Eman Casper. The crosstalk between Nrf2 and NF-κB pathways in coronary artery disease: Can it be regulated by SIRT6? Life Sci. Oct 1 :330:122007. 10.1016/j.lfs.2023.122007 . (2023). Epub 2023 Aug 5. Ma, P. J., Wang, M. M. & Wang, Y. Gut microbiota: A new insight into lung diseases. Biomed. Pharmacother 2022 Nov :155:113810. 10.1016/j.biopha.2022.113810 . Epub 2022 Oct 8. Additional Declarations No competing interests reported. Supplementary Files TableS1.doc WBrawdata20250121141859.pdf 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6166677","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":442406677,"identity":"0b9b4bbe-28c1-4c02-91f3-e25cdbb9a95f","order_by":0,"name":"Jin Xu","email":"","orcid":"","institution":"Luzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Xu","suffix":""},{"id":442406678,"identity":"2679e3b2-4898-4df4-bffe-8968a5b6b773","order_by":1,"name":"Chuankang Tang","email":"","orcid":"","institution":"the Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chuankang","middleName":"","lastName":"Tang","suffix":""},{"id":442406679,"identity":"f6897de6-99ec-4cca-bc0d-b21e92ab0c56","order_by":2,"name":"Qianyu He","email":"","orcid":"","institution":"Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qianyu","middleName":"","lastName":"He","suffix":""},{"id":442406680,"identity":"d47441ec-c091-4fdc-b830-c5193a957c19","order_by":3,"name":"Yu Lu","email":"","orcid":"","institution":"Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Lu","suffix":""},{"id":442406681,"identity":"c3e26ef1-2f5d-4a01-9fbb-b07ebffdf8f5","order_by":4,"name":"Pei Luo","email":"","orcid":"","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Luo","suffix":""},{"id":442406683,"identity":"863a21c5-b923-447f-a850-0253cb1c9d80","order_by":5,"name":"Jianbo Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYBACxhlgyoaHn7+BNC1pMpIzDhBrjQSYPGxj0JBApA7m2T1mEj93nOcxYDjA+OFjDjEOm3PGTLL3zG0ec+YGZsmZ24jRMiN3mzRj220ey4YDbMy8JGg5x2NwIIE0LQdI0TLn/GfL3rZkHskZB5uJ84vh7LbEGz/b7Oz5+ZsPfvhIlJYGhIUNOFWhAHnilI2CUTAKRsGIBgAeIzT+J4xc+AAAAABJRU5ErkJggg==","orcid":"","institution":"Southwest Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jianbo","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-03-06 03:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6166677/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6166677/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81413213,"identity":"26748972-9d76-44be-a666-fefcc1d687ae","added_by":"auto","created_at":"2025-04-25 21:43:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":534071,"visible":true,"origin":"","legend":"\u003cp\u003ePFOS reduced lung damage associated with DSS-induced enteritis. (A) Representative pictures of lung macrograph.(B) The most representative HE staining diagram (magnification 20× and 100×) of each experimental group showed typical lung tissue injury changes, including alveolar structure rupture and fusion, a large number of inflammatory cells and red blood cell infiltration, pulmonary interstitial hyperemia and edema, and diffuse thickening.The bar chart shows the pathological score of lung tissues.(C/D) Representative immunofluorescence staining of ZO-1 and Claudin-1 in the colon sections as indicated(bar, 20um and 10um). n = 5–8/group. Data shown as mean ± SEM. #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/20c13180e2938b4d9851d5ad.jpg"},{"id":81412764,"identity":"02901651-c473-45db-9c6d-d601b5e21be8","added_by":"auto","created_at":"2025-04-25 21:35:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":298331,"visible":true,"origin":"","legend":"\u003cp\u003ePFOS treatment reduced inflammatory response in lung tissues of colitis mice. (A) IHC-staining images of macrophage-related protein F4/80 in lung tissues (bar, 200um and 50um). (B) The MPO level in lung tissue (n = 5–6/group). (C-F) Impact of PFOS on inflammation-related genes expression (n = 5). Data shown as mean ± SEM. #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/199701e5784c796fc2df07be.jpg"},{"id":81412773,"identity":"4e61ed32-a904-466d-ae24-2b3ffb11ee00","added_by":"auto","created_at":"2025-04-25 21:35:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159083,"visible":true,"origin":"","legend":"\u003cp\u003ePFOS treatment reduced oxidative stress in lung tissues of colitis mice. (A) The activity of membrane lipid peroxidation (MDA) in lung tissue. (B-D) Changes in the activity of several typical antioxidant enzymes (SOD,GPX2,CAT) (n = 5/each group). (C-G) Oxidative stress genes amelioration by PFOS analysis by qPCR (n = 5/each group). Data shown as mean ± SEM. #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/feb33807d85641372fd32361.jpg"},{"id":81412763,"identity":"e9496da8-01f1-43b5-8b01-abbb50003206","added_by":"auto","created_at":"2025-04-25 21:35:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":562540,"visible":true,"origin":"","legend":"\u003cp\u003ePFOS administration suppressed NF-kB signaling pathway and up regulated Nrf2 signaling pathway to reduce inflammation and oxidative stress (A) WB bands and relative protein expressions of NFkB signal path in lung tissues. (n ≥3/each group) (B) IHC-staining images of IKK-β and IL-1β protein in lung tissues (bar, 200um and 50um)(n = 5-6/each group). (C) WB bands and relative protein expressions of Nrf2 signal path in lung tissues. (n ≥3/each group). (D) IHC-staining images of Nrf2 and HO-1 protein in lung tissues (bar, 200um and 50um)(n = 5-6/each group). Data shown as mean ± SEM. #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/39079336c325e6766f9f221a.jpg"},{"id":81413214,"identity":"7ab12897-7e32-4bed-a37d-798b566c8e45","added_by":"auto","created_at":"2025-04-25 21:43:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":705844,"visible":true,"origin":"","legend":"\u003cp\u003ePFOS inhibited LPS-induced epithelial barrier damages, inflammation and oxidative stress in lung epithelial cells (A549 cells) . (A) Schematic diagram of PFOS treatment in LPS-induced A549 cells. (B) Cell viability was detected by CCK8 assay (n = 3). (C) WB bands and relative protein expressions of epithelial barrier proteins ( ZO-1 and Claudin-1) in the cells . (D) Levels of MPO in the cell extract and TNF-α, IL-1β and IL-6 in the supernatants. (E) WB bands and relative protein expressions of NFkB signal path. (F) Activity of MDA and several antioxidant enzymes in cell extract. (G) WB bands and relative protein expressions of Nrf2 signal path. Data shown as mean ± SEM. #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/c5df602f10581519fc448ff0.jpg"},{"id":81413211,"identity":"86d0e01b-2d01-48d9-9a9f-fc8fa74b18ad","added_by":"auto","created_at":"2025-04-25 21:43:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":97625,"visible":true,"origin":"","legend":"\u003cp\u003eRaw data preprocessing and principal component analysis. (A/B) Cationic and anionic PCA analysis of fecal samples from normal control group (a), DSS model group (b) and flavispermine oligosaccharide pre-intervention group.\u003c/p\u003e","description":"","filename":"17.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/9337c6be5588a9a9c0795f52.jpg"},{"id":81412807,"identity":"85ab66af-dfa0-490a-9d06-df576d9e641a","added_by":"auto","created_at":"2025-04-25 21:35:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":288855,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of different metabolites between DSS model group and normal control group. A and B represent cationic and anionic OPLS-DA scores of normal control group (a) compared with DSS model group (b); C and D represent S-plots in positive and negative ion modes generated by differential metabolites according to p\u0026lt;0.05, VIP\u0026gt;1.0 conditions; E represents the volcanic map of the difference metabolites compared between the two groups, the blue dot indicates the difference metabolites down-regulated, the red dot indicates the difference metabolites up-regulated, the more the left and right side and the upper point the difference in expression is more significant; F represents KEGG pathway enrichment analysis of metabolites in normal control group was significantly different from DSS model group(The red and blue boxes indicate amino acid and lipid metabolic pathways, respectively).\u003c/p\u003e","description":"","filename":"18.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/89881329391c7d15f2e4d5dd.jpg"},{"id":81412767,"identity":"a77b8ed0-ebc3-4013-b6e4-56a7bfb628d3","added_by":"auto","created_at":"2025-04-25 21:35:36","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":315808,"visible":true,"origin":"","legend":"\u003cp\u003eMultivariate statistical analysis of fecal metabolites of mice in PFOS preintervention group and DSS model group. A and B represent the cationic and anionic OPLS-DA scores of the PFOS pre-intervention group (c) and the DSS model group (b). C and D represent S-plots in positive and negative ion modes generated by differential metabolites according to p\u0026lt;0.05, VIP\u0026gt;1.0 conditions; E represents the volcanic map of the difference metabolites compared between the two groups, the blue dot indicates the difference metabolites down-regulated, the red dot indicates the difference metabolites up-regulated, the more the left and right side and the upper point the difference in expression is more significant; F represents KEGG pathway enrichment analysis of metabolites in in PFOS preintervention group was significantly different from DSS model group (The red and blue boxes indicate amino acid metabolic pathways and inflammatory regulatory pathway, respectively).\u003c/p\u003e","description":"","filename":"19.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/285ea81d8bf117c5f26f6461.jpg"},{"id":81412780,"identity":"9ceabcfb-aa0b-47b2-a786-90d492879caf","added_by":"auto","created_at":"2025-04-25 21:35:37","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":229960,"visible":true,"origin":"","legend":"\u003cp\u003eThe two different metabolites were combined and analyzed by clustering of different metabolites.\u003c/p\u003e","description":"","filename":"110.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/71534ff3a4d729ba8934a3b2.jpg"},{"id":81412786,"identity":"ae39fd21-94d0-48e8-a97a-fd1a2110e817","added_by":"auto","created_at":"2025-04-25 21:35:37","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":277918,"visible":true,"origin":"","legend":"\u003cp\u003eThe efficacy and mechanism of PFOS on the lung inflammation and epithelial barrier damages in the treatment of lung injury linked to enteritis.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/70ca9bb9789d0718dbb89333.jpg"},{"id":82711953,"identity":"6238ec2f-2b2b-4000-89f0-7f6750d5ffa5","added_by":"auto","created_at":"2025-05-14 11:38:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4072552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/9db5eeec-d889-464d-934f-135750907219.pdf"},{"id":81412758,"identity":"afa17444-d069-43c4-83e3-53dc5f50b1f6","added_by":"auto","created_at":"2025-04-25 21:35:36","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22653,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.doc","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/d91fa988cf8042709cba0a83.doc"},{"id":81412776,"identity":"cb745e8d-2873-46c6-b87c-f2f8b0d17fa8","added_by":"auto","created_at":"2025-04-25 21:35:37","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2773675,"visible":true,"origin":"","legend":"","description":"","filename":"WBrawdata20250121141859.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6166677/v1/292b36f6ab09ff910a8e6575.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oligosaccharides from Polygonatum cyrtonema Hua ameliorate colitis-induced lung injury via inhibiting inflammation and oxidative stress","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe \"Lung-Intestinal Axis\" links the lungs and intestines through a shared developmental origin, common mucosal immune system, similar immunomodulatory mechanisms, and a shared molecular foundation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mechanisms like inflammatory mediator circulation, gut microbiota dysbiosis, and oxidative stress play crucial roles in this axis, linking lung and intestinal health[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These mechanisms have implications for understanding diseases that affect both the lungs and the intestines, such as those triggered by infections or chronic inflammation[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003ePolygonatum\u003c/em\u003e has long been recognized for its medicinal and nutritional value, being widely utilized as a traditional tonic food and medicinal ingredient across various countries, including China, Pakistan, Iran, Japan, and others, despite regional cultural differences [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The primary active compounds identified in \u003cem\u003ePolygonatum\u003c/em\u003e species include steroidal saponins, flavonoids, polysaccharides, and alkaloids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In our recent work, we successfully isolated and characterized oligosaccharides from \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e Hua (PFOS), and explored several of their biological activities[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Notably, PFOS were shown to mitigate intestinal damage induced by dextran sodium sulfate (DSS), with mechanisms involving anti-inflammatory and antioxidant effects [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, PFOS significantly reduced mortality in mice with lipopolysaccharide (LPS)-induced peritonitis and ameliorated the associated acute lung injury [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In clinical work, the interplay between gut and lung health has garnered increasing attention, particularly in conditions where symptoms manifest across both systems. For example, an analysis of clinical characteristics in 140 patients with SARS-CoV-2 infection in Wuhan revealed that 39.6% of patients exhibited gastrointestinal symptoms, underscoring the gut-lung axis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Similarly, data from the RHINE study indicated that individuals with inflammatory bowel disease (IBD), particularly females with ulcerative colitis (UC), exhibited a higher prevalence of asthma and respiratory symptoms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, researchers have demonstrated that DSS-induced colitis in genetically susceptible models is sufficient to induce lung inflammation and tissue damage, highlighting the potential for developing therapeutic strategies aimed at reducing comorbidities in IBD patients[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].Given our previous findings on the protective effects of PFOS in both gut and lung injuries, we sought to further investigate its potential effects on lung injury associated with enteritis.\u003c/p\u003e \u003cp\u003eResearches indicated that nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in inhibiting ferroptosis, thereby alleviating intestinal ischemia/reperfusion-induced acute lung injury[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, cigarette smoke has been found to promote the production of endogenous reactive oxygen species (ROS) in patients with chronic obstructive pulmonary disease (COPD), potentially exacerbating intestinal disorders[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These findings suggest that Nrf2 may have a pivotal role in diseases involving gut-lung crosstalk. Nrf2 is now widely recognized to regulate over 200 genes encoding enzymes involved in antioxidant defense mechanisms, including glutathione peroxidase (GPx), glutathione (GSH), and heme oxygenase-1 (HO-1) synthesis[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, its role in innate immunity and the regulation of immune-related gene expression remains less well understood. Emerging evidence indicates that activation of the Nrf2/HO-1 signaling pathway exerts significant anti-inflammatory effects[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Notably, sinomenine has been shown to modulate macrophage polarization toward the M2 phenotype in an Nrf2-dependent manner, while inhibiting IκBα phosphorylation and nuclear translocation of NF-κB, thus identifying the upstream target of sinomenine in modulating NF-κB activity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, basing on the findings from previous studies, we established a mouse model of DSS-induced enteritis-associated lung injury to investigate the effects of PFOS on lung pathology in this context. The study employed various biotechnological approaches to assess the efficacy and underlying mechanisms of PFOS, with a particular focus on oxidative stress and inflammatory responses. Additionally, we developed cellular models using lung epithelial cells to further validate the efficacy and elucidate the mechanisms of action of PFOS in this system.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cem\u003e2.1 Materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe preparation of PFOS was provided as the previous report[17]. Dextran sulfate sodium (DSS) was obtained from Yuanye Biotechnology Co. Ltd. (S14049, molecular weight: 50,000, Shanghai, China). CAT assay kits, SOD assay kits, MDA assay kits and GPX2 assay kits were purchased from JSBOSSEN Biotechnology Company (Nanjing, China) or from Jiancheng Biotechnology Company (Nanjing, China). \u0026nbsp;Human IL-1β, IL-6, TNF-α, IL-10 ELISA kits were purchased from JSBOSSEN. The primary antibodies against ZO-1,Claudin-1, p65,p-p65, IKK-β, Nrf2,HO-1,NQO-1, Keap-1were from Santa Cruz or Proteintech. Nrf2 inhibitor ML385 were purchased from MCE.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2 Animals and treatment\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; Ethical statement. The animal procedures in this study followed the“Principles of Laboratory Animal Care”\u0026nbsp;published by the National Society for Medical Research and the\u0026nbsp;“Guide for the Care and Use of Laboratory Animals”\u0026nbsp;developed by the Institute of Laboratory Animal Resources and published by the National Institutes of Health in 2011 (8th edition). All experimental procedures were conducted in strict accordance with the guidelines approved by the Animal Ethics Committee of Southwest Medical University (20240229-01). All the reported methods are in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003eMale C57BL/6 mice (6–8 weeks old) which were obtained from Chengdu Yaokang Biotechnology Co. Ltd were used for all the studies. All mice were maintained in the room with a 12 h/12 h light/ dark cycle, constant temperature (20–25◦C) and allowed access to water and food adlibitum. After an acclimatization period of one week, the mice were grouped according to weight. Mice were divided into 4 groups, Group 1 (normal control): no DSS exposure and no treatment; Group 2 (negative control): DSS exposure with the same volume PBS treatment; Group 3–4: DSS exposure and 0.5 and 2kg/day PFOS treatment. The mice were orally gavaged with the same volume of PBS or PFOS for two weeks. And then, acute colitis was induced by administering 3% DSS salt to the mice for 7 days. In brief, DSS was dissolved in drinking water to a concentration of 3%, and the mice were allowed to drink freely. The DSS solution was replaced daily.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3 Histological analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLung tissues were fixed in 4% neutral formaldehyde for 24 h, embedded in paraffin and cut into 2 um sections. Sections dewaxed to water were stained with hematoxylin and eosin (H\u0026amp;E) followed by microscopic observations. The Smith score was used to evaluate lung injury[18]. Briefly, the pathological injury score of lung tissue needs to evaluate the degree of hemorrhage and edema in 6 different fields of the same section: no injury, 0 score; Damage area less than 25%, 1 score; The damage area is 25%~50%, 2 points; The damage area is 50%~75%, 3 points; More than 75% of the damage. Four points. The average total score of each group was divided into lung injury scores.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4 Immunohistochemical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter the lung sections were repaired in citrate buffer at high temperature, H2O2 was added to block the removal of endogenous enzymes. After the first antibody 4 spent the night, the second antibody was added and incubated for 45min. Diluent cell signaling11724S with DAB(kit, sigalstainR DAB Diluent cell signaling11724S); hematoxylin staining.Pictures were obtained by the light microscope (Olympus, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5 Immunofluorescence\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe lung sections were fixed with paraformaldehyde for 10min, the EDTA was used for antigen repair, BSA was added, and the serum was blocked for 30min.The first antibody was incubated at 4°C overnight, and the second antibody was incubated at room temperature for 50min. DAPI dye solution was added to lung sections and incubated at room temperature for 10min away from light. The slices are slightly dried and sealed with an anti-fluorescence attenuation sealant. The images were observed and collected under a fluorescence microscope. According to the results of immunofluorescence experiments, the nuclei stained by DAPI were blue under ultraviolet excitation, and the positive expression was luciferin labeled green.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6 Cell culture and cell viability assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHuman normal lung epithelial cell A549(purchased from Guangzhou Rongman Biotechnology Co., LTD.) requires a complete culture medium consisting of 1% double antibody, 90%DMEM high sugar medium and 10%FBS, cultured in a 5%CO2 37℃ incubator.The optimal concentration of PFOS in LPS-induced A549 pneumonia was determined by CCK-8 method. In brief, logarithmic cells were collected and 96-well plates were inoculated at a cell density of 1,000 to 10,000 cells/well. After 24 hours of incubation, seven concentrations of PFOS were administered at 0.3125uM, 0.625uM, 1.5uM, 5uM, 10uM, 15uM and 20uM, respectively. Additionally, 10ug/ml LPS was administered for 24h. After 24h, take out the 96-well plate and add 10ul CCK-8 solution to each hole. Incubation was continued in the cell incubator, and the OD value of each well at 450nm was measured by enzyme-labeler after 1h and 2h incubation, respectively. According to OD value, the optimal concentration of oligosaccharides was selected to continue the sequence experiment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.7 Cell experimental\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA549 cells (2×10\u003csup\u003e5\u003c/sup\u003e cells/ml) were seeded in 6-well plates, and then the cells were cultured at 37.0 ◦C with 5 % CO2 for 24 h. Cells were treated with PFOS (5uM) for 12h, and intervened with ML385 (10uM, a specific inhibitor of Nrf2) and/or 10ug/ml LPS for 24 h. Cell suspension was collected and then stored at −80℃ for detecting antioxidant activity. Cell samples were collected for protein tests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.8 Western blot analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e1 ml RIPA pyrolysis liquid containing 10ul PMSA protease inhibitor was added into 100 mg lung tissue or cells extract, and ground in ice until the tissue was fully lysed. The lysed sample was centrifuged at 4 ℃, 12,000 rpm/min, 20 min and collected the supernatant. Add 25ul loading buffer (SDS) to the supernatant, 816 metal bath was heated at 95 ℃ and boiled for 8 min. An equal amount of protein (45 μg) was loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes(PVDF). Block the membrane with 5% skim milk powder solution for 1 h and then incubated overnight at 4 ◦C with specific primary antibodies. The horseradish peroxidase (HRP)-coupled secondary antibody was then added and incubated for 1.5 h at room temperature. Bands were then visualized through chemiluminescence by using Omni-ECL™Femto Light Chemiluminescenc Kit. Protein expression was then quantified with Image J software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.9 Enzyme-linked immunosorbent assay\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe levels of inflammatory cytokines in the supernatants of A549 cells subjected to LPS and the levels of oxidative stress related indicators in the cell extract were quantified using the human ELISA kits (JSBOSSEN, Nanjing, China) as per the manufacturer’s instructions. The level of MPO in lung tissue homogenate were quantified using the mouse ELISA kits (Jiancheng, Nanjing, China).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.10 Detection of oxidative stress related indicators activity in lung tissue\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The activity levels of SOD, GPX2, CAT, MDA in lung tissues were detected using biochemical detection kits according to the instructions of manufacturer, respectively.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003e2.11 Quantitative real-time PCR\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA of lung tissues was extracted by using TRIZOL reagent (Invitrogen, USA). cDNA synthesis was completed according to the PrimeScriptTM RT Reagent Kit (Takara, Japan)with gDNA Eraser (Perfect Real Time) Kit (Takara, Japan). Real-time PCR was performed using SYBR ® RT-qPCR kit (Takara, Japan) in strict accor[1]dance with the instructions. Reaction conditions: pre-denaturation (95 ℃, 2 min); Denaturation (95 ℃, 5 s); Annealing (55 ℃~ 60 ℃, 10 s); Amplification program (55 cycles); Extend (55 ℃ to 95◦℃) and analyze the melting point curve. All the primers used in this study are listed inSupplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.12 Non-targeted metabolomics analysis of cecal metabolites\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWeigh 50mg of each specimen into a 2 mL centrifuge tube and add a 6 mm diameter grinding bead. A 400μL mixture of methanol and water was prepared according to the 4:1 volume ratio, and 0.02 mg/mL of L-2-chlorophenylalanine was added to the mixture to prepare the extraction solution, and then added to the centrifuge tube. The sample solution was ground in a frozen tissue grinder for 6 min (-10℃, 50 Hz), and then extracted by ultrasound at low temperature for 30 min (5℃, 40 kHz). The sample was placed at -20℃ for 30 min, centrifuged for 15 min (4℃, 13000 g), and the supernatant was transferred to the injection vial with internal intubation for machine analysis. The samples were analyzed by LC-MS/MS using Thermo Field ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system. Chromatographic conditions were set: 3ul samples were separated by HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm) and then entered mass spectrometry for detection. Mobile phase A consists of 95% water +5% acetonitrile (containing 0.1% formic acid) and mobile phase B consists of 47.5% acetonitrile +47.5% isopropyl alcohol +5% water (containing 0.1% formic acid). The flow rate was 0.40 mL/min and the column temperature was 40℃. Set mass spectrum conditions: The sample quality spectrum signal is collected in positive and negative ion scanning mode, and the quality scanning range is 70-1050m/z. The flow rate of sheath gas is 50psi, the flow rate of auxiliary gas is 13psi, the heating temperature of auxiliary gas is 425℃, the positive mode ion spray voltage is set to 3500 V, the negative mode ion spray voltage is set to -3500 V, the ion transport tube temperature is 325℃, and the normalized collision energy is 20-40-60 V. The primary mass spectrometry resolution was 60000, the secondary mass spectrometry resolution was 7500, and the data were collected by DDA mode. After the completion of the computer, the LC-MS raw data was imported into the metabolomics processing software for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.13 Statistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were repeated for three or more times, and the data of each group were expressed as mean± SD or mean±SEM. SPSS21.0 was used for paired t test and one-way statistical analysis of variance. #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, indicating that pairwise comparison was statistically significant; ns p \u0026gt; 0.05, indicating no statistical significance in pairwise comparison.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1 PFOS reduced lung damage associated with DSS-induced enteritis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIntestinal barrier dysfunction is a known mechanism contributing to acute lung injury (ALI) in various extrapulmonary diseases[19,20]. Given that our previous research demonstrated the protective effects of PFOS on the intestinal barrier, this study aimed to evaluate its effectiveness in mitigating lung injury associated with enteritis in a DSS-induced mouse model. First, gross examination of lung specimens revealed notable differences among the experimental groups. As shown in Fig. 1A, lungs from the control group exhibited a glossy, uniform pink appearance and elasticity. In contrast, lungs from the DSS model group appeared dark red, dull, and swollen. However, pre-administration of two doses of PFOS in the DSS-induced colitis model led to a marked improvement in lung appearance, with lungs exhibiting a pink color with scattered dark red hemorrhagic spots but retaining relative glossiness. Histopathological analysis using HE staining further confirmed that PFOS administration significantly reduced lung damage. Lungs from the colitis group showed extensive inflammatory cell infiltration, whereas PFOS-treated mice displayed a notable decrease in the histopathological scores, indicating reduced lung injury compared to the DSS model group (Fig. 1B).\u003c/p\u003e\n\u003cp\u003eAdditionally, immunofluorescence analysis of mucosal epithelial barrier-associated proteins, ZO-1 and Claudin-1, revealed that PFOS pre-administration reduced the accumulation of ZO-1 and Claudin-1 droplets and partially restored the original ring-like structure of the cell membrane (Fig. 1C-D). Previous studies have shown that liquid-liquid phase separation of ZO proteins drives the polymerization of tight junction proteins into a continuous belt[21]. Therefore, we postulated that PFOS may enhance the formation of tight epithelial barrier junctions by promoting ZO-1 phase separation, thereby mitigating lung injury associated with enteritis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2 PFOS treatment reduced inflammatory response in lung tissues of colitis mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe further investigated the effect of PFOS on lung macrophages by analyzing the surface-specific antigen F4/80 through immunohistochemical staining. As shown in Fig. 2A, the positive rate of macrophages in the alveolar septum and peribronchus was significantly higher in the DSS group compared to the normal control group. However, PFOS administration markedly inhibited macrophage recruitment. Additionally, the accumulation of neutrophils, closely linked to increased myeloperoxidase (MPO) activity, was reflected by a dramatic elevation in MPO levels in the lung tissues of colitis mice. PFOS treatment significantly reduced these elevated MPO levels (Fig. 2B). Furthermore, mRNA expression analysis of inflammatory cytokines revealed that oral administration of DSS significantly increased IL-1\u0026beta;, IL-6, and TNF-\u0026alpha; levels in lung tissues compared to the control group. Consistent with its inhibitory effect on inflammatory cell infiltration, PFOS treatment (2 mg/kg) suppressed the expression of these proinflammatory factors (Fig. 2C-F). Collectively, these findings indicate that PFOS treatment effectively alleviates lung inflammation in colitis-induced mice.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3 PFOS treatment reduced the oxidative stress in lung tissues of colitis mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Several studies have reported that the active ingredients of natural medicines exert protective effects against acute lung injury via the Nrf2 signaling pathway[22]. Similarly, our previous studies also confirmed the antioxidant effects of PFOS[8]. In this study, we investigated whether PFOS could inhibit colitis-induced oxidative stress in the lungs. Malondialdehyde (MDA) is a key indicator of oxidative stress and the body\u0026apos;s antioxidant capacity. As shown in Fig. 3A, MDA levels were significantly elevated in the lung tissues of colitis mice, while PFOS treatment (2 mg/kg) substantially reduced these levels. Additionally, we found that PFOS treatment at both 0.5 mg/kg and 2mg/kg significantly restored the activity of key antioxidant enzymes, including SOD, CAT, and GPX2. PFOS also promoted the mRNA expression of these antioxidant enzymes (Fig. 3B-G). Collectively, these data indicate that PFOS attenuates colitis-induced lung injury through its antioxidant activity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4 PFOS administration suppressed NF-kB signaling pathway and up regulated Nrf2 signaling pathway to reduce inflammation and oxidative stress\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of various inflammatory cytokines is regulated by the NF-\u0026kappa;B pathway, while Nrf2 serves as a key transcription factor mediating antioxidant signaling. Therefore, we further investigated whether PFOS could modulate the NF-\u0026kappa;B/Nrf2 signaling pathways to reduce inflammatory responses and oxidative stress in lung tissues. As shown in Fig. 4A, oral administration of DSS in mice activated the expression of NF-\u0026kappa;B protein in lung tissues, leading to inflammation. However, PFOS treatment significantly inhibited NF-\u0026kappa;B expression, and phosphorylation of p65 was markedly reduced. Further analysis revealed that PFOS exerted its anti-inflammatory effects by decreasing the protein expression of IKK\u0026beta; following administration of PFOS at 5 mg/kg (Fig.4A), a result confirmed by immunohistochemical staining (Fig.4B). Additionally, immunohistochemistry detected IL-1\u0026beta;, a downstream pro-inflammatory factor of the NF-\u0026kappa;B pathway, and PFOS treatment significantly inhibited IL-1\u0026beta; expression, consistent with its anti-inflammatory effects (Fig. 4C).\u003c/p\u003e\n\u003cp\u003eWe also explored whether the antioxidant activity of PFOS was mediated through the regulation of the Nrf2 signaling pathway. DSS treatment significantly suppressed the expression of Nrf2, HO-1, and NQO-1 proteins in the lung tissues of colitis-induced mice (Fig.4D). However, PFOS treatment restored the expression of these Nrf2 pathway proteins. Interestingly, no statistically significant changes in Keap-1 expression were observed across all groups. Immunohistochemical staining further confirmed the expression of Nrf2 and HO-1 proteins in the lung tissues (Fig.4E-F).Taken together, these results demonstrate that PFOS alleviates lung injury by inhibiting the NF-\u0026kappa;B signaling pathway and upregulating the Nrf2 signaling pathway.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5 PFOS inhibited LPS-induced epithelial barrier damages,\u0026nbsp;inflammation and\u0026nbsp;oxidative stress in lung epithelial cells\u003c/em\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLPS is known to damage the epithelial barrier by inducing the rapid redistribution and reducing the expression of ZO-1 in tight junctions[23]. Additionally, LPS is commonly used to establish in vitro models of inflammation and oxidative stress[24]. In this study, we developed an in vitro model using LPS-induced lung epithelial cells (A549) to verify the efficacy and mechanisms of PFOS in mitigating epithelial barrier damage, inflammation, and oxidative stress in the lungs(Fig. 5A).\u003c/p\u003e\n\u003cp\u003eThe CCK8 assay was used to assess cell viability after PFOS treatment. The results showed that PFOS at a concentration of 10 mM displayed a downward trend in viability, with 20 mM demonstrating significant toxicity compared to the control group (Fig. 5B). Based on these findings, we selected 5 mM PFOS for subsequent experiments. Since Nrf2 is known to negatively regulate the NF-\u0026kappa;B pathway[25], we included an Nrf2 inhibitor (ML385) to preliminarily explore the crosstalk between Nrf2 and NF-\u0026kappa;B signaling. Consistent with previous research, LPS exposure significantly reduced the expression of the tight junction proteins ZO-1 and Claudin-1 in A549 cells (Fig. 5C). However, PFOS treatment partially restored the expression of these proteins, while the protective effect was significantly inhibited by the Nrf2 inhibitor (Fig. 5C).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 5D, LPS exposure also significantly increased the production of myeloperoxidase (MPO), a marker of neutrophil activation, as well as the proinflammatory cytokines TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6. PFOS treatment in LPS-stimulated A549 cells markedly reduced the levels of these cytokines and MPO. Given that the NF-\u0026kappa;B pathway is a critical regulator of inflammation, we investigated the changes in NF-\u0026kappa;B signaling through Western blot analysis. PFOS treatment significantly suppressed the expression of p65, p-p65, and IKK-\u0026beta; in LPS-stimulated A549 cells (Fig. 5E). Although the addition of the Nrf2 inhibitor resulted in a downward trend in NF-\u0026kappa;B-related protein expression, the changes were not statistically significant (Fig. 5E). These results suggest that the anti-inflammatory effects of PFOS are closely related to the inhibition of NF-\u0026kappa;B pathway activation, with potential signaling crosstalk between Nrf2 and NF-\u0026kappa;B, though this crosstalk may not involve direct negative regulation of NF-\u0026kappa;B by Nrf2.\u003c/p\u003e\n\u003cp\u003eTo further explore the antioxidant effects of PFOS, we measured the activity of several antioxidant enzymes in cell extracts. LPS exposure led to a decrease in the activities of SOD, GPX2, and CAT, while increasing malondialdehyde (MDA) levels in the cell supernatant (Fig. 5F). PFOS treatment significantly reduced MDA expression and enhanced the activity of SOD, GPX2, and CAT, though these effects were almost completely blocked by the Nrf2 inhibitor ML385 (Fig. 5F). Subsequent Western blot analysis showed that the antioxidant effect of PFOS was mediated by activation of the Nrf2 signaling pathway. As shown in Fig. 5G, LPS significantly inhibited the protein expression of Nrf2, HO-1, and NQO-1. In contrast, PFOS treatment restored the expression of these proteins to levels close to those of the normal control, and the use of ML385 confirmed that the antioxidant effects of PFOS depend on the Nrf2 pathway. Collectively, these results demonstrate that PFOS alleviates LPS-induced epithelial barrier damage, inflammation, and oxidative stress in A549 cells by inhibiting the NF-\u0026kappa;B pathway and upregulating the Nrf2 signaling pathway. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.6 Intestinal metabolites affected PFOS exhibits potential correlations to the lung inflammation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn previous studies, oligosaccharides were found to partially restore imbalances in the intestinal flora in a DSS-induced enteritis mouse model[6]. In the present study, non-targeted fecal metabolomics was employed to investigate the effects of PFOS pre-intervention on intestinal metabolites in mice, aiming to determine whether the pulmonary protective effects of PFOS are related to changes in intestinal metabolites.\u003c/p\u003e\n\u003cp\u003eFirst, principal component analysis (PCA) revealed varying degrees of inter-group differences among stool samples across different experimental groups. Notably, the distribution pattern of intestinal metabolites after PFOS intervention was more similar to that of the normal control group than the model group (Fig. 6A-B). Further pairwise comparisons were conducted using orthogonal partial least squares discriminant analysis (OPLS-DA). Permutation tests were performed, and differential metabolites were identified using selection criteria (p\u0026lt;0.05, VIP\u0026gt;1.0). These metabolites were visualized in S-plots and volcano plots, highlighting those with significant differences (Fig. 7A-E).\u003c/p\u003e\n\u003cp\u003eSubsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted for the significantly altered metabolites. As illustrated in Fig. 7F, compared with the normal control group, the metabolites in the model group were significantly enriched in pathways related to amino acid metabolism (five pathways) and lipid metabolism (three pathways). These results indicate that DSS intervention induced significant disturbances in amino acid and lipid metabolism in mice with enteritis. However, PFOS intervention ameliorated the DSS-induced disruptions in these metabolic processes (Fig. 8A-E).\u003c/p\u003e\n\u003cp\u003eIt is worth noting that PFOS intervention also resulted in the enrichment of differentially expressed metabolites in three key signaling pathways: cAMP, PPAR, and TRP, all of which are known to be involved in the regulation of NF-\u0026kappa;B and Nrf2 signaling pathways[26-28] (Fig. 8F). Cluster analysis of the significantly altered metabolites across all groups was performed, and a heatmap was generated to visually represent the expression levels of these compounds in each group. As shown in Fig. 9, the metabolic profile of the DSS model group was distinctly separated from that of the normal control group and the PFOS pre-intervention group. Furthermore, the metabolic profiles of the normal control and PFOS pre-intervention groups were similar, consistent with previous findings.In conclusion, PFOS intervention mitigates DSS-induced metabolic disorders in mice and may play a role in regulating inflammation and oxidative stress through alterations in key metabolites.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eUC is a systemic inflammatory condition known to affect various organs, including the lungs, with nearly half of UC patients exhibiting low-grade respiratory tract inflammation[29,30]. The gut-lung crosstalk is thought to involve microbial products, metabolites, mucosal barrier dysfunction, and lymphocyte homing[31,32]. In this study, a DSS-induced enteritis mouse model was used to assess lung tissue damage, confirming lung injury consistent with previous research[33].PFOS, previously shown to protect against intestinal and pulmonary inflammation[7,8], was administered as a pre-intervention. Results demonstrated that PFOS, particularly at higher doses, reduced lung injury scores in the mice (Figure 1B). DSS-induced disruption of tight junction proteins ZO-1 and Claudin-1, critical for epithelial barrier integrity[34,35], was mitigated by PFOS, which restored the membrane structure of these proteins and reduced droplet-like accumulations observed in DSS-treated mice (Figure 1C-D).\u003c/p\u003e\n\u003cp\u003eMoreover, PFOS increased the expression of ZO-1 and Claudin-1 in cell experiments compared to the LPS model group (Figure 5C), suggesting that it may enhance tight junction formation by regulating protein phase separation and expression. This, in turn, could restore epithelial barrier function and alleviate DSS-induced lung injury.\u003c/p\u003e\n\u003cp\u003eInflammation plays a critical role in compromising epithelial barriers in both the lungs and intestines. When these barriers are damaged, bacterial toxins like lipopolysaccharide (LPS) can enter the systemic circulation, intensifying immune responses in distant tissues such as the lungs[24]. This study focused on the effects of PFOS on lung inflammation associated with enteritis. In the DSS-induced colitis model, F4/80 immunohistochemical staining, a marker for macrophage infiltration, and myeloperoxidase (MPO), a neutrophil infiltration marker, were significantly elevated in the lung tissues of the model group. However, PFOS (2 mg/kg) treatment notably reduced the infiltration of both macrophages and neutrophils (Figure 2A-B). Additionally, PFOS significantly suppressed mRNA transcription of pro-inflammatory cytokines in lung tissues of mice and reduced the expression of pro-inflammatory proteins in human lung epithelial cell line A549 (Figure 2C-F). Given that NF-κB is a key transcription factor responsible for activating inflammatory mediators[36], we further explored the involvement of the NF-κB signaling pathway in the mechanism of PFOS. NF-κB activation typically depends on IκB kinase (IKK) activity[37]. Our findings showed that PFOS markedly decreased the expression of IKK, p65, and phosphorylated p65 (p-p65), thereby inhibiting NF-κB signaling activation in both the lung tissue of mice with enteritis-associated lung injury and LPS-induced lung epithelial cell injury (Figure 4A and Figure5E).Here, PFOS attenuates lung inflammation by downregulating the NF-κB/IKK signaling pathway, providing a protective effect against pneumonia in mice with colitis.\u003c/p\u003e\n\u003cp\u003eFrom a molecular pathology perspective, the accumulation of excessive reactive oxygen species (ROS) in lung tissue is often associated with the activation of pro-inflammatory signaling pathways[38]. In this study, both in vivo and in vitro experiments demonstrated that PFOS treatment significantly reduced the activity of the lipid peroxidation marker malondialdehyde (MDA). Additionally, PFOS intervention upregulated the transcription and activity of key antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase 2 (GPX2) (Figure 3 and Figure5F).Nuclear factor erythroid 2-related factor 2 (Nrf2), a central transcription factor involved in the regulation of the body’s antioxidant defense mechanisms, controls the expression of these antioxidant enzymes[39]. We further investigated the effect of PFOS on the Nrf2 signaling pathway. Our results confirmed that PFOS significantly increased the protein expression of Nrf2 and its downstream targets, heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO-1). Notably, PFOS-induced activation of Nrf2 was independent of Kelch-like ECH-associated protein 1 (Keap1), which traditionally regulates Nrf2. Previous studies have demonstrated that Nrf2 activation can occur independently of Keap1, with protein kinases playing a crucial role in modulating Nrf2 activity[40].These findings suggest that PFOS exerts its protective effects, in part, by activating the Nrf2 signaling pathway (Figure 4D and Figure5G). Furthermore, evidence indicates crosstalk between the Nrf2 and NF-κB pathways. The Nrf2 pathway can suppress NF-κB activation and inhibit NF-κB-mediated transcription, while NF-κB signaling can downregulate Nrf2 activity[41]. To explore this interaction further, we used the Nrf2 inhibitor ML385 to investigate whether PFOS affects both pathways. The results confirmed that PFOS-mediated lung protection is closely related to Nrf2 activation, and consistent with previous findings, Nrf2 negatively regulated NF-κB signaling (Figure5).\u003c/p\u003e\n\u003cp\u003eFinally, it is important to note that intestinal metabolites play a significant role in modulating immune responses in lung diseases[42]. In this study, non-targeted fecal metabolomics analysis revealed substantial changes in the metabolites of DSS-induced enteritis mice. KEGG pathway enrichment analysis showed that these altered metabolites were mainly involved in amino acid metabolism, lipid metabolism, and mineral absorption, indicating that DSS-induced enteritis caused disruptions in amino acid and lipid metabolism (Figure7F). After PFOS pre-intervention, these metabolic imbalances were ameliorated. Of particular interest (Figure8F), several of the significantly altered metabolites were enriched in pathways related to cAMP, peroxisome proliferator-activated receptor (PPAR), and transient receptor potential (TRP), all of which are known to regulate NF-κB and Nrf2 signaling[26-28]. Taken together, these findings suggest that PFOS not only affects intestinal metabolites but also modulates key signaling pathways, supporting the hypothesis that PFOS may function as a next-generation prebiotic. This is in line with previous research indicating that PFOS positively impacts the gut microbiota[6], further highlighting its potential therapeutic role.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, this study shows that PFOS reduces lung tissue damage associated with enteritis by protecting the epithelial barrier and regulating the Nrf2/NF-\u0026kappa;B signaling pathways (Fig.10). PFOS upregulates antioxidant proteins (Nrf2, HO-1, NQO-1) and suppresses pro-inflammatory markers (p65, p-p65, IKK-\u0026beta;), indicating its antioxidant and anti-inflammatory effects. Additionally, PFOS\u0026rsquo;s impact on intestinal metabolites may contribute to its regulation of inflammation and oxidative stress, further protecting lung tissue. These findings suggest PFOS as a potential therapeutic agent for lung injury linked to enteritis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript: \u0026nbsp;UC, ulcerative colitis; PFOS, Oligosaccharides from Polygonatum cyrtonema Hua; DSS, dextran sodium sulfate; LPS, lipopolysaccharide; IBD, inflammatory bowel disease; Nrf2, nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; COPD, chronic obstructive pulmonary disease; GPx, glutathione peroxidase; GSH, glutathione; HO-1, heme oxygenase-1; H\u0026amp;E, hematoxylin and eosin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride membranes; HRP. horseradish peroxidase; ALI ,acute lung injury; MPO, myeloperoxidase; MDA, Malondialdehyde; PCA, principal component analysis; OPLS-DA, orthogonal partial least squares discriminant analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; IKK, I\u0026kappa;B kinase, p-p65, phosphorylated p65; NQO-1, NAD(P)H quinone dehydrogenase 1; PPAR, peroxisome proliferator-activated receptor; TRP, transient receptor potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJin Xu:\u003c/strong\u003e Investigation, Data curation, Visualization, Writing original draft.\u003cstrong\u003e\u0026nbsp;Chuangkang Tang:\u003c/strong\u003e Investigation, Visualization, Writing original draft. \u003cstrong\u003eYuqian He:\u003c/strong\u003e Investigation, Data curation. \u003cstrong\u003eYu Lu:\u003c/strong\u003e Investigation,Visualization. \u003cstrong\u003ePei Luo:\u003c/strong\u003e Supervision, Writing - review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Jianbo Wu:\u0026nbsp;\u003c/strong\u003eFunding acqui[1]sition, Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing finan[1]cial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Sichuan Province Science and Technology Agency Grant (2024NSFSC0591)，and\u0026nbsp;Luzhou-Southwest Medical University \u0026nbsp;University's scientific research project (2018-ZRQN-159).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with Ethics Requirements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll Institutional and National Guidelines for the care and use of animals were followed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the fndings of this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKr\u0026ouml;ner, P. 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M. \u0026amp; Wang, Y. Gut microbiota: A new insight into lung diseases. \u003cem\u003eBiomed. Pharmacother 2022 Nov\u003c/em\u003e :155:113810. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2022.113810\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2022.113810\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2022 Oct 8.\u003c/span\u003e\u003c/li\u003e\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":"PFOS, colitis-induced lung injury, NF-kB/IKK-β, Nrf2/HO-1/NQO-1, mucosal barrier, intestinal metabolites","lastPublishedDoi":"10.21203/rs.3.rs-6166677/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6166677/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLung disease is one of the parenteral manifestations of ulcerative colitis (UC), and still requires attention to the impact on the lungs while treating UC. Oligosaccharides from Polygonatum cyrtonema Hua (PFOS) has the therapeutic potential for lung injury associated with enteritis.To investigate the mechanism of PFOS in treating colitis-induced lung injury through NF-kB/Nrf2 signaling pathway, this study constructed in vitro and in vivo injury models using DSS and LPS. PFOS was used for the drug treatment, and Nrf2 inhibitor ML385 was used to interfere with Nrf2/HO-1 signaling pathway in the cells. In vivo results showed that PFOS reduces lung tissue damage associated with enteritis by protecting the epithelial barrier and regulating the Nrf2/NF-κB signaling pathways. PFOS upregulates antioxidant proteins (Nrf2, HO-1, NQO-1) and suppresses pro-inflammatory markers (p65, p-p65, IKK-β), indicating its antioxidant and anti-inflammatory effects. In vitro results showed that PFOS suppressed epithelial barrier damages, inflammation and oxidative stress in lung epithelial cells by inhibiting the NF-kB pathway and upregulating the Nrf2 signaling pathway. Additionally, PFOS intervention ameliorated the DSS-induced amino acid and lipid metabolism disorder.In conclusion,the DSS induced enteritis associated lung injury model, PFOS inhibited epithelial barrier damages, inflammation and oxidative stress of lung suppressed NF-kB and up regulated Nrf2 signaling pathway. Additionally, PFOS\u0026rsquo;s impact on intestinal metabolites may contribute to further protect lung tissue.\u003c/p\u003e","manuscriptTitle":"Oligosaccharides from Polygonatum cyrtonema Hua ameliorate colitis-induced lung injury via inhibiting inflammation and oxidative stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 21:35:31","doi":"10.21203/rs.3.rs-6166677/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":"6db4d863-60d7-4c5f-a28b-4da1495372da","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":47095811,"name":"Biological sciences/Drug discovery"},{"id":47095812,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-05-14T11:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-25 21:35:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6166677","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6166677","identity":"rs-6166677","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}