The injury effect of osteopontin in sepsis-associated lung injury

The injury effect of osteopontin in sepsis-associated lung injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The injury effect of osteopontin in sepsis-associated lung injury Qian Wang, Zhicai Yu, Zhixing Song, Xuexue Lu, Zhu Li, Dandan Pi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5435730/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Journal of Inflammation → Version 1 posted 7 You are reading this latest preprint version Abstract Background Sepsis is a severe condition causing organ failure due to an abnormal immune reaction to infection, characterized by ongoing excessive inflammation and immune system issues. Osteopontin (OPN) is secreted by various cells and plays a crucial role in inflammatory responses and immune regulation. Nonetheless, the precise function of OPN in sepsis remains to be elucidated. Methods In the present study, we evaluated the levels of OPN in paediatric patients with sepsis and healthy individuals. We examined the impact of OPN on survival rates, systemic inflammation, and lung injury within an experimental sepsis model using cecal ligation and puncture (CLP). Furthermore, the pro-inflammatory effects and potential mechanisms of OPN in sepsis were investigated through Mouse Hemophagocytic Synuclein (MH-S) cells. Results The OPN level was found to be elevated in patients with sepsis (243.5 ± 204.5 pg/ml) compared to children with common infections (101.6 ± 30.67 pg/ml) (P < 0.005) and healthy individuals (44.71 ± 23.82 pg/ml) (P < 0.0001).The serum OPN level on the first day ( 243.5 ± 204.3 pg/ml) was significantly higher than that on the sceond day (69.58 ± 58.70 pg/ml) (P < 0.005) and the third day (60.42 ± 55.15 pg/ml) (P < 0.005) in pediatric patient with sepsis admission to pediatric intensive care unit(PICU). Intravenous administration of OPN inhibitor into the tail vein decreased the mortality rate (HR = 0.2695, p = 0.0015), suppressed systemic inflammatory responses and mitigated lung tissue damage. The concentration of TNF-α, IL-6 and IL-1β in serum of CLP mice treated with OPN inhibitor decreased compared with CLP mice. Within the sepsis mouse model, there was a marked increase in OPN expression in the lungs tissues compared to the sham group mice. This surge was accompanied by a significant accumulation of alveolar macrophages and an upregulation of inflammasome expression. Mechanistic investigations in MH-s cells revealed that OPN-SiRNA suppressed the LPS-induced macrophage inflammatory response by inhibiting caspase1-dependent classical pyroptosis signaling pathway. Conclusion This study reveals that OPN has an adverse impact on the host's immune response to sepsis. Suppressing OPN expression holds potential therapeutic value for the treatment of sepsis. Trial registration Study on the diagnostic value of osteopontin in children with sepsis. MR5024001771. Registered 22 January 2024. https//www.medicalresearch.org.cn. Sepsis lung injure Osteopontin Macrophage Pyroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection[ 1 ]. Sepsis-associated lung injury is the most common severe organ dysfunction in children with sepsis, significantly contributing to elevated morbidity and mortality rates[ 2 , 3 ]. The latest diagnostic criteria for sepsis in children emphasize the high incidence and importance of respiratory dysfunction, especially as a secondary organ dysfunction[ 4 ]. Research has revealed that 25 million cases of sepsis in children were reported in 2017,which resulted in over 3 million deaths[ 5 ]. Despite numerous clinical investigations into sepsis and lung injury, identifying effective therapeutic drugs remains a formidable challenge. Osteopontin (OPN), also referred to as early T lymphocyte activation 1 protein, and secreted phosphorylated protein 1 (SPP1), belongs to the family of small integrin-binding ligand N-linked glycoproteins. It exists in various forms and fragments, with a molecular weight ranging from 41 to 74 kilodaltons(kDa)[ 6 , 7 ]. OPN is secreted by a wide array of tissue cells throughout the body and plays a crucial role in inflammatory response and immune regulation[ 8 – 11 ]. Previous studies have found that OPN concentrations are significantly elevated in the serum of patients with sepsis[ 12 ]. Currently, the precise role and mechanism of OPN in sepsis remain to be elucidated. To explore the potential role of OPN in sepsis, we initially assessed serum OPN levels in pediatric patients diagnosed with the condition. Following this, we established a mouse model of sepsis through cecal ligation and puncture (CLP), and a cellular model of sepsis by co-culturing Lipopolysaccharide (LPS) with Mouse Hemophagocytic Synuclein (MH-S) cells, to examine the function and potential molecular mechanisms of OPN in the context of sepsis. Materials and methods Study population Serum samples were obtained from children fulfilling the inclusion criteria and hospitalized at the Children's Hospital of Chongqing Medical University from January 2024 through December 2024. 26 cases of sepsis were included, with the following criteria: a. Consistency with the 2024 International Consensus on Sepsis in Children[ 4 ]. During the same period, 16 cases of infection children were identified among children exhibiting signs of infection, yet these cases did not meet the diagnostic criteria for sepsis within the same age group at the same hospital. For the control group, comprising 27 healthy children, the subjects were of the same age and were examined at the same hospital's health examination center during a physical examination. This study protocol has received approval from the Institutional Review Board of Children's Hospital of Chongqing Medical University ( File No: (2021) Ethical Review Research No. 325-1). Informed consent has been obtained from all participants in accordance with the principles outlined in the Declaration of Helsinki. Experimental animals Male wild-type (WT) C57BL/6J mice, aged between 6 to 8 weeks, were procured from Chongqing Medical University. These mice were bred in a controlled environment characterized by a temperature range of 20–24°C and a 12-hour light/dark cycle. They were provided with unrestricted access to standard food and water. All animal experiments were conducted in compliance with the regulations approved by the Chongqing Experimental Animal Center and the Animal Committee of Children's Hospital (Approval No.: CHCMU-IACUC20231208004). To create a model of polymicrobial sepsis, the procedure of CLP was executed[ 13 ]. Mice were sedated using pentobarbital sodium at a dosage of 50 mg/kg. After sterilization, a 1-cm midline laparotomy was performed on the abdomen, the cecum was then ligated at 20% of its length and punctured with an 18-gauge needle, resulting in a slight extrusion of cecal contents. Subsequently, the cecum was repositioned into the abdominal cavity, and the incisions were sutured. Sham-operated animals underwent identical surgical procedures, except for the ligation and puncture of the cecum. After CLP surgery, the animals were resuscitated with an intraperitoneal injection of normal saline at a ratio of 1 ml per 20 g of body weight. Twenty-four hours post-surgery, the animals were humanely euthanized, after which serum and lung tissue samples were collected. Cells experiments MH-s cells (Procell, Wuhan, China) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, within a 37°C, 5% CO2 incubator, and subculture at intervals of 1 to 2 days. The cells were allocated into three distinct groups: negative control ( NC) group, NC + LPS group, and OPN-SiRNA + LPS group. A concentration of 100 nM of either NC or OPN-SiRNA was complexed with an equal volume of transfection reagent for 10–15 minutes. Subsequently, these complexes were added to MH-s cells in RPMI1640 medium supplemented with 10% FBS and subjected to incubation for a period ranging from 24 to 72 hours. Then, the medium was refreshed, and 100 ng/mL LPS was introduced to continue the culture for an additional 6 hours, Ultimately, RNA or protein was extracted from the cells. Inhibitor‑mediated blockade of OPN To counteract the activity of OPN in CLP, mice in the CLP + OPN inhibitor (OI) group were administered 100 µg of an OPN inhibitor (MCE, OPN expression inhibitor 1, HY-146064, USA), which was dissolved in 100 µl of phosphate buffered saline (PBS). In contrast, a control group of mice received an equivalent volume of sterile PBS as a vehicle control. Enzyme-linked immunosorbent assay (ELISA) The concentrations of OPN in human samples were quantified by using commercial ELISA kits provided by FineTest (EH0248, China). Likewise,, the levels of OPN (Jonin, JL10068, China) and various inflammatory cytokines in mice (with n = 5–9 per group) were evaluated. These cytokines encompassed tumor necrosis factor-alpha (TNF-α) (Neobioscience, EMC102a.96, China), interleukin (IL)-1β (Neobioscience, EMC001b.96, China), and IL-6 (Neobioscience, EMC004.96, China). Both serum and cell supernatant samples were analyzed with commercially available ELISA kits. The dry-to-wet (D/W) ratio of lung tissue The upper lobe of the right lung from the mice was excised. The blood on its surface was carefully absorbed using filter paper and then weighed. This weight was recorded as the wet weight (W). Subsequently, the tissue was placed in an oven set at 60°C for a duration of 48 hours. After that, it was weighed again, and the result was noted as the dry weight (D). Consequently, the dry-to-wet ratio of the lung tissue was calculated using the formula D/W. Immunofluorescence detection The paraffin sections of lung tissue were heated at 60°C in an oven for 1 hour. Subsequently, dewaxing and sodium citrate antigen retrieval were performed. Then, the sections were cooled and rinsed three times with 1× phosphate buffer saline with tween-20 (PBST) for 5 minutes each. Incubation with goat serum was performed at room temperature for 30 minutes. After drying the slides, they were placed in a humid chamber with the primary antibody and overnight at 4°C. On the next day, the slides were washed again with 1×PBST three times for 5 minutes each. Then, the secondary antibody was then added and incubated at room temperature in the dark for 1 hour. After washing three times with 1×PBST as before, a drop of 4′,6-diamidino-2-phenylindole (DAPI)-containing anti-fluorescence quenching mounting medium was applied to seal the slides, and images were captured using a fluorescence microscope. Histopathology Lung tissue was harvested, and hematoxylin and eosin (H&E) staining was utilized to evaluate pathological changes (n = 5). Fresh samples were rinsed with cold PBS and then fixed in 4% paraformaldehyde. Subsequently, the tissues were dehydrated, embedded in paraffin, sliced into 4 µm sections, and stained routinely. The pathology scores for the lung were determined based on the following aspects: alterations in lung histology, including edema, congestion, interstitial inflammation, and inflammatory cell infiltration. RNA extraction and quantitative real-time PCR Total RNA was extracted from cells by employing the RNA Isolation Kit (Beyotime, R0027, China). Subsequently, 1 µg of RNA was reverse transcribed into cDNA using the ABScript III RT Master Mix for qPCR, which includes a gDNA remover kit (ABclonal, RK20429, China). Gene expression was analyzed through real-time quantitative PCR (qPCR) on a Bio-Rad CFX ConnectTM Real-Time System (Bio-Rad, USA), utilizing SYBR Green (ABclonal, Rk21203, China). The primer sequences are detailed in Table 1 , The expression levels were quantified using the 2−△△Cq method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the internal control. Table 1 q-PCR Primer Sequences OPN Osteopontin, TNF tumour necrosis factor, IL interleukin, NLRP3 NOD-, LRR- and pyrin domain-containing 3, GSDMD Gasdermin D, ASC apoptosis-associated speck-like protein containing a CARD, GAPDH ; glyceraldehyde-3-phosphate dehydrogenase, Quantitative Real-time reverse transcriptase-polymerase chain reaction, qPCR. Primer Sequence OPN F-5’-TGACGATGATGATGACGATGGAGAC-3’ R-5’-TGTAGGGACGATTGGAGTGAAAGTG-3’ TNF-α F-5’-CACGCTCTTCTGTCTACTGAACTTC-3’ R-5’-CTTGGTGGTTTGTGAGTGTGAGG-3’ IL-6 F-5’-TTCTTGGGACTGATGCTGGTGAC-3’ R-5’-GTGGTATCCTCTGTGAAGTCTCCTC-3’ IL-1β F-5’-CTCGCAGCAGCACATCAACAAG-3’ R-5’-CCACGGGAAAGACACAGGTAGC-3’ NLRP3 F-5’-AGGAGGAAGAAGAAGAGAGGAGAGG-3’ R-5’-TTGAGAAGAGACCACGGCAGAAG-3’ GSDMD F-5’-GTGGACAGCCTGCGGAACTC-3’ R-5’-GGTTCTGGTTCTGGAGCACTGG-3’ caspase1 F-5’-CCTGGTCTTGTGACTTGGAGGAC-3’ R-5’-ATCAGCAGTGGGCATCTGTAGC-3’ ASC F-5’-GAAGTGGACGGAGTGCTGGATG-3’ R-5’-ATCTTGTCTTGGCTGGTGGTCTC-3’ IL-18 F-5’-AAAGTGCCAGTGAACCCCAGAC-3’ R-5’-AGAGAGGGTCACAGCCAGTCC-3’ GAPDH F-5’-GCAAATTCAACGGCACAGTCAAG-3’ R-5’-TCGCTCCTGGAAGATGGTGATG-3’ Western blotting analysis Proteins were extracted from cells by using a radioimmunoprecipitation assay (RIPA) lysis buffer (MCE, HY-K1001, USA) supplemented with 1% phenylmethanesulfonylfluoride (PMSF) (MCE, HY-B0496, USA), 1% protease inhibitor (MCE, HY-K0010, USA), and 1% phosphatase inhibitors (MCE, HY-K0021, USA). The protein concentration was quantified using a NanoDrop spectrophotometer (Thermo Fisher). Subsequently, the proteins were separated by sodium dodecyl sulfate‒polyacrylamide (SDS‒PAGE) gel (EpiZyme Biotechnology, PG112, China) electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010, USA). The membranes were blocked with NcmBlot blocking buffer (New Cell & Molecular Biotech, P30500, China) for 20 minutes and incubated with the appropriate primary antibodies overnight at 4°C. The primary antibodies employed were as follows: anti-Osteopontin (Abcam, ab283656, USA), anti-IL-1β (Proteintech, 16806-1-AP, China), anti-IL-18 (Proteintech, 10663-1-AP, China), NLRP3 Rabbit mAb (Abclonal, A24294, China), GSDMD Polyclonal antibody (Proteintech, 20770-1-AP, China), CASP1 Rabbit pAb (Abclonal, A20470, China), ASC Rabbit mAb (Abclonal, A22046, China), and anti-TNF-α (Proteintech, 17590-1-AP, China). Thereafter, the membranes were incubated with goat anti-rabbit IgG, HRP-conjugated polyclonal antibody (CoWin Bio, CW0103S, China) and goat anti-mouse HRP-conjugated polyclonal antibody (Proteintech, 66009-1-Ig, China) as secondary antibodies at room temperature for 1 hour, and the results were visualized using a Bio-Rad ChemiDocTM Touch Imaging System (Bio-Rad, California, USA). Statistical analysis Statistical analyses were performed using GraphPad Prism 9 software. All data are presented as the mean ± standard deviation (SD) from at least three independents experiments. Group differences were evaluated by either a t-test (Mann-Whitney U test) or a one-way analysis of variance (Tukey's multiple comparison test). A P value less than 0.05 was considered statistically significant, where "n" values indicate the number of cultures, tissue samples, or animals examined within each group. Results The concentration of OPN is significantly elevated in pediatric patients with sepsis. In the present study, ELISAs were employed to confirm the expression of OPN. The detailed characteristics of patients diagnosed with sepsis, infection, and healthy controls are outlined in Table 2 . The serum concentration of OPN was observed to be increased in pediatric patients with sepsis (243.5 ± 204.5 pg/ml) compared to both healthy individuals (44.71 ± 23.82 pg/ml) (P < 0.0001) and those with infections (101.6 ± 30.67 pg/ml) (P < 0.005) (Fig. 1 A). Furthermore, we revealed that the serum OPN levels on the first day ( 243.5 ± 204.3 pg/ml) were notably elevated compared to those on the second day ( 69.58 ± 58.70 pg/ml) (P < 0.005), and the third day ( 60.42 ± 55.15 pg/ml) (P < 0.005) (Fig. 1 B) among survivors of sepsis in the intensive care unit. Table 2 Characteristics of paediatric patients with sepsis, infections and healthy controls Characteristics Sepsis patients (n = 26) Infection patients (n = 16) healthy controls (n = 27) Sex (male/female) 16/10 8/8 19/8 Age (years) 4.59(0.94–6.12) 5.04(2.44–7.25) 6.90(3.00–10.00) CRP (mg/L) 75.58(25.72–124.0) 8.02(0.8-12.55) NA PCT (ng/ml) 31.39(1.09–66.95) 0.37(0.055–0.185) NA IL-6 2656(51.94–3009) NA NA IL-10 287.9(44.24–461.1) NA NA TNF-α 1.528(0.575–2.145) NA NA IL-1β 22.67(1.365–2.45 NA NA Organs dysfunction(n,%) Respiratory 22 NA NA Circulatory 9 NA NA Nervous 11 NA NA Blood 17 NA NA Gastrointestinal 9 NA NA Urinary 4 NA NA Infection site (Number of patients) Respiratory 18 12 NA Gastrointestinal 2 1 NA Nervous 3 0 NA Vascular 3 2 NA Skin 0 1 NA Bacteraemia Isolates (Number of patients) Gram positive 6 NA NA Gram negative 3 NA NA Mycoplasma pneumonia 7 NA NA Fungus 0 NA NA Virus 9 NA NA Miscellaneous 5 NA NA Other / NA NA Phoenix Sepsis Score 3.5(2–4) NA NA PICU stay(days) 12.68(6–17) NA NA Ventilation 19 NA NA Died/survived 0/26 NA NA The data are expressed as the median (interquartile range) unless otherwise indicated. CRP C-reactive protein, PCT procalcitonin, IL interleukin, TNF tumour necrosis factor, PICU paediatric intensive care unit, NA not applicable The OPN expression is elevated in the plasma and lungs following septic mice. To analyze variations in OPN levels at the 24-hour mark after CLP, serum and lung tissues from both Sham and CLP mice were analyzed using ELISA and immunofluorescence techniques. The ELISA results indicated a significant elevation of OPN in the serum of CLP mice compared with the Sham group (0.7312 ± 0.2263 vs 0.3765 ± 0.08419 ng/ml, P = 0.0001) (Fig. 2 A), mirroring observations in septic patients. Correspondingly, Immunofluorescence analysis showed that, compared with the Sham group, the expression of OPN in the lung tissue of the CLP group increased by 34.8%, P = 0.0039(Fig. 2BC). Administration of OPN inhibitor protects septic mice To elucidate the function of OPN in the development of sepsis, mice subjected to CLP were administered an OPN expression inhibitor 1. Notably, the survival rate of these CLP mice was increased (HR = 0.2695, p = 0.0015) (Fig. 3 A). This protective outcome might be ascribed to a diminished systemic inflammatory response following the onset of sepsis. We observed that compared with the Sham group, the levels of serum inflammatory factors in CLP mice were significantly increased, and OPN expression inhibitor 1 significantly reversed this change, such as IL-6 (17.17 ± 9.543 ng/ml vs 661.5 ± 513.3 ng/ml vs 125.1 ± 160.5 ng/ml), TNF-α (9.486 ± 10.21 pg/ml vs 158.2 ± 86.98 pg/ml vs 25.52 ± 9.279 pg/ml), and IL-1β ༈16.62 ± 7.695 pg/ml vs 33.41 ± 3.282 pg/ml vs 25.25 ± 1.541 pg/ml)(Fig. 3 B). Administration of OPN inhibitor protects against sepsis-associated lung injury in mice To elucidate the impact of the OPN expression inhibitor 1 on CLP-associated lung injury, we initially conducted a histological examination of lung tissue and evaluated the extent of injury using a standardized scoring system, as detailed in the Materials and Methods section. Representative histological images of lung tissue from mice treated with sham surgery, vehicle, and OPN inhibitor are depicted in Fig. 3 C. Compared with the sham group, the lung tissue from the CLP group exhibited pronounced morphological alterations, including edema, hemorrhage, alveolar collapse, and infiltration of inflammatory cells. Conversely, treatment with OPN inhibitors markedly attenuated these microscopic signs of deterioration when compared to the CLP group. As illustrated in Fig. 3 D, the histological injury score of lung tissue was significantly higher in the CLP group than the sham group (7.667 ± 0.5774 vs 2.200 ± 0.3801, P < 0.0001), whereas the OPN inhibitor-treated group exhibited a significant improvement in the histological injury score compared to the vehicle group (3.200 ± 0.3801 vs 7.667 ± 0.5774, P < 0.0001). Subsequently, we assessed the D/W ratio of lung tissue in mice. Our findings indicated that the D/W ratio in the CLP group was markedly lower than that of the sham-operated group (20.18%±0.7485% vs 22.85%±0.7066%, P = 0.0002); however, the administration of the OPN expression inhibitor 1 effectively reversed this trend (21.85%±0.7329% vs 20.18%±0.7485%, P = 0.0065) (Fig. 3 E). Beyond blood analysis, we also evaluated the expression of cytokines in lung tissue at the mRNA level. The levels of IL-6, TNF-α, and IL-1β in lung tissue were considerably elevated in the vehicle group compared to the sham group. Nevertheless, treatment with an OPN inhibitor resulted in a significant reduction of these mRNA levels by 80%, 87.5%, and 70%, respectively, compared to the vehicle mice (Fig. 3 F). The pathological sections of mouse lung tissue in this study revealed a substantial infiltration of inflammatory cells following CLP, with OPN inhibitors markedly decreasing their count. However, macrophages constitute the primary immune cells within the lungs. Certain studies suggest that OPN facilitates the migration of macrophages to areas of disease, although there remains a divergence of views on whether OPN exerts pro-inflammatory or anti-inflammatory effects. So we first examined changes in the number of macrophage infiltrates by F4/80 staining. Immunofluorescence staining showed great infiltration of F4/80-positive macrophages into lung tissue in the CLP group than in the sham group, and OPN inhibitors significantly reduced the number of F4/80-positive macrophages. Interestingly, we observed the same trend of NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome ( a hallmark of pyroptosis ) in lung tissue (Fig. 4ABC). This may elucidate the precise mechanism through which OPN mediates its deleterious effects on the lungs. OPN aggravates macrophage inflammatory response in vitro To elucidate the role of OPN in sepsis-associated lung injury in mice, we investigated the impact of OPN-SiRNA on LPS-treated MH-s cells in vitro. Initially, we identified the presence of OPN in the supernatant of LPS-stimulated MH-s cells and the mRNA expression of OPN in the control group. Our findings indicated that both the mRNA (6.761 ± 1.241 vs 1.211 ± 0.1833, P = 0.0016) and protein (2139 ± 1186 pg/ml vs 349.8 ± 60.05 pg/ml, P = 0.0236) levels of OPN were markedly elevated following LPS stimulation compared to the control group (Fig. 5AB). Then, we successfully employed OPN-SiRNA to suppress the expression of OPN at both the mRNA and protein levels in MH-s cells (Fig. 5CDE). Subsequently, we found that both the mRNA and protein levels of IL-6, TNF-αand IL-1β in MH-s cells were increased during LPS exposure, while silencing OPN could significantly reduce their levels (Fig. 6AB). This outcome further substantiates the pro-inflammatory and tissue-damaging role of OPN within the context of a sepsis model at the cellular level. OPN promotes pyroptosis in macrophages treated with LPS Pyroptosis, as a form of programmed cell death, plays a crucial role in initiating and advancing the inflammatory response through an amplification loop of inflammation and necrosis.[ 14 ]. In vivo experiments, we observed that OPN inhibitor can significantly reduce the expression of NLRP3 in the lungs of CLP mice, we hypothesize that there is a correlation between OPN and pyroptosis. We silenced the expression of OPN in MH-s cells, and then detected the expression of NLRP3, Gasdermin D ( GSDMD ), caspase1, apoptosis-related spot-like protein ( ASC ), IL-1β and IL-18. The results showed that silencing OPN expression significantly reduced both the mRNA and protein levels of NLRP3, GSDMD, caspase1, ASC, IL-1β and IL-18 in the LPS-induced pyroptosis pathway ( Fig. 7AB ). Overall, these results indicate that the OPN-mediated inflammatory response in MH-S cells is facilitated by the induction of caspase1-dependent classical pyroptosis. Discussion The findings revealed that circulating levels of OPN were significant elevated in children with sepsis compared to healthy group and infections group. Additionally, the administration of OPN inhibitors in septic mice demonstrated that survival rates were increased by suppressing the expression of inflammatory mediators (TNF-α, IL-1β, IL-6) and mitigating tissue damage, at least in the lung tissue. Moreover, in vitro experiments indicated that the silencing of OPN expression curtailed the inflammatory response by inhibiting the pyroptosis signaling pathway in LPS-stimulated MH-s cells. Federico Carbone and colleagues discovered that elevated early OPN levels can forecast mortality in patients suffering from septic shock, and,the OPN levels on the first day are associated with multiple organ dysfunction, prolonged hospital stays, extended durations for resolving infections, and various pro-inflammatory responses mediated by macrophages[ 15 ]. Additionally, research has indicated that plasma OPN detection could diagnose sepsis in patients[ 16 ]. However, these investigations have primarily concentrated on adult subjects, with limited data available on pediatric septic patients. In our study, we observed that the serum OPN levels in children with sepsis were significantly higher than those with infections and healthy controls. We also observed that the serum OPN levels on the first day were notably elevated compared to the second day and the third day among survivors of sepsis in the PICU. The concentration of OPN seems to diminish as sepsis severity improves. Vivo experiments revealed that mortality, systemic inflammatory response, organ damage were aggravated in CLP, which may be related to the increase of OPN concentration. When treated with an OPN inhibitor, we found that all those were improved. It is reasonable to infer that the overall improvement in sepsis outcomes through the inhibition of OPN may be attributed to the suppression of systemic inflammation and the amelioration of lung organ damage. In vitro experiments, we observed a positive correlation between OPN levels and the expression of cytokines TNF-α, IL-6 and IL-1β in the LPS-induced inflammatory response model of MH-s cells. The inhibition of OPN expression by using OPN-siRNA was found to counteract the elevation of TNF-α, IL-6 and IL-1β. These findings support the anti-inflammatory properties of OPN-siRNA. Bruemmer et al. have demonstrated that acute macrophage infiltration is significantly diminished in OPN −/− mice compared to WT counterparts in a dextran sodium sulfate (DSS)-induced colitis[ 17 ]. Then, Yohei et al. discovered that OPN significantly enhanceed neutrophil migration to inflammatory lung lesions by upregulating the mitogen-activated protein (MAP) kinase signaling pathway molecules p38 and extracellular signal-regulated protein kinase (ERK)[ 18 ]. In previous research, OPN has been demonstrated to function as a proinflammatory cytokine and chemotactic agent, facilitating the migration of immune cells to areas of inflammation[ 7 , 19 – 22 ]. Nonetheless, the majority of researches have focused on the role of OPN on migration and activation[ 18 , 23 , 24 ], there is a lack of investigation into whether there is a correlation between OPN and the programmed cell death of macrophages. In our study, we found that macrophage infiltration and NLRP3 inflammasome expression in the lungs of septic mice was elevated compared to that in the sham group, and the use of OPN inhibitors could significantly ameliorate this situation. Correspondingly, previous studies have indicated that macrophage pyroptosis plays a crucial role in sepsis-induced lung injury by propagating the pulmonary inflammatory response, vascular leakage, and facilitating neutrophil migration to the lungs[ 25 ]. We have presented the initial evidence indicating a connection between pyroptosis in sepsis-related lung injury and OPN. In line with our results, OPN has been shown to be up-regulated in bleomycin-induced pulmonary fibrosis and is associated with macrophage pyroptosis through elevated expression of multiple endocrine neoplasia type 1 (Men1) expression.[ 26 ]. Likewise, when researchers employed calcium oxalate monohydrate ( COM ) to establish a nephrolithiasis cell model of macrophages, they observed that the expression trends of OPN and macrophage pyroptosis signaling pathways were notably consistent[ 27 ]. However, it remains ambiguous whether OPN is associated with macrophage pyroptosis, and the precise mechanism remains unclear. In our study, we observed that silencing OPN expression in MH-s cells can significantly reduce both the mRNA and protein levels of NLRP3, GSDMD, caspase1, ASC, IL-1β and IL-18 in LPS-induced elevated pyroptosis pathway. Therefore, it can be concluded that preventing OPN-induced pyroptosis could potentially mitigate the inflammatory damage associated with sepsis. The present investigation has several limitations that deserve consideration. Firstly, it is a single-center study with a relatively small sample size of sepsis patients. Furthermore, there is an lack of comparative analysis of OPN levels between survivors and non-survivors, as well as between sepsis patients and those with other organ dysfunctions in the PICU, highlighting the necessity for extensive clinical trials and additional research to elucidate the specific differential role of OPN in sepsis patients. In summary, the findings of this study highlight the detrimental role of OPN in the context of sepsis. Moreover, the therapeutic administration of OPN inhibitors has been demonstrated to increase survival rates and attenuate lung injury in septic mice. Additionally, the protective mechanism of OPN inhibitors encompasses both anti-inflammatory effects and the suppression of pyroptosis. Consequently, OPN not only holds potential as a promising diagnostic marker for sepsis but also presents a novel therapeutic target for the development of sepsis treatment medications. Abbreviations OPN Osteopontin SPP1 Secreted phosphorylated protein 1 CLP Cecal ligation and puncture LPS Lipopolysaccharide MH-S Mouse Hemophagocytic Synuclein PICU Pediatric intensive care unit WT Wild-type FBS Fetal bovine serum NC Negative control SiRNA Small interfering RNA OI OPN inhibitor PBS Phosphate buffered saline ELISA Enzyme-linked immunosorbent assay TNF-α Tumor necrosis factor-alpha IL-6 Interleukin-6 IL-1β Interleukin-1 beta LPS Lipopolysaccharide D/W Dry-to-wet PBST Phosphate buffer saline with tween-20 DAPI 4′,6-diamidino-2-phenylindole H&E Hematoxylin and eosin qPCR Real-time quantitative PCR GAPDH Glyceraldehyde-3-phosphate dehydrogenase RIPA Radioimmunoprecipitation assay PMSF Phenylmethanesulfonylfluoride SDS-PAGE Sodium dodecyl sulfate‒polyacrylamide PVDF Polyvinylidene fluoride NLRP3 NOD-, LRR- and pyrin domain-containing 3 GSDMD Gasdermin D ASC Apoptosis-related spot-like protein Declarations Ethics approval and consent to participate All procedures performed in studies involving human participants were approved by the Clinical Research Ethics Committee of Institutional Review Board of Children’s Hospital of Chongqing Medical University (File No: (2021) Ethical Review Research No. 325-1). All animal experiments were conducted under the rules approved by the Ethics Committee of Chongqing Experimental Animal Center and the Animal Committee of Children’s Hospital (CHCMU-IACUC20231208004). Consent for publication All listed authors consent to the submission, and all data are used with the consent of the person generating the data. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was supported by the Key Program of Chongqing Clinical Medical Research (Grant No. NCRCCHD-2021-KP-03) and the Program For Youth Innovation in Future Medicine of Chongqing Medical University (W0124 to JL). Authors’ contributions XF and LJ contributed to the conception and design. QW performed the experiments, analysed data, and wrote the manuscript. ZCY helped to revise the manuscript. ZXS, XL, ZL, and DDP helped in the acquisition of clinical data. All authors read and approved the final manuscript. Acknowledgements Not applicable. Authors' information 1 Department of Critical Care Medicine, Children’s Hospital of Chongqing Medical University Chongqing, China. 2 National Clinical Research Center for Child Health and Disorders, Chongqing, China. 3 Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, China. 4 Chongqing Key Laboratory of Pediatrics, Chongqing, China. 5 Department of Pediatric Intensive Care Unit, First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China. 6 Department of Life Science Center, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China. 7 Clinical Laboratory of Children 's Hospital of Chongqing Medical University, Chongqing, China. 8 Department of Emergency, Children 's Hospital of Chongqing Medical University, Chongqing, China. 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Eur J Immunol. 2005;35:1510–20. Kawamura K, Iyonaga K, Ichiyasu H, Nagano J, Suga M, Sasaki Y. Differentiation, maturation, and survival of dendritic cells by osteopontin regulation. Clin Diagn Lab Immunol. 2005;12:206–12. Vaschetto R, Nicola S, Olivieri C, Boggio E, Piccolella F, Mesturini R, et al. Serum levels of osteopontin are increased in SIRS and sepsis. Intensive Care Med. 2008;34:2176–84. Dejager L, Pinheiro I, Dejonckheere E, Libert C. Cecal ligation and puncture: the gold standard model for polymicrobial sepsis? Trends in Microbiology. 2011;19:198–208. Linkermann A, Stockwell BR, Krautwald S, Anders H-J. Regulated cell death and inflammation: an auto-amplification loop causes organ failure. Nat Rev Immunol. 2014;14:759–67. Carbone F, Bonaventura A, Vecchiè A, Meessen J, Minetti S, Elia E, et al. Early osteopontin levels predict mortality in patients with septic shock. Eur J Intern Med. 2020;78:113–20. Castello LM, Baldrighi M, Molinari L, Salmi L, Cantaluppi V, Vaschetto R, et al. The Role of Osteopontin as a Diagnostic and Prognostic Biomarker in Sepsis and Septic Shock. Cells. 2019;8. Zhong J, Eckhardt ERM, Oz HS, Bruemmer D, de Villiers WJS. Osteopontin deficiency protects mice from dextran sodium sulfate-induced colitis. Inflamm Bowel Dis. 2006;12:790–6. Hirano Y, Aziz M, Yang W-L, Wang Z, Zhou M, Ochani M, et al. Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury. Crit Care. 2015;19:53. Clemente N, Raineri D, Cappellano G, Boggio E, Favero F, Soluri MF, et al. Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases. J Immunol Res. 2016;2016:7675437. Lamort A-S, Giopanou I, Psallidas I, Stathopoulos GT. Osteopontin as a Link between Inflammation and Cancer: The Thorax in the Spotlight. Cells. 2019;8. Intracellular osteopontin regulates homeostasis and function of natural killer cells - PubMed [Internet]. [cited 2023 Jun 17]. Available from: https://pubmed.ncbi.nlm.nih.gov/25550515/ Tan Y, Zhao L, Yang Y-G, Liu W. The Role of Osteopontin in Tumor Progression Through Tumor-Associated Macrophages. Front Oncol. 2022;12:953283. Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. Journal of Leukocyte Biology. 2002;72:752–61. Kim D, Haynes CL. The role of p38 MAPK in neutrophil functions: Single cell chemotaxis and surface marker expression. Analyst. 2013;138:6826. Qin X, Zhou Y, Jia C, Chao Z, Qin H, Liang J, et al. Caspase-1-mediated extracellular vesicles derived from pyroptotic alveolar macrophages promote inflammation in acute lung injury. Int J Biol Sci. 2022;18:1521–38. Lu Y, Zhao J, Tian Y, Shao D, Zhang Z, Li S, et al. Dichotomous Roles of Men1 in Macrophages and Fibroblasts in Bleomycin—Induced Pulmonary Fibrosis. Int J Mol Sci. 2022;23:5385. Ding T, Zhao T, Li Y, Liu Z, Ding J, Ji B, et al. Vitexin exerts protective effects against calcium oxalate crystal-induced kidney pyroptosis in vivo and in vitro. Phytomedicine. 2021;86:153562. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Jan, 2025 Read the published version in Journal of Inflammation → Version 1 posted Editorial decision: Revision requested 05 Dec, 2024 Reviews received at journal 21 Nov, 2024 Reviewers agreed at journal 16 Nov, 2024 Reviewers invited by journal 14 Nov, 2024 Editor assigned by journal 12 Nov, 2024 Submission checks completed at journal 12 Nov, 2024 First submitted to journal 11 Nov, 2024 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. 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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-5435730","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":381174970,"identity":"429d605d-c627-4762-8727-c59b552bdc0c","order_by":0,"name":"Qian Wang","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Wang","suffix":""},{"id":381174971,"identity":"962b1e23-dab5-4799-b9e8-bc364ed6892d","order_by":1,"name":"Zhicai Yu","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinxiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhicai","middleName":"","lastName":"Yu","suffix":""},{"id":381174972,"identity":"d2a9dea3-a39d-4759-864c-8d220b112897","order_by":2,"name":"Zhixing Song","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhixing","middleName":"","lastName":"Song","suffix":""},{"id":381174973,"identity":"2024b0be-2af6-4778-aa04-e309e175ed3f","order_by":3,"name":"Xuexue Lu","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuexue","middleName":"","lastName":"Lu","suffix":""},{"id":381174974,"identity":"aa5e3599-8180-4e1b-8127-c547be46940a","order_by":4,"name":"Zhu Li","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Li","suffix":""},{"id":381174975,"identity":"f3da1836-fe15-43fc-845b-c957e2c28683","order_by":5,"name":"Dandan Pi","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Pi","suffix":""},{"id":381174976,"identity":"9075d616-7a42-4f50-9f43-3cad9359b9ab","order_by":6,"name":"Feng Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFACxgdAwkaOn735wIEPP4jSwmwAJNKMJXuOJR6c2UO8lsOJG27kGB/mYCNCg8GNZMbHBb8OGxvcyPlwmIGHQZ5f7AB+LZIzkpmNZ/aly0meebvhcIEFg+HM2Qn4tfBL5B+T5u2xNuY7nrvh8AwehgSD2wS0sEkks//m7WFObDiQ8+AwDxsRWvglktmYeX44J044kcNAnBbJnsfM0rwN4EA2AAayBGG/GBxPZvzM8wcclY8/fPhhI88vTUALGDC2wZkSRCgHgz/EKhwFo2AUjIIRCQCbt0gzTpQ/cAAAAABJRU5ErkJggg==","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Feng","middleName":"","lastName":"Xu","suffix":""},{"id":381174977,"identity":"fd5c2fed-3cb7-4dda-94e4-1482c8fbe20e","order_by":7,"name":"Jing Li","email":"","orcid":"","institution":"Children’s Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-11-12 03:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5435730/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5435730/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12950-025-00430-4","type":"published","date":"2025-01-24T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71563470,"identity":"3677fc8c-693d-4ed4-926c-7f5b25bbd20b","added_by":"auto","created_at":"2024-12-16 17:18:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117456,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOPN levels are significantly elevated in pediatric patients with sepsis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) ELISAs were used to confirm the expression of OPN in 26 paediatric patients with sepsis, 27 healthy controls, and 16 common infections in clinical studies. (B) ELISAs was employed to identify the level of OPN in the serum of paediatric patients with sepsis at 1st, 2nd, 3rd day after staying in PICU.\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e p \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003e p \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eOsteopontin, OPN; The first day of pediatric patient with sepsis admission to PICU, sepsis-D1; The second day of pediatric patient with sepsis admission to PICU, sepsis-D2; The third day of pediatric patient with sepsis admission to PICU, sepsis-D3; enzyme-linked immunosorbent assay, ELISA.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/88e580c69bad8ea48788ecb8.jpg"},{"id":71563474,"identity":"393447ce-1146-4d3f-87ac-c1a48163a62d","added_by":"auto","created_at":"2024-12-16 17:18:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":278735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expressions of OPN are improved obviously in mice with CLP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Serum levels of OPN in sham mice and mice with CLP (n=8). (B) Immunofluorescence was employed to detect the expression of OPN in lung tissue (n=5). (C) Relative fluorescence intensity of OPN in the lung tissue of Sham and CLP mice.\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e p \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003e p \u003c/em\u003e\u0026lt;0.0001. Osteopontin, OPN; caecal ligation and puncture, CLP; 4′,6-diamidino-2-phenylindole, DAPI.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/8e92ac2c5f25454bf2b86254.jpg"},{"id":71564669,"identity":"5241c94b-49ff-4574-a95e-424aeb5a752d","added_by":"auto","created_at":"2024-12-16 17:26:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":621065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdministration of OPN inhibitor protects septic mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Survival of mice with CLP after treatment with OPN inhibitor (n=12). (B) Serum cytokine levels in theSham,CLP and CLP+OPN inhibitor groups at 24 h post-operative (n=5-6). (C) Lung tissues were stained with H\u0026amp;E in the Sham,CLP and CLP+OPN inhibitor groups (n=5). (D) Semiquantitative scores of lung tissues were calculated in the Sham,CLP and CLP+OPN inhibitor groups (n=5). (E) The D/W ratio of lung tissue in the Sham,CLP and CLP+OPN inhibitor groups (n=5). (F) The mRNA expression levels of IL-6, TNF-α and IL-1β in lung tissues were determined by qPCR (n=5).\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003e p \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003e p \u003c/em\u003e\u0026lt;0.0001. Osteopontin, OPN; OPN inhibitor, OI; caecal ligation and puncture, CLP; phosphate buffered saline, PBS; interleukin, IL; tumour necrosis factor, TNF; haematoxylin-eosin staining, HE; dry-to-wet, D/W; Quantitative Real-time reverse transcriptase-polymerase chain reaction, qPCR.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/dd6c45ff33e915b5aee7316e.jpg"},{"id":71564670,"identity":"9cef7d51-6566-4b5e-a60a-6e639d2a5c6a","added_by":"auto","created_at":"2024-12-16 17:26:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":691992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdministration of OPN inhibitor reduced macrophage infiltration and inflammasome expression in lung tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence was employed to detect the expression of F4/80 and NLRP3 in lung tissue (n=5). (B) Relative fluorescence intensity of F4/80 in the lung tissue of Sham,CLP and CLP+OPN inhibitor groups.(C) Relative fluorescence intensity of NLRP3 in the lung tissue of Sham,CLP and CLP+OPN inhibitor groups.\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e p \u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003e p \u003c/em\u003e\u0026lt;0.0001. Osteopontin, OPN; OPN inhibitor, OI; NOD-, LRR- and pyrin domain-containing 3, NLRP3; 4′,6-diamidino-2-phenylindole, DAPI; caecal ligation and puncture, CLP.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/907fd311e2ac1d1092a1def1.jpg"},{"id":71563480,"identity":"e6279f4d-dbf6-408b-adf7-373d5cc4aa55","added_by":"auto","created_at":"2024-12-16 17:18:19","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":309949,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOPN was increased in LPS-treated MH-s cells, and OPN-SiRNA could silence the expression of OPN.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The mRNA expression levels of OPN in MH-s cells were determined by qPCR. (B) ELISAs were used to detected the concentration of OPN in cell supernatant of MH-s cells. (C) Western blot analysis of the relative expression levels of OPN in MH-s cells.\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.001, **** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eOsteopontin, OPN; negative control, NC; lipopolysaccharide, LPS; Mouse alveolar macrophage cells, MH-s; Quantitative Real-time reverse transcriptase-polymerase chain reaction, qPCR; enzyme-linked immunosorbent assay, ELISA.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/098dada12a904698611abbae.jpg"},{"id":71563485,"identity":"59a9b1ce-a5e8-4df5-8af8-6a570421966c","added_by":"auto","created_at":"2024-12-16 17:18:19","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":370295,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOPN-SiRNA decreases inflammatory cytokine responses in LPS-treated MH-s cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The mRNA expression levels of TNF-α, IL-6 and IL-1β in MH-s cells were determined by qPCR. (B) Western blot analysis of the relative expression levels of TNF-α, IL-6 and IL-1β in MH-s cells.\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.001, **** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0001. Osteopontin, OPN; interleukin, IL; tumour necrosis factor, TNF; negative control, NC; lipopolysaccharide, LPS; Mouse alveolar macrophage cells, MH-s; Quantitative Real-time reverse transcriptase-polymerase chain reaction, qPCR.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/da46a5abc51cca0643bc8560.jpg"},{"id":71563483,"identity":"a9e47566-5951-44c1-b4ed-fc7735cf6faf","added_by":"auto","created_at":"2024-12-16 17:18:19","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":606578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOPN-SiRNA inhibits pyroptosis in MH-s cells treated with LPS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The mRNA expression levels of NLRP3, GSDMD, caspase1, ASC, IL-1β and IL-18 in MH-s cells. (B) Western blotting analysis of the relative expression of the NLRP3, GSDMD, caspase1, ASC, IL-1β and IL-18 in MH-s cells.\u003c/p\u003e\n\u003cp\u003eThe data arepresented as the means ± standard deviations (S.D.). “*” indicates a difference between groups. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.001, **** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eOsteopontin, OPN; NOD-, LRR- and pyrin domain-containing 3, NLRP3; Gasdermin D, GSDMD; apoptosis-associated speck-like protein containing a CARD, ASC; interleukin, IL; tumour necrosis factor, TNF; negative control, NC; lipopolysaccharide, LPS; Mouse alveolar macrophage cells, MH-s; Quantitative Real-time reverse transcriptase-polymerase chain reaction, qPCR.\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/846c7fcd59e2e0eaa2dd1da3.jpg"},{"id":74858311,"identity":"52b5112e-dbfe-4064-b010-b04a7cd8a8a0","added_by":"auto","created_at":"2025-01-27 16:07:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4411661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5435730/v1/99151724-9912-4ec1-8a09-08761a18d349.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The injury effect of osteopontin in sepsis-associated lung injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Sepsis-associated lung injury is the most common severe organ dysfunction in children with sepsis, significantly contributing to elevated morbidity and mortality rates[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The latest diagnostic criteria for sepsis in children emphasize the high incidence and importance of respiratory dysfunction, especially as a secondary organ dysfunction[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Research has revealed that 25\u0026nbsp;million cases of sepsis in children were reported in 2017,which resulted in over 3\u0026nbsp;million deaths[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite numerous clinical investigations into sepsis and lung injury, identifying effective therapeutic drugs remains a formidable challenge.\u003c/p\u003e \u003cp\u003eOsteopontin (OPN), also referred to as early T lymphocyte activation 1 protein, and secreted phosphorylated protein 1 (SPP1), belongs to the family of small integrin-binding ligand N-linked glycoproteins. It exists in various forms and fragments, with a molecular weight ranging from 41 to 74 kilodaltons(kDa)[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. OPN is secreted by a wide array of tissue cells throughout the body and plays a crucial role in inflammatory response and immune regulation[\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous studies have found that OPN concentrations are significantly elevated in the serum of patients with sepsis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Currently, the precise role and mechanism of OPN in sepsis remain to be elucidated.\u003c/p\u003e \u003cp\u003eTo explore the potential role of OPN in sepsis, we initially assessed serum OPN levels in pediatric patients diagnosed with the condition. Following this, we established a mouse model of sepsis through cecal ligation and puncture (CLP), and a cellular model of sepsis by co-culturing Lipopolysaccharide (LPS) with Mouse Hemophagocytic Synuclein (MH-S) cells, to examine the function and potential molecular mechanisms of OPN in the context of sepsis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eSerum samples were obtained from children fulfilling the inclusion criteria and hospitalized at the Children's Hospital of Chongqing Medical University from January 2024 through December 2024. 26 cases of sepsis were included, with the following criteria: a. Consistency with the 2024 International Consensus on Sepsis in Children[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. During the same period, 16 cases of infection children were identified among children exhibiting signs of infection, yet these cases did not meet the diagnostic criteria for sepsis within the same age group at the same hospital. For the control group, comprising 27 healthy children, the subjects were of the same age and were examined at the same hospital's health examination center during a physical examination.\u003c/p\u003e \u003cp\u003e This study protocol has received approval from the Institutional Review Board of Children's Hospital of Chongqing Medical University ( File No: (2021) Ethical Review Research No. 325-1). Informed consent has been obtained from all participants in accordance with the principles outlined in the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eMale wild-type (WT) C57BL/6J mice, aged between 6 to 8 weeks, were procured from Chongqing Medical University. These mice were bred in a controlled environment characterized by a temperature range of 20\u0026ndash;24\u0026deg;C and a 12-hour light/dark cycle. They were provided with unrestricted access to standard food and water. All animal experiments were conducted in compliance with the regulations approved by the Chongqing Experimental Animal Center and the Animal Committee of Children's Hospital (Approval No.: CHCMU-IACUC20231208004).\u003c/p\u003e \u003cp\u003eTo create a model of polymicrobial sepsis, the procedure of CLP was executed[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Mice were sedated using pentobarbital sodium at a dosage of 50 mg/kg. After sterilization, a 1-cm midline laparotomy was performed on the abdomen, the cecum was then ligated at 20% of its length and punctured with an 18-gauge needle, resulting in a slight extrusion of cecal contents. Subsequently, the cecum was repositioned into the abdominal cavity, and the incisions were sutured. Sham-operated animals underwent identical surgical procedures, except for the ligation and puncture of the cecum. After CLP surgery, the animals were resuscitated with an intraperitoneal injection of normal saline at a ratio of 1 ml per 20 g of body weight. Twenty-four hours post-surgery, the animals were humanely euthanized, after which serum and lung tissue samples were collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCells experiments\u003c/h2\u003e \u003cp\u003eMH-s cells (Procell, Wuhan, China) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, within a 37\u0026deg;C, 5% CO2 incubator, and subculture at intervals of 1 to 2 days. The cells were allocated into three distinct groups: negative control ( NC) group, NC\u0026thinsp;+\u0026thinsp;LPS group, and OPN-SiRNA\u0026thinsp;+\u0026thinsp;LPS group. A concentration of 100 nM of either NC or OPN-SiRNA was complexed with an equal volume of transfection reagent for 10\u0026ndash;15 minutes. Subsequently, these complexes were added to MH-s cells in RPMI1640 medium supplemented with 10% FBS and subjected to incubation for a period ranging from 24 to 72 hours. Then, the medium was refreshed, and 100 ng/mL LPS was introduced to continue the culture for an additional 6 hours, Ultimately, RNA or protein was extracted from the cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eInhibitor‑mediated blockade of OPN\u003c/h2\u003e \u003cp\u003eTo counteract the activity of OPN in CLP, mice in the CLP\u0026thinsp;+\u0026thinsp;OPN inhibitor (OI) group were administered 100 \u0026micro;g of an OPN inhibitor (MCE, OPN expression inhibitor 1, HY-146064, USA), which was dissolved in 100 \u0026micro;l of phosphate buffered saline (PBS). In contrast, a control group of mice received an equivalent volume of sterile PBS as a vehicle control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe concentrations of OPN in human samples were quantified by using commercial ELISA kits provided by FineTest (EH0248, China). Likewise,, the levels of OPN (Jonin, JL10068, China) and various inflammatory cytokines in mice (with n\u0026thinsp;=\u0026thinsp;5\u0026ndash;9 per group) were evaluated. These cytokines encompassed tumor necrosis factor-alpha (TNF-α) (Neobioscience, EMC102a.96, China), interleukin (IL)-1β (Neobioscience, EMC001b.96, China), and IL-6 (Neobioscience, EMC004.96, China). Both serum and cell supernatant samples were analyzed with commercially available ELISA kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe dry-to-wet (D/W) ratio of lung tissue\u003c/h2\u003e \u003cp\u003eThe upper lobe of the right lung from the mice was excised. The blood on its surface was carefully absorbed using filter paper and then weighed. This weight was recorded as the wet weight (W). Subsequently, the tissue was placed in an oven set at 60\u0026deg;C for a duration of 48 hours. After that, it was weighed again, and the result was noted as the dry weight (D). Consequently, the dry-to-wet ratio of the lung tissue was calculated using the formula D/W.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence detection\u003c/h2\u003e \u003cp\u003eThe paraffin sections of lung tissue were heated at 60\u0026deg;C in an oven for 1 hour. Subsequently, dewaxing and sodium citrate antigen retrieval were performed. Then, the sections were cooled and rinsed three times with 1\u0026times; phosphate buffer saline with tween-20 (PBST) for 5 minutes each. Incubation with goat serum was performed at room temperature for 30 minutes. After drying the slides, they were placed in a humid chamber with the primary antibody and overnight at 4\u0026deg;C. On the next day, the slides were washed again with 1\u0026times;PBST three times for 5 minutes each. Then, the secondary antibody was then added and incubated at room temperature in the dark for 1 hour. After washing three times with 1\u0026times;PBST as before, a drop of 4\u0026prime;,6-diamidino-2-phenylindole (DAPI)-containing anti-fluorescence quenching mounting medium was applied to seal the slides, and images were captured using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eLung tissue was harvested, and hematoxylin and eosin (H\u0026amp;E) staining was utilized to evaluate pathological changes (n\u0026thinsp;=\u0026thinsp;5). Fresh samples were rinsed with cold PBS and then fixed in 4% paraformaldehyde. Subsequently, the tissues were dehydrated, embedded in paraffin, sliced into 4 \u0026micro;m sections, and stained routinely. The pathology scores for the lung were determined based on the following aspects: alterations in lung histology, including edema, congestion, interstitial inflammation, and inflammatory cell infiltration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells by employing the RNA Isolation Kit (Beyotime, R0027, China). Subsequently, 1 \u0026micro;g of RNA was reverse transcribed into cDNA using the ABScript III RT Master Mix for qPCR, which includes a gDNA remover kit (ABclonal, RK20429, China). Gene expression was analyzed through real-time quantitative PCR (qPCR) on a Bio-Rad CFX ConnectTM Real-Time System (Bio-Rad, USA), utilizing SYBR Green (ABclonal, Rk21203, China). The primer sequences are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, The expression levels were quantified using the 2\u0026minus;△△Cq method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the internal control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eq-PCR Primer Sequences\u003c/b\u003e OPN Osteopontin, TNF tumour necrosis factor, IL interleukin, NLRP3 NOD-, LRR- and pyrin domain-containing 3, GSDMD Gasdermin D, ASC apoptosis-associated speck-like protein containing a CARD, GAPDH ; glyceraldehyde-3-phosphate dehydrogenase, Quantitative Real-time reverse transcriptase-polymerase chain reaction, qPCR.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eOPN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-TGACGATGATGATGACGATGGAGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-TGTAGGGACGATTGGAGTGAAAGTG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-CACGCTCTTCTGTCTACTGAACTTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-CTTGGTGGTTTGTGAGTGTGAGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eIL-6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-TTCTTGGGACTGATGCTGGTGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-GTGGTATCCTCTGTGAAGTCTCCTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eIL-1β\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-CTCGCAGCAGCACATCAACAAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-CCACGGGAAAGACACAGGTAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eNLRP3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-AGGAGGAAGAAGAAGAGAGGAGAGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-TTGAGAAGAGACCACGGCAGAAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eGSDMD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-GTGGACAGCCTGCGGAACTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-GGTTCTGGTTCTGGAGCACTGG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ecaspase1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-CCTGGTCTTGTGACTTGGAGGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-ATCAGCAGTGGGCATCTGTAGC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eASC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-GAAGTGGACGGAGTGCTGGATG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-ATCTTGTCTTGGCTGGTGGTCTC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eIL-18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-AAAGTGCCAGTGAACCCCAGAC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-AGAGAGGGTCACAGCCAGTCC-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eGAPDH\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF-5\u0026rsquo;-GCAAATTCAACGGCACAGTCAAG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR-5\u0026rsquo;-TCGCTCCTGGAAGATGGTGATG-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eProteins were extracted from cells by using a radioimmunoprecipitation assay (RIPA) lysis buffer (MCE, HY-K1001, USA) supplemented with 1% phenylmethanesulfonylfluoride (PMSF) (MCE, HY-B0496, USA), 1% protease inhibitor (MCE, HY-K0010, USA), and 1% phosphatase inhibitors (MCE, HY-K0021, USA). The protein concentration was quantified using a NanoDrop spectrophotometer (Thermo Fisher). Subsequently, the proteins were separated by sodium dodecyl sulfate‒polyacrylamide (SDS‒PAGE) gel (EpiZyme Biotechnology, PG112, China) electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010, USA). The membranes were blocked with NcmBlot blocking buffer (New Cell \u0026amp; Molecular Biotech, P30500, China) for 20 minutes and incubated with the appropriate primary antibodies overnight at 4\u0026deg;C. The primary antibodies employed were as follows: anti-Osteopontin (Abcam, ab283656, USA), anti-IL-1β (Proteintech, 16806-1-AP, China), anti-IL-18 (Proteintech, 10663-1-AP, China), NLRP3 Rabbit mAb (Abclonal, A24294, China), GSDMD Polyclonal antibody (Proteintech, 20770-1-AP, China), CASP1 Rabbit pAb (Abclonal, A20470, China), ASC Rabbit mAb (Abclonal, A22046, China), and anti-TNF-α (Proteintech, 17590-1-AP, China). Thereafter, the membranes were incubated with goat anti-rabbit IgG, HRP-conjugated polyclonal antibody (CoWin Bio, CW0103S, China) and goat anti-mouse HRP-conjugated polyclonal antibody (Proteintech, 66009-1-Ig, China) as secondary antibodies at room temperature for 1 hour, and the results were visualized using a Bio-Rad ChemiDocTM Touch Imaging System (Bio-Rad, California, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9 software. All data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independents experiments. Group differences were evaluated by either a t-test (Mann-Whitney U test) or a one-way analysis of variance (Tukey's multiple comparison test). A P value less than 0.05 was considered statistically significant, where \"n\" values indicate the number of cultures, tissue samples, or animals examined within each group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe concentration of OPN is\u003c/b\u003e significantly \u003cb\u003eelevated in pediatric patients with sepsis.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present study, ELISAs were employed to confirm the expression of OPN. The detailed characteristics of patients diagnosed with sepsis, infection, and healthy controls are outlined in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The serum concentration of OPN was observed to be increased in pediatric patients with sepsis (243.5\u0026thinsp;\u0026plusmn;\u0026thinsp;204.5 pg/ml) compared to both healthy individuals (44.71\u0026thinsp;\u0026plusmn;\u0026thinsp;23.82 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and those with infections (101.6\u0026thinsp;\u0026plusmn;\u0026thinsp;30.67 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, we revealed that the serum OPN levels on the first day ( 243.5\u0026thinsp;\u0026plusmn;\u0026thinsp;204.3 pg/ml) were notably elevated compared to those on the second day ( 69.58\u0026thinsp;\u0026plusmn;\u0026thinsp;58.70 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005), and the third day ( 60.42\u0026thinsp;\u0026plusmn;\u0026thinsp;55.15 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) among survivors of sepsis in the intensive care unit.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of paediatric patients with sepsis, infections and healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSepsis\u0026nbsp;patients\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;26)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eInfection\u0026nbsp;patients\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ehealthy\u0026nbsp;controls\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;27)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u0026nbsp;(male/female)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e16/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e8/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e19/8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u0026nbsp;(years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e4.59(0.94\u0026ndash;6.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e5.04(2.44\u0026ndash;7.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.90(3.00\u0026ndash;10.00)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCRP\u0026nbsp;(mg/L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e75.58(25.72\u0026ndash;124.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e8.02(0.8-12.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePCT\u0026nbsp;(ng/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e31.39(1.09\u0026ndash;66.95)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.37(0.055\u0026ndash;0.185)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e2656(51.94\u0026ndash;3009)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e287.9(44.24\u0026ndash;461.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e1.528(0.575\u0026ndash;2.145)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL-1β\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e22.67(1.365\u0026ndash;2.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOrgans\u0026nbsp;dysfunction(n,%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRespiratory\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCirculatory\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNervous\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBlood\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGastrointestinal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eUrinary\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInfection\u0026nbsp;site\u0026nbsp;(Number\u0026nbsp;of\u0026nbsp;patients)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRespiratory\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGastrointestinal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNervous\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVascular\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSkin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBacteraemia\u0026nbsp;Isolates\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(Number\u0026nbsp;of\u0026nbsp;patients)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGram\u0026nbsp;positive\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGram\u0026nbsp;negative\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMycoplasma\u0026nbsp;pneumonia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFungus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVirus\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMiscellaneous\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOther\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePhoenix\u0026nbsp;Sepsis\u0026nbsp;Score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e3.5(2\u0026ndash;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePICU\u0026nbsp;stay(days)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003e12.68(6\u0026ndash;17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eVentilation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDied/survived\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eThe data are expressed as the median (interquartile range) unless otherwise indicated. CRP C-reactive protein, PCT procalcitonin, IL interleukin, TNF tumour necrosis factor, PICU paediatric intensive care unit, NA not applicable\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe OPN expression is elevated in the plasma and lungs following septic mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo analyze variations in OPN levels at the 24-hour mark after CLP, serum and lung tissues from both Sham and CLP mice were analyzed using ELISA and immunofluorescence techniques. The ELISA results indicated a significant elevation of OPN in the serum of CLP mice compared with the Sham group (0.7312\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2263 vs 0.3765\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08419 ng/ml, P\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), mirroring observations in septic patients. Correspondingly, Immunofluorescence analysis showed that, compared with the Sham group, the expression of OPN in the lung tissue of the CLP group increased by 34.8%, P\u0026thinsp;=\u0026thinsp;0.0039(Fig.\u0026nbsp;2BC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAdministration of OPN inhibitor protects septic mice\u003c/h2\u003e \u003cp\u003eTo elucidate the function of OPN in the development of sepsis, mice subjected to CLP were administered an OPN expression inhibitor 1. Notably, the survival rate of these CLP mice was increased (HR\u0026thinsp;=\u0026thinsp;0.2695, p\u0026thinsp;=\u0026thinsp;0.0015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This protective outcome might be ascribed to a diminished systemic inflammatory response following the onset of sepsis. We observed that compared with the Sham group, the levels of serum inflammatory factors in CLP mice were significantly increased, and OPN expression inhibitor 1 significantly reversed this change, such as IL-6 (17.17\u0026thinsp;\u0026plusmn;\u0026thinsp;9.543 ng/ml vs 661.5\u0026thinsp;\u0026plusmn;\u0026thinsp;513.3 ng/ml vs 125.1\u0026thinsp;\u0026plusmn;\u0026thinsp;160.5 ng/ml), TNF-α (9.486\u0026thinsp;\u0026plusmn;\u0026thinsp;10.21 pg/ml vs 158.2\u0026thinsp;\u0026plusmn;\u0026thinsp;86.98 pg/ml vs 25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;9.279 pg/ml), and IL-1β ༈16.62\u0026thinsp;\u0026plusmn;\u0026thinsp;7.695 pg/ml vs 33.41\u0026thinsp;\u0026plusmn;\u0026thinsp;3.282 pg/ml vs 25.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.541 pg/ml)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAdministration of OPN inhibitor protects against sepsis-associated lung injury in mice\u003c/h2\u003e \u003cp\u003eTo elucidate the impact of the OPN expression inhibitor 1 on CLP-associated lung injury, we initially conducted a histological examination of lung tissue and evaluated the extent of injury using a standardized scoring system, as detailed in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eMaterials and Methods\u003c/span\u003e section. Representative histological images of lung tissue from mice treated with sham surgery, vehicle, and OPN inhibitor are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. Compared with the sham group, the lung tissue from the CLP group exhibited pronounced morphological alterations, including edema, hemorrhage, alveolar collapse, and infiltration of inflammatory cells. Conversely, treatment with OPN inhibitors markedly attenuated these microscopic signs of deterioration when compared to the CLP group. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the histological injury score of lung tissue was significantly higher in the CLP group than the sham group (7.667\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5774 vs 2.200\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3801, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), whereas the OPN inhibitor-treated group exhibited a significant improvement in the histological injury score compared to the vehicle group (3.200\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3801 vs 7.667\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5774, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Subsequently, we assessed the D/W ratio of lung tissue in mice. Our findings indicated that the D/W ratio in the CLP group was markedly lower than that of the sham-operated group (20.18%\u0026plusmn;0.7485% vs 22.85%\u0026plusmn;0.7066%, P\u0026thinsp;=\u0026thinsp;0.0002); however, the administration of the OPN expression inhibitor 1 effectively reversed this trend (21.85%\u0026plusmn;0.7329% vs 20.18%\u0026plusmn;0.7485%, P\u0026thinsp;=\u0026thinsp;0.0065) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Beyond blood analysis, we also evaluated the expression of cytokines in lung tissue at the mRNA level. The levels of IL-6, TNF-α, and IL-1β in lung tissue were considerably elevated in the vehicle group compared to the sham group. Nevertheless, treatment with an OPN inhibitor resulted in a significant reduction of these mRNA levels by 80%, 87.5%, and 70%, respectively, compared to the vehicle mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The pathological sections of mouse lung tissue in this study revealed a substantial infiltration of inflammatory cells following CLP, with OPN inhibitors markedly decreasing their count. However, macrophages constitute the primary immune cells within the lungs. Certain studies suggest that OPN facilitates the migration of macrophages to areas of disease, although there remains a divergence of views on whether OPN exerts pro-inflammatory or anti-inflammatory effects. So we first examined changes in the number of macrophage infiltrates by F4/80 staining. Immunofluorescence staining showed great infiltration of F4/80-positive macrophages into lung tissue in the CLP group than in the sham group, and OPN inhibitors significantly reduced the number of F4/80-positive macrophages. Interestingly, we observed the same trend of NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome ( a hallmark of pyroptosis ) in lung tissue (Fig.\u0026nbsp;4ABC). This may elucidate the precise mechanism through which OPN mediates its deleterious effects on the lungs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOPN aggravates macrophage inflammatory response in vitro\u003c/h2\u003e \u003cp\u003eTo elucidate the role of OPN in sepsis-associated lung injury in mice, we investigated the impact of OPN-SiRNA on LPS-treated MH-s cells in vitro. Initially, we identified the presence of OPN in the supernatant of LPS-stimulated MH-s cells and the mRNA expression of OPN in the control group. Our findings indicated that both the mRNA (6.761\u0026thinsp;\u0026plusmn;\u0026thinsp;1.241 vs 1.211\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1833, P\u0026thinsp;=\u0026thinsp;0.0016) and protein (2139\u0026thinsp;\u0026plusmn;\u0026thinsp;1186 pg/ml vs 349.8\u0026thinsp;\u0026plusmn;\u0026thinsp;60.05 pg/ml, P\u0026thinsp;=\u0026thinsp;0.0236) levels of OPN were markedly elevated following LPS stimulation compared to the control group (Fig.\u0026nbsp;5AB). Then, we successfully employed OPN-SiRNA to suppress the expression of OPN at both the mRNA and protein levels in MH-s cells (Fig.\u0026nbsp;5CDE). Subsequently, we found that both the mRNA and protein levels of IL-6, TNF-αand IL-1β in MH-s cells were increased during LPS exposure, while silencing OPN could significantly reduce their levels (Fig.\u0026nbsp;6AB). This outcome further substantiates the pro-inflammatory and tissue-damaging role of OPN within the context of a sepsis model at the cellular level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOPN promotes pyroptosis in macrophages treated with LPS\u003c/h2\u003e \u003cp\u003ePyroptosis, as a form of programmed cell death, plays a crucial role in initiating and advancing the inflammatory response through an amplification loop of inflammation and necrosis.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In vivo experiments, we observed that OPN inhibitor can significantly reduce the expression of NLRP3 in the lungs of CLP mice, we hypothesize that there is a correlation between OPN and pyroptosis. We silenced the expression of OPN in MH-s cells, and then detected the expression of NLRP3, Gasdermin D ( GSDMD ), caspase1, apoptosis-related spot-like protein ( ASC ), IL-1β and IL-18. The results showed that silencing OPN expression significantly reduced both the mRNA and protein levels of NLRP3, GSDMD, caspase1, ASC, IL-1β and IL-18 in the LPS-induced pyroptosis pathway ( Fig.\u0026nbsp;7AB ). Overall, these results indicate that the OPN-mediated inflammatory response in MH-S cells is facilitated by the induction of caspase1-dependent classical pyroptosis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings revealed that circulating levels of OPN were significant elevated in children with sepsis compared to healthy group and infections group. Additionally, the administration of OPN inhibitors in septic mice demonstrated that survival rates were increased by suppressing the expression of inflammatory mediators (TNF-α, IL-1β, IL-6) and mitigating tissue damage, at least in the lung tissue. Moreover, in vitro experiments indicated that the silencing of OPN expression curtailed the inflammatory response by inhibiting the pyroptosis signaling pathway in LPS-stimulated MH-s cells.\u003c/p\u003e \u003cp\u003eFederico Carbone and colleagues discovered that elevated early OPN levels can forecast mortality in patients suffering from septic shock, and,the OPN levels on the first day are associated with multiple organ dysfunction, prolonged hospital stays, extended durations for resolving infections, and various pro-inflammatory responses mediated by macrophages[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, research has indicated that plasma OPN detection could diagnose sepsis in patients[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, these investigations have primarily concentrated on adult subjects, with limited data available on pediatric septic patients. In our study, we observed that the serum OPN levels in children with sepsis were significantly higher than those with infections and healthy controls. We also observed that the serum OPN levels on the first day were notably elevated compared to the second day and the third day among survivors of sepsis in the PICU. The concentration of OPN seems to diminish as sepsis severity improves.\u003c/p\u003e \u003cp\u003eVivo experiments revealed that mortality, systemic inflammatory response, organ damage were aggravated in CLP, which may be related to the increase of OPN concentration. When treated with an OPN inhibitor, we found that all those were improved. It is reasonable to infer that the overall improvement in sepsis outcomes through the inhibition of OPN may be attributed to the suppression of systemic inflammation and the amelioration of lung organ damage. In vitro experiments, we observed a positive correlation between OPN levels and the expression of cytokines TNF-α, IL-6 and IL-1β in the LPS-induced inflammatory response model of MH-s cells. The inhibition of OPN expression by using OPN-siRNA was found to counteract the elevation of TNF-α, IL-6 and IL-1β. These findings support the anti-inflammatory properties of OPN-siRNA.\u003c/p\u003e \u003cp\u003eBruemmer et al. have demonstrated that acute macrophage infiltration is significantly diminished in OPN\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice compared to WT counterparts in a dextran sodium sulfate (DSS)-induced colitis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Then, Yohei et al. discovered that OPN significantly enhanceed neutrophil migration to inflammatory lung lesions by upregulating the mitogen-activated protein (MAP) kinase signaling pathway molecules p38 and extracellular signal-regulated protein kinase (ERK)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In previous research, OPN has been demonstrated to function as a proinflammatory cytokine and chemotactic agent, facilitating the migration of immune cells to areas of inflammation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Nonetheless, the majority of researches have focused on the role of OPN on migration and activation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], there is a lack of investigation into whether there is a correlation between OPN and the programmed cell death of macrophages. In our study, we found that macrophage infiltration and NLRP3 inflammasome expression in the lungs of septic mice was elevated compared to that in the sham group, and the use of OPN inhibitors could significantly ameliorate this situation. Correspondingly, previous studies have indicated that macrophage pyroptosis plays a crucial role in sepsis-induced lung injury by propagating the pulmonary inflammatory response, vascular leakage, and facilitating neutrophil migration to the lungs[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We have presented the initial evidence indicating a connection between pyroptosis in sepsis-related lung injury and OPN. In line with our results, OPN has been shown to be up-regulated in bleomycin-induced pulmonary fibrosis and is associated with macrophage pyroptosis through elevated expression of multiple endocrine neoplasia type 1 (Men1) expression.[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Likewise, when researchers employed calcium oxalate monohydrate ( COM ) to establish a nephrolithiasis cell model of macrophages, they observed that the expression trends of OPN and macrophage pyroptosis signaling pathways were notably consistent[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, it remains ambiguous whether OPN is associated with macrophage pyroptosis, and the precise mechanism remains unclear. In our study, we observed that silencing OPN expression in MH-s cells can significantly reduce both the mRNA and protein levels of NLRP3, GSDMD, caspase1, ASC, IL-1β and IL-18 in LPS-induced elevated pyroptosis pathway. Therefore, it can be concluded that preventing OPN-induced pyroptosis could potentially mitigate the inflammatory damage associated with sepsis.\u003c/p\u003e \u003cp\u003eThe present investigation has several limitations that deserve consideration. Firstly, it is a single-center study with a relatively small sample size of sepsis patients. Furthermore, there is an lack of comparative analysis of OPN levels between survivors and non-survivors, as well as between sepsis patients and those with other organ dysfunctions in the PICU, highlighting the necessity for extensive clinical trials and additional research to elucidate the specific differential role of OPN in sepsis patients.\u003c/p\u003e \u003cp\u003eIn summary, the findings of this study highlight the detrimental role of OPN in the context of sepsis. Moreover, the therapeutic administration of OPN inhibitors has been demonstrated to increase survival rates and attenuate lung injury in septic mice. Additionally, the protective mechanism of OPN inhibitors encompasses both anti-inflammatory effects and the suppression of pyroptosis. Consequently, OPN not only holds potential as a promising diagnostic marker for sepsis but also presents a novel therapeutic target for the development of sepsis treatment medications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOPN \u0026nbsp;Osteopontin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSPP1 \u0026nbsp;Secreted phosphorylated protein 1\u003c/p\u003e\n\u003cp\u003eCLP \u0026nbsp;Cecal ligation and puncture\u003c/p\u003e\n\u003cp\u003eLPS \u0026nbsp;Lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eMH-S \u0026nbsp;Mouse Hemophagocytic Synuclein\u003c/p\u003e\n\u003cp\u003ePICU \u0026nbsp;Pediatric intensive care unit\u003c/p\u003e\n\u003cp\u003eWT \u0026nbsp;Wild-type\u003c/p\u003e\n\u003cp\u003eFBS \u0026nbsp;Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eNC \u0026nbsp;Negative control\u003c/p\u003e\n\u003cp\u003eSiRNA \u0026nbsp;Small interfering RNA\u003c/p\u003e\n\u003cp\u003eOI \u0026nbsp;OPN inhibitor\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp;Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eELISA \u0026nbsp;Enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; \u0026nbsp;Tumor necrosis factor-alpha\u003c/p\u003e\n\u003cp\u003eIL-6 \u0026nbsp;Interleukin-6\u003c/p\u003e\n\u003cp\u003eIL-1\u0026beta; \u0026nbsp;Interleukin-1 beta\u003c/p\u003e\n\u003cp\u003eLPS \u0026nbsp;Lipopolysaccharide\u003c/p\u003e\n\u003cp\u003eD/W \u0026nbsp;Dry-to-wet\u003c/p\u003e\n\u003cp\u003ePBST \u0026nbsp;Phosphate buffer saline with tween-20\u003c/p\u003e\n\u003cp\u003eDAPI \u0026nbsp;4\u0026prime;,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E \u0026nbsp;Hematoxylin and eosin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eqPCR \u0026nbsp;Real-time quantitative PCR\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGAPDH \u0026nbsp;Glyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e\n\u003cp\u003eRIPA \u0026nbsp;Radioimmunoprecipitation assay\u003c/p\u003e\n\u003cp\u003ePMSF \u0026nbsp;Phenylmethanesulfonylfluoride\u003c/p\u003e\n\u003cp\u003eSDS-PAGE \u0026nbsp;Sodium dodecyl sulfate‒polyacrylamide\u003c/p\u003e\n\u003cp\u003ePVDF \u0026nbsp;Polyvinylidene fluoride\u003c/p\u003e\n\u003cp\u003eNLRP3 \u0026nbsp;NOD-, LRR- and pyrin domain-containing 3\u003c/p\u003e\n\u003cp\u003eGSDMD \u0026nbsp;Gasdermin D\u003c/p\u003e\n\u003cp\u003eASC \u0026nbsp;Apoptosis-related spot-like protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were approved by the Clinical Research Ethics Committee of Institutional Review Board of Children\u0026rsquo;s Hospital of Chongqing Medical University (File No: (2021) Ethical Review Research No. 325-1). All animal experiments were conducted under the rules approved by the Ethics Committee of Chongqing Experimental Animal Center and the Animal Committee of Children\u0026rsquo;s Hospital (CHCMU-IACUC20231208004). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll listed authors consent to the submission, and all data are used with the consent of the person generating the data. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Key Program of Chongqing Clinical Medical Research (Grant No. NCRCCHD-2021-KP-03) and the Program For Youth Innovation in Future Medicine of Chongqing Medical University (W0124 to JL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXF and LJ contributed to the conception and design. QW performed the experiments, analysed data, and wrote the manuscript. ZCY helped to revise the manuscript. ZXS, XL, ZL, and DDP helped in the acquisition of clinical data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eDepartment of Critical Care Medicine, Children\u0026rsquo;s Hospital of Chongqing Medical University Chongqing, China. \u003csup\u003e2\u003c/sup\u003e National Clinical Research Center for Child Health and Disorders, Chongqing, China. \u003csup\u003e3\u003c/sup\u003e Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, China. \u003csup\u003e4\u0026nbsp;\u003c/sup\u003eChongqing Key Laboratory of Pediatrics, Chongqing, China. \u003csup\u003e5\u003c/sup\u003e Department of Pediatric Intensive Care Unit, First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China. \u003csup\u003e6\u0026nbsp;\u003c/sup\u003eDepartment of Life Science Center, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, China. \u003csup\u003e7\u0026nbsp;\u003c/sup\u003eClinical Laboratory of Children \u0026apos;s Hospital of Chongqing Medical University, Chongqing, China. \u003csup\u003e8\u003c/sup\u003e Department of Emergency, Children \u0026apos;s Hospital of Chongqing Medical University, Chongqing, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSinger M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315:801\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eLiu R, Yu Z, Xiao C, Xu F, Xiao S, He J, et al. Epidemiology and clinical characteristics of pediatric sepsis in PICUs in southwest China: A prospective multicenter study. Pediatr Crit Care Med. 2024;25:425\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eWatson RS, Carrol ED, Carter MJ, Kissoon N, Ranjit S, Schlapbach LJ. The burden and contemporary epidemiology of sepsis in children. The Lancet Child \u0026amp; Adolescent Health. 2024;8:670\u0026ndash;81. \u003c/li\u003e\n\u003cli\u003eInternational Consensus Criteria for Pediatric Sepsis and Septic Shock - PMC [Internet]. [cited 2024 Aug 11]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10900966/\u003c/li\u003e\n\u003cli\u003eGlobal, regional, and national sepsis incidence and mortality, 1990\u0026ndash;2017: analysis for the Global Burden of Disease Study - PMC [Internet]. [cited 2024 Sep 10]. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970225/\u003c/li\u003e\n\u003cli\u003eIcer MA, Gezmen-Karadag M. The multiple functions and mechanisms of osteopontin. Clinical Biochemistry. 2018;59:17\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eBastos ACS da F, Gomes AVP, Silva GR, Emerenciano M, Ferreira LB, Gimba ERP. The Intracellular and Secreted Sides of Osteopontin and Their Putative Physiopathological Roles. Int J Mol Sci. 2023;24. \u003c/li\u003e\n\u003cli\u003eLund SA, Wilson CL, Raines EW, Tang J, Giachelli CM, Scatena M. Osteopontin mediates macrophage chemotaxis via \u0026alpha; \u003csub\u003e4\u003c/sub\u003e and \u0026alpha; \u003csub\u003e9\u003c/sub\u003e integrins and survival via the \u0026alpha; \u003csub\u003e4\u003c/sub\u003e integrin. J of Cellular Biochemistry. 2013;114:1194\u0026ndash;202. \u003c/li\u003e\n\u003cli\u003eZou C, Luo Q, Qin J, Shi Y, Yang L, Ju B, et al. Osteopontin promotes mesenchymal stem cell migration and lessens cell stiffness via integrin \u0026beta;1, FAK, and ERK pathways. Cell Biochem Biophys. 2013;65:455\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eMiyazaki T, Ono M, Qu W-M, Zhang M-C, Mori S, Nakatsuru S, et al. Implication of allelic polymorphism of osteopontin in the development of lupus nephritis in MRL/lpr mice. Eur J Immunol. 2005;35:1510\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eKawamura K, Iyonaga K, Ichiyasu H, Nagano J, Suga M, Sasaki Y. Differentiation, maturation, and survival of dendritic cells by osteopontin regulation. Clin Diagn Lab Immunol. 2005;12:206\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eVaschetto R, Nicola S, Olivieri C, Boggio E, Piccolella F, Mesturini R, et al. Serum levels of osteopontin are increased in SIRS and sepsis. Intensive Care Med. 2008;34:2176\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eDejager L, Pinheiro I, Dejonckheere E, Libert C. Cecal ligation and puncture: the gold standard model for polymicrobial sepsis? Trends in Microbiology. 2011;19:198\u0026ndash;208. \u003c/li\u003e\n\u003cli\u003eLinkermann A, Stockwell BR, Krautwald S, Anders H-J. Regulated cell death and inflammation: an auto-amplification loop causes organ failure. Nat Rev Immunol. 2014;14:759\u0026ndash;67. \u003c/li\u003e\n\u003cli\u003eCarbone F, Bonaventura A, Vecchi\u0026egrave; A, Meessen J, Minetti S, Elia E, et al. Early osteopontin levels predict mortality in patients with septic shock. Eur J Intern Med. 2020;78:113\u0026ndash;20. \u003c/li\u003e\n\u003cli\u003eCastello LM, Baldrighi M, Molinari L, Salmi L, Cantaluppi V, Vaschetto R, et al. The Role of Osteopontin as a Diagnostic and Prognostic Biomarker in Sepsis and Septic Shock. Cells. 2019;8. \u003c/li\u003e\n\u003cli\u003eZhong J, Eckhardt ERM, Oz HS, Bruemmer D, de Villiers WJS. Osteopontin deficiency protects mice from dextran sodium sulfate-induced colitis. Inflamm Bowel Dis. 2006;12:790\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eHirano Y, Aziz M, Yang W-L, Wang Z, Zhou M, Ochani M, et al. Neutralization of osteopontin attenuates neutrophil migration in sepsis-induced acute lung injury. Crit Care. 2015;19:53. \u003c/li\u003e\n\u003cli\u003eClemente N, Raineri D, Cappellano G, Boggio E, Favero F, Soluri MF, et al. Osteopontin Bridging Innate and Adaptive Immunity in Autoimmune Diseases. J Immunol Res. 2016;2016:7675437. \u003c/li\u003e\n\u003cli\u003eLamort A-S, Giopanou I, Psallidas I, Stathopoulos GT. Osteopontin as a Link between Inflammation and Cancer: The Thorax in the Spotlight. Cells. 2019;8. \u003c/li\u003e\n\u003cli\u003eIntracellular osteopontin regulates homeostasis and function of natural killer cells - PubMed [Internet]. [cited 2023 Jun 17]. Available from: https://pubmed.ncbi.nlm.nih.gov/25550515/\u003c/li\u003e\n\u003cli\u003eTan Y, Zhao L, Yang Y-G, Liu W. The Role of Osteopontin in Tumor Progression Through Tumor-Associated Macrophages. Front Oncol. 2022;12:953283. \u003c/li\u003e\n\u003cli\u003eWeber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Ashkar S. Phosphorylation-dependent interaction of osteopontin with its receptors regulates macrophage migration and activation. Journal of Leukocyte Biology. 2002;72:752\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eKim D, Haynes CL. The role of p38 MAPK in neutrophil functions: Single cell chemotaxis and surface marker expression. Analyst. 2013;138:6826. \u003c/li\u003e\n\u003cli\u003eQin X, Zhou Y, Jia C, Chao Z, Qin H, Liang J, et al. Caspase-1-mediated extracellular vesicles derived from pyroptotic alveolar macrophages promote inflammation in acute lung injury. Int J Biol Sci. 2022;18:1521\u0026ndash;38. \u003c/li\u003e\n\u003cli\u003eLu Y, Zhao J, Tian Y, Shao D, Zhang Z, Li S, et al. Dichotomous Roles of Men1 in Macrophages and Fibroblasts in Bleomycin\u0026mdash;Induced Pulmonary Fibrosis. Int J Mol Sci. 2022;23:5385. \u003c/li\u003e\n\u003cli\u003eDing T, Zhao T, Li Y, Liu Z, Ding J, Ji B, et al. Vitexin exerts protective effects against calcium oxalate crystal-induced kidney pyroptosis in vivo and in vitro. Phytomedicine. 2021;86:153562. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jinf","sideBox":"Learn more about [Journal of Inflammation](http://journal-inflammation.biomedcentral.com/)","snPcode":"12950","submissionUrl":"https://submission.nature.com/new-submission/12950/3","title":"Journal of Inflammation","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sepsis, lung injure, Osteopontin, Macrophage Pyroptosis","lastPublishedDoi":"10.21203/rs.3.rs-5435730/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5435730/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSepsis is a severe condition causing organ failure due to an abnormal immune reaction to infection, characterized by ongoing excessive inflammation and immune system issues. Osteopontin (OPN) is secreted by various cells and plays a crucial role in inflammatory responses and immune regulation. Nonetheless, the precise function of OPN in sepsis remains to be elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn the present study, we evaluated the levels of OPN in paediatric patients with sepsis and healthy individuals. We examined the impact of OPN on survival rates, systemic inflammation, and lung injury within an experimental sepsis model using cecal ligation and puncture (CLP). Furthermore, the pro-inflammatory effects and potential mechanisms of OPN in sepsis were investigated through Mouse Hemophagocytic Synuclein (MH-S) cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe OPN level was found to be elevated in patients with sepsis (243.5\u0026thinsp;\u0026plusmn;\u0026thinsp;204.5 pg/ml) compared to children with common infections (101.6\u0026thinsp;\u0026plusmn;\u0026thinsp;30.67 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and healthy individuals (44.71\u0026thinsp;\u0026plusmn;\u0026thinsp;23.82 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).The serum OPN level on the first day ( 243.5\u0026thinsp;\u0026plusmn;\u0026thinsp;204.3 pg/ml) was significantly higher than that on the sceond day (69.58\u0026thinsp;\u0026plusmn;\u0026thinsp;58.70 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and the third day (60.42\u0026thinsp;\u0026plusmn;\u0026thinsp;55.15 pg/ml) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.005) in pediatric patient with sepsis admission to pediatric intensive care unit(PICU). Intravenous administration of OPN inhibitor into the tail vein decreased the mortality rate (HR\u0026thinsp;=\u0026thinsp;0.2695, p\u0026thinsp;=\u0026thinsp;0.0015), suppressed systemic inflammatory responses and mitigated lung tissue damage. The concentration of TNF-α, IL-6 and IL-1β in serum of CLP mice treated with OPN inhibitor decreased compared with CLP mice. Within the sepsis mouse model, there was a marked increase in OPN expression in the lungs tissues compared to the sham group mice. This surge was accompanied by a significant accumulation of alveolar macrophages and an upregulation of inflammasome expression. Mechanistic investigations in MH-s cells revealed that OPN-SiRNA suppressed the LPS-induced macrophage inflammatory response by inhibiting caspase1-dependent classical pyroptosis signaling pathway.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study reveals that OPN has an adverse impact on the host's immune response to sepsis. Suppressing OPN expression holds potential therapeutic value for the treatment of sepsis.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003eStudy on the diagnostic value of osteopontin in children with sepsis. MR5024001771. Registered 22 January 2024. https//www.medicalresearch.org.cn.\u003c/p\u003e","manuscriptTitle":"The injury effect of osteopontin in sepsis-associated lung injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-16 17:18:14","doi":"10.21203/rs.3.rs-5435730/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-05T14:06:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-22T02:53:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294497344336866215124704161114554657044","date":"2024-11-16T11:00:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-14T12:34:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-12T05:03:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-12T05:01:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Inflammation","date":"2024-11-12T03:13:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jinf","sideBox":"Learn more about [Journal of Inflammation](http://journal-inflammation.biomedcentral.com/)","snPcode":"12950","submissionUrl":"https://submission.nature.com/new-submission/12950/3","title":"Journal of Inflammation","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"378d5038-199f-41ea-a454-8f2f2fc1b18b","owner":[],"postedDate":"December 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T15:59:54+00:00","versionOfRecord":{"articleIdentity":"rs-5435730","link":"https://doi.org/10.1186/s12950-025-00430-4","journal":{"identity":"journal-of-inflammation","isVorOnly":false,"title":"Journal of Inflammation"},"publishedOn":"2025-01-24 15:57:06","publishedOnDateReadable":"January 24th, 2025"},"versionCreatedAt":"2024-12-16 17:18:14","video":"","vorDoi":"10.1186/s12950-025-00430-4","vorDoiUrl":"https://doi.org/10.1186/s12950-025-00430-4","workflowStages":[]},"version":"v1","identity":"rs-5435730","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5435730","identity":"rs-5435730","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}