Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 levels are elevated in both BALF and lung tissues of LPS‐induced ARDS Rats. (A, D, E) ELISA analysis of ORM1, TF, and PAI‐1 concentrations in bronchoalveolar lavage fluid (BALF) from control and LPS‐treated rats at 24 h post‐induction. (B, E, H) qRT‐PCR analysis of ORM1, TF, and PAI‐1 mRNA expression levels in lung tissues of control and LPS‐treated rats at 24 h post‐induction. (J) Western blot analysis of ORM1, TF, and PAI‐1 protein expression in lung tissues of LPS‐induced ARDS rats at 24 h post‐induction, with quantitative densitometry shown in (G, C, I). (K) Immunofluorescence co‐staining for ORM1 (red) and alveolar type II cell marker surfactant protein C (SP‐C, green). Yellow signal (white arrow) indicates ORM1/SP‐C co‐localization. Scale bar: 300 μm. Data represent mean ± SD ( n = 6 per group). Significance in (A–J) was calculated using an independent t test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Marker

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: Downregulation of ORM1 Significantly Mitigates LPS‐Induced Lung Injury in ARDS Rats. (A) Hematoxylin and eosin (H&E) staining revealssubstantial histopathological damage in lung tissues at 24 h post‐LPS induction and significant attenuation of LPS‐induced pulmonary injury following ORM1 knockdown. Scale bars: 200 μm. (B) Histopathological injury scores corresponding to H&E staining for lung injury assessment. (C) Pulmonary edema assessed by lung wet/dry weight ratio. Data represent mean ± SD ( n = 6). Significance in (B) and (C) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. Statistical significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Staining, Knockdown, Control

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Suppression Ameliorates Alveolar Hypercoagulation and Fibrinolysis Inhibition In Vivo. (A–C) ELISA quantification of ORM1, TF, and PAI‐1 concentrations in bronchoalveolar lavage fluid (BALF) showing: Significant elevation of all three biomarkers at 24 h post‐LPS airway nebulization inhalation compared to controls, and marked reduction in their levels following ORM1 knockdown. (D–F) qRT‐PCR analysis reveals: Significant elevation of ORM1, TF, and PAI‐1 mRNA levels in LPS‐induced ARDS lung tissues compared to control, Marked downregulation of these transcripts following ORM1 knockdown. (J) Representative Western blots showing upregulated ORM1, TF, and PAI‐1 protein expression in lung tissues at 24 h post‐LPS induction, with significant reduction following ORM1 knockdown, Quantitative densitometric analysis of (G) ORM1, (H) TF, and (I) PAI‐1 protein levels normalized to GAPDH. (K) Collagen III deposition in lung parenchyma visualized by immunohistochemistry (brown staining indicates positive expression). Scale bars: 300 μm. Data represent mean ± SD ( n = 6), Significance in (A–I) were calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Inhibition, In Vivo, Enzyme-linked Immunosorbent Assay, Knockdown, Quantitative RT-PCR, Control, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Enhances NF‐κB Signaling Pathway Activation In Vivo. (A) The Western blot method was used to detect the protein expression levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), and total p65 in the lung tissues of rats in each group, with quantitative densitometry showing the expression levels of total IKKβ (B) and total p65 (D) in the lung tissues of each group of rats were not statistically significant, and the expression levels of phosphorylated IKKβ (C) and phosphorylated p65 (E) in the lung tissues of rats induced by LPS increased, while those in the lung tissues of rats induced by LPS decreased after knockdown of ORM1. Data represent mean ± SD ( n = 6). Significance in (B–E) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: Not significant ( p > 0.05).

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Activation Assay, In Vivo, Western Blot, Expressing, Knockdown

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Promotes TF and PAI‐1 Expression in LPS‐Stimulated AEC II Cells. (A–C) qRT‐PCR analysis showed that, compared with the control group, the mRNA levels of ORM1, TF, and PAI‐1 in AEC II cells induced by LPS for 24 h were significantly increased, and the levels of these indicators were down‐regulated after the knockdown of ORM1. (D) Representative Western blot results showed that the protein expressions of ORM1, TF, and PAI‐1 in AEC II cells were upregulated after 24 h of LPS induction and significantly decreased after the knockdown of ORM1. Quantitative density analysis normalized the protein levels of (E) ORM1, (F) TF, and (G) PAI‐1 to GAPDH. Data represent mean ± SD ( n = 6). Significance in (A–C, E–G) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Western Blot

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Enhances TF and PAI‐1 expression maybe through the NF‐κB pathway. (A) Protein levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), and total p65 were assessed using Western Blot analysis, followed by semiquantitative analysis (B–E) after ORM1 knockdown. Protein levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), total p65, TF, and PAI‐1 were assessed using Western Blot analysis (F), followed by semiquantitative analysis (G–N) after NF‐κB inhibitor benzoxathiole treatment. Data represent mean ± SD ( n = 3). Significance in (B–E, G–N) was calculated using one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns: Not significant ( p > 0.05).

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Expressing, Western Blot, Knockdown

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Directly Activates the NF‐κB Pathway in LPS‐Exposed AEC II Cells. (A) Cell immunofluorescence co‐staining of ORM1 (red) and phosphorylated IKKβ (p‐IKKβ, green). The yellow signal (white arrow) indicates the co‐localization of ORM1/p‐IKKβ. After 24 h of LPS induction, the expression and co‐expression of ORM1 and p‐IKKβ in AEC II cells increased. After knockout of ORM1, In LPS‐induced AECII cells, the expressions of ORM1, p‐IKKβ and their co‐expressions were all decreased. (B) Co‐expression of ORM1 and p‐IKKβ in LPS‐Induced AEC II cells following NF‐κB pathway inhibitor (benzoxathiole) treatment were decreased. The yellow areas, indicated by white arrows, represent regions of co‐localization between ORM1 and p‐IKKβ (scale bar: 30 μm). (C and D) The immunoprecipitation results show that in LPS‐induced AEC II cells, ORM1 interacts with p‐IKKB.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Immunofluorescence, Staining, Expressing, Knock-Out, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: Concentrations of ORM1 in BALF Are Significantly Elevated in Patients with ARDS. (A) ORM1, (B) TF, and (C) PAI‐1 concentrations in BALF from patients with ARDS ( n = 28) and non‐ARDS ( n = 26) were measured using specific human ORM1, TF, and PAI‐1 enzyme‐linked immunosorbent assay (ELISA) kits. (D) Oxygenation index, expressed as the ratio of arterial oxygen partial pressure to oxygen concentration (P/F), in ARDS patients ( n = 28) and non‐ARDS patients ( n = 26). Correlations between BALF concentrations of ORM1 and those of (E) TF, (F) PAI‐1, and (G) P/F ratio in patients with ARDS are shown. Data represent mean ± SD. Significance in (A–D) was calculated using an independent t test; significance in (A–D) was calculated using one‐way analysis of variance (ANOVA) with Scheffé's post hoc test, and (E–G) were evaluated with Pearson's correlation coefficients. **** p < 0.0001.

Article Snippet: Lung tissue sections were blocked with sheep serum (Sorabio, Beijing, China) for 15 min at room temperature, and for double immunofluorescence staining with ORM1 (1:100 dilution, Proteintech, Wuhan, China) and SP‐C antibodies (1:100 dilution, Affinity Biosciences, USA) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 levels are elevated in both BALF and lung tissues of LPS‐induced ARDS Rats. (A, D, E) ELISA analysis of ORM1, TF, and PAI‐1 concentrations in bronchoalveolar lavage fluid (BALF) from control and LPS‐treated rats at 24 h post‐induction. (B, E, H) qRT‐PCR analysis of ORM1, TF, and PAI‐1 mRNA expression levels in lung tissues of control and LPS‐treated rats at 24 h post‐induction. (J) Western blot analysis of ORM1, TF, and PAI‐1 protein expression in lung tissues of LPS‐induced ARDS rats at 24 h post‐induction, with quantitative densitometry shown in (G, C, I). (K) Immunofluorescence co‐staining for ORM1 (red) and alveolar type II cell marker surfactant protein C (SP‐C, green). Yellow signal (white arrow) indicates ORM1/SP‐C co‐localization. Scale bar: 300 μm. Data represent mean ± SD ( n = 6 per group). Significance in (A–J) was calculated using an independent t test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Marker

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: Downregulation of ORM1 Significantly Mitigates LPS‐Induced Lung Injury in ARDS Rats. (A) Hematoxylin and eosin (H&E) staining revealssubstantial histopathological damage in lung tissues at 24 h post‐LPS induction and significant attenuation of LPS‐induced pulmonary injury following ORM1 knockdown. Scale bars: 200 μm. (B) Histopathological injury scores corresponding to H&E staining for lung injury assessment. (C) Pulmonary edema assessed by lung wet/dry weight ratio. Data represent mean ± SD ( n = 6). Significance in (B) and (C) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. Statistical significance: ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control group.

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Staining, Knockdown, Control

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Suppression Ameliorates Alveolar Hypercoagulation and Fibrinolysis Inhibition In Vivo. (A–C) ELISA quantification of ORM1, TF, and PAI‐1 concentrations in bronchoalveolar lavage fluid (BALF) showing: Significant elevation of all three biomarkers at 24 h post‐LPS airway nebulization inhalation compared to controls, and marked reduction in their levels following ORM1 knockdown. (D–F) qRT‐PCR analysis reveals: Significant elevation of ORM1, TF, and PAI‐1 mRNA levels in LPS‐induced ARDS lung tissues compared to control, Marked downregulation of these transcripts following ORM1 knockdown. (J) Representative Western blots showing upregulated ORM1, TF, and PAI‐1 protein expression in lung tissues at 24 h post‐LPS induction, with significant reduction following ORM1 knockdown, Quantitative densitometric analysis of (G) ORM1, (H) TF, and (I) PAI‐1 protein levels normalized to GAPDH. (K) Collagen III deposition in lung parenchyma visualized by immunohistochemistry (brown staining indicates positive expression). Scale bars: 300 μm. Data represent mean ± SD ( n = 6), Significance in (A–I) were calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Inhibition, In Vivo, Enzyme-linked Immunosorbent Assay, Knockdown, Quantitative RT-PCR, Control, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Enhances NF‐κB Signaling Pathway Activation In Vivo. (A) The Western blot method was used to detect the protein expression levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), and total p65 in the lung tissues of rats in each group, with quantitative densitometry showing the expression levels of total IKKβ (B) and total p65 (D) in the lung tissues of each group of rats were not statistically significant, and the expression levels of phosphorylated IKKβ (C) and phosphorylated p65 (E) in the lung tissues of rats induced by LPS increased, while those in the lung tissues of rats induced by LPS decreased after knockdown of ORM1. Data represent mean ± SD ( n = 6). Significance in (B–E) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: Not significant ( p > 0.05).

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Activation Assay, In Vivo, Western Blot, Expressing, Knockdown

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Promotes TF and PAI‐1 Expression in LPS‐Stimulated AEC II Cells. (A–C) qRT‐PCR analysis showed that, compared with the control group, the mRNA levels of ORM1, TF, and PAI‐1 in AEC II cells induced by LPS for 24 h were significantly increased, and the levels of these indicators were down‐regulated after the knockdown of ORM1. (D) Representative Western blot results showed that the protein expressions of ORM1, TF, and PAI‐1 in AEC II cells were upregulated after 24 h of LPS induction and significantly decreased after the knockdown of ORM1. Quantitative density analysis normalized the protein levels of (E) ORM1, (F) TF, and (G) PAI‐1 to GAPDH. Data represent mean ± SD ( n = 6). Significance in (A–C, E–G) was calculated using the one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Western Blot

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Enhances TF and PAI‐1 expression maybe through the NF‐κB pathway. (A) Protein levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), and total p65 were assessed using Western Blot analysis, followed by semiquantitative analysis (B–E) after ORM1 knockdown. Protein levels of phosphorylated IKKβ (p‐IKKβ), total IKKβ, phosphorylated p65 (p‐p65), total p65, TF, and PAI‐1 were assessed using Western Blot analysis (F), followed by semiquantitative analysis (G–N) after NF‐κB inhibitor benzoxathiole treatment. Data represent mean ± SD ( n = 3). Significance in (B–E, G–N) was calculated using one‐way analysis of variance (ANOVA) with Scheffé's post hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns: Not significant ( p > 0.05).

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Knockdown

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: ORM1 Directly Activates the NF‐κB Pathway in LPS‐Exposed AEC II Cells. (A) Cell immunofluorescence co‐staining of ORM1 (red) and phosphorylated IKKβ (p‐IKKβ, green). The yellow signal (white arrow) indicates the co‐localization of ORM1/p‐IKKβ. After 24 h of LPS induction, the expression and co‐expression of ORM1 and p‐IKKβ in AEC II cells increased. After knockout of ORM1, In LPS‐induced AECII cells, the expressions of ORM1, p‐IKKβ and their co‐expressions were all decreased. (B) Co‐expression of ORM1 and p‐IKKβ in LPS‐Induced AEC II cells following NF‐κB pathway inhibitor (benzoxathiole) treatment were decreased. The yellow areas, indicated by white arrows, represent regions of co‐localization between ORM1 and p‐IKKβ (scale bar: 30 μm). (C and D) The immunoprecipitation results show that in LPS‐induced AEC II cells, ORM1 interacts with p‐IKKB.

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Immunofluorescence, Staining, Expressing, Knock-Out, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Orosomucoid 1 Participates in Alveolar Hypercoagulation and Fibrinolytic Inhibition Involving NF ‐ κB Signaling Pathway in LPS ‐Induced ARDS

doi: 10.1096/fj.202502459RR

Figure Lengend Snippet: Concentrations of ORM1 in BALF Are Significantly Elevated in Patients with ARDS. (A) ORM1, (B) TF, and (C) PAI‐1 concentrations in BALF from patients with ARDS ( n = 28) and non‐ARDS ( n = 26) were measured using specific human ORM1, TF, and PAI‐1 enzyme‐linked immunosorbent assay (ELISA) kits. (D) Oxygenation index, expressed as the ratio of arterial oxygen partial pressure to oxygen concentration (P/F), in ARDS patients ( n = 28) and non‐ARDS patients ( n = 26). Correlations between BALF concentrations of ORM1 and those of (E) TF, (F) PAI‐1, and (G) P/F ratio in patients with ARDS are shown. Data represent mean ± SD. Significance in (A–D) was calculated using an independent t test; significance in (A–D) was calculated using one‐way analysis of variance (ANOVA) with Scheffé's post hoc test, and (E–G) were evaluated with Pearson's correlation coefficients. **** p < 0.0001.

Article Snippet: The slides were blocked with goat serum (Solarbio, Beijing, China) at room temperature for 1 h and incubated with ORM1 antibody (1:100 dilution; Proteintech Group Inc., Wuhan, China) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Proteomic analysis of the synovial fluid (SF) samples from patients with different stages of temporomandibular joint osteoarthritis (TMJOA). A) Flow diagram for patient inclusion and grouping. B) Photograph showing joint fluid collection during arthroscopic surgery. C) Flow diagram for the proteomic analysis of SF samples. D) Principal component analysis of SF samples. E) Top 10 genes that showed an upward trend and top 10 genes that showed a downward trend in the three groups. F) Orosomucoid 1 (ORM1) expression in patients with different grades of TMJOA, including Mild (n = 21), Moderate (n = 27), and Severe (n = 15) groups. Data is expressed as mean ± SD and analyzed using one‐way analysis of variance (ANOVA) followed by Tukey's post‐hoc test. G) ORM1 expression in patients with pain (n = 30) or without pain (n = 26). Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed). H) ORM1 expression in patients with different degrees of maximum interincisal opening (MIO). Data in MIO ≤ 25 mm (n = 10), MIO 26–35 mm (n = 20), and MIO ≥ 36 mm (n = 29) groups are expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test. I) ORM1 expression in patients with sleep bruxism (n = 26) or without sleep bruxism (n = 23). Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed). J) ORM1 concentration in SF samples detected by ELISA assay in Mild (n = 5), Moderate (n = 5), and Severe (n = 5) groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test. FOT, fraction of the total; * p < 0.05.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Expressing, Two Tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Proteomic analysis of the synovial fluid (SF) samples from patients with different stages of temporomandibular joint osteoarthritis (TMJOA). A) Flow diagram for patient inclusion and grouping. B) Photograph showing joint fluid collection during arthroscopic surgery. C) Flow diagram for the proteomic analysis of SF samples. D) Principal component analysis of SF samples. E) Top 10 genes that showed an upward trend and top 10 genes that showed a downward trend in the three groups. F) Orosomucoid 1 (ORM1) expression in patients with different grades of TMJOA, including Mild (n = 21), Moderate (n = 27), and Severe (n = 15) groups. Data is expressed as mean ± SD and analyzed using one‐way analysis of variance (ANOVA) followed by Tukey's post‐hoc test. G) ORM1 expression in patients with pain (n = 30) or without pain (n = 26). Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed). H) ORM1 expression in patients with different degrees of maximum interincisal opening (MIO). Data in MIO ≤ 25 mm (n = 10), MIO 26–35 mm (n = 20), and MIO ≥ 36 mm (n = 29) groups are expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test. I) ORM1 expression in patients with sleep bruxism (n = 26) or without sleep bruxism (n = 23). Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed). J) ORM1 concentration in SF samples detected by ELISA assay in Mild (n = 5), Moderate (n = 5), and Severe (n = 5) groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test. FOT, fraction of the total; * p < 0.05.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Expressing, Two Tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Histopathological and microcomputed tomography (micro‐CT) analysis showing the effect of ORM1 on maintaining cartilage homeostasis in rats with TMJOA. A) Immunofluorescence analysis of ORM1 in the condylar cartilage of rats with or without TMJOA. Data are expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed), n = 3, * p < 0.05. B) Subcellular localization of exogenous ORM1 conjugated with fluorescein isothiocyanate (FITC). C) Hematoxylin–eosin and Safranin O‐fast green staining of the condylar cartilage of rats in various treatment groups. D) Thickness of the condylar cartilage of rats in various treatment groups based on the Hematoxylin–eosin staining. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group. E) Osteoarthritis Research Society International (OARSI) scores of the condylar cartilage of rats in various treatment groups based on the Safranin O‐fast green staining. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group. F) Micro‐CT images of condylar tissue samples from various treatment groups. Comparison of percent bone volume (BV/TV) (G), bone surface/volume ratio (BS/BV) (H), trabecular number (Tb.N) (I), trabecular separation (Tb.Sp) (J), and bone mineral density (BMD) K) among various treatment groups. NS, normal saline; UAC, unilateral anterior crossbite. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Tomography, Micro-CT, Immunofluorescence, Two Tailed Test, Staining, Comparison, Saline

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Bulk RNA sequencing in C28/I2 cells treated with ORM1, and the inhibitory effect of ORM1 on MMP13 and MMP3. A) Administration on C28/I2 cells and the principal component analysis; B) Volcano map showing the differentially expressed genes (DEGs) of interest; C) Top 20 DEGs rescued after ORM1 treatment; D) Expression change of genes related to osteoarthritis in various treatment groups; E) The transcripts per million (TPM) of MMP13, MMP3, MMP12, and MMP9 in the three groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the NC group, * p < 0.05. F) GO analysis of DEGs that were upregulated after IL‐1β treatment and downregulated after ORM1 treatment. G) mRNA expression level of MMP13 and MMP3 in both natural and inflammation environments in C28/I2 and SW1353 cells after treatment with ORM1 protein. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the NC group, * p < 0.05. H) Western blot showing the protein level of MMP13 and MMP3 in both natural and inflammatory environments in C28/I2 and SW1353 cells after treatment with ORM1 protein. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the natural control (NC) group, * p < 0.05. I, J) Immunofluorescence analysis showing the effect of ORM1 on MMP13 and MMP3 in vitro. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, # p < 0.05 compared with the NC group, * p < 0.05. K) Immunohistochemical staining of MMP13 and MMP3 in the condylar cartilage of rats among various treatment groups. L) Comparison of MMP13‐ and MMP3‐ positive cells in rats among various treatment groups. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with UAC+NS group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Immunofluorescence, In Vitro, Immunohistochemical staining, Staining, Comparison

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Identification of the interaction between ORM1 and vimentin (VIM). Co‐immunoprecipitation (CoIP) was used to detect binding between ORM1 and VIM in HEK‐293T (A) and C28/I2 cells (B). An immunocolocalization assay was used to examine the colocalization of VIM and exogenous ORM1 (C) or endogenous ORM1 (D) in C28/I2 cells. Data are expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed), n = 3, * p < 0.05. Immunocolocalization (E) and quantification (F) of ORM1 and VIM in the condylar cartilage of rats in the three treatment groups, with the yellow lines showing the articular surface and the interface between bone and cartilage. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05. Western blot (G) and immunofluorescence (H) showing the protein level of VIM in both natural and inflammation environments in C28/I2 and SW1353 cells after treatment with ORM1 protein. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the NC group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Immunoprecipitation, Binding Assay, Two Tailed Test, Western Blot, Immunofluorescence

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Inhibitory effect of ORM1 on VIM/MAPK/MMP signaling pathway. A) Effect of ORM1 treatment on the phosphorylation of ERK, JNK, and p38 in C28/I2 and SW1353 cells was detected using western blot. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the NC group. Expression of MMP13 and MMP3 (B), and the phosphorylation of extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (p38) (C) after overexpression of VIM with or without ORM1 treatment in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the Myc group. Expression of MMP13 and MMP3 (D), and the phosphorylation of ERK, JNK, and p38 (E) after knockdown of VIM in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using unpaired Student's t test (two‐tailed), n = 3, * p < 0.05, # p < 0.05 compared with the NC siRNA group. F) Expression of MMP13 and MMP3 after overexpression of VIM with or without inhibition of ERK, JNK, and p38 in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05. G) The phosphorylation levels of ERK, JNK, and p38 in condylar cartilage of rats were detected by immunofluorescence, with the yellow lines showing the articular surface and the interface between bone and cartilage. H) Quantification of the phosphorylation levels of ERK, JNK, and p38 in condylar cartilage of rats based on the immunofluorescence. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 5, * p < 0.05 compared with the UAC+NS group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Phospho-proteomics, Western Blot, Expressing, Over Expression, Knockdown, Two Tailed Test, Inhibition, Immunofluorescence

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: The role of ORM1‐VIM binding on the activities of MAPK pathway and the expression of MMPs. A) Immunocolocalization of ORM1 truncations and VIM in C28/I2 cells. B) CoIP of ORM1 truncations and VIM in C28/I2 cells. C) Expression of MMP13 and MMP3 after overexpression of VIM without or with the ORM1 truncations in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the Myc+Flag group. D) Phosphorylation of ERK, JNK, and p38 after overexpression of VIM without or with the ORM1 truncations in C28/I2 and SW1353 cells. Data is expressed as mean ± SD and analyzed using one‐way ANOVA followed by Tukey's post‐hoc test, n = 3, * p < 0.05, # p < 0.05 compared with the Myc+Flag group.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: Binding Assay, Expressing, Over Expression, Phospho-proteomics

Journal: Advanced Science

Article Title: Orosomucoid 1 Ameliorates Temporomandibular Joint Osteoarthritis by Maintaining Cartilage Homeostasis

doi: 10.1002/advs.202500028

Figure Lengend Snippet: Schematic diagram of the role of ORM1 in maintaining cartilage homeostasis via suppressing VIM/MAPK/MMP signaling.

Article Snippet: Enzyme‐linked immunosorbent assay (ELISA) was performed to measure the concentration of ORM1 in SF samples by an ELISA kit (KE00137; Proteintech).

Techniques: