Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: The expression of F2/Rho, HMGB1/RAGE/p38MAPK/NF-κB signal pathway-related proteins increases in the lung tissues of rats simulated high-altitude hypoxia. (A) Western blot analysis was performed to examined the protein expression levels of F2, ROCK1, RhoA, HMGB1, RAGE, p38 MAPK and (NF-κB) p65 in the rat lung tissue. (B) Relative protein expression was normalized to that of the respective control, β-actin. (C-E) Immunohistochemistry of RAGE and HMGB1 in lung tissues of rats. The staining results of the images revealed that the expression of RAGE and HMGB1 in the lung tissues of rats exposed to hypoxia was significantly increased compared with that of the control rats. The left and right dark and bright images in each group is the comparison of the results before and after molecular staining. The positive results in the bright pictures refer to the immunostaining of HMGB1 and RAGE. * P<0.05 vs. the control group. F2, prothrombin; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1; C, control; H, hypoxia.

Article Snippet: Anti-RAGE (cat. no. orb622096), anti-F2 (cat. no. orb539413) and anti-rabbit IgG (H&L; CF488A; cat. no. orb216207-CF488A) were from Biorbyt, Ltd.; anti-p38 (cat. no. R25239), anti-phosphorylated (p-) p38 (cat. no. 310091), anti-NF-κB-p65 (cat. no. 340830), anti-p-NF-κBp-p65 (cat. no. 340830), anti-IκBα (cat. no. 380682), anti-p-IκBα (cat. no. R24672), anti-RhoA (cat. no. 346086), anti-ROCK1 (cat. no. R25607), anti-β-actin (cat. no. 380624) and anti-rabbit IgG (H&L; HRP-conjugated; cat. no. 511203) were from Chengdu Zen Bioscience Co., Ltd.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Comparison, Immunostaining

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: The expression of F2/Rho, HMGB1/RAGE/p38MAPK/NF-κB signaling pathway-related proteins increased in the NR8383 cells with exposed to hypoxia. (A) Western blot analysis was performed to examine the protein expression levels of F2, ROCK1, RhoA, HMGB1, RAGE, p38MAPK and (NF-κB) p65. (B-H) Relative protein expression was normalized to that of the respective control, β-actin. Protein expression exhibited variable changes; * P<0.05, vs. the control group. C, control; H, hypoxia; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1.

Article Snippet: Anti-RAGE (cat. no. orb622096), anti-F2 (cat. no. orb539413) and anti-rabbit IgG (H&L; CF488A; cat. no. orb216207-CF488A) were from Biorbyt, Ltd.; anti-p38 (cat. no. R25239), anti-phosphorylated (p-) p38 (cat. no. 310091), anti-NF-κB-p65 (cat. no. 340830), anti-p-NF-κBp-p65 (cat. no. 340830), anti-IκBα (cat. no. 380682), anti-p-IκBα (cat. no. R24672), anti-RhoA (cat. no. 346086), anti-ROCK1 (cat. no. R25607), anti-β-actin (cat. no. 380624) and anti-rabbit IgG (H&L; HRP-conjugated; cat. no. 511203) were from Chengdu Zen Bioscience Co., Ltd.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: The cells were randomly divided into four groups as follows: The control group, control + siRAGE group, hypoxia + siRAGE group and hypoxia group, and cultured according to the culture conditions (the concentration of siRAGE was 100 nM). (A) Western blot analysis was performed to examine the protein expression levels of RAGE in the different groups. (B) The relative protein expression gray scale statistics of RAGE. (C) Western blot analysis was performed to examine the protein expression levels of RAGE, HMGB1, p-p38, p-p65, HIF-1α and β-actin in NR8383 cells in the control, control + siRAGE, hypoxia + siRAGE and hypoxia groups. (D) The relative protein expression was normalized to that of the respective control, β-actin. Data are presented as the mean ± standard deviation. *** P<0.001 and **** P<0.0001, comparison between control group and hypoxia group; ### P<0.001 and #### P<0.0001, comparison between the hypoxia group and hypoxia + siRAGE group; ns, not significant; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1.

Article Snippet: Anti-RAGE (cat. no. orb622096), anti-F2 (cat. no. orb539413) and anti-rabbit IgG (H&L; CF488A; cat. no. orb216207-CF488A) were from Biorbyt, Ltd.; anti-p38 (cat. no. R25239), anti-phosphorylated (p-) p38 (cat. no. 310091), anti-NF-κB-p65 (cat. no. 340830), anti-p-NF-κBp-p65 (cat. no. 340830), anti-IκBα (cat. no. 380682), anti-p-IκBα (cat. no. R24672), anti-RhoA (cat. no. 346086), anti-ROCK1 (cat. no. R25607), anti-β-actin (cat. no. 380624) and anti-rabbit IgG (H&L; HRP-conjugated; cat. no. 511203) were from Chengdu Zen Bioscience Co., Ltd.

Techniques: Cell Culture, Concentration Assay, Western Blot, Expressing, Standard Deviation, Comparison

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: Pre-treatment with SB203580 inhibits the activation of the NF-кB signaling pathway. NR8383 cells were pre-treated with SB203580 (10 µ M) for 1 h and then exposed to hypoxia for 24 h. Pre-treatment with SB203580 intercepted the phosphorylation of p65 protein. (A) Western blot analysis was performed to examine the protein expression levels of p38, p-p38, RhoA, IкBα, p-IкBα, p65 and p-p65 in NR8383 cells in the control and hypoxia groups. (B) The relative protein expression was normalized to that of the respective control, β-actin. Data are presented as the mean ± standard deviation. * P<0.05 and ** P<0.01, comparison between control group and hypoxia group; # P<0.05, comparison between the hypoxia group and hypoxia + SB203580 group.

Article Snippet: Anti-RAGE (cat. no. orb622096), anti-F2 (cat. no. orb539413) and anti-rabbit IgG (H&L; CF488A; cat. no. orb216207-CF488A) were from Biorbyt, Ltd.; anti-p38 (cat. no. R25239), anti-phosphorylated (p-) p38 (cat. no. 310091), anti-NF-κB-p65 (cat. no. 340830), anti-p-NF-κBp-p65 (cat. no. 340830), anti-IκBα (cat. no. 380682), anti-p-IκBα (cat. no. R24672), anti-RhoA (cat. no. 346086), anti-ROCK1 (cat. no. R25607), anti-β-actin (cat. no. 380624) and anti-rabbit IgG (H&L; HRP-conjugated; cat. no. 511203) were from Chengdu Zen Bioscience Co., Ltd.

Techniques: Activation Assay, Western Blot, Expressing, Standard Deviation, Comparison

Journal: Molecular Medicine

Article Title: Depression of lncRNA MINCR antagonizes LPS-evoked acute injury and inflammatory response via miR-146b-5p and the TRAF6-NFkB signaling

doi: 10.1186/s10020-021-00367-3

Figure Lengend Snippet: MINCRT affected the progression of acute lung injury (ALI) through the miR-146b-5p/TRAF6 axis. A TRAF6 was predicted to be a target gene of miR-146b-5p. B analysis of luciferase activity in TRF6-wild type (WT) or TRAF6-Mut vector co-transfected cells. C protein expression levels of TRAF6 in small airway epithelial cells (SAECs). D protein expression levels of TRAF6, P65, and p-P65 in SAECs was detected by Western blotting assay. E protein levels of TRAF6, p65, and p-P65 in lung tissue treated with lipopolysaccharide (LPS) was detected by Western blotting assay. * P < 0.05, N = 5

Article Snippet: Thereafter, proteins were separated using 10% polyacrylamide gel electrophoresis and blocked with 5% BSA for 1 h. After transferring onto PVDF membrane, the membrane was incubated with anti-TRAF6 (1:1000, Amyjet, Wuhan, China), anti-p65 (1:1000, Amyjet, Wuhan, China), anti-p-p65 (1:1000, Amyjet, Wuhan, China), anti-IL-10 (1:1000, Amyjet, Wuhan, China), anti-IL-6 (1:1000, Amyjet, Wuhan, China), anti-TNF-α (1:1000, Amyjet, Wuhan, China), and anti-GAPDH (1:1000, Amyjet, Wuhan, China) overnight, followed by incubation with anti-rabbit secondary antibody (1:1000, Amyjet, Wuhan, China) for 1 h (Sanaa and Seada ).

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Transfection, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Neurotransmitter 5-HT Further Promotes LL-37-Induced Rosacea-like Inflammation Through HTR3A

doi: 10.3390/ijms26073156

Figure Lengend Snippet: Roles of HTR3A in L5-induced THP-1-derived macrophages. ( A , B ) After treatment with SGR, TPS or SB-269970 for 1 h and stimulation by L5 for 24 h, the ELISA results of the expressions of IL-1β and TNF-α of control (black), L5 (blue), L5 + SGR (purple), L5 + TPS (red), and L5 + SB-269970 (pink) treated cell groups. ( C ) After treatment for 24 h, the qPCR analysis of mRNA expressions of HTR3A of siNC (black), siHTR3A#1 (blue), and siHTR3A#2 treated cell groups. ( D , E ) After stimulation by L5 for 24 h in transfected cells, the qPCR analysis of mRNA expressions of IL-1β and TNF-α of siNC (black), siNC + L5 (blue), si HTR3A #1 + L5 (purple), and si HTR3A #2 + L5 (red) treated cell groups. ( F ) After treatment with TPS for 1 h and stimulation by L5 for 1 h, the expressions of p-p65 in THP-1-derived macrophages were tested by Western blot analysis. ( G ) The relative protein levels of p-p65/p65 of control (black), TPS (blue), L5 (purple), and L5 + TPS (red) treated cell groups in Western blot analysis. ( H ) The immunofluorescence analysis of p65 after incubation with TPS for 1 h and stimulation by L5 for 4 h. Scale bar 50 μm. Data represent mean ± SEM for three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance.

Article Snippet: The used primary antibodies were listed as follows: anti-p65 (Beyotime, China), anti-p-p65 (ser563, Beyotime, China), anti-β-actin (Beyotime, China), anti-β-tubulin (Proteintech, Wuhan, China), and anti-GAPDH (Proteintech, China).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Control, Transfection, Western Blot, Immunofluorescence, Incubation