il 1β  (Elabscience Biotechnology)

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: Activation of the NLRP3 inflammasome pathway in myocardial tissues of T1DM mice. (a) Representative Western blot images showing the protein expression levels of NLRP3, caspase‐1 P20, and N‐GSDMD in myocardial tissues. (b–d) Semiquantitative analysis of the protein expression levels of (b) NLRP3, (c) caspase‐1 P20, and (d) N‐GSDMD. β ‐actin was used as a loading control, n = 6. (e,f) Serum concentration of (e) IL‐1 β and (f) IL‐18 measured by ELISA, n = 6. (g) Representative immunofluorescence images showing ASC speck formation (red) in myocardial tissues. Nuclei were stained with DAPI (blue). Scale bar: 20 μ m. (h) Quantification of ASC fluorescence intensity, n = 6.

Article Snippet: ASC (Cat#A1170), N‐GSDMD (Cat# A24476 ), the IL‐1 β (Cat#RK00006), and IL‐18 (Cat# SEKM‐0019) enzyme‐linked immunosorbent assay (ELISA) kits were from ABclonal (Beijing, China).

Techniques: Activation Assay, Western Blot, Expressing, Analysis, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: AGEs activate the NLRP3 inflammasome pathway in primary cultured cardiomyocytes. (a) Representative Western blot images showing the protein levels of NLRP3, caspase‐1 P20, and N‐GSDMD in cardiomyocytes treated with or without AGEs, n = 4. (b–d) Semiquantitative analysis of (b) NLRP3, (c) caspase‐1 P20, and (d) N‐GSDMD protein expression. β ‐actin was used as a loading control, n = 4. (e,f) Concentration of (e) IL‐1 β and (f) IL‐18 in the cell culture supernatant detected by ELISA, n = 4. (g) Representative immunofluorescence images showing ASC speck formation (red) in cardiomyocytes. Nuclei were stained with DAPI (blue). Scale bar: 10 μ m. (h) Quantification of ASC fluorescence intensity, n = 4.

Article Snippet: ASC (Cat#A1170), N‐GSDMD (Cat# A24476 ), the IL‐1 β (Cat#RK00006), and IL‐18 (Cat# SEKM‐0019) enzyme‐linked immunosorbent assay (ELISA) kits were from ABclonal (Beijing, China).

Techniques: Cell Culture, Western Blot, Analysis, Expressing, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: AGEs activate the NLRP3 inflammasome in cardiomyocytes via the RAGE/ROS/NF‐ κ B signaling axis. (a–k) Primary neonatal mouse cardiomyocytes were pretreated with or without the RAGE inhibitor FPS‐ZM1 (20 μ M) or vehicle for 1 h, followed by stimulation with AGEs (200 μ g/mL) for 24 h. (a) Representative Western blots of phospho‐NF‐ κ B p65 (pp65), total NF‐ κ B p65, NLRP3, caspase‐1 P20, and N‐GSDMD. (b–e) Quantitative analysis of the protein levels of (b) pp65/NF‐ κ B p65, (c) NLRP3, (d) caspase‐1 P20, and (e) N‐GSDMD, n = 4. (f,g) Concentrations of (f) I L‐1 β and (g) IL‐18 in the cell culture supernatant were measured by ELISA, n = 4. (h,i) Intracellular ROS levels were detected using the DCFHDA fluorescent probe. (h) Representative fluorescence images. Scale bar, 50 μ m. (i) Quantitative analysis of ROS fluorescence intensity, n = 4. (j,k) ASC oligomerization was assessed by immunofluorescence. (j) Representative images showing ASC specks. Scale bar, 10 μ m. (k) Quantification of ASC fluorescence intensity, n = 4. (l–v) Cardiomyocytes were pretreated with or without the ROS scavenger NAC (10 μ M) or vehicle for 1 h, followed by stimulation with AGEs (200 μ g/mL) for 24 h. (l) Representative immunoblots of the indicated proteins. (m–p) Quantitative analysis of (m) pp65/NF‐ κ B p65, (n) NLRP3, (o) caspase‐1 P20, and (p) N‐GSDMD, n = 4. (q,r) ELISA analysis of (q) IL‐1 β and (r) IL‐18 levels in the supernatant, n = 4. (s,t) Intracellular ROS levels were measured as above. (s) Representative images. Scale bar, 50 μ m. (T) Fluorescence quantification, n = 4. (u,v) Assessment of ASC speck formation. (u) Representative images. Scale bar, 10 μ m. (v) Quantification of ASC fluorescence intensity, n = 4.

Article Snippet: ASC (Cat#A1170), N‐GSDMD (Cat# A24476 ), the IL‐1 β (Cat#RK00006), and IL‐18 (Cat# SEKM‐0019) enzyme‐linked immunosorbent assay (ELISA) kits were from ABclonal (Beijing, China).

Techniques: Western Blot, Analysis, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunofluorescence

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: Clinical association and in vivo characterization of the AGEs/RAGE/NLRP3 inflammasome axis in T1DM cardiomyopathy. (a,b) Analysis of publicly available transcriptomic data (GEO Accession: GSE123658 ) from whole blood samples of T1DM patients and healthy controls. Scatter plots show a significant positive correlation between AGER (RAGE) mRNA expression and transcript levels of (a) IL‐1 β and (b) IL‐18. Pearson′s correlation coefficients ( r ) and p values are indicated in the figure, n = 39. Each point represents an individual subject. (c–f) Protein analysis in myocardial tissues from control and T1DM mice. (c) Representative Western blots showing protein levels of RAGE, NLRP3, and N‐GSDMD. (d) Quantitative analysis of RAGE protein expression normalized to β ‐actin, n = 6. (e,f) Scatter plots showing individual‐level expression of RAGE protein versus (e) NLRP3 and (f) N‐GSDMD in T1DM mouse hearts. Each point represents an individual mouse. A linear trend line is shown to illustrate the overall pattern of the data, n = 6.

Article Snippet: ASC (Cat#A1170), N‐GSDMD (Cat# A24476 ), the IL‐1 β (Cat#RK00006), and IL‐18 (Cat# SEKM‐0019) enzyme‐linked immunosorbent assay (ELISA) kits were from ABclonal (Beijing, China).

Techniques: In Vivo, Analysis, Blood/Plasma/Serum Samples, Expressing, Control, Western Blot

Journal: Journal of Diabetes Research

Article Title: The AGEs/RAGE Axis Contributes to Myocardial Cell Death in Type 1 Diabetic Cardiomyopathy via the ROS/NF‐ κ B/NLRP3 Inflammasome Pathway

doi: 10.1155/jdr/6438008

Figure Lengend Snippet: NLRP3 is required for AGEs‐induced inflammatory responses in cardiomyocytes. Primary neonatal mouse cardiomyocytes were pretreated with or without the NLRP3 inhibitor MCC950 (10 μ M) or vehicle for 1 h, followed by stimulation with AGEs (200 μ g/mL) for 24 h. (a) Representative Western blots of NLRP3, caspase‐1 p20, and N‐GSDMD. (b–d) Quantitative analysis of the protein levels of (b) NLRP3, (c) caspase‐1 P20, and (d) N‐GSDMD, n = 4. (e,f) Concentrations of (e) IL‐1 β and (f) IL‐18 in the cell culture supernatant were measured by ELISA, n = 4.

Article Snippet: ASC (Cat#A1170), N‐GSDMD (Cat# A24476 ), the IL‐1 β (Cat#RK00006), and IL‐18 (Cat# SEKM‐0019) enzyme‐linked immunosorbent assay (ELISA) kits were from ABclonal (Beijing, China).

Techniques: Western Blot, Analysis, Cell Culture, Enzyme-linked Immunosorbent Assay