Journal: International Journal of Biological Sciences
Article Title: Gastrodin alleviates high fructose-induced podocyte mitochondria-mediated apoptosis by inhibiting NLRP6 to facilitate TRIM7-triggered Bok mRNA degradation
doi: 10.7150/ijbs.120307
Figure Lengend Snippet: Pharmacological inhibition of NLRP6 by gastrodin ameliorates high fructose-induced glomerular podocyte mitochondria-mediated apoptosis. (A) Molecular docking shows interactions between gastrodin and NLRP6. The hydrogen-bonding interactions are shown in yellow dashed lines. (B) The binding affinity of gastrodin to NLRP6 was measured by MST, ( n = 3). (C) Western blot detection of NLRP6 in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 6). (D) Western blot detection of NLRP6 in MPC5, which was stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 5-6). (E) Western blot detection of TRIM7, BOK, and FAM213A in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 6). (F) Western blot detection of TRIM7, BOK, and FAM213A in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 6-8). (G) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 6-7). (H) Western blot detection of cleaved Caspase 3 and cleaved Caspase 9 in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 5). (I) Flow cytometry analysis of activated Caspase 3 in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 4). (J) ROS levels were considered as the fluorescence intensity of fluorogenic probe DCFH 2 -DA, measured via microplate reader in MPC5. MPC5 were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 10). (K) Mitochondrial ROS production was considered as the fluorescence intensity of fluorogenic probe MitoSOX, measured by flow cytometry in MPC5. MPC5 were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 3-4). (L) OCR detection in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 8). (M) TEM images and quantification of podocyte in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg). Scale: 1 μm (Top), 250 nm (Bottom), ( n = 3). (N) Western blot detection of Cyto C performed on mitochondrial and cytosolic fractions in control mouse and HFrD-fed mouse glomeruli with or without gastrodin treatment (25, 50, and 100 mg/kg), ( n = 3). (O) Western blot detection of Cyto C was performed on mitochondrial and cytosolic fractions in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 3). (P) Flow cytometry analysis of mitochondrial membrane potentials using JC-10 dye in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 4). (Q) Representative IF images of TUNEL assay in control mouse and HFrD-fed mouse glomeruli with or without the treatment of gastrodin (25, 50, and 100 mg/kg), Scale: 20 μm. (R) Flow cytometry analysis of apoptotic cells through Annexin V-FITC/PI staining in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, ( n = 5). (S) Representative IF images of TUNEL assay in MPC5, which were stimulated with 5 mM fructose as well as gastrodin (25, 50, and 100 μM) or not, Scale: 100 μm. Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.
Article Snippet: Podocyte Caspase 3 activity level was assessed by the commercially available kit (C1168M, Beyotime).
Techniques: Inhibition, Binding Assay, Western Blot, Control, Flow Cytometry, Fluorescence, Membrane, TUNEL Assay, Staining
