Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 4. Astrocytic Kir6.1 deletion aggravates astrocyte injury and NLRP3-related pyroptosis in the hippocampus. A-B Immunofluorescent staining of GFAP-positive astrocytes in hippocampal sections (A) with quantification (B, n = 6). C-D ELISA of IL-1β (C) and IL-18 (D) from hippocampus of WT and CKO mice after CSDS (n = 6). E Representative immunoblots of NLRP3, caspase-1, and GSDMD-N from mice hippocampus. F-H Quantification of NLRP3 (F), caspase-1 (G), and GSDMD-N (H) (n = 5). The data shown are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs corresponding control (Con) group; ##p < 0.01, ###p < 0.001 vs WT CSDS groups.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 5. Astrocytic Kir6.1 ablation promotes pyroptosis of astrocytes in the hippocampus. A, C, E Representative magnification images showing the co-localization of GFAP (red) and NLRP3 (green) (A), GFAP (red) and caspase-1 (green) (C), and GFAP (red) and GSDMD (green) (E) in a part of mice hippocampus region after CSDS. White arrows show example of GFAP+/NLRP3+, GFAP+/caspase-1+, and GFAP+/GSDMD+ cells. B, D, F Quantification of the percentage of GFAP positive cells that are NLRP3 positive (B), caspase-1 positive (D), and GSDMD positive (F) in the hippocampus (n = 5). The data shown are the mean ± SEM. **p < 0.01, ***p < 0.001 vs corresponding control (Con) group; ###p < 0.001 vs WT CSDS groups.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: Control

Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 6. Kir6.1 is a negative regulator of NLRP3-mediated astrocytic pyroptosis. Primary astrocytes prepared from WT and CKO mice were treated with 10 µM VX765 for 1 h, followed by stimulation with LPS plus ATP. A The viability of cells was assessed by the CCK-8 assay (n = 4). B LDH in supernatants was measured by LDH kit (n = 5). C ELISA of IL-1β in the supernatants (n = 5). D-G Representative immunoblots of the cleaved caspase-1 in the supernatants (SN) and the NLRP3, pro-caspase-1, full-length GSDMD and GSDMD N-terminal in cell lysate (D) and quantification of cleaved caspase-1 (E, n = 3), NLRP3 (F, n = 3) and GSDMD-N (G, n = 5). H Treated astrocytes were stained with YO-PRO-1 (green) and Eth-D2 (red) to visualize the discrete membrane pores. DAPI stains nucleus (blue). I Quantification of YO-PRO-1+ Eth-D2- cells (n = 5). The data shown are the mean ± SEM. **p < 0.01, ***p < 0.001 vs corresponding control (Con) group; $$p < 0.01, $$$p < 0.001 vs WT LPS+ATP groups; ##p < 0.01, ###p < 0.001 vs corresponding LPS+ATP group.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Membrane, Control

Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 7. Kir6.1 interacts with NLRP3 and inhibits NLRP3 inflammasome assembly. A Representative images showing the localization of NLRP3 (red) and Kir6.1 (green) in astrocytes. B The interaction between Kir 6.1 and NLRP3 in astrocytes was measured by co-IP. C The association of ASC with NLRP3, and procaspase-1 in LPS+ATP treated astrocytes isolated from WT and CKO mice was assessed by co-IP. D-E Quantification of data shown in (C, n = 3). The data shown are the mean ± SEM. **p < 0.01.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: Co-Immunoprecipitation Assay, Isolation

Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 8. The mitochondrial ROS is required for NLRP3-mediated pyroptosis in Kir6.1 knockout astrocytes. A-B Representative images and quantification of mitochondrial ROS levels by flow cytometry (n = 3). C-D Representative images and quantification of MitoSOX fluorescence intensity (n = 3). The nucleus was stained with DAPI (blue). ***p < 0.001 vs control (Con) group; ###p < 0.001 vs WT LPS+ATP groups. Primary astrocytes from CKO mice were pretreated with 5 mM ROS inhibitor NAC for 1 h and then stimulated with LPS plus ATP. E-H Representative immunoblots of the cleaved caspase-1 and mature IL-1β in the supernatants and the GSDMD and GSDMD N-terminal in cell lysates (E) and quantification of caspase-1 cleavage (F), IL-1β (G), and GSDMD-N (H). n = 3, *p < 0.05 vs control (Con) group; #p < 0.05, ##p < 0.01 vs LPS+ATP groups.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: Knock-Out, Flow Cytometry, Fluorescence, Staining, Control, Western Blot

Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 9. Inhibiting NLRP3 inflammasome rescues pyroptosis of astrocytes and depressive behaviors in CKO mice. CKO mice were intraperitoneally injected with VX765 (100 mg/kg) once daily for 10 consecutive days. A Representative magnification images showing the co-localization of GFAP (red) and GSDMD (green) in a part of mice hippocampus region after CSDS. White arrows show example of GFAP+/GSDMD+ cells. B-C Quantification of GFAP+ cells number (B, n = 6) and percentage of GFAP positive cells that are GSDMD positive (C, n = 5) in the DG area of hippocampus. **p < 0.01, ***p < 0.001. D-E Representative immunoblot (D) and quantitative analysis of GSDMD in hippocampus of mice (E, n = 5). F ELISA of IL-1β from hippocampus of mice (n = 6). G-I Sucrose preference (G, n = 8), total immobility time in TST (H, n = 9) and in FST (I, n = 9) of mice. ***p < 0.001 vs control (Con) group; ##p < 0.01, ###p < 0.001 vs CSDS groups.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Theranostics

Article Title: Kir6.1/K-ATP channel in astrocytes is an essential negative modulator of astrocytic pyroptosis in mouse model of depression.

doi: 10.7150/thno.77455

Figure Lengend Snippet: Figure 10. Proposed model depicting the crucial role of Kir6.1, via its interaction with NLRP3 and inhibition of mtROS generation, in preventing the assembly and activation of NLRP3 inflammasome, consequently, inhibiting the pyroptosis of astrocytes in depression.

Article Snippet: Astrocytes were primed with lipopolysaccharide (LPS, 100 ng ml-1, Sigma, USA) for 24 h and then pulsed with 5 mM ATP (Sigma, USA) for 30 min. For pharmacological measurements, NLRP3 inflammasome inhibitor VX765 (10 μM, Selleck, China) or ROS inhibitor N-acetyl-cysteine (NAC, 5 mM, Sigma, USA) was added to the cell culture medium 1 h before LPS plus ATP stimulation.

Techniques: Inhibition, Activation Assay

Journal: Diabetologia

Article Title: Targeting the NLRP3 inflammasome–IL-1β pathway in type 2 diabetes and obesity

doi: 10.1007/s00125-024-06306-1

Figure Lengend Snippet: Schematic overview of canonical IL-1β production and secretion. TLR2, TLR4 and IL-1 signalling prime myeloid cells, starting the transcription of inflammasome components (e.g. PYCARD [ASC], NLRP3 ) and IL-1-responsive genes (e.g. IL1B , IL1RN [IL-1RA], CXCL8 [IL-8], IL6 ). Glucose, K + efflux, and DAMPs and PAMPs trigger intracellular stimuli that act as a second signal activating the NLRP3 inflammasome. Elevated cytosolic levels of reactive oxygen species (ROS), islet amyloid polypeptide (IAPP) and ATP activate NLRP3 receptors. ROS and crystalline substances such as cholesterol and uric acid disrupt the lysosomal membrane in phagocytes, promoting leakage of lysosome cargo into the cytosol, releasing epitopes that are recognised by the LRR domain of the NLRP3 receptor. The NLRP3 inflammasome is composed of the sensor NLRP3, the adaptor protein ASC and the effector molecule caspase-1. On receiving a second stimulus, the NLRP3 inflammasome is assembled in the cytosol, leading to the activation of caspase-1 and subsequent secretion of active IL-1β via the gasdermin D pore. After being secreted, IL-1β binds to the IL-1R1, amplifying intracellular inflammatory signalling and transcription of NLRP3 inflammasome components. TCA, tricarboxylic acid. This figure is available as part of a downloadable slideset

Article Snippet: Olatec provides the authors with an NLRP3 inhibitor used for an ongoing clinical study funded by a European Union/Swiss grant.

Techniques: Membrane, Activation Assay

Journal: Diabetologia

Article Title: Targeting the NLRP3 inflammasome–IL-1β pathway in type 2 diabetes and obesity

doi: 10.1007/s00125-024-06306-1

Figure Lengend Snippet: Role of the NLRP3 inflammasome–IL-1β pathway in the pathogenesis of metabolic diseases and comorbidities. Metabolic stress induced by NEFAs, glucose, cholesterol, uric acid and islet amyloid polypeptide (IAPP), along with ageing and changes in the microbiome, activate NLRP3 with subsequent cleavage of pro-IL-1β. In turn, IL-1β contributes to impaired insulin secretion, insulin resistance, MAFLD, atherosclerosis, heart failure, retinopathy, nephropathy, neuropathy, obesity, Alzheimer’s disease, fatigue, testosterone deficiency, polycystic ovary syndrome, rheumatoid arthritis and gout. Blocking IL-1 signalling or inhibition of NLRP3 counteracts pathologies associated with metabolic diseases. This figure is available as part of a downloadable slideset

Article Snippet: Olatec provides the authors with an NLRP3 inhibitor used for an ongoing clinical study funded by a European Union/Swiss grant.

Techniques: Blocking Assay, Inhibition

Journal: Diabetologia

Article Title: Targeting the NLRP3 inflammasome–IL-1β pathway in type 2 diabetes and obesity

doi: 10.1007/s00125-024-06306-1

Figure Lengend Snippet: Clinical studies of NLRP3 inhibition or IL-1 antagonism for the treatment of type 2 diabetes and its complications

Article Snippet: Olatec provides the authors with an NLRP3 inhibitor used for an ongoing clinical study funded by a European Union/Swiss grant.

Techniques: Inhibition, Drug discovery