Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A , B , Representative immunoblots ( A ) and quantification ( B ) of NLRP3, ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 in the heart of WT mice subjected to either ND or HFD consumption for 24 wks (n=6 per group). ( C ) Representative images of wheat germ agglutinin staining (green) and immunofluorescence staining of NLRP3 (red) in the heart of WT mice fed an HFD or ND for 24 wks. D , E , Protein of IL‐1β ( D ) and IL‐18 ( E ) in the heart of WT mice fed an HFD or ND for 24 wks. IL‐1β and IL‐18 protein were detected by ELISA. Values were normalized to total protein level (n=6 per group). F , Representative images of immunohistochemical staining of IL‐1β in the heart of WT mice subjected to either ND or HFD consumption for 24 wks. ( G ) Representative immunoblots of NLRP3, ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 protein in NRVMs treated with BSA or BSA+PA for 24 h. H , Representative images of immunofluorescence staining of NLRP3 (red) and F‐Actin (green) in NRVMs treated with BSA or BSA+PA. Significance was assessed by Student t test. Data are shown as the mean±SEM. ASC indicates apoptosis‐associated speck like protein; HFD, high‐fat diet; BSA, Bovine Serum Albumin; IL, interleukin; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; PA, palmitic acid; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Western Blot, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A , Representative M‐mode echocardiographic images of left ventricle from WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks. B , C , LV ejection fraction and fractional shortening were measured by echocardiography (n=12 per group). D , E , Representative images of transmitral flow obtained by Doppler echocardiography and ratio between mitral E wave and A wave in WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks. E wave DT is indicated by red bars (n=10 per group). F , E wave DT, a marker of diastolic function, evaluated by transmitral flow Doppler echocardiography in indicated mice (n=10 per group). G , LV mass was measured by echocardiography in WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks (n=10 per group). H , Heart weight normalized by tibia length, a marker of cardiac hypertrophy. I , Representative images of cardiac H&E, WGA staining, Oil Red O staining and Masson trichrome staining in perivascular and interstitial area. J , Quantification of cardiomyocyte cross‐sectional area by WGA in indicated hearts (n=5 per group). K , Quantification of left ventricle collagen volume by Masson trichrome staining (n=6 per group). L , Quantification of myocardial lipid accumulation by Oil Red O staining (n=5 per group). DT indicates deceleration time; H&E, hematoxylin and eosin; HFD, high‐fat diet; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; ORO, Oil Red O; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Marker, Staining, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through L , Wild‐type mice treated with NS or MCC950 for 12 wks were subjected to ND or HFD feeding for 24 wks. A , Representative M‐mode echocardiographic images of left ventricle from the indicated mice. B , C , LV ejection fraction and fractional shortening were measured by echocardiography (n=12 per group). D , E , Representative images of transmitral flow obtained by Doppler echocardiography and ratio between mitral E wave and A wave. E wave DT is indicated by red bars (n=10 per group). F , E wave DT, a marker of diastolic function, evaluated by transmitral flow Doppler echocardiography in indicated mice (n=10 per group). G , LV mass was measured by echocardiography in indicated mice subjected to NS or MCC950 for 12 wks (n=10 per group). H , Heart weight normalized by tibia length, a marker of cardiac hypertrophy (n=10 per group). I , Representative images of cardiac H&E, WGA staining, Oil Red O staining and Masson trichrome staining in perivascular and interstitial area. J , Quantification of cardiomyocyte cross‐sectional area by WGA in indicated hearts (n=6 per group). K , Quantification of LV collagen volume by Masson trichrome staining (n=6 per group). L , Quantification of myocardial lipid accumulation by Oil Red O staining (n=6 per group). M , N , Mitochondrial oxidative capacity was measured in real time after MCC950 treatment or BSA+PA incubation in NRVMs. Quantification of maximal respiration in ( L ) (n=6 independent experiments). DT indicates deceleration time; FCCP, Trifluoromethoxy carbonylcyanide phenylhydrazone; H&E, hematoxylin and eosin; HFD, high‐fat diet; BSA, Bovine Serum Albumin; HW/TL, heart weight/tibia length ratio; ND, normal diet; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; NS, normal saline; OCR, oxygen consumption rate; ORO, Oil Red O; PA, palmitic acid; and WGA, wheat germ agglutinin.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Marker, Staining, Incubation, Binding Assay, Saline

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through D , Wild‐type mice treated with NS or MCC950 for 12 wks were subjected to ND or HFD feeding for 24 wks. A , qRT‐PCR detection of indicated genes related to NLRP3 inflammasome components and the proinflammatory cytokine IL‐1β and IL‐18 in hearts from indicated mice (n=5 per group). B , Immunohistochemical staining for NF‐κB in indicated murine heart. C , D , Representative images of immunoblot and quantitative analysis of NF‐κB in heart tissues from indicated mice. E through F , NRVMs treated with NS or MCC950 were incubated with BSA or BSA+PA medium for 24 h. E , Representative images of immunofluorescence staining of NF‐κB (red) and F‐Actin (green) in NRVMs. F , Immunoblotting analysis of NF‐κB protein in whole‐cell lysates and cytoplasmic and nuclear components in the indicated NRVMs. HFD indicates high‐fat diet; IL, interleukin; ND, normal diet; NF‐κB, nuclear factor kappa B; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; ns, not significant; NS, normal saline; p‐NF‐κB, phosphorylated nuclear factor kappa B; PA, palmitic acid; and qRT‐PCR, quantitative real‐time polymerase chain reaction.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot, Incubation, Immunofluorescence, Binding Assay, Saline, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A , Representative immunoblots of TXNIP in the whole‐cell lysate of the heart at different time points following HFD consumption in mice. B , qRT‐PCR detection of TXNIP in hearts of mice fed an ND or an HFD for different durations (n=5 per group). C , Protocol and schematics of establishment of TXNIP overexpressing mouse model in WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks. D through N , WT or NLRP3 ‐/‐ mice received rAAV9‐cTnT‐null or rAAV9‐cTnT‐TXNIP were subjected to ND or HFD feeding for 24 wks. D , Representative images of M‐model echocardiography (upper) and transmitral flow obtained by Doppler echocardiography (lower). E wave deceleration time is indicated by red bars. E , F , LV ejection fraction ( E ) and fractional shortening ( F ) were measured by echocardiography in indicated mice (n=10 per group). G , H , Histogram of ratio between mitral E wave and A wave and E wave deceleration time, evaluated by transmitral flow Doppler echocardiography in indicated mice (n=10 per group). I , J , Histogram of LV mass ( I ) and HW/TL ( J ) in indicated mice (n=10 per group). K , Representative images of cardiac WGA staining, Oil Red O staining and Masson trichrome staining in perivascular and interstitial area (n=10 per group). L , Quantification of cardiomyocyte cross‐sectional area by WGA (n=5 per group). M , Quantification of LV collagen volume in interstitial area by Masson trichrome staining (n=5 per group). N , Quantification of myocardial lipid accumulation by Oil Red O staining (n=5 per group). cTnT indicates cardiac troponin T; HFD, high‐fat diet; HW/TL, heart weight/tibia length ratio; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; ORO, Oil Red O; qRT‐PCR, quantitative real‐time polymerase chain reaction; rAAV9, recombinant adeno‐associated virus serotype 9 encoding TXNIP; TXNIP, thioredoxin‐interacting protein; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Western Blot, Quantitative RT-PCR, Staining, Binding Assay, Real-time Polymerase Chain Reaction, Recombinant, Virus

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through H , NRVMs treated with PBS or MitoTEMPO were incubated with BSA or BSA+PA medium for 24 h. A , Schematic diagram showing the procedure of NRVMs. B , Intracellular total ROS using DCFH‐DA staining (upper) and mitochondrial ROS using MitoSOX staining (lower) were determined in NRVMs. C , Cell lysates of NRVMs were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and TRX antibodies. NRVMs were treated with siTXNIP to knockdown of TXNIP. D , Triple immunofluorescence staining for TXNIP (red), NLRP3 (green), and nuclei (DAPI, blue) was performed in indicated NRVMs. E , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 protein in NRVMs from indicated groups. F , Oil Red O staining of NRVMs from indicated groups. Red indicates lipid droplets, blue indicates nuclei. G , qRT‐PCR detection of indicated genes related to fatty acid transport genes Cd36 and fatty acid oxidation genes Cpt1b , Acadl , and Acadvl in hearts from indicated mice (n=6 independent experiments). H , Expression level of hypertrophic marker gene BNP was determined by RT‐PCR and normalized to that of GAPDH (n=6 independent experiments). ASC indicates apoptosis‐associated speck like protein; BNP, B‐type natriuretic peptide; BSA, Bovine Serum Albumin; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; PA, palmitic acid; qRT‐PCR, quantitative real‐time polymerase chain reaction; and ROS, reactive oxygen species.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Incubation, Staining, Immunoprecipitation, Western Blot, Knockdown, Immunofluorescence, Quantitative RT-PCR, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through L , WT mice received NS or MitoTEMPO were subjected to ND or HFD feeding for 24 wks. A , Experimental schematic diagram showing the process of HFD‐induced obesity cardiomyopathy. After 12 wks of HFD feeding, MitoTEMPO was injected into obese mice at a dose of 10 mg/kg per day for 12 wks. B , C , LV fractional shortening ( B ) and ratio between mitral E wave and A wave ( C ) were measured by echocardiography in indicated mice (n=12 per group). D , Histogram of HW/TL in indicated mice. HW indicated wet weight (n=12 per group). E , Representative images of cardiac WGA staining, Oil Red O staining, and Masson trichrome staining in perivascular and interstitial area in indicated heart. F , Quantification of cardiomyocyte cross‐sectional area by WGA (n=6 per group). G , Quantification of LV collagen volume in interstitial area by Masson trichrome staining (n=6 per group). H , Quantification of myocardial lipid accumulation by Oil Red O staining (n=6 per group). I , Tissue lysate in indicated heart were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and thioredoxin antibodies. J , K , Protein of IL‐1β ( J ) and IL‐18 ( K ) in the heart of WT mice fed an HFD or ND for 24 wks with or without MitoTEMPO treatment. IL‐1β and IL‐18 protein were detected by ELISA. Values were normalized to total protein level (n=6 per group). L , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 in the indicated heart. ASC indicates apoptosis‐associated speck like protein; HFD, high‐fat diet; HW/TL, heart weight/tibia length ratio; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; ORO, Oil Red O; NS, normal saline; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Injection, Staining, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Saline

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Galectin‐3 expression is abundant in human and mouse atherosclerotic lesions. (A) Ten main cell types are visualised in atherosclerotic core (AC) and proximal adjacent (PA) tissues by tSNE (t‐distributed stochastic neighbour embedding). (B) The macrophage population significantly increased in AC relative to PA. (C) Biaxial scatter plots show the expression pattern of galectin‐3 in total cell types between AC and PA. The colour scale represents expression levels in biaxial scatter plots (grey: low; pink: high). (D) Galectin‐3‐positive macrophages expanded in AC in comparison with PA. (E) Five macrophage subtypes are visualised in AC and PA tissues by tSNE. (F) My.0 and My.1 account for 34.1% and 47.6% of macrophages in AC, respectively. My.2 significantly increased in AC relative to PA. (G) Biaxial scatter plots exhibit the expression pattern of galectin‐3 in macrophage subtypes between AC and PA. (H) Galectin‐3‐positive My.0 and My.1 account for 35.8% and 47.5% of galectin‐3‐positive macrophages in AC, respectively. Galectin‐3‐positive My.2 expands in AC in comparison with PA. (I) Representative Western blots and relative quantitative analysis of galectin‐3 in human atherosclerotic lesions and peripheral normal artery. (J) Triple immunofluorescence staining for galectin‐3 (red), NLRP3 (green), CD68 (pink) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the colocalisation of galectin‐3 and NLRP3 in CD68‐positive macrophages. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of galectin‐3 in the aortas of ApoE −/− mice fed with an HFD or normal diet. (L) Triple immunofluorescence staining for galectin‐3 (red), NLRP3 (green), CD68 (pink) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals that galectin‐3 and NLRP3 are colocalised in CD68‐positive macrophages. Scale bar: 50 µm. (M) Cell lysates from ox‐LDL‐treated macrophages are immuno‐precipitated with anti‐galectin‐3 or anti‐NLRP3 antibodies, and blotted with anti‐NLRP3 or anti‐galectin‐3 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Expressing, Comparison, Western Blot, Immunofluorescence, Staining, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages concurrently exist in human atherosclerotic lesions. (A–C) Necrotic core formation, lipid deposition and extracellular fibrosis are observed in lower extremity and carotid atherosclerotic lesions, but not in histologically normal artery by means of HE, Oil Red O, and Movat's staining. Scale bar: 1000 µm. (D) Immunohistochemical staining for CD68, a marker for macrophages, is conducted on histologically normal arteries as well as on lower extremity and carotid atherosclerotic lesions. Scale bar: 100 µm. (E–G) Compared with histologically normal artery (E), lower extremity (F) and carotid atherosclerotic lesions (G) show the pyroptotic, apoptotic and necroptotic characteristics of macrophages, including discontinuity of plasma membrane (red arrows), chromatin condensation, and electron‐light zones in transmission electron microscopy images. Scale bar: 2.5 µm. (H) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in human atherosclerotic lesions and peripheral normal artery. (I) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in human atherosclerotic lesions and peripheral normal artery. (J) The capacity of caspase‐3 in human atherosclerotic lesions and peripheral normal artery. (K and L) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in human atherosclerotic lesions and peripheral normal artery. (M) Representative Western blots and relative quantitative analysis of TLR4, MyD88 and phospho‐NF‐kB in human atherosclerotic lesions and peripheral normal artery. (N) Human atherosclerotic lesions release a significant amount of TNF‐1α, IL‐1β, IL‐18 and IL‐6, whereas the peripheral normal artery releases less. (O) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (yellow) and DAPI (blue) in human atherosclerosis and peripheral normal artery reveals the co‐existence of pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Triple‐positive cells are shown by the arrows. Scale bar: 50 µm. (P–R) Double immunofluorescence staining for GSDMD (M)/caspase‐3 (N)/RIPK3 (O) (red), CD68 (green), and DAPI (blue) in human atherosclerosis and peripheral normal artery demonstrates the presence of pyroptotic, apoptotic and necroptotic markers in macrophages, as indicated by the colocalisation of caspase‐3/GSDMD/RIPK3 and CD68 (a macrophage marker). Double‐positive cells are shown by the arrows. Scale bar: 50 µm. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Staining, Immunohistochemical staining, Marker, Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Double Immunofluorescence Staining, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Silencing galectin‐3 downregulated TLR4/MyD88/NF‐kB expression and attenuated ox‐LDL induced pyroptotic, apoptotic, and necroptotic cell death in macrophages. (A) Electron microscopy ultrastructural analysis of control and ox‐LDL‐induced macrophages. Control macrophages have a normal‐looking cellular structure, whereas ox‐LDL‐induced macrophages show loss of cell plasma integrity, chromatin condensation or fragmentation, and electron‐light zone. Scale bar: 2.5 µm. (B) Confocal microscopy with double immunofluorescence staining for caspase‐3 (red) and RIPK3 (green) in macrophages show the colocalisation of apoptotic and necroptotic components. Confocal microscopy analysis of double immunofluorescence labelling is indicative of overlapping expression of caspase‐3 (red) and GSDMD (green) in macrophages. Scale bar: 25 µm. (C) Representative Western blots and relative quantitative analysis of galectin‐3 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (D and E) Flow cytometry (E) and quantification analysis (F) with annexin V/PI double staining show that ox‐LDL increased the percentage of apoptotic cells in macrophages, which is alleviated by silencing galectin‐3. (F–H) Flow cytometry (F) and quantification analysis (G) with PI/Hoechst staining (H) show that ox‐LDL enhanced PI uptake in macrophages, which is markedly blocked by silencing galectin‐3. Scale bar: 50 µm. (I) Silencing galectin‐3 abrogated LDH release in macrophages ignited by ox‐LDL. (J) Ox‐LDL induced the accumulation of intracellular lipid droplets in macrophages, which are potently reversed by silencing galectin‐3. Scale bar: 50 µm. (K) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (L) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (M and N) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (O) Ox‐LDL treatment promotes the release of proinflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) from macrophages, which is markedly rescued by silencing galectin‐3. (P and Q) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐kB and phospho‐NF‐kB in control macrophages and cells treated with ox‐LDL, ox‐LDL plus siControl RNA, and ox‐LDL plus siGalectin‐3 RNA. (R) Cell lysates from ox‐LDL‐treated macrophages are immunoprecipitated with anti‐TLR4 or anti‐MyD88 antibodies, and blotted with anti‐TLR4 or anti‐MyD88 antibodies. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Expressing, Electron Microscopy, Control, Clinical Proteomics, Confocal Microscopy, Double Immunofluorescence Staining, Immunofluorescence, Western Blot, Flow Cytometry, Double Staining, Staining, Immunoprecipitation, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: NLRP3 agonist nigericin counteracted the inhibitory effect of silencing galectin‐3 on pyroptosis, apoptosis and necroptosis in macrophages. (A) Confocal microscopy with double immunofluorescence staining for galectin‐3 (red) and NLRP3 (green) in macrophages reveals the colocalisation of galectin‐3 with NLRP3. Scale bar: 25 µm. (B and C) Flow cytometry (B) and quantification analysis (C) with annexin V/PI double staining show that silencing galectin‐3 decreases the percentage of apoptotic cells in ox‐LDL‐induced macrophages, and nigericin robustly blunts the inhibitory effect of siGalectin‐3. (D–F) Flow cytometry (D) and quantification analysis (E) with PI/Hoechst staining (F) show that silencing galectin‐3 diminishes the percentage of PI‐positive cells in ox‐LDL‐induced macrophages, and nigericin mostly abolishes the protective effect of siGalectin‐3. Scale bar: 50 µm. (G) Silencing galectin‐3 suppresses the LDH release in ox‐LDL‐induced macrophages, which is largely abrogated by nigericin. (H) Silencing galectin‐3 lessens the intracellular lipid droplet in ox‐LDL‐induced macrophages, while nigericin exerts the opposite effect. Scale bar: 50 µm. (I) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (J) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase‐8 and cleaved caspase‐8 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (K) The activity of caspase‐3 in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (L and M) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in macrophages treated with ox‐LDL, ox‐LDL plus galectin‐3 siRNA, ox‐LDL plus nigericin, and ox‐LDL plus galectin‐3 siRNA plus nigericin. (N) Silencing galectin‐3 inhibits the release of inflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) in ox‐LDL‐induced macrophages, and nigericin effectively blocks the role of siGalectin‐3. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Confocal Microscopy, Double Immunofluorescence Staining, Flow Cytometry, Double Staining, Staining, Western Blot, Activity Assay, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Pyroptosis, apoptosis and necroptosis in macrophages coordinately occurred in ApoE −/− mice fed an HFD, which are alleviated by galectin‐3 deficiency, and conversely are aggravated by NLRP3 agonist nigericin. (A) Pyroptosis, apoptosis and necroptosis of macrophages are identified in the aortas of ApoE −/− mice fed an HFD, as evidenced by plasma membrane pore (red arrows), chromatin condensation (red arrows), and electron‐light zone (red arrows) by transmission electron microscopy. Scale bar: 2.5 µm. (B) Triple immunofluorescence staining for GSDMD (green), caspase‐3 (red), RIPK3 (pink) and DAPI (blue) in the aortas of ApoE −/− mice fed HFD or normal diet reveals the potential crosstalk among pyroptosis, apoptosis and necroptosis as evidenced by the colocalisation of GSDMD, caspase‐3 and RIPK3. Three‐positive cells are shown by the arrows. Scale bar: 50 µm. (C–E) Dual immunofluorescence staining for caspase‐3 (C)/GSDMD (D)/RIPK3 (E) (red), F4/80 (green), and DAPI (blue) in the aortas of ApoE −/− mice fed an HFD or normal diet demonstrate that GSDMD/caspase‐3/RIPK3 immunoreactivity colocalises with macrophage marker CD68. Scale bar: 50 µm. (F) Representative Western blots and relative quantitative analysis of NLRP3, GSDMD and GSDMD‐N in the aortas of ApoE −/− mice fed with a normal diet or HFD, NLRP3 agonist nigericin‐treated ApoE −/− mice fed with an HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (G) Representative Western blots and relative quantitative analysis of caspase‐3, cleaved caspase‐3, caspase 8 and cleaved caspase 8 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with HFD. (H) The activity of caspase‐3 in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (I and J) Representative Western blots and relative quantitative analysis of RIPK3, MLKL and phospho‐MLKL in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. (K and L) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐κB and phospho‐NF‐κB in the aortas of ApoE −/− mice fed with a normal diet or HFD, nigericin‐treated ApoE −/− mice fed with HFD, and Galectin‐3 −/− / ApoE −/− mice fed with an HFD. n = 4–8 mice per group. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Clinical Proteomics, Membrane, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Marker, Western Blot, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: TLR4 silencing inhibits the development of atherosclerotic lesions via the downstream MyD88/NF‐kB/NLRP3 signalling molecules. (A) The mRNA level of TLR4 is markedly reduced in the atherosclerotic aorta of HFD‐fed ApoE −/− mice administered by AAV‐F4/80‐shTLR4. (B–D) Representative Western blots and relative quantitative analysis of TLR4, MyD88, NF‐kB, phospho‐NF‐kB and NLRP3 in the aortas of HFD‐fed ApoE−/− mice, empty vector‐treated HFD‐fed ApoE−/− mice and shTLR4‐treated HFD‐fed ApoE−/− mice. (E) Representative en face Oil Red O‐stained images of the entire aorta are obtained from HFD‐fed ApoE−/− mice, empty vector‐treated HFD‐fed ApoE−/− mice and shTLR4‐treated HFD‐fed ApoE−/− mice. Scale bar: 50 mm. (F) En face lesion area is quantified as a percentage of the total aortic surface area. After 16 weeks of HFD feeding, en face lesion areas are significantly smaller in HFD‐fed ApoE −/− mice treated with shTLR4 compared with those treated with the empty vector. (G) Representative sections of HE, Oil Red O and Movat's staining in the aortic sinuses are acquired from three different groups of mice. Scale bar: 1 mm. (H) Aortic sinus plaque lesion area, lipid lesion area and mucin area are represented as total area in µm 2 . Plaque area, lipid lesion area (red) and mucin area (blue‐green) are much smaller in HFD‐fed ApoE −/− mice treated with shTLR4 compared with those treated with empty vector. (I) Necrotic core area is calculated as the total area in µm 2 . Fibrous cap thickness is designated as average thickness in µm. TLR4 silencing diminishes the necrotic core area and fibrous cap thickness in vivo. (J and K) The silencing efficacy of TLR4 in ox‐LDL‐treated RAW 264.7 macrophages is verified by western blotting and RT‐ qPCR. (L and M) Representative Western blots and relative quantitative analysis of MyD88, NF‐kB, phospho‐NF‐kB and NLRP3 in ox‐LDL‐induced macrophages transduced by shTLR4 with or without co‐treatment by the NF‐κB activator berubicin. (N and O) Representative Western blots and relative quantitative analysis of MyD88, NF‐κB, phospho‐NF‐κB and NLRP3 in ox‐LDL‐induced macrophages transduced by shTLR4 with or without co‐treatment by the NLRP3 activator nigericin. n = 4–8 mice per group. Data are derived from three to five independent experiments. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Western Blot, Plasmid Preparation, Staining, In Vivo, Quantitative RT-PCR, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: Macrophage‐derived galectin‐3 contributes to pyroptosis, apoptosis and necroptosis through TLR4/MyD88/NF‐κB/NLRP3 during atherosclerosis

doi: 10.1002/ctm2.70637

Figure Lengend Snippet: Galectin‐3 genetic deficiency or knockdown reduced and, conversely, NLRP3 agonist nigericin augmented atherosclerotic lesions in HFD‐fed ApoE −/− mice. (A) The knockout efficacy of galectin‐3 in the aorta is verified by Western blotting and RT‐qPCR. (B) Schematic diagram of animal study design. Galectin‐3 −/− /ApoE −/− mice and ApoE −/− mice are fed an HFD for 16 weeks, and ApoE −/− mice are intraperitoneally administered with NLRP3 agonist nigericin. (C) Representative images of en face Oil Red O staining in the entire aortas are obtained from ApoE −/− control mice, HFD‐fed ApoE −/− mice, HFD‐fed ApoE −/− mice treated with nigericin, and HFD‐fed Galectin‐3 −/− /ApoE −/− mice. Scale bar: 50 mm. (D) En face lesion area is quantified as a percentage of the total area of the aorta. Compared with those in HFD‐fed ApoE −/− mice, en face lesion areas are significantly smaller in HFD‐fed Galectin‐3 −/− /ApoE −/− mice and, conversely, are markedly bigger in HFD‐fed ApoE −/− mice treated with nigericin. (E) Representative sections of HE, Oil Red O and Movat's staining in the aortic sinuses are acquired from four different groups of mice. Scale bar: 1 mm. (F) Aortic sinus plaque lesion area, lipid lesion area and mucin area are represented as total area in µm 2 . Plaque area, lipid lesion area (red) and mucin area (blue‐green) are much bigger in HFD‐fed ApoE −/− mice treated with nigericin and, conversely, are relatively smaller in HFD‐fed Galectin‐3 −/− /ApoE −/− mice in comparison with HFD‐fed ApoE −/− mice. (G) The levels of inflammatory cytokines in the aortas are measured from four different groups of mice. (H) Schematic illustration of experimental protocol in ApoE −/− mice receiving the injection of AAV‐F4/80 shGalectin‐3/empty vector at the age of 4 weeks. After 2 weeks of a normal diet for rest, these mice are treated with HFD for 16 weeks. (I) The knockdown efficacy of shGalectin‐3 in the aorta is confirmed through Western blotting and RT‐qPCR. (J) Representative images of en face Oil Red O staining in the entire aortas are obtained from HFD‐fed ApoE −/− mice, empty vector‐treated HFD‐fed ApoE −/− mice and shGalectin‐3‐treated HFD‐fed ApoE −/− mice. Scale bar: 50 mm. (K) En face lesion area, quantified as a percentage of total area of the aorta, is significantly smaller in shGalectin‐3‐treated HFD‐fed ApoE −/− mice than in empty vector‐treated HFD‐fed ApoE −/− mice. (L) Representative sections of HE, Oil Red O and Movat's staining in the aortic sinuses are acquired from three different groups of mice. Scale bar: 1 mm. (M) Aortic sinus plaque lesion area, lipid lesion area (red) and mucin area (blue‐green), represented as total area in µm 2 , are much bigger in shGalctin‐3‐treated HFD‐fed ApoE −/− mice than in empty vector‐treated HFD‐fed ApoE −/− mice. (N) The levels of inflammatory cytokines (TNF‐1α, IL‐1β, IL‐18 and IL‐6) in the aortas are measured from three different groups of mice. n = 4–8 mice per group. * p ˂.05, ** p ˂.01, *** p ˂.001 by Student's t test. ns: not significant.

Article Snippet: Galectin‐3 and NLRP3 expression levels are elevated in both human and murine atherosclerotic lesions.

Techniques: Knockdown, Knock-Out, Western Blot, Quantitative RT-PCR, Staining, Control, Comparison, Injection, Plasmid Preparation

Journal: Journal of animal science and biotechnology

Article Title: Methionine deficiency inhibited pyroptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella): possibly via activating the ROS-AMPK-autophagy axis.

doi: 10.1186/s40104-024-01069-6

Figure Lengend Snippet: Fig. 5 Effect of Met on pyroptosis of primary hepatocytes of grass carp. Gene expression of NLRP3, ASC, caspase-1, GSDME, IL-1β (A); relative protein expression of NLRP3, cleaved-caspase-1, GSDME, cleaved-IL-1β (B) and quantification (C) after with or without 1 mmol/L Met treatment for 48 h followed by 100 ng/mL lipopolysaccharide (LPS) treatment for 4 h and then 0.5 μmol/L Nig treatment for 0.5 h. The results were expressed as mean ± SD of 3 or 6 independent observations (WB: n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, n.s: no significance. Met, methionine; Nig, nigericin sodium salt; NLRP3, NOD-like receptor thermal protein domain associated protein 3; ASC, apoptosis-associated speck-like protein containing a CARD; CASP-1, cysteinyl aspartate specific proteinase-1; GSDME, gasdermin E; IL-1β, interleukin-1β

Article Snippet: The following reagents were used in the experiments: LPS (L2880, Sigma-Aldrich, St. Louis, Missouri, USA), NLRP3 activator nigericin sodium salt (Nig) (S6653, Selleck Chemicals, Houston, TX, USA), autophagy inhibitor chloroquine (CQ) (S6999, Selleck Chemicals), AMPK inhibitor compound C (CC) (S7840, Selleck Chemicals), ROS scavenger N-acetyl-L-cysteine (NAC) (T0875, TargetMol Chemicals, Boston, MA, USA).

Techniques: Gene Expression, Expressing

Journal: Journal of animal science and biotechnology

Article Title: Methionine deficiency inhibited pyroptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella): possibly via activating the ROS-AMPK-autophagy axis.

doi: 10.1186/s40104-024-01069-6

Figure Lengend Snippet: Fig. 6 MD inhibited pyroptosis of primary hepatocytes of grass carp through increasing autophagy. Relative protein expression of LC3 II and p62 (A) and quantification (B) after with or without 1 mmol/L Met and CQ co-treatment for 48 h; relative protein expression of NLRP3, cleaved-caspase-1, cleaved-IL-1β (C) and quantification (D) after with or without 1 mmol/L Met and 20 μmol/L CQ co-treatment for 48 h followed by 100 ng/mL LPS treatment for 4 h and then 0.5 μmol/L Nig treatment for 0.5 h. The results were expressed as mean ± SD of 3 independent observations. *P < 0.05, **P < 0.01, ***P < 0.001, n.s: no significance. Met, methionine; CQ, chloroquine; LC3, microtubule-associated protein 1 light chain 3; p62, sequestosome 1; LPS, lipopolysaccharide; Nig, nigericin sodium salt; NLRP3, NOD-like receptor thermal protein domain associated protein 3; CASP-1, cysteinyl aspartate specific proteinase-1; IL-1β, interleukin-1β

Article Snippet: The following reagents were used in the experiments: LPS (L2880, Sigma-Aldrich, St. Louis, Missouri, USA), NLRP3 activator nigericin sodium salt (Nig) (S6653, Selleck Chemicals, Houston, TX, USA), autophagy inhibitor chloroquine (CQ) (S6999, Selleck Chemicals), AMPK inhibitor compound C (CC) (S7840, Selleck Chemicals), ROS scavenger N-acetyl-L-cysteine (NAC) (T0875, TargetMol Chemicals, Boston, MA, USA).

Techniques: Expressing

Journal: Journal of animal science and biotechnology

Article Title: Methionine deficiency inhibited pyroptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella): possibly via activating the ROS-AMPK-autophagy axis.

doi: 10.1186/s40104-024-01069-6

Figure Lengend Snippet: Fig. 9 Mechanism of MD induced autophagy but inhibited pyroptosis. MD promoted autophagy to inhibit pyroptosis through the ROS-AMPK signaling pathway of primary hepatocyte in grass carp. ROS, reactive oxygen species; LKB1, liver kinase B1; p-AMPK, phosphorylated-AMP-activated protein kinase; ULK1, Unc-51-like kinase 1; LC3, microtubule-associated protein 1 light chain 3; p62, sequestosome 1; NLRP3, NOD-like receptor thermal protein domain associated protein 3; ASC, apoptosis-associated speck-like protein containing a CARD; CASP-1, cysteinyl aspartate specific proteinase-1; IL-1β, interleukin-1β; GSDME, gasdermin E

Article Snippet: The following reagents were used in the experiments: LPS (L2880, Sigma-Aldrich, St. Louis, Missouri, USA), NLRP3 activator nigericin sodium salt (Nig) (S6653, Selleck Chemicals, Houston, TX, USA), autophagy inhibitor chloroquine (CQ) (S6999, Selleck Chemicals), AMPK inhibitor compound C (CC) (S7840, Selleck Chemicals), ROS scavenger N-acetyl-L-cysteine (NAC) (T0875, TargetMol Chemicals, Boston, MA, USA).

Techniques:

Journal: Cells

Article Title: The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Exhibits Anti-Inflammatory Activity in LPS-Stimulated Organotypic Hippocampal Cultures

doi: 10.3390/cells10061524

Figure Lengend Snippet: The effects of LPS and/or MR-39 and WRW4 on the levels of proteins involved in the NLRP3 inflammasome signaling pathway ( a —NLRP3; b —Caspase-1; c —ASC, d —GSDMD) in organotypic hippocampal cultures (OHCs) derived from the offspring of wild-type (WT) and FPR2−/− (KO) mice. OHCs obtained from WT and KO mice were pretreated with the FPR2 antagonist WRW4 (10 µM) for 30 min. Afterward, MR-39 (1 µM) was added and incubated for 1 h, and then OHCs were stimulated with lipopolysaccharide (LPS; 1 μg/mL) for 24 h. Control cultures were treated with the appropriate vehicle. The results are presented as the means ± SEM. The data were obtained from three independent experiments. The results were statistically evaluated using a factorial analysis of variance (ANOVA) with Duncan’s post hoc test to assess the differences between the treatment groups. Significant differences are indicated by * p < 0.05. ASC—apoptosis-associated speck-like protein containing a caspase recruitment domain; NLRP3—Nod-like receptor pyrin-containing 3 subunit; GSDMD—gasdermin D. +—with LPS, MR or WRW treatment; -—without LPS, MR or WRW treatment.

Article Snippet: Levels of the NLRP3 ((NACHT, LRR and PYD domains-containing protein 3 ELISA kit), NF-кB (nuclear factor-kappa B; both ELISA kits were from Cusabio, Houston, TX, USA), MyD88 (Myeloid differentiation primary response 88), CASP-1 (Caspase-1), PYCARD (Pyrin Domain-containing Protein 3, ELISA kit for PYD and CARD Domain-containing Protein), TRAF6 (TNF Receptor-associated Factor 6), GSDMD (Mouse Gasdermin D ELISA kit); all ELISA kits from ELK Biotechnology, Wuhan, China) and pNF-кB (NF-кB p65 (phospho) InstantOne ELISA kit, Thermo Fisher, Waltham, MA, USA) proteins in the OHCs lysates were measured using commercially available ELISA kits according to the manufacturers’ instructions.

Techniques: Derivative Assay, Incubation, Control

Journal: Frontiers in Pharmacology

Article Title: TCM and related active compounds in the treatment of gout: the regulation of signaling pathway and urate transporter

doi: 10.3389/fphar.2023.1275974

Figure Lengend Snippet: Anti-gout active ingredients of Chinese botanical drug.

Article Snippet: The utilization of Isatis indigotica Fortune ex Lindl. polysaccharide (can lead to a reduction in the levels of NLRP3, ASC, and caspase-1.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: TCM and related active compounds in the treatment of gout: the regulation of signaling pathway and urate transporter

doi: 10.3389/fphar.2023.1275974

Figure Lengend Snippet: Regulation effect of botanical drug on uric acid transporters in early-onset of gout.

Article Snippet: The utilization of Isatis indigotica Fortune ex Lindl. polysaccharide (can lead to a reduction in the levels of NLRP3, ASC, and caspase-1.

Techniques: In Vivo, In Vitro, Saline

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: NLRP3 expression is upregulated by obesity. A The mRNA levels of NLRP3 in the testes of obese mice ( n = 6). B Western blot analysis and quantitative results of NLRP3 and ASC in the testes of obese mice ( n = 6). (C) NLRP3 mRNA levels in cell populations isolated from the testes of obese mice ( n = 6). D The mRNA level of IL-1β in primary SCs isolated from HFD mice ( n = 6). E Caspase 1 activity in primary SCs isolated from HFD mice ( n = 6). F The mRNA levels of NLRP3 in primary SCs ( n = 6). G Western blot analysis and quantitative results of NLRP3 and ASC in primary SCs ( n = 6). H , I The mRNA levels of IL-1β and caspase 1 activity in primary SCs ( n = 6). J NLRP3 mRNA levels in cell populations isolated from the human testis ( n = 5). K Western blot analysis and quantitative results of NLRP3 and ASC in human primary SCs ( n = 5). Data are presented as the mean ± SD. For A and F , the statistical analysis was carried out by Student’s two-tailed t test; for others, the statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Expressing, Western Blot, Isolation, Activity Assay, Two Tailed Test, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: NLRP3 deficiency attenuated palmitic acid-induced impairment of SCs in vitro. A NLRP3 expression was detected in TM4 cells at 24 h after siNLRP3 infection ( n = 6). B , C The IL-1β mRNA and protein expression in TM4 cells after PA incubation for 24 h ( n = 6). D Caspase 1 activity in TM4 cells after PA incubation for 24 h ( n = 6). E The protein expression of ZO-1 and occludin in TM4 cells after PA incubation for 24 h ( n = 6). F Transepithelial electrical resistance was detected in TM4 cells after PA incubation for 48 h ( n = 6). G The IL-1β mRNA in primary SCs isolated from NLRP3-deficient mice ( n = 4–5). H Transepithelial electrical resistance was detected in primary SCs isolated from NLRP3-deficient mice ( n = 4–5). I NLRP3 expression was detected in TM4 cells at 48 h after NLRP3 overexpression ( n = 6). J The IL-1β mRNA and protein expression in TM4 cells at 48 h after NLRP3 overexpression ( n = 6). K Caspase 1 activity in TM4 cells at 48 h after NLRP3 overexpression ( n = 6). L , M The protein expression of ZO-1 and occludin in TM4 cells at 48 h after NLRP3 overexpression ( n = 6). N Transepithelial electrical resistance was detected in TM4 cells at 48 h after NLRP3 overexpression ( n = 6). Data are presented as the mean ± SD. For A and I – N , the statistical analysis was carried out by Student’s two-tailed t test; for others, the statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: In Vitro, Expressing, Infection, Incubation, Activity Assay, Isolation, Over Expression, Two Tailed Test, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: NLRP3 regulated the expression of occludin via NF-κB/MMP-8 in vitro. A The nuclear accumulation of NF-κB was detected in TM4 cells after PA incubation for 24 h ( n = 6). B The nuclear accumulation of NF-κB was detected in TM4 cells at 48 h after NLRP3 overexpression ( n = 6). C The luciferase assay revealed the binding of NF-κB to the promoter of MMP-8 in TM4 cells after PA incubation for 24 h ( n = 6). D – E MMP-8 mRNA and expression in TM4 cells after PA incubation for 24 h ( n = 6). F MMP-8 expression in isolated NLRP3-deficient SCs after PA incubation for 24 h ( n = 4–5). G , H MMP-8 mRNA and expression in TM4 cells after NLRP3 overexpression ( n = 6). I The protein expression of occludin in TM4 cells at 48 h after SN50 or MMP-8 blocking peptide incubation ( n = 6). J Transepithelial electrical resistance was detected in TM4 cells at 48 h after SN50 or MMP-8 blocking peptide incubation ( n = 6). Data are presented as the mean ± SD. For B, C and G , H , the statistical analysis was carried out by Student’s two-tailed t test; for others, statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Expressing, In Vitro, Incubation, Over Expression, Luciferase, Binding Assay, Isolation, Blocking Assay, Two Tailed Test, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: NLRP3 deficiency protected against HFD-induced subfertility in male mice. A Statistical results of the ratio of testis weight and tibia length in the indicated groups ( n = 12). B , C HE staining of testes and tubular diameter from the indicated groups ( n = 6). Black arrows indicate destruction of the BTB. D Sperm count, viability, and motility of the indicated groups ( n = 6). E Serum testosterone concentration of the indicated groups ( n = 6). F , G The mRNA of steroidogenic enzymes in the testes of obese mice ( n = 6). H The expression of IL-1β in the testes of obese mice ( n = 6). I Caspase 1 activity in the testes of obese mice ( n = 6). J The nuclear accumulation of NF-κB was detected in the testes of obese mice ( n = 6). K MMP-8 expression in the testes of obese mice ( n = 6). L Immunofluorescence of occludin in the testes of obese mice ( n = 5). M The protein expression of ZO-1 and occludin in the testes of obese mice ( n = 6). Data are presented as the mean ± SD. Statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs WT/ND; # P < 0.05 vs WT/HFD

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Staining, Concentration Assay, Expressing, Activity Assay, Immunofluorescence

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: ROS production regulates NLRP3 activation in the testis during obesity. A , B The ROS production and MDA content in the testes of the indicated groups ( n = 6). C , D The ROS production and MDA content in PA-treated TM4 cells ( n = 6). E The ROS production in TM4 cells that were treated with various inhibitors ( n = 6). F The mRNA levels of gp91phox and p67phox in the testes of the indicated groups ( n = 6). G The phosphorylation of p47phox in the testes of the indicated groups ( n = 6). H The activity of NADPH oxidase in the testes of the indicated groups ( n = 6). I The mRNA levels of gp91phox and p67phox in cultured TM4 cells at 24 h after PA incubation ( n = 6). J The activity of NADPH oxidase in PA-treated TM4 cells ( n = 6). K , L The expression of IL-1β in PA-treated TM4 cells ( n = 6). M Caspase 1 activity in PA-treated TM4 cells ( n = 6). N The mRNA level of MMP-8 in PA-treated TM4 cells ( n = 6). O Nuclear NF-κB and NLRP3 activation was detected in PA-treated TM4 cells ( n = 6). P The protein expression of occludin in PA-treated TM4 cells ( n = 6). Data are presented as the mean ± SD. For A – D and F – J , the statistical analysis was carried out by Student’s two-tailed t test; for others, the statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Cell Culture, Incubation, Expressing, Two Tailed Test, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: Constitutively active AMPKα attenuated palmitic acid-induced impairment of SCs in vitro. A , B The protein expression of AMPKα and ACC ( n = 6). C The protein expression of p47phox in PA-treated TM4 cells at 24 h after caAMPKα infection ( n = 6). D , E The production of ROS and NADPH oxidase activity in PA-treated TM4 cells at 24 h after caAMPKα infection ( n = 6). F – H The NLRP3 and IL-1β levels and caspase 1 activity in PA-treated TM4 cells at 24 h after caAMPKα infection ( n = 6). I The nuclear NF-κB accumulation in PA-treated TM4 cells at 24 h after caAMPKα infection ( n = 6). J , K MMP-8 expression in PA-treated TM4 cells at 24 h after caAMPKα infection ( n = 6). L The protein expression of occludin in PA-treated TM4 cells at 24 h after caAMPKα infection ( n = 6). Data are presented as the mean ± SD. For A , B , the statistical analysis was carried out by Student’s two-tailed t test; for others, the statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: In Vitro, Expressing, Infection, Activity Assay, Two Tailed Test, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: miR-451 inhibition suppressed palmitic acid-induced impairment of SCs in vitro. A , B The miR-451 level in HFD mouse testes ( n = 6). C , D The miR-451 level in PA-treated mouse and human primary SCs ( n = 6). E The luciferase assay in cultured TM4 cells ( n = 6). F – J Cab mRNA and protein expression in cultured TM4 cells ( n = 6). K The protein expression of AMPKα and ACC in PA-treated TM4 cells at 24 h after miR-451 inhibition ( n = 6). L , M ROS production and NADPH oxidase activity in PA-treated TM4 cells at 24 h after miR-451 inhibition ( n = 6). N The protein expression of NLRP3 and NF-κB in PA-treated TM4 cells at 24 h after miR-451 inhibition ( n = 6). O – Q The IL-1β level, caspase 1 activity and MMP-8 in PA-treated TM4 cells at 24 h after miR-451 inhibition ( n = 6). Data are presented as the mean ± SD. For A – E and G – J , the statistical analysis was carried out by Student’s two-tailed t test; for others, the statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Inhibition, In Vitro, Luciferase, Cell Culture, Expressing, Activity Assay, Two Tailed Test, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: miR-451 inhibition lost protection in AMPKα-deficient mice. A Statistical results of the ratio of testis weight and tibia length in the indicated groups ( n = 10). B HE staining of the testes and the tubular diameter from the indicated groups ( n = 6). C ROS production in the testes of obese mice ( n = 6). D NLRP3 and occludin protein expression in the testes of obese mice ( n = 6). E MMP-8 level in the testes of obese mice ( n = 6). Data are presented as the mean ± SD. The statistical analysis was carried out by one-way ANOVA. * P < 0.05 vs the matched control

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Inhibition, Staining, Expressing, Control

Journal: Inflammation and Regeneration

Article Title: Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

doi: 10.1186/s41232-022-00203-z

Figure Lengend Snippet: The proposed mechanism of NLRP3 in obesity-induced subfertility. Free fatty acids (FFAs) induce miR-451 expression, which impairing AMPKα activity via targeting Cab39 and thus result in NADPH oxidases-dependent ROS production. NLRP3 activation caused by excessive ROS promotes IL-1β secretion from SCs to impair testosterone synthesis and sperm performance, activates NF-κB and increases MMP-8 expression to degrade occludin, thus affecting the BTB and spermatogenesis

Article Snippet: To exclude the direct effect of NLRP3 on sperm, the sperm were incubated with an NLRP3 inhibitor (MCC950 Sodium, Selleck, 0.01 μmol/L) for 12 h, and then sperm viability and motility were assessed according to our previous studies [ , , ].

Techniques: Expressing, Activity Assay, Activation Assay