Journal: Neuroreport

Article Title: Effects of electroacupuncture on neural function and the expression of inflammation-related proteins NLRP3/caspase-1 in rats with ischemic stroke

doi: 10.1097/WNR.0000000000002167

Figure Lengend Snippet: NLRP3/GSDMD expression in the brains of rats with ischemia-reperfusion injury. (a–d) Immunohistochemical analysis was conducted on the 1st and 7th days postelectroacupuncture (EA) intervention to evaluate the expression and localization of NLRP3 (a and c) and GSDMD (b and d) in each group ( *** P < 0.001, # P < 0.05, ## P < 0.01, and ### P < 0.001). Both NLRP3 and GSDMD were predominantly localized in the cytoplasm. The extent of positive cell area correlated with protein expression levels, with red arrows indicating positive cells. Scale bar=20 μm. Data were expressed as mean ± SD ( n = 3 rats per group). (e) Immunofluorescence staining showed the expression and localization of NLRP3 and GSDMD in each group. NLRP3 and GSDMD were mainly expressed in the cytoplasm and costained with DAPI. Double immunostaining showed the coexistence of NLRP3 and GSDMD (red arrows point to positive cells). ( *** P < 0.001, ## P < 0.01, ### P < 0.001). Scale bar = 40 μm. Data were expressed as mean ± SD ( n = 3 rats per group). (f) Immunofluorescence staining showed the expression and localization of NLRP3 and GSDMD in each group. NLRP3 and GSDMD were mainly expressed in the cytoplasm and costained with DAPI. Double immunostaining showed the coexistence of NLRP3 and GSDMD (red arrows point to positive cells). ( *** P < 0.001, ### P < 0.001). Scale bar = 40 μm. Data were expressed as mean ± SD ( n = 3 rats per group).

Article Snippet: The reagents and instruments used in this research included a digital medical image analysis system (MOTIC Asia Optical Technology Co. Ltd., China); EA equipment (HANS-200A, Jisheng Medical instrument Co., Ltd., China); stainless steel disposable acupuncture needles (Hwato, 0.18 × 13 mm, Suzhou Medical Equipment Factory, China); Bicinchoninic acid (BCA) protein quantification test kit (Beyotime Institute of Biotechnology, China); primary antibody diluent for Western blotting (GTX28208) (Beyotime Institute of Biotechnology, China); NLRP3 antibody (TA336883) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:1000), interleukin-1β antibody (TA506440) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:4000), interleukin-18 antibody (10663-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:2000), caspase-1 antibody (TA336409) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:1000), GSDMD antibody (20770-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:2000); β-actin antibody (20536-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:4000); goat antimouse IgG H&L (Alexa Fluor488) (ab150113) (Abcam, China, 1:5000); goat antirabbit IgG H&L (Alexa Fluor647) (ab150079) (Abcam, China, 1:5000); antimouse IgG (H + L) Antibody (5220-0341) (Seracare, China, 1:5000); antirabbit IgG (H + L) antibody (5450-0010) (Seracare, China, 1:5000); ammonium persulfate (MYM Biological Technology Co. Ltd., USA); polyvinylidene fluoride membrane (Invitrogen Reagent Corporation, Carlsbad, USA); IL-1β, IL-18 ELISA Research Kit (Jiangsu Meimian industria Co. Ltd, Jiang Su, China); and reagents for glycine, 56-40-6 (Biosharp Biotechnology life science Co. Ltd., China); 30% acrylamide and BIO-BEST 300 M gel imaging analysis system (Solarbio Science and Biotechnology Co., Ltd., China); Ultra High Sensitivity ECL chemiluminescence kit, batch number: 50018 (Beyotime Institute of Biotechnology, China); and MCC950 powder and DMSO (MedChemExpress LLC, China).

Techniques: Expressing, Immunohistochemical staining, Immunofluorescence, Staining, Double Immunostaining

Journal: Neuroreport

Article Title: Effects of electroacupuncture on neural function and the expression of inflammation-related proteins NLRP3/caspase-1 in rats with ischemic stroke

doi: 10.1097/WNR.0000000000002167

Figure Lengend Snippet: The expression levels of NLRP3 and GSDMD genes as well as IL-1β and IL-18 in rats on the 1st and 7th days after intervention. (a–d) Real-time fluorescence quantitative detection of NLRP3 (a, b) and GSDMD (c, d) gene expression levels in the brain tissue of rats with ischemia-reperfusion injury after 1 day and 7 days of intervention. The MCAO group demonstrated significantly increased expression levels of NLRP3 and GSDMD compared with the sham group. *** P < 0.001 vs. the sham group. The expression levels of NLRP3 and GSDMD in the MCAO+EA group and MCAO+MCC950 group were significantly lower than those in the MCAO group. ### P < 0.001 vs. the MCAO group. Data were expressed as mean ± SD ( n = 3 rats per group). (e–h) IL-1β (e, f) and IL-18 (g, h) expression levels on days 1 and 7, respectively; the expression of IL-1β and IL-18 was significantly lower in the MCAO group compared to the sham group. *** P < 0.001 vs. the sham group. The MCAO+EA group and MCAO+MCC950 group showed significantly lower expression of IL-1β and IL-18 than the MCAO group. ### P < 0.001 vs. the MCAO group. Data were expressed as mean ± SD ( n = 3 rats per group). EA, electroacupuncture; MCAO, middle cerebral artery occlusion.

Article Snippet: The reagents and instruments used in this research included a digital medical image analysis system (MOTIC Asia Optical Technology Co. Ltd., China); EA equipment (HANS-200A, Jisheng Medical instrument Co., Ltd., China); stainless steel disposable acupuncture needles (Hwato, 0.18 × 13 mm, Suzhou Medical Equipment Factory, China); Bicinchoninic acid (BCA) protein quantification test kit (Beyotime Institute of Biotechnology, China); primary antibody diluent for Western blotting (GTX28208) (Beyotime Institute of Biotechnology, China); NLRP3 antibody (TA336883) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:1000), interleukin-1β antibody (TA506440) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:4000), interleukin-18 antibody (10663-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:2000), caspase-1 antibody (TA336409) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:1000), GSDMD antibody (20770-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:2000); β-actin antibody (20536-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:4000); goat antimouse IgG H&L (Alexa Fluor488) (ab150113) (Abcam, China, 1:5000); goat antirabbit IgG H&L (Alexa Fluor647) (ab150079) (Abcam, China, 1:5000); antimouse IgG (H + L) Antibody (5220-0341) (Seracare, China, 1:5000); antirabbit IgG (H + L) antibody (5450-0010) (Seracare, China, 1:5000); ammonium persulfate (MYM Biological Technology Co. Ltd., USA); polyvinylidene fluoride membrane (Invitrogen Reagent Corporation, Carlsbad, USA); IL-1β, IL-18 ELISA Research Kit (Jiangsu Meimian industria Co. Ltd, Jiang Su, China); and reagents for glycine, 56-40-6 (Biosharp Biotechnology life science Co. Ltd., China); 30% acrylamide and BIO-BEST 300 M gel imaging analysis system (Solarbio Science and Biotechnology Co., Ltd., China); Ultra High Sensitivity ECL chemiluminescence kit, batch number: 50018 (Beyotime Institute of Biotechnology, China); and MCC950 powder and DMSO (MedChemExpress LLC, China).

Techniques: Expressing, Fluorescence, Gene Expression

Journal: Neuroreport

Article Title: Effects of electroacupuncture on neural function and the expression of inflammation-related proteins NLRP3/caspase-1 in rats with ischemic stroke

doi: 10.1097/WNR.0000000000002167

Figure Lengend Snippet: The expression levels of proteins associated with the NLRP3/caspase-1 inflammatory pathway were assessed at 1 day and 7 days postelectroacupuncture treatment. (a–f) On the first day after intervention, the WB experiment showed that the expression levels of NLRP3, GSDMD, caspase-1, IL-1β, and IL-18 were significantly higher in the MCAOl group compared to the sham group. *** P < 0.001 vs. the sham group. Compared to the MCAO group, the expression levels of NLRP3, GSDMD, caspase-1, IL-1β, and IL-18 were significantly lower in the MCAO+EA group and the MCAO+MCC950 group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the MCAO group. Data were expressed as mean ± SD ( n = 3 rats per group). (g–l) After 7 days of intervention, the WB experiment showed that the expression levels of NLRP3, GSDMD, caspase-1, IL-1β, and IL-18 were significantly higher in the MCAO group compared to the sham group. ** P < 0.01, *** P < 0.001 vs. the sham group. Compared to the MCAO group, the expression levels of NLRP3, GSDMD, caspase-1, IL-1β, and IL-18 were significantly lower in the MCAO+EA group and the MCAO+MCC950 group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. the MCAO group. Data were expressed as mean ± SD ( n = 3 rats per group). EA, electroacupuncture; MCAO, middle cerebral artery occlusion.

Article Snippet: The reagents and instruments used in this research included a digital medical image analysis system (MOTIC Asia Optical Technology Co. Ltd., China); EA equipment (HANS-200A, Jisheng Medical instrument Co., Ltd., China); stainless steel disposable acupuncture needles (Hwato, 0.18 × 13 mm, Suzhou Medical Equipment Factory, China); Bicinchoninic acid (BCA) protein quantification test kit (Beyotime Institute of Biotechnology, China); primary antibody diluent for Western blotting (GTX28208) (Beyotime Institute of Biotechnology, China); NLRP3 antibody (TA336883) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:1000), interleukin-1β antibody (TA506440) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:4000), interleukin-18 antibody (10663-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:2000), caspase-1 antibody (TA336409) (Beijing Zhongshan Jinqiao Company, China, mouse-derived antibody, 1:1000), GSDMD antibody (20770-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:2000); β-actin antibody (20536-1-AP) (Wuhan Sanying Biotechnology Co., Ltd., China, rabbit-derived antibody, 1:4000); goat antimouse IgG H&L (Alexa Fluor488) (ab150113) (Abcam, China, 1:5000); goat antirabbit IgG H&L (Alexa Fluor647) (ab150079) (Abcam, China, 1:5000); antimouse IgG (H + L) Antibody (5220-0341) (Seracare, China, 1:5000); antirabbit IgG (H + L) antibody (5450-0010) (Seracare, China, 1:5000); ammonium persulfate (MYM Biological Technology Co. Ltd., USA); polyvinylidene fluoride membrane (Invitrogen Reagent Corporation, Carlsbad, USA); IL-1β, IL-18 ELISA Research Kit (Jiangsu Meimian industria Co. Ltd, Jiang Su, China); and reagents for glycine, 56-40-6 (Biosharp Biotechnology life science Co. Ltd., China); 30% acrylamide and BIO-BEST 300 M gel imaging analysis system (Solarbio Science and Biotechnology Co., Ltd., China); Ultra High Sensitivity ECL chemiluminescence kit, batch number: 50018 (Beyotime Institute of Biotechnology, China); and MCC950 powder and DMSO (MedChemExpress LLC, China).

Techniques: Expressing

Journal: Cells

Article Title: Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia

doi: 10.3390/cells13090783

Figure Lengend Snippet: Sequences of primers.

Article Snippet: Antibody used were for NLRP3 (cat MAB7578-SB from R&D Systems), ASC (cat # NBP1-78978 from Novus), and Casp1 (Cat # ab74279), respectively, with appropriate dilution according to instructions from the suppliers.

Techniques:

Journal: Cells

Article Title: Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia

doi: 10.3390/cells13090783

Figure Lengend Snippet: Ang II infusion induces activation of NLRP3 inflammasomes. Mice were infused with Ang II as described in Materials and Methods. After 4 weeks of infusion, hearts from each group of animals were collected. The expression levels of NLRP3 ( A ), Casp1 ( B ), and ASC ( C ) were quantified by qRT-PCR. Data are presented as the mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 Ang II-infused vs. saline-infused animals. @@@@ p < 0.0001 Ang II-infused vehicle-treated vs. Ang II-infused WFA-treated animals.

Article Snippet: Antibody used were for NLRP3 (cat MAB7578-SB from R&D Systems), ASC (cat # NBP1-78978 from Novus), and Casp1 (Cat # ab74279), respectively, with appropriate dilution according to instructions from the suppliers.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Saline

Journal: Cells

Article Title: Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia

doi: 10.3390/cells13090783

Figure Lengend Snippet: Ang II infusion induces expression of NLRP3, ASC, and Caspase 1 proteins. Mice were infused with Ang II as described in Materials and Methods. After 4 weeks of infusion, hearts from each group of animals were collected. The expression levels of NLRP3, ASC, and Caspase 1 proteins in cardiac tissues were evaluated by immunofluorescence technique. Data are presented as the mean ± SD (n = 5). * p < 0.05, *** p < 0.001, and **** p < 0.0001 Ang II-infused vs. saline-infused groups. Scale Bar = 50 µm.

Article Snippet: Antibody used were for NLRP3 (cat MAB7578-SB from R&D Systems), ASC (cat # NBP1-78978 from Novus), and Casp1 (Cat # ab74279), respectively, with appropriate dilution according to instructions from the suppliers.

Techniques: Expressing, Immunofluorescence, Saline

Journal: Journal of cellular and molecular medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Fig. 1 Effects of Nlrp3 gene silencing on visfatin-induced disruption of junction pro- teins in mouse vascular endothelial cells (MVECs). MVECs were transfected scram- ble (Scr) or Nlrp3 shRNA (Nlrp3sh) plas- mids by Nucleofection and then stimulated with or without visfatin (Visf: 4 lg/ml) for 24 hrs. (A) Immunofluores- cence stainings were performed with Alex- a555-conjugated antibodies against ZO-1, ZO-2, occludin or VE-Cadherin (VE-Cad) for determination of the expression of these junction proteins. Representative images show the cell membrane of fluo- rescence of ZO-1, ZO-2, occludin or VE- Cadherin (red) are representative of at least three independent experiments. (B– F) Representative Western blot gel docu- ment and summarized data showing the protein expression of ZO-1, ZO-2, occlu- din, VE-Cadherin and b-actin expression in the microsomes of MVECs (n = 3–4). *P < 0.05 versus Scr; #P < 0.05 versus Scr+Visf.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Disruption, Transfection, shRNA, Expressing, Membrane, Western Blot

Journal: Journal of cellular and molecular medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Fig. 2 (A) Western blot documents and summarized data showing the effect of control or visfatin (0 or 4 lg/ml for 24 hrs) on the expression of high mobility group box protein 1 (HMGB1) or b-actin in either cell culture medium (Medium) or homogenized cytoplasm (Homo) of mouse vascular endothelial cells (MVECs; n = 6). *P < 0.05 versus control. (B) MVECs transfected with scramble shRNA (Scr) or Nlrp3 shRNA (Nlrp3sh) plasmids and then stimulated with or without visfatin (Visf: 4 lg/ml) for 24 hrs. Summarized data show the concentration of HMGB1in cul- ture medium of MVECs as analysed by ELISA (n = 6). *P < 0.05 versus Scr; #P < 0.05 versus Scr+Visf. (C) Cells were incubated with fresh culture medium before addition of recombinant HMGB1 (15 lg/ml) for 24 hrs. Representative Western blot gel document and summa- rized data show the effect of recombinant HMGB1 (n = 3) on protein expression of ZO-1, ZO-2, occludin, VE-Cadherin and b-actin in the microsomes of MVECs. *P < 0.05 versus control.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Western Blot, Control, Expressing, Cell Culture, Transfection, shRNA, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

Journal: Journal of cellular and molecular medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Fig. 5 Nlrp3 gene deletion inhibits high-fat diet (HFD)-induced disassembly of tight junction and adherens junction proteins in mouse coronary arterial endothelium. Wild-type (Nlrp3+/+) or Nlrp3 knockout (Nlrp3/) Mice were fed with either a normal diet (ND), or a high-fat diet (HFD) for 6 weeks. (A) Frozen sections of mouse hearts were stained with Alexa555-conju- gated antibodies against ZO-1, ZO-2, Occludin, or VE-Cadherin; scale bar = 50 lm. (B) The summarized data show the fluorescence intensity of the endothelial layer (n = 4–6). *P < 0.05 versus ND on Nlrp3+/+; #P < 0.05 versus Nlrp3+/+ with HFD.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Knock-Out, Staining

Journal: Journal of cellular and molecular medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Fig. 6 Nlrp3 deficiency gene deletion inhi- bits high-fat diet (HFD)-induced inflamma- some activation in mouse coronary arterial endothelium. Wild-type (Nlrp3+/+) or Nlrp3 knockout (Nlrp3/) Mice were fed with normal diet (ND) and high-fat diet (HFD) for 6 weeks. Frozen sections of mouse hearts were stained with FLICA, a green fluorescent probe specific for active caspase-1, and Alexa555-conjugated anti- bodies against an endothelium marker vWF in coronary arteries. (A) The merged images displayed yellow dots or patches indicating the colocalization of FLICA (green) with vWF (red). Enlarged images of area of interest (AOI) in merged images are shown; scale bar = 50 lm. (B) The summarized data show the colocalization coefficient of FLICA with vWF (n = 5–7). *P < 0.05 versus Nlrp3+/+ with ND; #P < 0.05 versus Nlrp3+/+ with HFD.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Activation Assay, Knock-Out, Staining, Marker

Journal: Journal of cellular and molecular medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Fig. 7 Nlrp3 deficiency inhibits high-fat diet (HFD)-induced increases of high mobility group box protein 1 (HMGB1) release in coronary arterial endothelium. Wild-type (Nlrp3+/+) or Nlrp3 knockout (Nlrp3/) Mice were fed with normal diet (ND) and high-fat diet (HFD) for 6 weeks. Frozen sections of mouse hearts were used for confocal immunofluorescent analysis. (A) Representative confocal fluo- rescence images of HMGB1 with endothe- lium marker vWF in coronary arteries of mice. Enlarged images of area of interest (AOI) in merged images are shown; scale bar = 50 lm. (B) The summarized data show the colocalization coefficient (PCC) of HMGB1 with vWF (n = 4–8). *P < 0.05 versus Nlrp3+/+ with ND; #P < 0.05 versus Nlrp3+/+ with HFD.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Knock-Out, Marker

Journal: Journal of cellular and molecular medicine

Article Title: Instigation of endothelial Nlrp3 inflammasome by adipokine visfatin promotes inter-endothelial junction disruption: role of HMGB1.

doi: 10.1111/jcmm.12657

Figure Lengend Snippet: Fig. 8 Nlrp3 deficiency blocks high-fat diet (HFD)-induced T cell adhe- sion and infiltration in coronary arterial wall. Wild-type (Nlrp3+/+) or Nlr- p3 knockout (Nlrp3/) Mice were fed with either a normal diet (ND), or a high-fat diet (HFD) for 6 weeks. (A) Frozen sections of mouse hearts were stained with T cell marker (CD43). The fluorescence images displayed red dots indicating the CD43-positive cells infiltrated into the arterial wall. (B) Summarized data show the fluorescence intensity of the arterial wall area (n = 4). *P < 0.05 versus Nlrp3+/+ with ND; #P < 0.05 versus Nlrp3+/+ with HFD.

Article Snippet: The plasmid encoding shRNA for mouse Nlrp3 gene was obtained from Origene (#TG510752; Rockville, MD, USA).

Techniques: Knock-Out, Staining, Marker