Journal: Physiological Reports

Article Title: Glutamine enhances endothelial cell survival and vasodilation by increasing glutathione to reduce oxidative stress

doi: 10.14814/phy2.70737

Figure Lengend Snippet: Glutamine increased vasodilation ex vivo but did not impact eNOS phosphorylation or nitric oxide in vitro. (a) Mouse carotid artery vasodilation in response to acetylcholine with an intact endothelium. Isolated mouse carotid arteries were incubated at 4°C overnight in physiological saline containing either 0 or 2 mM glutamine. Vasodilation was measured the following day by pressure myography. n = 4–5 arteries per condition. (b) representative Western blot with quantification of eNOS and p‐eNOS in HCAEC cultured in 0 and 2 mM glutamine. n = 10–11 samples per condition. (c) HCAECs were treated with 0, 0.5, and 2 mM glutamine in 5.5 and 15 mM glucose. L‐glucose at 2 mM and 11.5 mM was included as positive control for glutamine and glucose respectively. After 24 h cells were treated with 20 μM of yoda1 and then the nitrites in the culture media was measured using a Griess assay. n = 9 samples. Data shown as mean ± standard deviation. Statistical significance determined by repeated measures ANOVA (a), Mann–Whitney nonparametric test (b), and two‐way ANOVA followed by Tukey's post hoc test.

Article Snippet: Endothelial‐independent vasodilation was measured by preincubating carotid arteries with eNOS inhibitor NG‐Nitroarginine methyl ester hydrochloride (L‐NAME, 10 −5 M; HY‐18729A; Med Chem Express) for 1 h. Vasodilation to acetylcholine was then measured as previously described.

Techniques: Ex Vivo, Phospho-proteomics, In Vitro, Isolation, Incubation, Saline, Western Blot, Cell Culture, Positive Control, Griess Assay, Standard Deviation, MANN-WHITNEY

Journal: Communications Biology

Article Title: GDF11 alleviates glucocorticoid-induced osteonecrosis of the femoral head by regulating angiogenesis via the PI3K-AKT-eNOS pathway

doi: 10.1038/s42003-025-09078-5

Figure Lengend Snippet: A Microarray heatmap of HUVECs from the MP groups with or without GDF11 treatment (n = 3 per group). B Bubble plots of differentiated pathways in indicated groups. C GO enrichment analysis of DEGs was conducted comparing MP groups with versus without GDF11 supplementation. D GSEA demonstrated significant positive enrichment in gene sets involved in cell migration, circulatory system process, and blood vessel morphogenesis after GDF11 treatment. E , F Immunofluorescence staining of p-PI3K and p-eNOS in HUVECs, with quantitative analysis of mean fluorescence intensity in control (untreated), MP-treated, and MP + GDF11-treated groups (n = 3 per group). Scale bar = 20 μm. G Western blotting was performed to detect PI3K, AKT, eNOS, p-PI3K, p-AKT, and p-eNOS protein expression in control (untreated), MP-treated, and MP + GDF11-treated groups. H Quantification of Western blotting analysis in different groups (n = 3 per group). I NO levels in control (untreated), MP-treated, and MP + GDF11-treated groups (n = 3 per group). Significant differences are indicated as follows: ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Article Snippet: HUVECs were treated with MP (100 μM) or MP (100 μM) + GDF11 (10 ng/ml) in the presence or absence of the PI3K inhibitor LY294002 (10 μM, A10547, Adooq, China) or eNOS inhibitor L-NAME (100 μM, HY-18729A, MCE, USA).

Techniques: Microarray, Migration, Immunofluorescence, Staining, Fluorescence, Control, Western Blot, Expressing

Journal: Communications Biology

Article Title: GDF11 alleviates glucocorticoid-induced osteonecrosis of the femoral head by regulating angiogenesis via the PI3K-AKT-eNOS pathway

doi: 10.1038/s42003-025-09078-5

Figure Lengend Snippet: A Western blotting was performed to detect expression of PI3K, AKT, eNOS, p-PI3K, p-AKT, and p-eNOS in the following groups: MP, MP + GDF11-treated, MP + GDF11 + LY294002-treated, and MP + GDF11 + L-NAME-treated cells. B Quantification of Western blotting analysis in different groups (n = 3 per group). C NO levels in MP, MP + GDF11-treated, MP + GDF11 + LY294002-treated, and MP + GDF11 + L-NAME-treated groups (n = 3 per group). D Representative immunofluorescence images of MP, MP + GDF11-treated, MP + GDF11 + LY294002-treated, and MP + GDF11 + L-NAME-treated groups showing red (CD31), green (phalloidine), blue (nuclei). Scale bar = 20 μm. E Quantitative analysis of mean fluorescence in different groups (n = 3 per group). F Western blotting was performed to detect expression of CD31 and VEGFA in the following groups: MP, MP + GDF11-treated, MP + GDF11 + LY294002-treated, and MP + GDF11 + L-NAME-treated cells. G Quantification of Western blotting analysis in different groups (n = 3 per group). H , I Scratch assay for 0, 12, 24, and 48 h and quantitative analysis in different groups (n = 3 per group). Scale bar = 200 μm. J Transwell assays were performed in four groups: MP, MP + GDF11-treated, MP + GDF11 + LY294002-treated, and MP + GDF11 + L-NAME-treated cells. Scale bar = 100 μm. K Quantification of migration cells of Transwell assay (n = 3 per group). L Tube formation assays were performed in four groups: MP, MP + GDF11-treated, MP + GDF11 + LY294002-treated, and MP + GDF11 + L-NAME-treated cells. Scale bar = 200 μm. M – O Quantification of Tube formation assay (n = 3 per group). Significant differences are indicated as follows: ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Article Snippet: HUVECs were treated with MP (100 μM) or MP (100 μM) + GDF11 (10 ng/ml) in the presence or absence of the PI3K inhibitor LY294002 (10 μM, A10547, Adooq, China) or eNOS inhibitor L-NAME (100 μM, HY-18729A, MCE, USA).

Techniques: Western Blot, Expressing, Immunofluorescence, Fluorescence, Wound Healing Assay, Migration, Transwell Assay, Tube Formation Assay

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The neuroprotective effect of propofol may be associated with promoting eNOS to produce nitric oxide (NO). (A) Representative immunoblots of eNOS and α‐tubulin proteins from four groups of mice. (B) Comparative analysis of eNOS protein expression among the four groups of mice ( n = 6). (C) Comparison of NO concentrations in brain tissue among the four groups of mice ( n = 6). Data are presented as mean ± standard deviation and were statistically analyzed using ANOVA, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Western Blot, Expressing, Comparison, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the early neuroprotective effects of propofol on TBI. (A) Representative images of Perls staining from two groups of mice are displayed. (B) A comparison of the number of positively stained cells in Perls staining between the two groups of mice ( n = 6 per group) is presented. (C) Representative images of Nissl staining from two groups of mice are shown, with scale bars at 50 or 10 µm. (D, E) Bar graphs illustrate the statistical analysis of Nissl body count and positive area per field of view for four groups of mice ( n = 6 per group). (F) Representative TUNEL fluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are displayed. Red labeling indicates neurons, green labeling indicates apoptotic cells, and blue labeling indicates cell nuclei, with a scale bar at 50 µm. (G) A comparison of the number of apoptotic neurons in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region among four groups of mice ( n = 6 per group) is presented. (H) Representative immunoblots of Gpx4, 4‐HNE, Bak, Bcl‐2, and α‐tubulin proteins from two groups of mice are shown. (I–K) Comparisons of Gpx4 protein expression, 4‐HNE protein expression, and the Bak/Bcl‐2 ratio between the two groups of mice ( n = 6 per group) are provided. (L) Comparison of the expression levels of FTH1 mRNA between the two groups of mice ( n = 6 per group). Data are presented as mean ± standard deviation and statistically analyzed using Student's t ‐test, with p values on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Staining, Comparison, TUNEL Assay, Fluorescence, Labeling, Western Blot, Expressing, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the effect of propofol in improving long‐term outcomes after TBI. (A, B) Representative GFAP and Iba1 immunofluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are shown. (C, D) Comparisons of GFAP‐positive area and Iba1‐positive cell counts in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region between two groups of mice are presented ( n = 6 per group). (E) The escape latencies during the MWM training period for two groups of mice were compared, and two‐way repeated measures ANOVA was used for statistical analysis, with **** p < 0.0001. (F) Representative traces of the mice in the MWM are shown. (G, H) Comparisons of platform crossing times and time spent in the target quadrant between two groups of mice are presented ( n = 6 per group). (I) Representative traces of the mice in the NOR test are shown, with N representing the novel object and F representing the familiar object. (J) A comparison of the discrimination index between two groups of mice is presented ( n = 6 per group). Data are expressed as mean ± standard deviation and were statistically analyzed using Student's t ‐test, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Immunofluorescence, Comparison, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The neuroprotective effect of propofol may be associated with promoting eNOS to produce nitric oxide (NO). (A) Representative immunoblots of eNOS and α‐tubulin proteins from four groups of mice. (B) Comparative analysis of eNOS protein expression among the four groups of mice ( n = 6). (C) Comparison of NO concentrations in brain tissue among the four groups of mice ( n = 6). Data are presented as mean ± standard deviation and were statistically analyzed using ANOVA, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Western Blot, Expressing, Comparison, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the early neuroprotective effects of propofol on TBI. (A) Representative images of Perls staining from two groups of mice are displayed. (B) A comparison of the number of positively stained cells in Perls staining between the two groups of mice ( n = 6 per group) is presented. (C) Representative images of Nissl staining from two groups of mice are shown, with scale bars at 50 or 10 µm. (D, E) Bar graphs illustrate the statistical analysis of Nissl body count and positive area per field of view for four groups of mice ( n = 6 per group). (F) Representative TUNEL fluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are displayed. Red labeling indicates neurons, green labeling indicates apoptotic cells, and blue labeling indicates cell nuclei, with a scale bar at 50 µm. (G) A comparison of the number of apoptotic neurons in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region among four groups of mice ( n = 6 per group) is presented. (H) Representative immunoblots of Gpx4, 4‐HNE, Bak, Bcl‐2, and α‐tubulin proteins from two groups of mice are shown. (I–K) Comparisons of Gpx4 protein expression, 4‐HNE protein expression, and the Bak/Bcl‐2 ratio between the two groups of mice ( n = 6 per group) are provided. (L) Comparison of the expression levels of FTH1 mRNA between the two groups of mice ( n = 6 per group). Data are presented as mean ± standard deviation and statistically analyzed using Student's t ‐test, with p values on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Staining, Comparison, TUNEL Assay, Fluorescence, Labeling, Western Blot, Expressing, Standard Deviation

Journal: Brain and Behavior

Article Title: Propofol Suppresses Ferroptosis via Modulating eNOS/NO Signaling Pathway to Improve Traumatic Brain Injury

doi: 10.1002/brb3.70187

Figure Lengend Snippet: The eNOS protein inhibitor L‐NAME can reverse the effect of propofol in improving long‐term outcomes after TBI. (A, B) Representative GFAP and Iba1 immunofluorescence images from the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region of two groups of mice are shown. (C, D) Comparisons of GFAP‐positive area and Iba1‐positive cell counts in the damaged cortical area, hippocampal CA1 region, CA3 region, and DG region between two groups of mice are presented ( n = 6 per group). (E) The escape latencies during the MWM training period for two groups of mice were compared, and two‐way repeated measures ANOVA was used for statistical analysis, with **** p < 0.0001. (F) Representative traces of the mice in the MWM are shown. (G, H) Comparisons of platform crossing times and time spent in the target quadrant between two groups of mice are presented ( n = 6 per group). (I) Representative traces of the mice in the NOR test are shown, with N representing the novel object and F representing the familiar object. (J) A comparison of the discrimination index between two groups of mice is presented ( n = 6 per group). Data are expressed as mean ± standard deviation and were statistically analyzed using Student's t ‐test, with p values indicated on the bar charts.

Article Snippet: In subsequent experiments, before the application of propofol, eNOS protein inhibitor L‐NAME (S2877, Selleck, USA) was administered intraperitoneally at a dose of 30 mg/kg, or an equivalent volume of solvent was injected as a control.

Techniques: Immunofluorescence, Comparison, Standard Deviation