Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a , b Heat maps revealing differential gene expression in FMT-DSS vs FMT-CON, FMT-DSS + SP vs FMT-DSS groups. n = 4 mice/group. The pathway-related genes were selected (log2 fold change at least > 1, p < 0.05). c , d KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS group compared with the FMT-CON group (Top 20). n = 4 mice/group. e , f KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS + SP group vs the FMT-DSS group (Top 20). n = 4 mice/group. g Quantitative real-time PCR analysis of mRNA expressions of NF-κB signaling pathway genes ( p65 and IKBα ) in the hippocampus. n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). h The protein expression of p-p65 and p-IKBα in hippocampus tissue, as determined by western blotting. n = 3 independent experiments. i Quantitative real-t i me PCR analysis of mRNA expressions of GABA receptor and Ca 2+ signaling genes ( Gabra1 , Gabra3 , Gabrg2 , Gabrb2 and Camk2d ). n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). j Protein expressions of GABA A R, GABA B R, GAD65 and CaMKII were measured in hippocampus tissue by western blot. n = 3 independent experiments. k Representative immunofluorescence images of double-labeling for p-IKBα (red)/Iba1 (green) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. I Representative immunofluorescence images of double-labeling for GABA A R (green)/GFAP (red) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. m Double immunofluorescence staining for CaMKII (green)/GFAP (red) in hippocampus tissues and statistical analysis. Scale bar = 50 μm. n = 3 independent experiments. n Differential gene expressions were presented by the heatmap in microglia of three groups, and GSEA analysis of microglia from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. o Heat maps of differential gene expression in astrocytes from three groups and GSEA analysis of astrocytes from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Labeling, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Diagram illustrating the mouse model of colitis employed in this study, and inositol treatments are indicated (Some schematic elements were created by Figdraw.com). b Daily body weight changes. n = 8 mice/group. c Diseases activity index. n = 6 mice/group. d Macroscopic pictures of colons. e H&E-stained colon sections. Scale bar = 100 μm. n = 5 independent experiments. f Immunofluorescence staining for E-cadherin (red)/DAPI (blue) in colon tissues. Scale bar = 100 μm. n = 4 independent experiments. g The mRNA level of inflammatory cytokines ( IL-1β , IL-6 and TNF-α ) in the colon. n = 4 mice/group. h , i Representative movement tracks in the open field test and related bar graphs (Distance traveled, distance of center region and speed). n = 6 mice/group. j , k Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms and percentage of times entering the open arms. n = 6 mice/group. l Representative H&E-stained hippocampus sections of three groups. Scale bar = 100 μm. n = 6 independent experiments. m Photomicrographs of Nissl staining in the hippocampus. Scale bar = 100 μm. n = 6 independent experiments. n The mRNA expressions of downstream cytokines ( IL-6 , IL-1β , TNF-α and IL-10 ) in the hippocampus tissue. n = 5 mice/group. o Representative immunohistochemistry images of Iba-1 in hippocampus, Scale bar = 100 μm. n = 3 independent experiments. p The concentration of GABA in hippocampus tissue. n = 6 mice/group. q The mRNA expression of GABA A Rα1 in hippocampus. n = 5 mice/group. r The protein level of GABA B R detected by western blot. n = 3 independent experiments. s Double immunofluorescence staining for GFAP (red)/GAT1 (green) in the DG and CA1 regions of hippocampus tissue. Scale bar = 50 μm. n = 3 independent experiments. Data were presented as means ± SD. For body weight change, two-way repeated-measures ANOVA was performed and the rest of the statistics was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Activity Assay, Staining, Immunofluorescence, Immunohistochemistry, Concentration Assay, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Schematic illustration of experimental design (Some schematic elements were created by Figdraw.com). b , c Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms, percentage of distance traveled in the open arms and percentage of times entering the open arms. n = 6 mice/group. d , e Track plot of the elevated zero maze, and statistical analysis including percentage of time spent in the open zones and percentage of times entering the open zones. n = 6 mice/group. f Statistical analysis, including percentage of distance traveled in the light area, percentage of time spent in the light area, and times of transitions in the light-dark box test. n = 6 mice/group. g Representative images of H&E-staining for the hippocampus sections. Scale bar = 100 μm. n = 6 independent experiments. h Representative images of Nissl staining for the hippocampus sections. Scale bar = 100 μm. n = 4 independent experiments. i The content of inositol in hippocampus tissue tested by ELISA. n = 8 mice/group. j The content of inositol in serum tested by ELISA. n = 8 mice/group. k–n The mRNA expression of TNF-α , IL-1β , IL-6 and iNOS in BV-2 cells. n = 3 mice/group. o, p The mRNA expression of Gabra3 and Camk2d in C8D1A cells. n = 3 mice/group. q The protein level of GABA A R, CaMKII, GABA B R detected by western blot. n = 3 independent experiments. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

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

Journal: Journal of Neuroinflammation

Article Title: Mesenchymal-derived extracellular vesicles enhance microglia-mediated synapse remodeling after cortical injury in aging Rhesus monkeys

doi: 10.1186/s12974-023-02880-0

Figure Lengend Snippet: The expression of inhibitory synaptic markers in perilesional M1 and PMC. a Box-and-whisker plots with vertical scatter plots of individual cases showing the particle density and average size of VGAT+ puncta in perilesional M1 and in PMC (Ctr: n = 3; Veh group: n = 5; EV group: n = 5 monkeys). b Representative maximum-projection confocal images of VGAT immuno-label in M1 and PMC. c Box-and-whisker plots with vertical scatter plots of individual cases of the density and average size of GABA a α1+ puncta in perilesional M1 and in PMC. The density of GABA a α1 subunit in perilesional M1 was significantly lower in both veh and EV group as compared with non-lesion controls (one-way ANOVA, main effect, p = 0.004; Fisher’s LSD post hoc, ctr. vs. Veh, p = 0.038; ctr. vs. EV, p = 0.009). The density of GABA a α1 in PMC was lower in veh and EV group (one-way ANOVA, main effect, p = 0.016; Fisher’s LSD post hoc, ctr. vs. veh, p = 0.05; ctr. vs. EV, p = 0.02). The size of GABA a α1 in M1 was significantly smaller in the EV group ( p < 0.001). d Representative maximum-projection confocal images of GABA a α1 receptor subunit immuno-label in M1 and PMC. e Box-and-whisker plots with vertical scatter plots of individual cases showing the particle density and average size of GABA b R2+ puncta in perilesional M1 and PMC. f Representative maximum-projection confocal images of GABA b R2 subunit immuno-label in M1 and PMC. For c-f Ctr: n = 3; Veh group: n = 4; EV group: n = 5 monkeys. Scale bar: 20 µm. Box-and-whisker plots: bars show interquartile range and median (horizontal line) with error bars = 95% confidence interval; * p < 0.05, ** p < 0.01

Article Snippet: Sections were incubated in primary antibodies in carrier solution (0.2% BSA, 1% NDS, 0.1% Triton X-100 in 0.1 M PB) at 4 °C for 72 h. We used primary antibodies to goat VGLUT1 (1: 500, Synaptic Systems, Cat# 135,307, RRID: AB_2619821), guinea pig VGLUT2 (1:1000, Synaptic Systems, Cat# 135,404, RRID: AB_887884), rabbit GLUR2/3 (1:500, Millipore Cat# 07–598, RRID:AB_11213931), guinea pig VGAT (1:400, Synaptic Systems, Cat# 131,004, RRID: AB_887873), rabbit GABA a α1 (1:500, Abcam, AB33299, RRID: AB_732498), mouse GABA b R2 (1:1000, LSBio, LS-C285897).

Techniques: Expressing, Whisker Assay

Journal: Journal of Neuroinflammation

Article Title: Mesenchymal-derived extracellular vesicles enhance microglia-mediated synapse remodeling after cortical injury in aging Rhesus monkeys

doi: 10.1186/s12974-023-02880-0

Figure Lengend Snippet: Glutamate and GABA receptors subunit mRNA expression in perilesional cortex. a Box-and-whisker plots with vertical scatter plots of individual cases showing relative fold changes in glutamate receptor subunit gene expression. The gene expression of GRIA2 (GLUR2) was significantly higher in the veh group, as compared with non-lesion controls ( t -test, p < 0.001) and the EV group ( p = 0.01). b Fold changes of GABA receptor subunit gene expression. The gene expression of GABRD (GABA a ∂) was significantly higher in the EV group as compared with non-lesion controls (t -test, p = 0.004). Gene names: GRIA1 (AMPA GLUR1), GRIA2 (AMPA GLUR2), GRIN1 (NMDA NR1), GRIN2B (NMDA NR2B), GABRA1 (GABAa α1), GABRA2 (GABA a α2), GABRA5 (GABA a α5), GABRD (GABA a ∂), GABBR2 (GABA b R2). Ctr: n = 2; Veh: n = 5; EV: n = 4 monkeys. c MDS plot showing clustering of cases based on mRNA expression profiles of Glu and GABA receptor subunits. The proximity of points indicates the relative similarity-based pair-wise correlation of these multiple mRNA expression variables. Ctr: n = 2; Veh: n = 5; EV: n = 3 monkeys. Box-and-whisker plots: bars show interquartile range and median (horizontal line) with error bars = 95% confidence interval; * p < 0.05, ** p < 0.01

Article Snippet: Sections were incubated in primary antibodies in carrier solution (0.2% BSA, 1% NDS, 0.1% Triton X-100 in 0.1 M PB) at 4 °C for 72 h. We used primary antibodies to goat VGLUT1 (1: 500, Synaptic Systems, Cat# 135,307, RRID: AB_2619821), guinea pig VGLUT2 (1:1000, Synaptic Systems, Cat# 135,404, RRID: AB_887884), rabbit GLUR2/3 (1:500, Millipore Cat# 07–598, RRID:AB_11213931), guinea pig VGAT (1:400, Synaptic Systems, Cat# 131,004, RRID: AB_887873), rabbit GABA a α1 (1:500, Abcam, AB33299, RRID: AB_732498), mouse GABA b R2 (1:1000, LSBio, LS-C285897).

Techniques: Expressing, Whisker Assay