Fig 1: Differentially expressed genes and recovery correlated gene analysis in ipsilateral and contralateral motor cortex. (A,B) Heat map of hierarchical clustering using z-scores for normalized gene expression values (log2 based) in the ipsilateral (A) and contralateral (B) motor cortex across the experimental groups (C, S, ST, SA, SAT; n = 2/group). The color scale represents relative gene expression, with red indicating upregulation and blue indicating downregulation. Bar graphs represent the number of differentially expressed genes (DEGs) in the ipsilateral (A) and contralateral (B) hemispheres for each group compared to the control. (C) Spearman correlation analysis between DEG expression in the contralateral motor cortex and behavioral recovery, identifying nine genes with significant correlation. (D) Fold change analysis of Npff expression in the contralateral motor cortex across the experimental groups. Results are presented as the mean ± SEM (n = 2/group). ** p < 0.01 compared to the S group. (E) Quantitative RT-PCR validation of Npff expression, confirming fold changes observed in prior analyses. Results are presented as the mean ± SEM (n = 4/group). * p < 0.05 compared to the C group. C, sham control group; S, stroke control group; ST, stroke group treated with task-specific training (TST); SA, stroke group treated with 5-Aza-dC; SAT, stroke group treated with both 5-Aza-dC and TST. DEGs, differentially expressed genes; RT-PCR, reverse transcription polymerase chain reaction; NPFF, neuropeptide FF.
Fig 2: Effects of rNPFF on neuronal injury and recovery in an OGD model. (A) Quantification of LDH release in the following groups: control (C), OGD, and OGD treated with different concentrations of rNPFF (rNPFF 50 ng/mL, N50; rNPFF 100 ng/mL, N100; rNPFF 200 ng/mL, N200; rNPFF 400 ng/mL, N400). Results are presented as the mean ± SEM (n = 6/group). *** p < 0.001 compared to control; ## p < 0.01 compared to the OGD group. (B) Western blot analysis showing protein expression levels of NPFFR1, NPFFR2, and TUJ1 across groups (C, OGD, and OGD groups with NPFF treatment). (C–E) Densitometric analysis of NPFFR1 (C), NPFFR2 (D), and TUJ1 (E) expression levels, normalized to control levels. Results are presented as the mean ± SEM (n = 4/group). *** p < 0.001 compared to the C group; # p < 0.05 compared to the OGD group. (F,G) Representative immunofluorescence images of TUJ1 (red) (F) and MAP2 (green) (G), with Topro3 (blue) staining in the C, OGD, and OGD groups treated with N400. Scale bars = 20 μm. (H,I) Quantification of TUJ1 (H) and MAP2 (I) fluorescence intensity relative to control levels. Results are presented as the mean ± SEM (n = 4/group). ** p < 0.01 compared to the C group; # p < 0.05 compared to the OGD group for TUJ1; *** p < 0.001 compared to the C group; ## p < 0.01 compared to the OGD group for MAP2. C, sham control group; OGD, oxygen–glucose deprivation; rNPFF, recombinant neuropeptide FF protein; LDH, lactate dehydrogenase; SEM, standard error of mean.
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