Figure 7.

Mt-encoded peptide HN promotes antioxidative mechanisms, upregulates synaptogenesis-related genes, and stimulates neuronal growth under ICH-like stress. A, Representative Western blot image and quantitating bar graph showing phosphorylation of STAT3 at Y705 in cultured neurons pretreated with 100 ng/ml recombinant HN protein for 24 h. The significant changes in p-STAT3/β-actin protein levels were assessed by two-tailed unpaired t test (n = 7 per group), **p < 0.01 (p < 0.0001, CON vs HN), t value (t = 7.561). CON, Control. B, Representative Western blot image and quantitating bar graph showing Mn-SOD protein levels in cultured neurons pretreated with 100 ng/ml recombinant HN protein for 24 h, followed by exposure to ICH-like injury for 48 h. The significant changes in Mn-SOD/β-actin protein levels were assessed by one-way ANOVA/Fisher's LSD test (n = 9 per group), **p < 0.01 (p < 0.0001, CON vs ICH-like injury), t value (t = 4.993); **p < 0.01 (p = 0.0050, ICH-like injury vs HN plus ICH-like injury), t value (t = 3.092). C, ROS generation in cultured neurons pretreated with 100 ng/ml recombinant HN protein for 24 h, followed by exposure to ICH-like injury for 12 h. The significant changes in ROS generation were assessed by one-way ANOVA/Fisher's LSD test (n = 16 per group), **p < 0.01 (p < 0.0001, CON vs ICH-like injury), t value (t = 4.535); *p < 0.05 (p = 0.0140, ICH-like injury vs HN plus ICH-like injury), t value (t = 2.556). D, Neurite outgrowth quantification in cultured neurons (5.0 × 10⁴ neurons/well) pretreated with 100 ng/ml recombinant HN protein for 24 h, followed by exposure to ICH-like injury (RBC lysates, 1.7 × 10⁶ RBCs/well) for 96 h. The six individual neurites in culture from each treatment group (3 independent replicates for each treatment condition) were manually traced, and the length of each neurite was measured using ImageJ software. The significance in the neurite length changes was assessed by one-way ANOVA/Fisher's LSD test (3 different colors represent 1 of 3 independent experiments-biological replicates, n = 3). Data are shown as mean ± SEM. The mean was calculated by averaging three values for each experiment; **p < 0.01 (p = 0.0023, CON vs ICH-like injury), t value (t = 5.073); *p < 0.05 (p = 0.0146, ICH-like injury vs HN plus ICH-like injury), t value (t = 3.394). E, Representative Western blot images and quantitating bar graphs showing synapsin 1/2 and PSD-95 protein levels in neurons pretreated with 100 ng/ml recombinant HN for 24 h, followed by ICH-like injury for 48 h. The significant changes in synapsin 1/2/β-actin and PSD-95/β-actin protein levels were assessed by one-way ANOVA/Fisher's LSD test (n = 10 in synapsin 1/2 and 9 in PSD-95), **p < 0.01 (p = 0.0087, CON vs ICH-like injury in synapsin 1/2/β-actin), t value (t = 2.828); **p < 0.01 (p = 0.0058, ICH-like injury vs HN plus ICH-like injury in synapsin 1/2/β-actin), t value (t = 2.996); **p < 0.01 (p < 0.0001, CON vs ICH-like injury in PSD-95/β-actin), t value (t = 5.361); **p < 0.01 (p < 0.0001, ICH-like injury vs HN plus ICH-like injury in PSD-95/β-actin), t value (t = 5.031). Data are shown as mean ± SEM.