Figure 1.
Body weight (A) and blood glucose levels (B).
The body weight (A) and blood glucose levels (B) of C57BL/6 mice were measured once a week. Blood samples were collected from the tails of mice under fasting condition. The body weight was measured in gram, and the blood glucose was measured using a strip-operating blood glucose sensor. The data expressed as the mean ± SEM (n = 12). Abbreviations: con: normal control group; DM: diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; con + shRNA/DM + shRNA: control/diabetes mellitus GIGYF2-knockdown group.
Figure 2.
The expression levels of target gene mRNA.
The levels of Grb10 Interacting GYF Protein 2 (GIGYF2) mRNA (A), growth factor receptor-bound protein 10 (Grb10) mRNA (B) and insulin-like growth factor-1 receptor (IGF1R) mRNA (C) were detected by using the real-time polymerase chain reaction and quantified from three independent experiments, and average levels in septum of each group mice were shown in the graphs. Regulation of Grb10 Interacting GYF Protein 2 (GIGYF2) levels using a lentiviral vector carrying GIGYF2-short hairpin (sh) RNA. Histogram represents the gene expression of target genen, compared to the control group. The data expressed as the mean ± SEM (n = 3). (*p<0.05, **p<0.01 vs con; #p<0.05, vs DM + shRNA) Abbreviations: con: normal control group; DM: diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; con + shRNA/DM + shRNA: control/diabetes mellitus GIGYF2-knockdown group.
Figure 3.
The expression levels of target protein.
The protein levels of Grb10 Interacting GYF Protein 2 (GIGYF2) (A), growth factor receptor-bound protein 10 (Grb10) (B), insulin-like growth factor-1 receptor (IGF1R) (C) and phosphorylated IGF1R (D) were measured by using western blotting and quantified from three independent experiments, and average levels in septum of each group mice were showed in the graphs. Band intensities of phosphorylated IGF1R were normalized to total IGF1R. Except of phosphorylated IGF1R, band intensities were normalized to β-actin. The data expressed as the mean ± SEM (n = 3). (*p<0.05, **p<0.01) Abbreviations: con, normal control group; DM, diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; con + shRNA/DM + shRNA: control/diabetes mellitus GIGYF2-knockdown group.
Figure 4.
The expression levels of Akt and ERK1/2.
The protein levels of serine/threonine kinase (Akt) (A), extracellular signal-regulated kinase (ERK1/2) (B) were measured by Western blotting. The graphs showed the relative density of phosphorylated AKT or ERK1/2 to the total AKT or ERK1/2. The bars represented results from three independent experiments. Band intensities of phosphorylated protein were normalized to total protein. The data expressed as the mean ± SEM (n = 3). (**p<0.01 vs con) Abbreviations: con, control group; DM, diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; DM + shRNA: diabetes mellitus GIGYF2-knocked down group.
Figure 5.
The results of Morris water maze (MWM) test.
Morris water maze (MWM) test was performed before intrahippocampal injection surgery, 1 week after surgery and 10 weeks after surgery in adult male mice. Learning acquisition curve showing the effects of streptozotocin and a single surgery on spatial learning (Aa, b) and the protective effects of Grb10 Interacting GYF Protein 2 (GIGYF2)-short hairpin (sh) RNA (Ac) using the mean escape time to reach the hidden platform (escape latency) over consecutive trials in the MWM task. For the probe trials, the platform crossings (B) and time spent in the target quadrant (C) was recorded. Data are expressed as the mean ± SEM (n = 12 per group). (*p<0.01 vs con; #p<0.01 vs DM + shRNA) Abbreviations: con: normal control group; DM: diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; con + shRNA/DM + shRNA: control/diabetes mellitus GIGYF2-knockdown group.
Figure 6.
Haematoxylin and eosin staining (HE) of hippocampal tissue.
Arrows show senescent neurons (A, inset windows) and cell disorder arrangement (A) in the DM group compared to the healthier hippocampal neurones in the DM + shRNA group and the normal ones in the con group. Immunohistochemistry of hippocampus tissue indicated the place and expression levels of Grb10 Interacting GYF Protein 2 (GIGYF2). Integrated optical density (IOD) values were measured by Image-Pro Plus, version 6.0, and expressed as the mean ± SEM (n = 3) of two independent experiments. (*p<0.05 vs con; #p<0.05 vs DM + shRNA) Abbreviations: con: normal control group; DM: diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; DM + shRNA: diabetes mellitus GIGYF2-knockdown group.
Figure 7.
Morphology and spine synapse density.
Morphologically intact synapse (arrows) are indicated and do not change by the effect of Grb10 Interacting GYF Protein 2 (GIGYF2)-short hairpin (sh) RNA. By contrast, in the DM group, hyperglycaemia results in a significant decrease in synapse density and swollen axons at 10 weeks after streptozotocin injection. Values of the quantity of the spine density are determined by pictures of 8000 times amplification using stereological technique in the Disector Countor, version 1.0 software, and the data are expressed as the mean ± SEM (n = 3). (*p<0.05 vs con; #p<0.05 vs DM + shRNA) Abbreviations: con: normal control group; DM: diabetes mellitus group; DM +0: diabetes mellitus sham-knocked down group; DM + shRNA, diabetes mellitus GIGYF2-knockdown group.