Table 1.
PCR Primers used in this study.
Figure 1.
Body weight, blood glucose and MCR expression in STZ-induced diabetic rats.
The body weight of the diabetic rats was significantly lower than that of the normal counterparts (A). The blood glucose levels of the diabetic rats averaged at 25 mM at both 3 d and 4 w after diabetic induction and were significantly higher than the normal rats (B). The expression of MC1R and MC5R (C), MC3R and MC4R (D) were detected with the expected size by regular RT-PCR. M: 1 kb DNA ladder, two arrows designate the position of 750 bp and 1 kb fragment in the ladder. E: retina from the eye; L: liver; H: hypothalamus; (-): negative control. *** p<0.001.
Figure 2.
Intravitreal injections of α-MSH normalized the ROS levels and the total anti-oxidative abilities in early diabetic retinas.
The levels of H2O2 (A) and ROS (B), and the total anti-oxidative abilities (C) were measured and normalized to total protein concentrations in the retina samples from experimental groups. n = 5–10/group, * p<0.05, ** p<0.01.
Figure 3.
α-MSH administration substantially reduced the number of apoptotic cells in the trypsin-digested diabetic retina preparation.
TUNEL staining images were merged with those of DAPI counterstaining. The positive and negative controls for TUNEL staining are shown in (A). The TUNEL staining images of normal control (normal), diabetes (DM), and diabetes with α-MSH intervention (α-MSH+DM) are shown in (B); the insets are the amplified portion of the images for better visualization. n = 8/group, scale bar, 20 μm.
Figure 4.
α-MSH significantly reduced the number of apoptotic cells in the neuroretina of diabetic rats.
Retinal cryosections were subjected to TUNEL staining. The merged images of TUNEL staining and DAPI counterstaining are shown in (A). The positive and negative controls for TUNEL staining are shown in the upper panel of (A). Representative images for normal, DM and α-MSH+DM groups are shown in the lower panel of (A). The apoptotic cells are indicated by white arrows. The estimated representation of the number of apoptotic cells per section is shown in (B). n = 5–6/group, *** p<0.001. Scale bar, 50 μm.
Figure 5.
α-MSH ameliorated ultrastructural changes in the early diabetic retina.
TEM images of the normal retina (A–D), the diabetic retina (E–H), and the diabetic retina with α-MSH intervention (I–L) are shown. In the diabetic retina (E–H), collagen fibers of the Bruch's membrane were broken and protruded to the capillary lumens (E, black arrow head), basement membranes of the choroid capillaries appeared thickened and laminated (E, black arrow), and the capillary lumen was congested with blood cells (E, white arrows). Bubbles (F, black arrow) and medullary scale bodies (G, black arrows) were observed in the photoreceptor outer segments and outer nuclear layer, respectively. The lumen of the inner retina capillary was severely congested (H, black arrow) by the deformed red blood cells (H, white arrow head), and the basement membrane appeared swelled (H, black arrow head). The ultrastructures of the α-MSH-treated diabetic retinas were greatly improved (J–M) and similar to those in the normal controls (A–D), n = 5/group. Scale bar, 500 nm.
Figure 6.
Intravitreal injections of α-MSH corrected aberrant gene expression in the diabetic retina.
The relative mRNA expression levels of eNOS (A), iNOS (B), nNOS (C), ICAM-1 (D), and TNF-α (E) in the retinal samples were examined by real-time RT-PCR and normalized to those of GAPDH. n = 5–7/group, * p<0.05, ** p<0.01, *** p<0.001.
Figure 7.
α-MSH inhibited the up-regulation of Foxo4 in the retina of diabetic rats.
The relative expression of Foxo1, Foxo3, Foxo4, and Foxo6 at the transcript level in the diabetic and normal retinas were examined by real-time RT-PCR and normalized to those of GAPDH. The expression of Foxo4, but not the other Foxo genes, was significantly up-regulated in diabetic retinas compared with the normal controls, and α-MSH treatments inhibited the up-regulation. n = 5–7/group, * p<0.05, ** p<0.01.
Figure 8.
α-MSH inhibited Foxo4 up-regulation in retinal vascular endothelial cells.
The RF/6A cells, a monkey retinal microvessel endothelial cell line, were treated with HG (25 mM) alone or with different concentrations of α-MSH 30 min before and during the HG stimulation. Eight hours following treatment, HG stimulation induced a reduced cell viability that was moderately but significantly boosted by α-MSH at 0.1 μM (A). Then, the cells were exposed to 25 mM HG for 8 h, the mRNA level of Foxo4, but not Foxo1 and 3, was significantly up-regulated, and this up-regulation was inhibited by incubating the cells with 0.1 μM α-MSH 30 min before and during HG stimulation (B). n = 6/group, * p<0.05, ** p<0.01, *** p<0.001.
Figure 9.
Foxo4 overexpression abolished anti-oxidative effects of α-MSH in retinal vascular endothelial cells.
In the CM-H2DCFDA assay, the fluorescence intensities of cell groups was measured and cell images were captured under a fluorescence microscope. Representative images are shown in (A). The fluorescence intensity was normalized to the total protein concentration of each sample (B). n = 5–10/group, * p<0.05, ** p<0.01, *** p<0.001.
Figure 10.
Foxo4 overexpression abrogated anti-apoptotic effects of α-MSH in retinal vascular endothelial cells.
Following various treatments, the cell groups were stained with Alexa 488-annexin V and analyzed by flow cytometry. The dot plot (A) shows the gate selection based on the forward scatter (abscissa) and side scatter (ordinate) of the RF/6A cells, so that the dead cells and cell debris were excluded. The histograms (B) are representative images of the flow cytometry analyses. The abscissa and ordinate represent the fluorescence intensity of Alexa 488-annexin V and cell counts, respectively. The distribution of the non-stained RF/6A cells cultured under the complete culture media was included as a negative control (red line) and used to set the marker M1. The distributions of the treated and stained cells are also shown (black line). The cell populations inside M1 were considered positive for Alexa 488-annexin V staining. The number above M1 indicates the percentage of the cells positive for the Alexa 488-annexin V staining in each representative picture. The Alexa 488-annexin V staining positivity in each treatment group was compared in (C). n = 6/group, ** p<0.01, *** p<0.001.