Melanocortin 1 receptor attenuates early brain injury following subarachnoid hemorrhage by controlling mitochondrial metabolism via AMPK/SIRT1/PGC-1α pathway in rats

Abstract

Mitochondria-mediated oxidative stress and apoptosis contribute greatly to early brain injury (EBI) following subarachnoid hemorrhage (SAH). This study hypothesized that activation of melanocortin 1 receptor (MC1R), using BMS-470539, attenuates EBI by controlling mitochondrial metabolism after SAH. Methods: We utilized BMS-470539, MSG-606, selisistat, and PGC-1α to verify the neuroprotective effects of MC1R. We evaluated short- and long-term neurobehavior after SAH. Western blotting, immunofluorescence, and Golgi staining techniques were performed to assess changes in protein levels. Results: The results of western blotting suggested that the expression of SIRT1 and PGC-1α were increased, reaching their peaks at 24 h following SAH. Moreover, BMS-470539 treatment notably attenuated neurological deficits, and also reduced long-term spatial learning and memory impairments caused by SAH. The underlying neuroprotective mechanisms of the BMS-470539/MC1R system were mediated through the suppression of oxidative stress, apoptosis, and mitochondrial fission by increasing the levels of SIRT1, PGC-1α, UCP2, SOD, GPx, Bcl-2, cyto-Drp1, and ATP, while decreasing the levels of cleaved caspase-3, Bax, mito-Drp1, ROS, GSH/GSSG, and NADPH/NADP+ ratios. The neuroprotective effects of the BMS-470539/MC1R system were significantly abolished by MSG-606, selisistat, and PGC-1α siRNA. Conclusions: The activation of MC1R with BMS-470539 significantly attenuated EBI after SAH by suppressing the oxidative stress, apoptosis, and mitochondrial fission through the AMPK/SIRT1/PGC-1α signaling pathway.

Keywords: melanocortin 1 receptor; mitochondrial fission; oxidative stress; peroxisome proliferator-activated receptor gamma coactivator 1-alpha; subarachnoid hemorrhage.

Figure 1

The expression of MC1R, SIRT1 and PGC-1α. (A) Representative western blot images and quantitative analyses of SIRT1 and PGC-1α of the time course study in samples obtained from the left hemisphere after SAH; n = 6 for each group. Data were shown as means ± SD, and were compared by 1-way ANOVA followed by the Tukey post-hoc test. *p < 0.05 vs. sham; (B) Representative microphotographs of immune-fluorescence staining, showing colocalization of MC1R (green) with NeuN, iba-1, and GFAP (all red) 24 h in the sham and SAH (24 h) groups. (n = 2 for each group). Scale bar = 50 μm.

Figure 2

Effects of BMS-470539 on short-term and long-term behaviors of rats subjected to SAH. (A) Beam balance and modified Garcia scores for each group, n = 6 per group; (B) Rotarod test of 5 RPM and 10 RPM, n = 10 per group. (C) Velocity, escape latency, and swim distance of water maze test, n = 10 per group; (D) Probe quadrant duration and typical traces of water maze test, n = 10 per group. Data of beam balance scores were shown as the medians with interquartile range, and were compared using the Kruskal-Wallis test followed by the Dunn post-hoc test. Data of rotarod test, escape latency and swim distance were shown as means ± SD and compared by 2-way ANOVA test. Other data were shown as means ± SD, and were compared using 1-way ANOVA followed by the Tukey post-hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle.

Figure 3

Neuroprotective effects of BMS-470539 on rats subjected to SAH. (A-B) Representative western blot images and quantitative analyses of cleaved caspase-3 (cc-3), Bcl-2 and Bax, n = 6 per group; (C) The levels of ROS and ATP, n = 6 per group; (D) Representative images of immunofluorescence staining of TUNEL and DHE, n = 4 per group, Scale bar = 50 μm; (E) Transmission electron microscopy images of the morphometric changes of the brain tissues in sham, SAH + vehicle, and SAH + BMS-470539 groups (scale bar = 2 μm for 4200 x; scale bar = 0.5 μm for 11500 x), n = 3 per group. The data were shown as means ± SD, and were compared using 1-way ANOVA followed by the Tukey post-hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle.

Figure 4

Representative images and quantitative analysis of Nissl staining of hippocampus (DG, CA 1, CA 2, and CA 3 areas) and cerebral cortex demonstrating reduced injury in BMS-470539 treated rats. Scale bar = 200 μm (general) and 50 μm (regions), n = 4 per group; The data were shown as means ± SD and compared by 2-way ANOVA test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle.

Figure 5

Inhibition of MC1R with MSG-606 abolished the neuroprotective effects of BMS-470539 at 24 h after SAH. (A) Representative western blot images; (B) Quantitative analyses of MC1R, p-AMPK, SIRT1, PGC-1α, UCP2, GPx, SOD, CC-3, Bcl-2, Bax; (C) ROS and ATP levels at 24 h after SAH. n = 6 for each group. Data were shown as means ± SD and compared by 1-way ANOVA followed by the Tukey post-hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle, #p < 0.05 vs. SAH + BMS-470539.

Figure 6

SIRT1 inhibitor, selisistat reversed the anti-oxidative stress and anti-apoptotic effects of MC1R at 24 h after SAH. (A) Representative western blot images; (B) Quantitative analyses of MC1R, p-AMPK, SIRT1, PGC-1α, UCP2, GPx, SOD, CC-3, Bcl-2, Bax. Data were shown as means ± SD and compared by 1-way ANOVA followed by the Tukey post-hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle, #p < 0.05 vs. SAH + BMS-470539.

Figure 7

SIRT1 and PGC-1α were involved in MC1R-mediated neuroprotection at 24 h after SAH. Representative images and quantification of immunofluorescence and Golgi staining of (A, B) DHE; (C, D) FJC; (E, F) TUNEL; (G, H) Golgi. n = 4 for each group. Scale bar = 50 μm. Data were shown as means ± SD and compared by 1-way ANOVA, followed by the Tukey post-hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle, #p < 0.05 vs. SAH + BMS-470539.

Figure 8

AMPK inhibitor decreased the levels of SIRT1, cellular apoptosis and oxidative stress at 24 h after SAH, which inversely stimulated the activation of AMPK. (A, B) Representative western blot images and quantitative analyses of p-AMPK and SIRT1; (C, D) Representative western blot images and quantitative analyses of p-AMPK, SIRT1, PGC-1α, GPx, SOD. n = 6 for each group; (E) Levels of ROS at 24 h after SAH. n = 6 for each group. Data were shown as means ± SD, and were compared using 1-way ANOVA followed by the Tukey post hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle, #p < 0.05 vs. SAH + BMS-470539.

Figure 9

Knockout of PGC-1α with siRNA aggravates oxidative stress and apoptosis following SAH. (A) Representative western blot images; (B) Quantitative analyses of MC1R, p-AMPK, SIRT1, PGC-1α, UCP2, GPx, SOD, CC-3, Bcl-2, Bax. Data were shown as means ± SD, and were compared using 1-way ANOVA followed by the Tukey post hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle, #p < 0.05 vs. SAH + BMS-470539.

Figure 10

The effects of PGC-1α siRNA was partly mediated by the suppression of Drp1. (A) Representative western blot images; (B) Representative western blot images and quantitative analyses of PGC-1α, cyto-Drp1, CC-3, GPx, and mito-Drp1. n = 6 for each group; (C-D) Levels of ROS, ATP, GSH/GSSG ratio and NADPH/NADP⁺ ratio at 24 h after SAH. n = 6 for each group; (E-F) Representative images and quantitative analysis of immunofluorescence staining of TUNEL and DHE, n = 4 per group. Scale bar = 50 μm; Data were shown as means ± SD, and were compared using 1-way ANOVA followed by the Tukey post hoc test. *p < 0.05 vs. sham, &p < 0.05 vs. SAH + vehicle, #p < 0.05 vs. SAH + BMS-470539.