ACEA Attenuates Oxidative Stress by Promoting Mitophagy via CB1R/Nrf1/PINK1 Pathway after Subarachnoid Hemorrhage in Rats

Abstract

Method: Endovascular perforation was performed to establish a SAH model of rats. ACEA was administered intraperitoneally 1 h after SAH. The CB1R antagonist AM251 was injected intraperitoneally 1 h before SAH induction. Adenoassociated virus- (AAV-) Nrf1 shRNA was infused into the lateral ventricle 3 weeks before SAH induction. Neurological tests, immunofluorescence, DHE, TUNEL, Nissl staining, transmission electron microscopy (TEM), and Western blot were performed.

Results: The expression of CB1R, Nrf1, PINK1, Parkin, and LC3II increased and peaked at 24 h after SAH. ACEA treatment exhibited the antioxidative stress and antiapoptosis effects after SAH. In addition, ACEA treatment increased the expression of Nrf1, PINK1, Parkin, LC3II, and Bcl-xl but repressed the expression of Romo-1, Bax, and cleaved caspase-3. Moreover, the TEM results demonstrated that ACEA promoted the formation of mitophagosome and maintained the normal mitochondrial morphology of neurons. The protective effect of ACEA was reversed by AM251 and Nrf1 shRNA, respectively.

Conclusions: This study demonstrated that ACEA alleviated oxidative stress and neurological dysfunction by promoting mitophagy after SAH, at least in part via the CB1R/Nrf1/PINK1 signaling pathway.

Figure 1

Time course expression of CB1R, Nrf1, PINK1, Parkin, and LC3II and cellular localization of CB1R after SAH. (a) Representative Western blot images of time course and (b) quantitative analyses of CB1R, Nrf1, PINK1, Parkin, and LC3II. n = 6 per group. Data were represented as mean ± SD. ^∗p < 0.05 vs. the Sham group. (c) Representative picture indicates the location of immunofluorescence staining (small black box). (d) Representative microphotographs of immunofluorescence staining for CB1R (green) with neurons (NeuN, red), astrocytes (GFAP, red), and microglia (Iba-1, red) in the left temporal cortex at 24 h after SAH. Nuclei were stained with DAPI (blue). n = 3 per group. Scale bar = 50 μm.

Figure 2

ACEA attenuated long-term neurological deficits and hippocampal neuronal degeneration after SAH. Rotarod test of 5 RPM (a) and 10 RPM (b) in the first, second, and third week after SAH, n = 6 per group. (c) Foot fault test during the three weeks after SAH, n = 6 per group. (d) Representative microphotographs of Nissl staining in the hippocampal CA1, CA3, and DG regions. Scale bar = 50 μm. (e) Interest areas of the CA1, CA3, and DG region in the left hippocampus. Scale bar = 200 μm. (f) Quantification of the Nissl-positive neurons, n = 6 per group. Data of the rotarod test were represented as the median with interquartile range. Other data were represented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 vs. the Sham group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+vehicle group.

Figure 3

ACEA attenuated neurological deficits and neuronal apoptosis, which was reversed by AM251. (a) Modified Garcia and (b) beam balance scores, n = 6 per group. (c) Representative microphotographs of TUNEL staining and quantification of TUNEL-positive neurons. Scale bar = 100 μm. n = 3 per group. (d) Representative Western blot images. (e) Quantitative analyses of Bcl-xl, Bax, and cleaved caspase-3. n = 6 per group. Data of Modified Garcia and beam balance scores were represented as the median with interquartile range. Other data were represented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 vs. the Sham group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+vehicle group; ^@p < 0.05 and ^@@p < 0.01 vs. the SAH+ACEA group.

Figure 4

Nrf1 shRNA abolished the neuroprotective and antiapoptotic effects of ACEA. (a) Modified Garcia and (b) beam balance scores, n = 6 per group. (c) Representative microphotographs of TUNEL staining and quantification of TUNEL-positive neurons. Scale bar = 100 μm. n = 3 per group. (d) Representative Western blot images. (e) Quantitative analyses of Bcl-xl, Bax, and cleaved caspase-3. n = 6 per group. Data of Modified Garcia and beam balance scores were represented as the median with interquartile range. Other data were represented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 vs. the SAH+vehicle group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+ACEA+scrambled shRNA group.

Figure 5

ACEA attenuated oxidative stress, which was reversed by AM251. (a) Representative Western blot images and quantitative analysis of Romo-1, n = 6 per group. (b) Quantification of the levels of MDA, SOD, GSH-Px, and GSH/GSSG ratio in the cortex of ipsilateral hemisphere, n = 6 per group (c) Representative microphotographs of DHE staining and quantification of DHE-positive cells. Scale bar = 50 μm. n = 3 per group. Data were represented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 vs. the Sham group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+vehicle group; ^@p < 0.05 and ^@@p < 0.01 vs. the SAH+ACEA group.

Figure 6

Nrf1 shRNA abolished the antioxidative stress effect of ACEA. (a) Representative Western blot images and quantitative analysis of Romo-1, n = 6 per group. (b) Quantification of the levels of MDA, SOD, GSH-Px, and GSH/GSSG ratio in the cortex of ipsilateral hemisphere, n = 6 per group (c) Representative microphotographs of DHE staining and quantification of DHE-positive cells. Scale bar = 50 μm. n = 3 per group. Data were represented as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 vs. the SAH+vehicle group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+ACEA+scrambled shRNA group.

Figure 7

ACEA promoted mitophagy and improved mitochondrial morphology, which was reversed by AM251. (a) Representative immunofluorescence colocalization of Tomm20 (mitochondrial marker, green) with LC3 (autophagosome marker, red) and quantification of the ratio of LC3-associated Tomm20 to total Tomm20. Scale bar = 50 μm. n = 3 per group. (b) Neuronal and mitochondrial structures were observed by TEM. Red arrow: normal mitochondria; red triangle: swollen mitochondria; red circle: mitophagosome; red star: mitochondrial vacuolization. Scale bar = 1 μm. (c) Representative Western blot images. (d) Quantitative analyses of CB1R, Nrf1, PINK1, Parkin, and LC3II. n = 6 per group. Data were expressed as mean ± SD. ^∗p < 0.05 and ^∗∗p < 0.01 vs. the Sham group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+vehicle group; ^@p < 0.05 and ^@@p < 0.01 vs. the SAH+ACEA group.

Figure 8

Nrf1 shRNA abolished the promoting effect of ACEA on mitophagy. (a) Representative immunofluorescence colocalization of Tomm20 (mitochondrial marker, green) with LC3 (autophagosome marker, red) and quantification of the ratio of LC3-associated Tomm20 to total Tomm20. Scale bar = 50 μm. n = 3 per group. (b) Neuronal and mitochondrial structures were observed by TEM. Red arrow: normal mitochondria; red triangle: swollen mitochondria; red circle: mitophagosome; red star: mitochondrial vacuolization. Scale bar = 1 μm. (c) Representative Western blot images. (d) Quantitative analyses of CB1R, Nrf1, PINK1, Parkin, and LC3II. n = 6 per group. Data were expressed as mean ± SD.^∗p < 0.05 and ^∗∗p < 0.01 vs. the SAH+vehicle group; ^#p < 0.05 and ^##p < 0.01 vs. the SAH+ACEA+scrambled shRNA group.

Figure 9

The graphical abstract. ACEA treatment attenuates oxidative stress by promoting mitophagy through the CB1R/Nrf1/PINK1 signaling pathway after SAH.