Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats

Fig. 1. Experimental design and animal groups

Fig. 2. Effects of exogenous apelin-13 on neurological functions, brain edema, and BBB leakage. a Representative pictures of brains in Sham and SAH groups. b SAH grade of each group. c The quantification of brain water content 24 h after SAH. d The quantification of Evans blue dye extravasation 24 h after SAH (n = 6 for each group). e, f Modified Garcia score and beam balance score; the effects of apelin-13 on long-term neurobehavioral outcomes after SAH. h Escape latency and i swim distance of Morris water maze on days 21 to 25 after SAH. j Probe quadrant duration. The bars represent the mean ± SD. *P < 0.05 versus sham, #P < 0.05 versus SAH + vehicle at 24 h, n = 10 per group

Fig. 3. Expression of apelin-13, APJ, and p-AMPK. a Representative Western blot images and quantitative analyses of apelin-13 time-course from the left hemisphere after SAH. b Representative Western blot images and quantitative analyses of APJ time course from the left hemisphere after SAH. c Representative Western blot images and quantitative analyses of p-AMPK time course from the left hemisphere after SAH; n = 6 for each group. The bars represent the mean ± SD. *P < 0.05 versus sham. d Representative microphotographs of immunofluorescence staining showing localization of APJ (green) with NeuN, iba-1, and GFAP (red) 24 h after SAH (N = 2 for each group). Scale bar = 50 μm

Fig. 4. Knockdown of APJ abolished the neuroprotective effects of exogenous apelin-13 24 h after SAH. a, b Modified Garcia score and beam balance score. c Representative Western blot images. d Quantitative analyses of APJ, apelin-13, p-AMPK, TXNIP, NLRP3, Bip, IL-1β, TNFα, cleaved caspase-1, and MPO. n = 6 for each group. The bars represent the mean ± SD. *P < 0.05 versus sham, #P < 0.05 versus SAH + vehicle, &P < 0.05 versus SAH + apelin-13. e The level of ROS

Fig. 5. AMPK inhibitor, dorsomorphin, reversed the anti-inflammatory effects of apelin-13 24 h after SAH. a, b Modified Garcia score and beam balance score. c Representative Western blot images. d Quantitative analyses of APJ, apelin-13, p-AMPK, TXNIP, NLRP3, Bip, IL-1β, TNFα, cleaved caspase-1, and MPO. n = 6 for each group. The bars represent the mean ± SD. *P < 0.05 versus sham, #P < 0.05 versus SAH + vehicle, &P < 0.05 versus SAH + apelin-13

Fig. 6. The effects of apelin-13 on neutrophil infiltration after SAH. a Representative images of immunofluorescence staining of MPO (green) with DAPI (blue) from the left hemisphere. b Quantitative analyses of MPO-positive cells from the left hemisphere 24 h after SAH. n = 5 for each group

Fig. 7. The effects of apelin-13 on microglia/macrophage activation after SAH. a Representative images of immunofluorescence staining of Iba-1 (green) with DAPI (blue) from the left hemisphere. b Quantitative analyses of Iba-1-positive cells from the left hemisphere 24 h after SAH. n = 5 for each group

Fig. 8. The effects of dorsomorphin on the rats with SAH. a Representative Western blot images. b Quantitative analyses of p-AMPK, IL-1β, TNFα, cleaved caspase-1, MPO. n = 6 for each group. The bars represent the mean ± SD. *P < 0.05 versus sham, #P < 0.05 versus SAH + vehicle

Fig. 9. The potential molecular mechanisms of apelin-13/APJ system-mediated anti-inflammatory and anti-oxidative effects via suppression of ER stress-associated AMPK/TXNIP/NLRP3 inflammasome activation