The Neuroprotective Effects of Necrostatin-1 on Subarachnoid Hemorrhage in Rats Are Possibly Mediated by Preventing Blood-Brain Barrier Disruption and RIP3-Mediated Necroptosis

Abstract

Despite the substantial efforts to elucidate the role of early brain injury in subarachnoid hemorrhage (SAH), an effective pharmaceutical therapy for patients with SAH continues to be unavailable. This study aims to reveal the role of necroptosis after SAH, and explore whether the disruption of the blood-brain barrier (BBB) and RIP3-mediated necroptosis following SAH in a rat SAH model are altered by necrostatin-1 via its selective inhibition of receptor-interacting protein kinase 1 (RIP1). Sixty-five rats were used in the experiments. The SAH model was established using endovascular perforation. Necrostatin-1 was intracerebroventricularly injected 1 h before SAH induction. The neuroprotective effects of necrostatin-1 were evaluated with multiple methods such as magnetic resonance imaging (MRI) scanning, immunohistochemistry, propidium iodide (PI) labeling, and western blotting. Pretreatment with necrostatin-1 attenuated brain swelling and reduced the lesion volume on T2 sequence and ventricular volume on MRI 72 h after SAH induction. Albumin leakage and the degradation of tight junction proteins were also ameliorated by necrostatin-1 administration. In addition, necrostatin-1 decreased the number of PI-positive cells in the basal cortex, reduced the levels of the RIP3 and MLKL proteins, and inhibited the production of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. Based on the findings from the present study, the selective RIP1 inhibitor necrostatin-1 functioned as a neuroprotective agent after SAH by attenuating brain swelling and BBB disruption. Moreover, the necrostatin-1 pretreatment prevented SAH-induced necroptosis by suppressing the activity of the RIP3/MLKL signaling pathway. These results will provide insights into new drugs and pharmacological targets to manage SAH, which are worth further study.

Keywords: blood–brain barrier; early brain injury; necroptosis; necrostatin-1; subarachnoid hemorrhage.

Figure 1.

Representative images of brain samples from each group and neurological scores, SAH grade and mortalities at 72 h after SAH. (a) Typical brains from sham, SAH + vehicle, and SAH + Nec-1 group. (b) The quantification of neurological scores in 24, 48, and 72 h after SAH induction. (c) The quantification of SAH severity. (d) The quantification of mortality. The bars represent the mean ± SEM. n = 14. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle.

Figure 2.

Necrostatin-1 attenuated brain swelling, reduced the lesion volume and ventricular volume on T2 sequences after SAH. (a) Representative T2-weighted MRI images of the brains from sham, SAH + vehicle, and SAH + Nec-1 group. (b) Brain swelling was calculated as ((volume of ipsilateral hemisphere – volume of contralateral hemisphere)/volume of contralateral hemisphere) × 100%. (c) T2 lesion volume was presented as the volume ratio to the ipsilateral hemisphere. (d) Ventricular volume was presented as the volume ratio to the average volume of the sham group. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle.

Figure 3.

Necrostatin-1 decreased albumin leakage after SAH. (a) Histological panels showed the albumin immunostaining in the perivascular regions of the ipsilateral basal cortex from sham, SAH + vehicle, and SAH + Nec-1 group. (b) Western blot assay for the level of albumin in the ipsilateral basal cortex in sham, SAH + vehicle, and SAH + Nec-1 groups at 72 h after SAH induction. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle.

Figure 4.

Necrostatin-1 Prevented the Disruption of Tight Junction Proteins after SAH. (a) Representative Western blots showing levels of ZO-1, occludin and claudin-5 in the ipsilateral cortex in each group 72 h after SAH induction. (b–d) The relative band densities of ZO-1, occluding, and claudin-5. The densities of the protein bands were analyzed and normalized to β-actin, and compared with the mean value of the sham group. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle. (e) Representative microphotographs of immunofluorescence staining showing the levels of claudin-5 in each group. White arrows indicate the claudin-5-positive parts. Scale bar = 50 μm.

Figure 5.

Effect of necrostatin-1 pretreatment on MMP-9 levels and acitvities 72 h after SAH induction. (a) Representative Western blots showing levels of MMP-9 in the ipsilateral cortex in each group 72 h after SAH induction. (b) Representative gelatin zymography bands showing activities of MMP-9 and MMP-2 in the ipsilateral cortex in each group 72 h after SAH induction. (c) The relative band densities of MMP-9. The densities of the protein bands were analyzed and normalized to β-actin, and compared with the mean value of the sham group. (d) The relative activity of MMP-9. (e) The relative activity of MMP-2. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle.

Figure 6.

Effect of necrostatin-1 on cell injury in the ipsilateral basal cortex 72 h after SAH induction. (a) Representative microphotographs showed the co-localization of DAPI (blue) with PI (red) positive cells in the ipsilateral basal cortex at 72 h after SAH induction. White arrows indicate the PI-positive cells. (b) Quantitative analysis of PI-positive cells showed that necrostatin-1 could significantly reduce the percentage of PI-positive cells in the ipsilateral basal cortex at 72 h after SAH induction. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle. Scale bar = 50 μm.

Figure 7.

Necrostatin-1 down-regulated RIP3/MLKL signaling pathway after SAH induction. (a) Representative microphotographs of immunofluorescence staining showing the localization of RIP3 (green) with caspase-3 (red) in ipsilateral basal cortex at 72 h after SAH induction. White arrows indicate the RIP3-positive and caspase-3 negative cells. (b) Representative Western blots showing levels of RIP3, MLKL, and cleaved caspase-8 in the ipsilateral cortex in each group 72 h after SAH induction. (c–e) The relative band densities of RIP3, MLKL, and cleaved caspase-8. The densities of the protein bands were analyzed and normalized to β-actin, and compared with the mean value of the sham group. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle. Scale bar = 50 μm.

Figure 8.

Necrostatin-1 attenuated neuroinflammation after SAH induction. (a) Representative western blots showing levels of IL-1β, IL-6, and TNF-α in the ipsilateral cortex in each group 72 h after SAH induction. (b–d) The relative band densities of IL-1β, IL-6, and TNF-α. The densities of the protein bands were analyzed and normalized to β-actin, and compared with the mean value of the sham group. The bars represent the mean ± SEM. n = 6. *p < 0.05 versus sham, ^#p < 0.05 versus SAH + vehicle.