Selective-cerebral-hypothermia-induced neuroprotection against-focal cerebral ischemia/reperfusion injury is associated with an increase in SUMO2/3 conjugation

Highlights

• Ischemia-reperfusion (I/R) injury increased SUMO2/3 conjugation and decreased SENP3 expression.

• Selective cerebral hypothermia significantly attenuated brain I/R injury by reduce cell apoptosis.

• Selective cerebral hypothermia could increase SUMO2/3 modification mainly via down-regulating the expression of SENP3.

Abstract

Selective cerebral hypothermia is considered an effective treatment for neuronal injury after stroke and avoids the complications of general hypothermia. Several recent studies hanve suggested that SUMO2/3 conjugation occurs following cerebral ischemia/reperfusion (I/R) injury. However, the relationship between the cerebral protective effect of selective cerebral hypothermia and SUMO2/3 conjugation remains unclear. In this study, we investigated the effect of selective cerebral hypothermia on SUMO2/3 conjugation during focal cerebral I/R injury. A total of 140 Sprague-Dawley rats were divided into four groups. In the sham group, only the carotid artery was exposed. The endoluminal filament technique was used to induce middle cerebral artery occlusion in the other three groups. After 2 h of occlusion, the filaments were slowly removed to allow blood reperfusion in the I/R group. In the hypothermia (HT) group and normothermia (NT) group, normal saline at 4 °C and 37 °C, respectively , was perfused through the carotid artery, followed by the restoration of blood flow. The results of the modified neurological severity score (mNSS), 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining demonstrated that selective cerebral hypothermia significantly decreased I/R-induced neuronal injury (mNSS, n = 8, 24 h, HT (5.88 ± 2.36) vs. I/R (8.63 ± 3.38), P < 0.05. 48 h, HT (5.75 ± 2.25) vs. I/R (8.5 ± 2.88), P < 0.05. Cerebral infarct volume percentages, n = 5, HT (18.71 ± 2.13) vs. I/R (41.52 ± 2.90), P < 0.01. Cell apoptosis rate, n = 5, 24 h, HT (21.28 ± 2.61) vs. I/R (43.72 ± 4.30), P < 0.05. 48 h, HT (20.50 ± 2.53) vs. I/R (38.94 ± 2.93), P < 0.05). The expression of Ubc9 and conjugated SUMO2/3 proteins was increased at 24 and 48 h after reperfusion in the 3 non-sham groups, and hypothermia further upregulated the expression of Ubc9 and conjugated SUMO2/3 proteins in the HT group. The expression of SENP3 was increased in the NT group and I/R group, while it was decreased in the HT group at 24 and 48 h after reperfusion (Relative quantities, n = 5, Ubc9, 24 h, HT (2.44 ± 0.22) vs. I/R (1.55 ± 0.39), P < 0.05. 48 h, HT (2.69 ± 0.16) vs. I/R (2.25 ± 0.33), P < 0.05. SENP3, 24 h, HT (0.47 ± 0.15) vs. I/R (2.18 ± 0.43), P < 0.05. 48 h, HT (0.72 ± 0.06) vs. I/R (1.51 ± 0.19), P < 0.05. conjugated SUMO2/3 proteins, 24 h, HT (2.84 ± 0.24) vs. I/R (2.51 ± 0.20), P < 0.05. 48 h, HT (2.73 ± 0.13) vs. I/R (2.44 ± 0.13), P < 0.05). Further analysis showed that the variation in SENP3 expression was more obvious than that in Ubc9 under hypothermia intervention in the HT group. These findings suggest that selective cerebral hypothermia could increase SUMO2/3 modification mainly via down-regulating the expression of SENP3, and then exert neuroprotective effects in rats with cerebral I/R injury.

Introduction

Cerebrovascular disease has a serious social and economic burden owing to its high morbidity, high disability rate, high mortality, and multiple complications, of which ischemic stroke accounts for approximately 80% of cases (Dubuc et al., 2014). The restoration of blood flow after ischemia is the key to the recovery of cerebral tissue after ischemia, but, it also exacerbates tissue damage; this phenomenon is known as cerebral ischemia/reperfusion (I/R) injury (Aronowski et al., 1997, Kalogeris et al., 2016).

SUMO, a small ubiquitin-like modified protein, belongs to a protein family that exists widely in eukaryotes and is highly conserved (Eifler and Vertegaal, 2015). Currently, four subtypes of SUMO proteins are found, SUMO1, SUMO2, SUMO3 and SUMO4, of which SUMO2/3 (SUMO2 and SUMO3 have 95% sequence identity and are denoted SUMO2/3) mainly modifies stress proteins and plays a more important role in the tolerance of cerebral ischemia and neuronal protection than SUMO 1 and SUMO 4 (Lee et al., 2014). SUMO and its substrate proteins continuously combine and dissociate through complex enzymatic reactions in dynamic and reversible modification processes, known as SUMOylation and deSUMOylation (Dohmen, 2004). Three key enzymes are involved in SUMOylation: E1 activating enzyme, E2 binding enzyme and E3 ligase. E2 (Ubc9), as the only specific binding enzyme during the SUMOylation process, increases with the level of SUMOylation (Lee et al., 2007). DeSUMOylation is mediated through SUMO-specific proteases (the SENP family), of which SUMO-specific protease 3 (SENP3) mainly induces the deconjugation of SUMO2/3, and the inhibition of the expression of SENP3 will enhance the level of SUMOylation (Guo et al., 2013).

Hypothermia, including systemic hypothermia and selective hypothermia, is considered an effective modality for the treatment of ischemic stroke (Huber et al., 2019). Selective cerebral hypothermia (SCH) exerts brain protection by affecting multiple pathological events in ischemic stroke, such as activating autophagy, promoting the polarization transition of microglia, inhibiting cell apoptosis, necrosis and inflammation (Liu et al., 2018, Ohmura et al., 2005, Tang et al., 2020, Tu et al., 2019, Zhao et al., 2020). SCH achieves mild brain hypothermia faster than systemic hypothermia and has few systemic side effects, such as shivering, hypotension, arrhythmia, and infection, which can complicate the clinical management of these patients (Vieites-Prado et al., 2016). SCH is strongly recommended to be the first choice for the treatment of ischemic stroke (Almekhlafi et al., 2019, Dumitrascu et al., 2016, Kurisu et al., 2016). However, it is unclear whether SCH reduces I/R injury by regulating SUMO2/3 conjugation. In this study, we investigated the degree of SUMO2/3 conjugation and the changes in Ubc9 and SENP3 under SCH, aiming to further explore the brain protection mechanism of SCH.