Activation of GPR30 with G1 attenuates neuronal apoptosis via src/EGFR/stat3 signaling pathway after subarachnoid hemorrhage in male rats.

Neuron apoptosis plays a vital role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies showed that the activation of G protein-coupled receptor 30 (GPR30) with GPR30 agonist G1 was anti-apoptotic after experimental trauma brain injury and global cerebral ischemia in male rats or mice. However, the role of GPR30 activation with G1 has not been clarified in SAH. The aim of this study was to investigate the anti-apoptotic effect of GPR30 activation and the underlying mechanism of src/EGFR/stat3 signaling pathway in a male rat model of SAH. A total of 215 male rats and 18 female rats were used. SAH was induced by intravascular perforation. G1 was administrated intravenously 1 h after SAH. For mechanism study, the GPR30 antagonist G15 or epidermal growth factor receptor (EGFR) antagonist AG1478 was administrated intravenously 1 h before SAH, small interfering ribonucleic acid (siRNA) for GPR30 and EGFR were administered intracerebroventricularly 48 h before SAH. Post-SAH assessments included SAH Grade, neurological deficits, western blot, terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) staining, Fluoro-Jade C (FJC) staining, Nissl staining and immunofluorescence. The expression of endogenous GPR30 in male rats was increased at 3 h and peaked at 24 h after SAH, which mainly co-localized with neurons, but there was no significant increase in intact female rats at 24 h after SAH. The G1 post-treatment significantly reduced the short-term and long-term neurological deficit as well as neuronal apoptosis in male rats, but it did not significantly improve the short-term outcome of intact female rats. Mechanistic studies indicated that G15 or GPR30 siRNA and AG1478 or EGFR siRNA reversed the anti-neuronal apoptosis effects of G1 and its effects on protein expressions of src/EGFR/stat3 signaling pathway. G1 reduced EBI through attenuating neuronal apoptosis after SAH in male rats, partly via activating src/EGFR/stat3/signaling pathway. G1 may provide a promising therapeutic strategy for SAH patients.

Peng J ，Zuo Y ，Huang L ，Okada T ，Liu S ，Zuo G ，Zhang G ，Tang J ，Xia Y ，Zhang JH ... -  《-》

Activation of TGR5 with INT-777 attenuates oxidative stress and neuronal apoptosis via cAMP/PKCε/ALDH2 pathway after subarachnoid hemorrhage in rats.

Oxidative stress and neuronal apoptosis play important roles in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). The activation of TGR5, a novel membrane-bound bile acid receptor, possesses anti-oxidative stress and anti-apoptotic effects in hepatobiliary disease and kidney disease. The present study aimed to explore the neuroprotective effect of TGR5 activation against EBI after SAH and the potential underlying mechanisms. The endovascular perforation model of SAH was performed on 199 Sprague Dawley rats to investigate the beneficial effects of TGR5 activation after SAH. INT-777, a specific synthetic TGR5 agonist, was administered intranasally at 1 h after SAH induction. TGR5 CRISPR and ALDH2 CRISPR were administered intracerebroventricularly at 48 h before SAH to illuminate potential mechanisms. The SAH grade, short-term and long-term neurobehavioral tests, TUNEL staining, Fluoro-Jade C staining, Nissl staining, immunofluorescence staining, and western blots were performed at 24 h after SAH. The expressions of endogenous TGR5 and ALDH2 gradually increased and peaked at 24 h after SAH. TGR5 was expressed primarily in neurons, as well as in astrocytes and microglia. The activation of TGR5 with INT-777 significantly improved the short-term and long-term neurological deficits, accompanied by reduced the oxidative stress and neuronal apoptosis at 24 h after SAH. Moreover, INT-777 treatment significantly increased the expressions of TGR5, cAMP, phosphorylated PKCε, ALDH2, HO-1, and Bcl-2, while downregulated the expressions of 4-HNE, Bax, and Cleaved Caspase-3. TGR5 CRISPR and ALDH2 CRISPR abolished the neuroprotective effects of TGR5 activation after SAH. In summary, the activation of TGR5 with INT-777 attenuated oxidative stress and neuronal apoptosis via the cAMP/PKCε/ALDH2 signaling pathway after SAH in rats. Furthermore, TGR5 may serve as a novel therapeutic target to ameliorate EBI after SAH.

Zuo G ，Zhang T ，Huang L ，Araujo C ，Peng J ，Travis Z ，Okada T ，Ocak U ，Zhang G ，Tang J ，Lu X ，Zhang JH ... -  《-》

Intranasal administration of recombinant Netrin-1 attenuates neuronal apoptosis by activating DCC/APPL-1/AKT signaling pathway after subarachnoid hemorrhage in rats.

Neuronal apoptosis is a crucial pathological process in early brain injury after subarachnoid hemorrhage (SAH). The effective therapeutic strategies to ameliorate neuronal apoptosis are still absent. We intended to determine whether intranasal administration of exogenous Netrin-1 (NTN-1) could attenuate neuronal apoptosis after experimental SAH, specifically via activating DCC-dependent APPL-1/AKT signaling cascade. Two hundred twenty-five male Sprague-Dawley rats were subjected to the endovascular perforation model of SAH. Recombinant human NTN-1 (rNTN-1) was administered intranasally. NTN-1 small interfering RNA (siRNA), APPL-1 siRNA, and AKT inhibitor MK2206 were administered through intracerebroventricular (i.c.v.) injection. SAH grade, neurological score, neuronal apoptosis assessed by cleaved caspase-3 (CC-3) expression and Fluoro-Jade C (FJC) staining, double immunofluorescence staining, and Western blot were examined. Our results revealed that endogenous NTN-1 level was increased after SAH. Administration of rNTN-1 improved neurological outcomes at 24 h and 72 h after SAH, while knockdown of endogenous NTN-1 worsened neurological impairments. Furthermore, exogenous rNTN-1 treatment promoted APPL-1 activation, increased phosphorylated-AKT and Bcl-2 expression, as well as decreased apoptotic marker CC-3 expression and the number of FJC-positive neurons, thereby alleviated neuronal apoptosis. Conversely, APPL-1 siRNA and MK2206 abolished the anti-apoptotic effect of exogenous rNTN-1 at 24 h after SAH. Collectively, intranasal administration of exogenous rNTN-1 attenuated neuronal apoptosis and improved neurological function in SAH rats, at least in apart via activating DCC/APPL-1/AKT signaling pathway.

Xie Z ，Huang L ，Enkhjargal B ，Reis C ，Wan W ，Tang J ，Cheng Y ，Zhang JH ... -  《-》

GPR30 alleviated subarachnoid hemorrhage-induced blood-brain barrier dysfunction by activating the PI3K/Akt and Nrf2/HO-1 pathways.

Peng J ，He J ，Hu X ，Xia Y ... -  《-》

AVE 0991 attenuates oxidative stress and neuronal apoptosis via Mas/PKA/CREB/UCP-2 pathway after subarachnoid hemorrhage in rats.

Oxidative stress and neuronal apoptosis have been demonstrated to be key features in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies have indicated that Mas receptor activation initiates an anti-oxidative and anti-apoptotic role in the brain. However, whether Mas activation can attenuate oxidative stress and neuronal apoptosis after SAH remains unknown. To investigate the beneficial effect of Mas on oxidative stress injury and neuronal apoptosis induced by SAH, a total of 196 rats were subjected to an endovascular perforation model of SAH. AVE 0991 (AVE), a selective agonist of Mas, was administered intranasally 1 h after SAH induction. A779, a selective inhibitor of Mas, and small interfering ribonucleic acid (siRNA) for UCP-2 were administered by intracerebroventricular (i.c.v) injection at 1 h and 48 h before SAH induction respectively. Neurological tests, immunofluorescence, TUNEL, Fluoro-Jade C, DHE staining, and Western blot experiments were performed. We found that Mas activation with AVE significantly improved neurobehavioral scores and reduced oxidative stress and neuronal apoptosis in SAH+AVE group compared with SAH+vehicle group. Moreover, AVE treatment significantly promoted phosphorylation of CREB and the expression UCP-2, as well as upregulated expression of Bcl-2 and downregulation of Romo-1 and Bax. The protective effects of AVE were reversed by i.c.v injection of A779 and UCP-2 siRNA in SAH+AVE+A779 and SAH+AVE+UCP-2 siRNA groups, respectively. In conclusion, our data provides evidence that Mas activation with AVE reduces oxidative stress injury and neuronal apoptosis through Mas/PKA/p-CREB/UCP-2 pathway after SAH. Furthermore, our study indicates that Mas may be a novel therapeutic treatment target in early brain injury of SAH.

Mo J ，Enkhjargal B ，Travis ZD ，Zhou K ，Wu P ，Zhang G ，Zhu Q ，Zhang T ，Peng J ，Xu W ，Ocak U ，Chen Y ，Tang J ，Zhang J ，Zhang JH ... -  《-》