Abstract
Xingnaojing (XNJ) injection, derived from traditional Chinese medicine formulation, has a protective effect against stroke, but the underlying mechanism is unclear, which severely limited its clinical application. This research aims to elucidate the role and mechanism of XNJ in reducing cerebral ischemic reperfusion (I/R) injury. Rats received 2 h cerebral ischemia followed by reperfusion of 24 h and were intraperitoneally given 5, 10, or 15 ml/kg XNJ 24 h before ischemia and at the onset of reperfusion, respectively. TTC staining, HE staining, and neurological score were implied to evaluate the effectiveness of XNJ. The protein expressions of PI3K/Akt and eNOS signaling were measured. Experiments were further performed in human brain microvascular endothelial cells (HBMECs) to investigate the protective mechanisms of XNJ. HBMECs were subjected to 3 h oxygen and glucose deprivation following 24 h of reoxygenation (OGD) to mimic cerebral I/R in vitro. PI3K inhibitor LY294002 was added with or without the preconditioning of XNJ. Multiple methods including western blot, immunofluorescence, DAPI staining, JC-1, and flow cytometry were carried out to evaluate the effect of XNJ on HBMECs. XNJ could improve rat cerebral ischemic injury and OGD induced HBMECs apoptosis. In vivo and in vitro researches indicated that the mechanism might be relevant to the activation of PI3K/Akt/eNOS signaling.
Highlights
Stroke remains an important major health problem with high morbidity, high disability, and high mortality worldwide, with 87% being ischemic stroke [1, 2]
To evaluate the protective effects of XNJ against cerebral ischemic reperfusion (I/R) damage, we examined neurological scores and infarct size 24 h after reperfusion in cerebral I/R injury rats
The findings above declared that PI3K/Akt/Endothelial nitric oxide synthase (eNOS) signaling pathway was an important mediator in regulating the protective effect of XNJ against apoptosis induced by Oxygen-Glucose Deprivation (OGD) in previous studies suggested that XNJ was protective in stroke, the effects and mechanisms have yet to be investigated
Summary
Stroke remains an important major health problem with high morbidity, high disability, and high mortality worldwide, with 87% being ischemic stroke [1, 2]. Cerebral ischemia/reperfusion (I/R) injury occurs after effective thrombolysis therapy of stroke and can cause more serious secondary damage to brain tissue. It is urgent for us to find the exact mechanisms as well as the effective therapeutic drug for cerebral I/R injury. The pathophysiological mechanisms of cerebral I/R injury are very complex [3]. Apoptosis has been shown to play a role in cerebral I/R injury [6,7,8,9]; inhibition of apoptosis is a potential therapeutic target in stroke patients. In regulating eNOS activity, Ser1177 phosphorylation is identified as one of the most important factors among the specific sites. A study of cerebral ischemia in rat showed that the phosphorylation of eNOS (p-eNOS) at Ser1177 regulates cerebral blood flow, represses apoptosis, and ameliorates the ischemic injury [10]
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