Abstract
A depressive or hibernation-like effect of chlorpromazine and promethazine (C + P) on brain activity was reported to induce neuroprotection, with or without induced-hypothermia. However, the underlying mechanisms remain unclear. The current study evaluated the pharmacological function of C + P on the inhibition of neuroinflammatory response and inflammasome activation after ischemia/reperfusion. A total of 72 adult male Sprague-Dawley rats were subjected to 2h middle cerebral artery occlusion (MCAO) followed by 6 or 24h reperfusion. At the onset of reperfusion, rats received C + P (8mg/kg) with temperature control. Brain cell death was detected by measuring CD68 and myeloperoxidase (MPO) levels. Inflammasome activation was measured by mRNA levels of NLRP3, IL-1β, and TXNIP, and protein quantities of NLRP3, IL-1β, TXNIP, cleaved-Caspase-1, and IL-18. Activation of JAK2/STAT3 pathway was detected by the phosphorylation of STAT3 (p-STAT3) and JAK2 (p-JAK2), and the co-localization of p-STAT3 and NLRP3. Activation of the p38 pathway was assessed with the protein levels of p-p38/p38. The mRNA and protein levels of HIF-1α, FoxO1, and p-FoxO1, and the co-localization of p-STAT3 with HIF-1α or FoxO1 were quantitated. As expected, C + P significantly reduced cell death and attenuated the neuroinflammatory response as determined by reduced CD68 and MPO. C + P decreased ischemia-induced inflammasome activation, shown by reduced mRNA and protein expressions of NLRP3, IL-1β, TXNIP, cleaved-Caspase-1, and IL-18. Phosphorylation of JAK2/STAT3 and p38 pathways and the co-localization of p-STAT3 with NLRP3 were also inhibited by C + P. Furthermore, mRNA levels of HIF-1α and FoxO1 were decreased in the C + P group. While C + P inhibited HIF-1α protein expression, it increased FoxO1 phosphorylation, which promoted the exclusion of FoxO1 from the nucleus and inhibited FoxO1 activity. At the same time, C + P reduced the co-localization of p-STAT3 with HIF-1α or FoxO1. In conclusion, C + P treatment conferred neuroprotection in stroke by suppressing neuroinflammation and NLRP3 inflammasome activation. The present study suggests that JAK2/STAT3/p38/HIF-1α/FoxO1 are vital regulators and potential targets for efficacious therapy following ischemic stroke.
Highlights
Acute ischemic stroke causes irreversible cell damage, leading to severe mortality and morbidity worldwide [1,2,3]
The present study suggests that Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3)/p38/hypoxia-induced factor-1α (HIF-1α)/FoxO1 are vital regulators and potential targets for efficacious therapy following ischemic stroke
This may be due to its attenuation of the neuroinflammatory response and inflammasome activation following ischemic stroke, shown by reduced CD68 and MPO and attenuated expressions of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3), IL1β, thioredoxin-interacting protein (TXNIP), cleaved-Caspase-1, and IL-18
Summary
Acute ischemic stroke causes irreversible cell damage, leading to severe mortality and morbidity worldwide [1,2,3]. The interruption and reperfusion of blood flow in brain tissue trigger the infiltration of inflammatory cells and cause a robust inflammatory response, thereby inducing neuronal apoptosis and death [10]. Neuroprotection with C + P has been identified to be related with inhibition of the inflammatory response and NLRP3 inflammasome activation after ischemic stroke [11, 12]. It has been reported that the NLRP3 inflammasome activation was a vital mediator of inflammatory responses after ischemic stroke [18]. Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and Forkhead box transcription factor O1 (FoxO1) are related to the inflammatory response and inflammasome activation[25, 18, 26]. The current study further investigated the pharmacological function of C + P on the inhibition of neuroinflammatory response and inflammasome activation after ischemia/reperfusion through JAK2/STAT3/p38/HIF-1α/FoxO1 regulation
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