Chlorpromazine and Promethazine (C+P) Reduce Brain Injury after Ischemic Stroke through the PKC-δ/NOX/MnSOD Pathway

Abstract

Cerebral ischemia-reperfusion (I/R) incites neurologic damage through a myriad of complex pathophysiological mechanisms, most notably, inflammation and oxidative stress. In I/R injury, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) produces reactive oxygen species (ROS), which promote inflammatory and apoptotic pathways, augmenting ROS production and promoting cell death. Inhibiting ischemia-induced oxidative stress would be beneficial for reducing neuroinflammation and promoting neuronal cell survival. Studies have demonstrated that chlorpromazine and promethazine (C+P) induce neuroprotection. This study investigated how C+P minimizes oxidative stress triggered by ischemic injury. Adult male Sprague-Dawley rats were subject to middle cerebral artery occlusion (MCAO) and subsequent reperfusion. 8 mg/kg of C+P was injected into the rats when reperfusion was initiated. Neurologic damage was evaluated using infarct volumes, neurological deficit scoring, and TUNEL assays. NOX enzymatic activity, ROS production, protein expression of NOX subunits, manganese superoxide dismutase (MnSOD), and phosphorylation of PKC-δ were assessed. Neural SHSY5Y cells underwent oxygen-glucose deprivation (OGD) and subsequent reoxygenation and C+P treatment. We also evaluated ROS levels and NOX protein subunit expression, MnSOD, and p-PKC-δ/PKC-δ. Additionally, we measured PKC-δ membrane translocation and the level of interaction between NOX subunit (p47phox) and PKC-δ via coimmunoprecipitation. As hypothesized, treatment with C+P therapy decreased levels of neurologic damage. ROS production, NOX subunit expression, NOX activity, and p-PKC-δ/PKC-δ were all significantly decreased in subjects treated with C+P. C+P decreased membrane translocation of PKC-δ and lowered the level of interaction between p47phox and PKC-δ. This study suggests that C+P induces neuroprotective effects in ischemic stroke through inhibiting oxidative stress. Our findings also indicate that PKC-δ, NOX, and MnSOD are vital regulators of oxidative processes, suggesting that C+P may serve as an antioxidant.

Modulation of NLRP3 inflammasome-related-inflammation via RIPK1/RIPK3-DRP1 or HIF-1α signaling by phenothiazine in hypothermic and normothermic neuroprotection after acute ischemic stroke

“…Our findings support the hypothesis that suppressing the NLRP3 inflammasome can mitigate brain damage by modulating the inflammatory response [ 25 ]. In addition, we have demonstrated that C + P therapy diminishes ROS levels post-ischemia, contributing to a reduction in oxidative stress and cellular damage [ 29 ].…”

“…We incubated the cells at 5 % CO 2 and 37 °C in a humidified incubator. As previously described [ 29 ], we first prepared an anaerobic chamber by flushing it with a 95 % N 2 and 5 % CO 2 (v/v) mixture, maintaining the temperature at 37 °C. The cell culture medium was then replaced with deoxygenated, glucose-free DMEM to initiate a 2-h OGD phase.…”

“…The values were averaged and for statistical analysis. The percentage of co-localization cells (%) was calculated as (co-localization cells/total cells) × 100 % [ 29 ].…”

“…Coimmunoprecipitation (Co-IP). Co-IP of HIF-1α and NLRP3 was performed as described by us [ 29 ]. Immunoprecipitation lysis buffer was used to collect brain tissue lysates.…”

Identification of key potassium channel genes of temporal lobe epilepsy by bioinformatics analyses and experimental verification

“…There is little exacerbation of preexisting epileptic foci by enflurane, the only exception being the case of certain myoclonic-type epilepsies, such as progressive myoclonic epilepsy and photosensitive epilepsy (76). Promethazine reduces brain injury after ischemic stroke through PKC-δ/NOX/MnSOD and RIP1-RIP3 regulated activation of the NLRP3 inflammasome following ischemic stroke (77,78). Miconazole exerts disease-modifying effects in epilepsy by suppressing neuroinflammation via the NF-κB pathway and iNOS production (79).…”