In the present study, we elucidated that C + P with temperature control reduced brain damage after 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 NLRP3, IL-1β, TXNIP, cleaved-Caspase-1, and IL-18. The phosphorylation activation of JAK2/STAT3 and p38 pathways and co-localization of p-STAT3 with NLRP3 were also inhibited by C + P. Furthermore, C + P reduced the expression of HIF-1α protein and decreased the activity of FoxO1 through promoting its nuclear release and inhibiting its activation. At the same time, C + P reduced the co-localization of p-STAT3 with HIF-1α and p-STAT3 with FoxO1. Taken together, these results suggest C + P repressed JAK2/STAT3 and p38 signaling pathways as well as FoxO1 and HIF-1α in a hypothermia-independent manner, leading to reduced neuroinflammatory response and inflammasome activation, which ultimately attenuated brain damage.
In our previous study, we found that the administration of the classic neuroleptic phenothiazines, C + P, conferred neuroprotection in models of severe stroke by suppressing the damaging cascade of metabolic events, which was partially dependent on drug induced hypothermia [6, 30]. Chlorpromazine, a phenothiazine, was shown to suppress the production of proinflammatory cytokines including TNF-α, IL-1β, and IL-6 [31–33]. Moreover, the neuroprotective role of C + P in ischemic stroke seemed to be dependent on its amelioration of hyperglycolysis , blood–brain barrier disruption , inflammation , inflammasome activation , and apoptosis . C + P can induce hypothermia, but its neuroprotective effect post-ischemic stroke was partially independent of its hypothermic effect [6, 8, 11, 12]. In the present study, we verified how C + P exerted pharmacological and neuroprotective effects without hypothermia, namely, the inhibitory effects of C + P on the inflammatory response and inflammasome activation [11, 12].
Macrophages and neutrophils participate in brain inflammation after ischemic stroke and high levels of these cells are associated with poor prognosis . Myeloperoxidase (MPO) is a heme containing peroxidase that is expressed in the aforementioned inflammatory cells . MPO has been observed to be elevated in the context of ischemic strokes, in both animal and clinical studies [37, 38]. CD68 is an inflammatory marker specific to activated microglia that is usually not expressed in surveillant microglia [39, 40]. It is also associated with age dependent neuroinflammation . In the context of ischemic stroke, MPO and CD68 were representative of apoptotic cell death  and were attenuated by HIF-1α inhibition, indicating that they serve as accurate markers of inflammation. In the present study, C + P reduced MPO and CD68 levels, which represented suppression of inflammation through inhibition of immune cell infiltration and apoptotic cell death.
C + P repressed the MCAO induced increases of NLRP3, IL-1β, IL18, TXNIP, and cleaved-Caspase 1, resulting in the inhibition of inflammasome activation. NLRP3 is an indispensable inflammasome of the NLR family and is activated under ischemic conditions. In addition, its levels are related to increased infarction and apoptotic cell death that accompanies ischemic injury . The up-regulation of caspase-1 activated by NLRP3 inflammasome promotes the maturation of IL-1β and IL-18, which are pyrogens involved in inflammatory apoptosis . During the cerebral ischemia reperfusion injury, TXNIP dissociates from the Trx1/TXNIP complex and enters the cytoplasm to activate the NLRP3 inflammasome .
Chlorpromazine has been reported to decrease the phosphorylation of STAT3 . Treatment with chlorpromazine may induce the expression of distinct genes against apoptosis progression via the JAK-STAT signaling pathway . JAK/STAT signaling pathway is important for the progression of neurological diseases including stroke, traumatic brain injury, brain tumors, and neurodegenerative diseases [46, 47]. Many evidences have indicated that the JAK2/STAT3 signaling is phosphorylation activated during cerebral ischemia and mediates oxidative stress, inflammatory response, and neuronal apoptosis [46, 20, 19]. In both MCAO and OGD experimental models, the expression of p-STAT3 increased, which resulted in the activation of inflammasomes . It has been reported that the expression of p-STAT3 was co-localized with NLRP3-positive cells and upregulated NLRP3 via STAT3-dependent histone acetylation . p38 mitogen-activated protein kinase (MAPK) activity is involved in the inflammatory response during stroke, as supported by the observation that p-p38 expression is upregulated in the ischemia area [23, 50]. p38 MAPK signaling pathway induced the activation of the NLRP3 inflammasome and macrophage pyroptosis . Consistent with previous findings, we found that the activation of JAK2/ STAT3 and p38 and the co-localization of p-STAT3 with NLRP3 were significantly increased following postischemia, but attenuated by C + P therapy.
When cells are exposed to hypoxia, there is an increase in hypoxia-induced factor-1α (HIF-1α) expression . There is also an elevated expression of HIF-1α during periods of ischemia/reperfusion in neurons . HIF-1α mediates inflammatory response after cerebral ischemia/reperfusion injury . Moreover, our previous study reported that HIF-1α mediated NLRP3 inflammasome dependent pyroptotis following ischemic stroke . The activation of p38 through phosphorylation stabilizes HIF-1α, which in turn may be involved in the increased production of IL-1β . Phosphorylated STAT3 stimulates and binds to HIF-1α, promoting its stability during hypoxia . Mammalian FoxO proteins are assigned to the O class of the forkhead box class transcription factors . It has been reported that the expression of FoxO1 increased after ischemia/reperfusion [56, 57]. Additionally, after stroke, the phosphorylation of FoxO1, which represents its inactive form, was decreased, underwent nuclear translocation, and activated its target genes including inflammation pathways . Activated p38 mediates the translocation of FoxO1 into the nucleus and the binding of FoxO1 to the promoter of TXNIP, promoting the upregulation of the TXNIP protein and further increasing inflammasome activation [58–61]. FoxO1 can act as a coactivator of STAT3, correlating with the physical association of their co-localization in the nuclear regions . Moreover, HIF-1α drives FoxO1 expression by binding directly to the hypoxia-responsive elements within its promoter region [63, 64]. In the present study, we found that C + P suppressed the expression of HIF-1α after exposure to ischemic stroke. C + P also repressed the activation of FoxO1 through the inhibition of FoxO1 and increasing the phosphorylation thereof, leading to its exclusion from the nucleus. Moreover, we confirmed that MCAO induced the co-localization of p-STAT3 with HIF-1α or FoxO1, but C + P significantly reduced this trend. In summary, we observed that C + P induced pharmacological neuroprotection through inhibition of the NLRP3 inflammasome expression, which is mediated by HIF-1α and FoxO1 and may be related to the JAK2/STAT3 and p38 pathway.
In conclusion, the results of the present study indicated that C + P treatment conferred neuroprotection and rescued brain tissue after ischemia/reperfusion by suppressing neuroinflammatory responses and NLRP3 inflammasome activation in the absence of hypothermia induction. These therapeutic effects are associated with alterations of the JAK2/STAT3 and p38 pathways and subsequent inactivation of HIF-1α and FoxO1. The present study suggests that the JAK2/STAT3/p38/HIF-1α/FoxO1 pathway is a key regulator of ischemic stroke, and hence a potential therapeutic target.