Background
Subarachnoid hemorrhage (SAH) refers to a consequence of bleeding within the subarachnoid space. Cerebral aneurysm rupture is the most frequent etiology of SAH. Although SAH accounts for only 5% of stroke cases, the mortality rate could be as high as 67% in the first few months [
1,
2]. The significant mortality and morbidity may result from both the early brain injury and delayed cerebral ischemia [
1]. Therapies targeted toward delayed cerebral ischemia have had limited success, which has led to a focus on mechanisms of early brain injury [
3,
4]. In the past few decades, many pathological mechanisms of early brain injury have been proposed. A ruptured aneurysm leads to many physiological derangements such as elevated intracranial pressure (ICP), decreased cerebral blood flow (CBF), and decreased cerebral perfusion pressure (CCP). These events initiate various cascades of injuries such as inflammation, oxidative stress, blood brain barrier dysfunction, brain edema, and apoptosis [
5,
6].
The inflammasome is a multiprotein complex that regulate the innate immune inflammatory response. Several inflammasomes, such as NLRP1, NLRP3, and AIM2, have been described in the central nervous system(CNS) and associated with brain injury [
7,
8]. Among them, NLRP3 inflammasome is the most studied one, especially in SAH [
9,
10]. Inflammasome activation triggers activation of caspase-1 and mature of inflammatory cytokines, such as IL-1β and IL-18, activate the immune response eventually [
11]. In addition, caspase-1 activation also induces the regulated necrotic cell death, which is characterized by cell membrane rupture and subsequent inflammation [
12]. Accumulating evidence have demonstrated that inhibiting NLRP3 inflammasome activation attenuates neuroinflammation and cell death in early brain injury and improves neurological function following SAH [
9,
10,
13]. Moreover, recent studies have uncovered that autophagy activation inhibited inflammasomes activation [
14‐
16]. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is widely used to treat depression, obsessive-compulsive disorder, bulimia, and panic disorder [
17]. Recent studies have indicated that fluoxetine exhibits beneficial effects in neurological disorders, including ischemic stroke [
18], brain injury [
19], and spinal cord injury [
20]. In fact, it is reported that fluoxetine induces the autophagy pathway through FK506 binding protein 51(FKBP51) [
21]. Furthermore, fluoxetine inhibits NLRP3 inflammasome activation in depression [
22]. However, the effect of fluoxetine on NLRP3 inflammasome activation and potential mechanisms in early brain injury after SAH remains unclear.
In the present study, we investigated the role of NLRP3 inflammasome and caspase-1 activation in early brain injury after SAH and the effect of fluoxetine on NLRP3 inflammasome and caspase-1 activation and the underlying possible mechanisms in early brain injury after SAH. Our findings indicated that caspase-1 activation was induced at the early stages of SAH and promoted necrotic cell death and expression of inflammatory cytokines (IL-1β and IL-18) in early brain injury after SAH. In addition, our study showed that fluoxetine reduces NLRP3 inflammasome and caspase-1 activation in early brain injury after SAH, at least partly, by activating autophagy, providing potential therapeutic interventions for SAH.
Discussion
The present study provided the evidence that caspase-1 activation-induced necrotic neural cell death and inflammatory cytokines IL-1β and IL-18 contributed to the early brain injury after SAH. In addition, our report found that fluoxetine attenuated NLRP3 inflammasome and caspase-1 activation and subsequent inflammatory cytokines expression as well as necrotic cell death, reduced the brain edema, and improved neurological deficits. Moreover, autophagy activation was involved in these beneficial effects of fluoxetine, which were abolished by autophagy inhibitor 3-MA. Taken together, our findings suggested that fluoxetine administration, targeting NLRP3 inflammasome and caspase-1 activation, might be an effective therapeutic strategy after SAH.
Caspase-1 is an inflammatory caspase that is well known for the function of cleaving the preforms of inflammatory cytokines IL-1β and IL-18 into their active forms [
29]. Both IL-1β and IL-18 are the important inflammatory mediator in early brain injury after SAH. These inflammatory cytokines induced matrix metalloproteinase (MMP)-9 expression and disruption of the blood brain barrier (BBB), aggravating brain edema and neurological deficits after SAH [
24]. Clinical studies have demonstrated that caspase-1 and subsequent inflammatory cytokines elevated in cerebrospinal fluid (CSF) of patients with SAH [
30,
31]. In the current study, we found that cleaved caspase-1 was increased and peaked at 24 h after SAH. Besides, increasing studies indicated that caspase-1 also induced the necrotic cell death by cleaving adermin-D (GSDMD), which lyses the liposomes and forms the pores on the membranes [
32‐
34]. Our study indicated that caspase-1 activation was accompanied with increased necrotic neural cells. In addition, most of these necrotic neural cells were neurons, which is consistent with previous studies [
35]. And some of them were microglia and astrocytes. Additionally, the ultrastructural features of necrotic cell death were identified by using a transmission electron microscope. DNA fragmentation, swollen mitochondria, damaged cytoplasmic organelles, cytoplasmic hyper-vacuolization, and cytomembrane rupture were observed. Intriguingly, autophagic vacuoles were also observed, indicating that autophagy may be involved in the pathology of caspase-1-induced necrotic cell death. These features are consistent with previous studies [
36,
37]. Caspase-1 inhibitor AC-YVAD-CMK downregulated the expression of mature IL-1β and IL-18, reduced the number of necrotic cell death, and improved neurological function in SAH rats. Taken together, our study indicated that caspase-1 activation contributes to the early brain injury after SAH.
Caspase-1 activation requires a multiprotein platform, namely inflammasome. To date, NLRP1b, NLRP3, and NLRC4, as well as the cytosolic DNA sensor absent from in melanoma2(AIM2), are well-defined inflammasomes [
38]. Our previous studies demonstrated NLRP3 inflammasome contribute to the neuroinflammation in early brain injury after SAH [
9,
14]. A recent clinical study demonstrated NLRP3 inflammasome and caspase-1 were elevated in CSF of SAH patients and associated with functional outcome of these patients [
30]. Therapies targeting NLRP3 inflammasome activation attenuated early brain injury and improved neurological function after SAH in rats [
9,
10,
39]. Traditionally, fluoxetine is a common treatment for major depression due to its safer profile, greater tolerability, and fewer side effects. Recently, the protective pharmacological effects of fluoxetine have been demonstrated in clinical practice in the treatment of neurological diseases, such as ischemic [
18], Alzheimer’s disease [
40], and traumatic brain injury [
41]. Emerging evidence suggests that fluoxetine has anti-inflammatory properties [
42,
43]. But the precise mechanism is uncertain. In our present study, fluoxetine was found to reduce the expression of NLRP3, cleaved caspase-1, IL-1β, and IL-18, alleviate the neural necrotic cell death and brain edema, and improve the neurological function after SAH. These data indicated that fluoxetine alleviates early brain injury after SAH through the inhibition of NLRP3 inflammasome activation.
Autophagy is an intracellular process for cellular homeostasis and recycling of damaged organelles and proteins, as well as the destruction of intracellular pathogens [
44]. Activated autophagy pathway has been demonstrated in experimental SAH [
26,
45,
46]. More and more studies have demonstrated the regulatory roles of autophagy in inflammasome activation. The loss of proteins that are essential for autophagy activation, such as Atg16L1 and Atg7, resulting in caspase-1 activation as well as increased production of IL-1β and IL-18 in macrophages [
47,
48]. In addition, autophagy can also negatively regulate inflammasome activation through removing damaged mitochondria to preventing the release of mtROS and mtDNA into the cytoplasm, and ultimately limiting inflammasome assembly [
49,
50]. Additionally, assembled inflammasomes can be degraded by autophagosomes through the autophagic protein p62 [
51,
52]. Fluoxetine upregulated the expression of beclin-1, which is compatible with previous foundings that fluoxetine activates autophagic pathways via FKBP51 [
21]. Moreover, autophagy inhibitor 3-MA reversed the neuroprotective effects of fluoxetine in our present study. These data suggest that fluoxetine provides potential therapeutic interventions for EBI after SAH through the inhibition of NLRP3 inflammasome activation by enhancing autophagy.
It is important to note that there are several limitations in our present study. First, the antiapoptotic and antioxidant property of fluoxetine has been described; therefore, we cannot exclude the possibility that these properties also play a role in the neuroprotective effect of fluoxetine. Second, our study showed that caspase-1 activation induced neural necrotic cell death, but whether these necrotic cell death are pyroptosis needs further study. In addition, we studied the short-term effects of fluoxetine after SAH, but the side-effects of fluoxetine, such as myeloid response, warrant further study.