Background
Subarachnoid haemorrhage (SAH) is a serious neurological emergency associated with high morbidity and mortality [
1]. Current studies show that early brain injury (EBI) is the overriding factor and that both neuroinflammation and apoptosis have been confirmed to contribute to EBI [
2,
3]. EBI refers to the direct brain damage occurring within 72 h after SAH, including increased intracranial pressure, cerebral perfusion pressure disorder, blood–brain barrier (BBB) destruction and brain edema [
2,
3].
Neuroinflammation is associated with various acute and chronic neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and SAH [
3,
4]. Increased levels of cytokines have been found in the cerebrospinal fluid of SAH patients and are correlated with poor neurological outcomes [
5]. Once bleeding occurs, neural immunocytes (microglia and astrocytes) rapidly react to the extravascular blood components that enter the cerebral parenchyma [
6,
7]. The activated immunocytes then release various inflammation mediators such as Toll-like receptor 4 and interleukin-1β (IL-1β), thereby aggravating the secondary brain injury [
8‐
10]. Various pro-apoptotic mechanisms are also reported to contribute to EBI after SAH and involve not only the activation of pro-apoptotic proteins but also the inhibition of anti-apoptotic factors [
2,
3,
11]. Although some anti-inflammatory and anti-apoptotic strategies have been tested in preclinical and clinical trials, the mortality and disability burdens of SAH remain high, reminding us that the identification of appropriate and effective targets is still a major obstacle.
Thioredoxin-interacting protein (TXNIP), also known as thioredoxin-binding protein-2 or vitamin D
3-upregulated protein 1, is a natural antagonist of thioredoxin (TRX) in vivo. Recent research has confirmed that TXNIP is the critical link between nod-like receptor protein 3 (NLRP3) inflammasome activation and inflammatory amplification [
12]. Moreover, TXNIP can directly bind to and inhibit TRX function, leading to the activation of downstream apoptosis signalling pathways [
13]. However, whether the effects of TXNIP influence the pathogenesis of SAH remains unclear. Previously, our work revealed that downstream factors in endoplasmic reticulum stress (ER stress) can induce neuronal apoptosis after SAH [
14]. Intriguingly, recent studies have demonstrated that TXNIP expression can be significantly induced by ER stress at the transcriptional and post-transcriptional levels [
15,
16]. The ER is the major site of protein folding, post-translational modification and assembly [
17]. Although ER stress is primarily a self-protective signal transduction pathway, high-level ER stress could culminate in cell death via the activation of inflammation and apoptosis [
18,
19]. Moreover, persistent and maladaptive ER stress is implicated in various human neurodegenerative diseases [
20]. However, whether the induction of TXNIP by ER stress is involved in EBI after SAH remains unknown. Based on these findings, we utilized a TXNIP inhibitor, small interfering RNA (siRNA) and specific inhibitors of ER stress sensors to suppress TXNIP expression and ER stress–TXNIP signalling in order to assess the role of TXNIP in the progression of EBI after SAH.
Discussion
The present study proves that both pharmacological inhibition and gene knockdown of TXNIP significantly attenuated brain injury and improved early prognosis after SAH. We found that these effects were closely associated with TXNIP pro-inflammatory and pro-apoptotic signalling pathways. TXNIP was extensively co-localized with TUNEL-positive cells in both the rat hippocampus and subcortex after SAH. We also found for the first time that TXNIP is localized in microglia and astrocytes. These results represent critical evidence to support the pro-inflammatory and pro-apoptotic effects of TXNIP after SAH. PERK and IRE1α inhibition resulted in significant TXNIP suppression and attenuation of some prognostic indicators. More generally, we once again showed the important role of ER stress in EBI after SAH, highlighting the tight relationships between SAH, TXNIP and ER stress.
SAH results in severe neurological deficits, and there are few therapeutic drug targets available. Inflammation and apoptosis are regarded as the main causes in EBI after SAH. Although suppressing inflammation is generally neuroprotective in animal experiments, little success has been seen in clinical trials to date. Numerous apoptotic proteins are increased during this process, and many anti-apoptotic strategies have been developed for EBI treatment. Nevertheless, the mortality and disability of SAH remain unacceptably high, reminding us that there are some unknown mechanisms involved. Several studies recently reported that TXNIP plays dual pro-inflammatory and pro-apoptotic roles in ischaemic diseases [
23,
39], but the relationship between TXNIP and SAH has not been elucidated. Kaya and colleagues reported that SAH induced TXNIP mRNA expression [
40]. Therefore, we speculated that TXNIP may also participate in the pathogenesis of EBI. We found that TXNIP protein levels were significantly elevated and expressed in microglia and astrocytes during the initial phases of our SAH experiments. As described above, the neural immunocytes (microglia and astrocytes) play a major role in promoting neuroinflammation and secondary brain damage [
33,
34]. Excessive microglial activation can aggravate cerebral haemorrhage-induced brain injury by inflammatory amplification [
41,
42]. Inflammatory mediators could further activate astrocytes to induce secondary inflammatory responses [
43]. In combination with that NLRP3 inflammasome is reported to be expressed in microglia cells and participates in the inflammatory brain injury of SAH [
44,
45], our results provide a supplementary histological evidence to support that TXNIP may play a possible effect on EBI after SAH through NLRP3 inflammasome activation and inflammatory amplification.
Recent studies have found that TXNIP can interact closely with the leucine-rich repeats (LRRs) of the NLRP3 inflammasome, further inducing Caspase-1 and IL-1β activation under stress conditions and eventually promoting inflammatory amplification [
12]. The NLRP3 inflammasome, as the best characterized inflammasome, consists of an NLRP3 scaffold, apoptosis-associated speck-like protein containing a CARD (ASC) and pro-Caspase-1 [
46]. Under stress conditions, NLRP3 recruits ASC and pro-Caspase-1, further causing Caspase-1 autocatalytic activation and the downstream transformation of pro-IL-1β and pro-IL-18 into their cleaved and secreted forms [
46]. In addition, TXNIP can directly bind to the active cysteine residue of TRX and further disturb the TRX/ASK-1 inhibitory complex, leading to ASK-1 release and pro-apoptotic effects [
47]. TRX1 is a TRX protein that interacts closely with the N-terminal portion of ASK-1 and inhibits ASK-1-dependent apoptosis [
48]. Researchers have found that TRX inhibition can induce neuronal apoptosis after cerebral ischaemia [
38]. Likewise, we found that TRX1 expression was inhibited after SAH, which may be due to the co-localization of TXNIP with TRX1 and inhibition of TRX1 transcription [
38]. We also found that TXNIP was widely expressed in SAH rat brain neurons. More importantly, TUNEL staining revealed that TXNIP was widely co-localized with apoptotic cells in the hippocampus and cortex after SAH. Brain cell apoptosis was reduced, and prognostic indicators were attenuated with inhibition of TXNIP. These results could support our speculation that TXNIP participates in EBI after SAH.
Recently, Oslowski and colleagues found that PERK significantly promotes TXNIP expression via the ATF-5 and ChREBP transcription factors under ER stress [
16]. It has also been shown that hyperactivated IRE1α improves TXNIP mRNA stability through selective degradation of TXNIP-destabilizing microRNA via IRE1α endoribonuclease activity, eventually promoting TXNIP expression at the post-transcriptional level. When ER stress reaches a certain threshold, IRE1α selectively degrades four pre-microRNAs, including four microRNAs (miR-17, miR-34a, miR-96 and miR-125b) [
49]. Among these, miR-17 has been confirmed to function as a TXNIP mRNA-destabilizing microRNA [
15]. There are also some other mechanisms involved in regulating TXNIP expression, such as the p38 MAPK and forkhead box O1 transcriptional factor (FOXO1) pathways [
50]. Of course, more research is still needed to identify additional potential mechanisms of TXNIP and ER stress in SAH.
Meanwhile, there are also some limitations of this current study and were listed as follows: (1) firstly, we did not detect a dose-dependent change of the mRNA, protein and immunofluorescence levels of TXNIP and downstream-associated factors after treatment with TXNIP siRNA, RES, GSK2656157 and STF083010; (2) secondly, we only tested the PERK phosphorylation levels. And a more thorough method is to detect the total-PERK levels and then calculate the ratios between the phosphorylation of PERK and total PERK, which will provide more favourable evidence to support our results; (3) in addition, ER stress can also occur in other pro-inflammatory immune or supporting cell types of the neurovascular unit, such as oligodendrocytes, vascular smooth muscle and endothelial cells. It is also possible that ER stress might induce pro-inflammatory activation of other cell types, which then can mediate indirect activation of TXNIP in neuronal cells, microglia and astrocytes in a paracrine matter; and (4) our Western blot experiments showed that TXNIP expression was the most obvious at 48 h. However, the immunolocalization experiment in Fig.
2 and the follow-up Western blot study for TXNIP was not done at the same 48 h time point used. Due to these, we should choose 48 h as the best testing point, while, combined with our found clinical and experimental observations, clinical symptoms appear and are significant at 24 h in patients and animals after intracranial aneurysm bleeding and SAH. So at the beginning of the experiment design, we aimed to observe the immunofluorescence and protein detection at 24 h. At the same time, TXNIP expression at 24 h was also significant when compared with that in the sham group. Therefore, the results of immunofluorescence and protein detection at 24 h may also be powerful to support the conclusions. These are limitations of our study design, and more work needs to be done in the future to address them.