Introduction
Delayed postoperative paraplegia induced by spinal cord ischemia-reperfusion injury (SCII) is still one of the critical severe and catastrophic complications of thoracoabdominal aortic surgery. The incidence of paraplegia in patients undergoing that procedure is as high as 14% [
1]. A period of ischemia and blood reperfusion induces progressive damage and aggravation of nerve function [
2].
Numerous studies have demonstrated that, following the primary mechanical injury to the spinal cord, oxidative stress occurs that destroys the normal balance of redox states and consequently contributes to the production of reactive oxygen species (ROS) [
3,
4]. Furthermore, spinal cord mitochondria have an intrinsically higher risk of oxidative damage and overload with calcium than brain mitochondria [
5]. Moreover, NADPH oxidase 2 (NOX2) is a major contributor to oxidative stress in spinal cord injury [
6]. In addition, the production of large quantities of proinflammatory cytokines could also induce neuronal apoptosis and even death [
7]. Growing evidence has indicated that mitochondria-derived ROS and inflammation are linked through a redox sensor known as nod-like receptor pyrin domain-containing 3 (NLRP3) [
8]. Moreover, the maturation and secretion of proinflammatory cytokines require the activation of caspase-1, which together with the activation of the NLRP3 inflammasome, induces the inflammatory cascade and subsequently recruits apoptosis-associated proteins [
9]. Unfortunately, despite associated studies that have been reported over 30 years, markedly effective therapy to provide neuroprotection following SCII remains lacking.
Astaxanthin (AST), a naturally occurring carotenoid pigment, is widespread in living organisms such as salmon, shrimp, microalgae, and yeast [
10,
11]. AST possesses excellent biological effects, such as antioxidant activity, anti-inflammatory activity, modulation of immune responses, anti-cancer activity, and other neuroprotective properties [
12,
13]. It was reported that AST treatment not only inhibited brain hemorrhagic injury across the blood-brain barrier [
14] but also exhibited significant neuroprotective effects following compression spinal cord injury against neuropathic pain associated with multiple molecular targets [
15,
16]. AST was also able to activate the cAMP/PKA/CREB signaling pathway in brain tissues and
reduced isoflurane-induced neuroapoptosis via the phosphoinositide 3-kinases (PI3K) and their downstream target, protein kinase B (Akt) pathway, ultimately promoting axonal regeneration in the cerebral cortex and improving motor function [
17], which is similar to our previous report on the neuroprotective effect of AST in mice subjected to repeated cerebral ischemia-reperfusion [
18]. In addition, PI3K/Akt pathway is involved in the regulation of oxidation, inflammatory responses, and apoptosis [
19]. Activation of PI3K/Akt/GSK-3β signaling pathway has been demonstrated to result in the attenuation of myocardial injury [
20]. However, to our knowledge, the neuroprotective effects of AST on SCII have not been thoroughly investigated to date.
Thus, we aimed to elucidate the role of AST pretreatment in SCII injury in rats and its molecular mechanism. The current study indicated that AST pretreatment attenuates SCII injury via activation of the oxidative stress-mediated PI3K/Akt/GSK-3β pathway.
Discussion
In the present study, we observed that AST pretreatment effectively improved motor functions and ameliorated pathological damages by reversing histological changes in the spinal cord. Furthermore, AST significantly inhibited oxidative stress and attenuated mitochondria swelling degree following the SCII. More importantly, PI3K/Akt/GSK-3β signaling pathway was found to be associated with the protective effects of AST on SCII-treated rats.
The segmental blood supply of the spinal cord and relatively poor collateral circulation facilitate ischemia damage. SCII may further aggravate spinal cord damage. Various studies introduced a pathophysiological mechanism, associated with increases in oxidative stress and inflammatory processes with apoptosis, which plays a pivotal role in SCII [
2,
22,
24]. Meanwhile, oxidative stress and mitochondrial dysfunction not only result from spinal cord injury, but may directly contribute to SCII [
25]. Furthermore, complex pathological mechanism changes during reperfusion injury are correlated with time and compliance [
2], suggesting the importance of the involvement of early intervention in secondary damage after spinal cord injury to impede the aggressive apoptosis process.
The cell membranes of spinal cord neurons are readily susceptible to oxygen free radical attacks. After SCII, the lipid peroxidation reaction was dramatically activated, while antioxidative activities strikingly decreased [
26]. Therefore, decreasing MDA levels and increasing SOD activity have been shown to significantly attenuate SCII. In addition, an obvious inflammatory response develops within several hours after injury and is characterized by the activation of proinflammatory cytokines and infiltration of neutrophils [
27]. Numerous studies have reported that typical proinflammatory cytokines, such as IL-1β, TNF-α, and IL-18, are major mediators of spinal cord injury or in the pathological mechanisms of secondary damage [
28]. Moreover, IL-10 also mediates the anti-inflammatory response in the spinal cord [
29]. Hence, the aforementioned factors may easily contribute to spinal cord injury during the reperfusion period. However, thus far, neuroprotective agents with respect to SCII have not been systematically investigated.
AST, a dietary supplement with no significant adverse effects, is being investigated individually across a broad range of clinical applications, including cardiovascular health, acute pancreatitis, and neuropathic pain, all of which are associated with oxidative stress and inflammation in their pathogenesis [
10,
16]. Besides, AST has a therapeutic role in preserving cognitive function by promoting or maintaining neural plasticity in aged patients and in neurodegenerative disease [
30]. It has also been shown to maintain mitochondrial integrity and function and ameliorate oxidative stress in skeletal muscle injury [
31]. Recent studies that reported AST shows prominent antioxidant and/or anti-inflammatory properties in different models of ischemia and reperfusion injury confirmed its protective action, such as in steatotic liver, muscle, and brain tissue [
18,
32‐
34]. Based on the above characteristics of AST, it seemed to be a desirable candidate for further exploration to elucidate its therapeutic potential in SCII.
Given the numerous lines of evidence have implicated PI3K/Akt signaling pathway in modifying oxidative stress, GSK3β is a key active enzyme associated with downstream of Akt. Phosphorylation of GSK3β via Akt maintains this enzyme an inactive state and protects against tissue ischemic injury [
35]. Thus, we hypothesize that PI3K/Akt signaling may act as an important antioxidant mechanism for regulating AST-induced neuroprotection following SCII.
As expected, we observed there were worse hind limb motor scores and that fewer neurons survived at 72 h after reperfusion in SCII rats compared to the sham group. Furthermore, oxidative products (MDA, XO, and mitochondrial ROS) and proinflammatory cytokines (IL-1β, TNF-α, and IL-18) were dramatically elevated in SCII rats, which is similar to our previous reports [
22]. Interestingly, AST pretreatment substantially attenuated SCII-induced neurological dysfunction at various time points after reperfusion and mitigated neuronal apoptosis in the spinal cord while this effect was partially reversed when AST and LY294002 (a PI3K inhibitor) combined, which indicates that ROS-induced apoptosis after SCII is probably regulated by PI3K/Akt signaling pathway. In addition, the beneficial effects of AST pretreatment against SCII were associated with decreased levels of oxidative products (MDA, XO, and mitochondrial ROS) and proinflammatory cytokines (TNF-α), as well as increased activities of endogenous antioxidant enzymes (SOD and GSH). As to the other inflammatory cytokines, although a reverse tendency was demonstrated in the AST-treated group, such as a decrease in the IL-1β and IL-18 levels and an increase in the anti-inflammatory cytokine IL-10 level, there were no significant differences when compared with the SCII group.
Moreover, we also evaluated NOX2 and NLRP3 protein expressions, which are important factors that prompt the expressions of many oxidant stress and inflammation [
36,
37]. Although we found that SCII indeed caused NOX2 and NLRP3 activation in SCII rats, AST pretreatment did not simultaneously inhibit NOX2 and NLRP3 inflammasome activation, which is consistent with our results for the above proinflammatory cytokines. Therefore, the current results further confirmed our speculation that AST suppresses SCII-stimulated ROS production, perhaps mainly because the inhibition of NADPH oxidase activity enhances antioxidative stress capabilities rather than inhibiting the NLRP3-related inflammatory pathway. The aforementioned results suggest that AST might produce a protective effect against SCII in rats, primarily via antioxidative stress activity rather than the simultaneous anti-inflammatory response.
In order to further explore the possible molecular mechanism underlying the improved motor function of SCII rats pretreated with AST, we administered the PI3K inhibitor LY294002 as a comparative study and detected the levels of PI3K/Akt pathway-related proteins, including the ratios of p-Akt/Akt and p-GSK-3β
/GSK-3β. Similarly, these therapeutic effects were partially abolished when AST was combined with LY294002. This result might be explained that the nerve tissue had the capability of synthetizing and secreting some antioxidant enzymes to resist SCII-induced oxidative damage, although this capability did not satisfied the demand of cellular antioxidant defense [
38]. After administration of AST to the SCII rats, the PI3K/Akt/GSK-3β signaling pathway associated with antioxidant stress was activated, which further enhanced antioxidative capability and then promoted nerve regeneration. Taken together, AST-induced nerve functional recovery may be mediated via the activation of PI3K/Akt/GSK-3β pathway.
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