Postoperative cognitive dysfunction (POCD) was first identified in 1954 and is commonly seen in elderly patients over 65 years of age who undergo surgery and presents as a reversible acute neurological disorder[1]. The incidence of POCD can reach 25–40% in elderly patients[18]. The pathogenesis of POCD is still unclear, and the possible mechanisms are thought to include neuroinflammation, mitochondrial dysfunction (oxidative stress), blood-brain barrier disruption, neurotrophic support disorders, and disruption of synaptic structures[19–22]. Of these, neuroinflammation has received the most attention, and this appears to be the same pathology that underlies other neurodegenerative diseases basis. Many studies have focused on the search for effective amelioration of neuroinflammation. Our previous study has found that surgery-induced downregulation of hippocampal SIRT1 is involved in postoperative cognitive impairment through inhibition of autophagy processes. However, the mechanism of its downstream regulation of how neuroinflammation progresses is still unclear. In the present study, the relationship between SIRT1, autophagy, and NLRP3 inflammasomes was explored by both in vivo and in vitro experiments.
A wealth of evidence indicates that SIRT1 plays an important role in neurodegenerative diseases. As a deacetylase, SIRT1 has been shown to regulate a variety of physiological and pathological processes including brain development, aging, stress, and inflammation [9] [23, 24]. In the brain, SIRT1 is closely associated with synaptic plasticity, and SIRT1 expression in the brain gradually decreases with increasing age[25]. Furthermore, this decrement is closely related to cognitive decline. Although studies have shown that SIRT1 is predominantly expressed in neurons in the brain, the decline is also seen in microglia[10]. In our experiments, surgery induced a postoperative cognitive decline in aged mice accompanied by a reduction in SIRT1 expression in the hippocampal region. SRT1720 upregulates SIRT1 expression and successfully rescues cognitive decompensation in aged mice. However, how this change in SIRT1 is involved in neuroinflammation deserves to be explored.
Autophagy is a process that eliminates abnormal and dysfunctional cellular components and maintains cellular homeostasis, which is essential for proper cellular function[26]. It acts like a well-orchestrated cell manager, deciding which proteins and organelles are destroyed. Several genetic studies from yeast to mammals have identified more than 30 autophagy-associated genes (ATG)[27]. These genes are sequentially activated to regulate the initiation, formation, and elongation of autophagosomes and their fusion with lysosomes to produce autophagosomes. ATG activity decreases with aging, suggesting that autophagy is associated with aging as well as age-related deterioration and disease[28, 29]. A growing number of studies have shown that SIRT1 can deacetylate ATG to affect the autophagic process[30–32]. In addition, SIRT1 as a histone deacetylase seems to regulate the level of autophagy via epigenetics[33]. In the brain, impaired autophagy of neurons leads to the inability of neurons to clear abnormal accumulation of proteins or damaged organelles and eventually neuronal apoptosis[34]. Recent studies have demonstrated that autophagy-deficient microglia do not decrease in number, but rather increase the expression of pro-inflammatory factors[35]. In the present study, aged mice underwent splenectomy with decreased SIRT1 expression, which subsequently caused a decrease in Beclin-1, LC3II/I ratio, and p62 accumulation as markers of decreased autophagy levels. Moreover, autophagic defects increase NLRP3 inflammasome activation and promote POCD. Decreased autophagy induced by surgery can be ameliorated by increasing SIRT1 expression, exhibiting inhibition of NLRP3 inflammasome. Collectively, our findings reveal that SIRT1 is involved in the regulation of NLRP3 inflammasome through the autophagic pathway.
NLRP3 inflammasome is a multiprotein complex that activates caspase-1 and thereby causes the cutting and release of IL-1β and pyroptosis[DOI: 10.3390/ijms20133328]. Recent studies have greatly improved our understanding of NLRP3 inflammasome in neurodegenerative diseases[36, 37]. In addition, previous a study demonstrated that the NLRP3 inflammasome was in primed status in hippocampal tissue of aged mice but did not found in young mice[38]. This primed state explained the presence of an excessive neuroinflammatory response in aged mice, which is a common pathology of several neurodegenerative diseases. And evidence of NLRP3 inflammasome activation in the brain was demonstrated only in microglia[39]. We therefore also focused on the alteration of NLRP3 inflammasome in POCD. Surgery induced upregulation of NLRP3 expression, caspase-1 cleavage, and increased IL-1β in hippocampal tissue of aged mice, which alteration is all caused by reduced SIRT1 expression and impaired autophagy. We further observed that NLRP3 inflammasome activation could be significantly inhibited by SIRT1-dependent upregulation of autophagy levels. In vitro experiments, we also found an increase in ASC oligomerization specks in the LPS + ATP-induced inflammation model. This oligomerization of ASC proteins is also considered one of the markers of NLRP3 inflammasome activation.
While our results demonstrate that SIRT1 can affect the activation process of NLRP3 through autophagy, the more specific mechanism involved is poorly understood. Shi CS et al. found that autophagy can alleviate inflammation by eliminating active NLRP3 inflammasomes, which depend on ubiquitination of ASC and recruitment of p62, which translocates to autophagosomes[40]. This result implies that inflammasomes can be degraded through the autophagic pathway. IL-1β is a secretory protein, mostly secreted through the multivesicular body (MVB) and autophagosomes, and a small part is released directly after cell lysis. In one study, confocal immunofluorescence was used to demonstrate the presence of IL-1β in the intermembrane space of the double-membrane structure of autophagosomes[41]. HSP90 also played a key role in this translocation process. Zhang et al. found that when autophagy was inhibited, interleukin-1b accumulated intracellularly and could not be secreted[42]. From these points, we can easily see that the extracellular secretion of the pro-inflammatory factor IL-1β cannot be accomplished without autophagy.
Our study also has some limitations. First, SIRT1 was involved in a variety of intracellular physiological and biochemical processes, and here we did not determine whether SIRT1 functions directly by interacting with components of the NLRP3 inflammasome. Second, we used only male mice as study subjects to avoid the effects of estrogen and progesterone on the experiments. Therefore, we were unable to determine the role of estrogen and progesterone in POCD on NLRP3 inflammasomes. Third, we used BV-2 cells instead of microglia in the brain, and then the two were still somewhat different. Although some studies have shown no significant difference between these two in the activation of NLRP3 inflammasome[37, 43]. Although many studies have shown that NLRP3 inflammasomes are predominantly in microglia and therefore we also focused on microglia responses, we did not observe the role of SIRT1 in senescent microglia for autophagy and NLRP3 inflammasomes[39].
Taken together, our findings show that surgery downregulates hippocampal SITR1 expression, causing impaired autophagy and thus NLRP3 inflammasome activation. In other words, SIRT1 can be involved in the activation of NLRP3 inflammasomes through the regulation of autophagy to improve postoperative cognition in aged mice. Our study provides a new molecular mechanism for clinical intervention and treatment of POCD, clarifying the relationship between SIRT1, autophagy, and NLRP3 inflammasomes.