Dexmedetomidine Inhibits Nlrp3 Inflammasome Priming in BV-2 microglia through the NF-κB Pathway and the ROS-Nlrp3-IL-1β Signaling Axis


 In the brain, the NOD-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome is mostly expressed in microglia and is considered to be the primary cause of perioperative neurocognitive dysfunction (PND). Dexmedetomidine (Dex), a novel kind of clinical anesthetic with anti-inflammatory properties, has been shown to be effective in preventing PND in surgical patients. However, the mechanism of its anti-neuroinflammatory activity is still quite unclear. We examined the impact of Dex administration on Nlrp3 priming in activated BV-2 cells in this research. To investigate the mechanism by which Dex impacts Nlrp3 priming, we employed the inhibitors pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) to block the NF-κB p65 and the reactive oxygen species (ROS)-Nlrp3-interleukin (IL)-1β signaling axis, respectively. The results showed that Dex substantially decreased the expression of Nlrp3 and p65 and significantly inhibited the levels of the inflammatory factors IL-1β and tumor necrosis factor (TNF)-α in BV-2 cells stimulated with lipopolysaccharide (LPS). Additionally, when the NF-κB pathway was inhibited by PDTC, Dex could aggravate the downregulation of Nlrp3 and IL-1β in BV-2 cells. What is more, Dex negatively regulated the expression of Nlrp3 and IL-1β in activated BV-2 cells when NAC was added. These results showed that Dex inhibited Nlrp3 priming in LPS-induced BV-2 cells, presumably via blocking the NF-κB pathway and the ROS-Nlrp3-IL-1β signaling axis.


Introduction
Perioperative neurocognitive dysfunction (PND) refers to changes in cognitive function before or after surgery, and the clinical manifestations include abnormalities in learning, memory, language, thinking, spirit and emotion [1]. Microglia, which are resident macrophages in the central nervous system (CNS), can not only continuously monitor the brain microenvironment but also regulate various cellular responses [2]. There is evidence that surgery may activate microglia and enhance proin ammatory factor expression in the brain [3,4]. In the CNS, uncontrolled microglial activation and the accompanying in ammation are thought to lead to the development of PND [5,6]. Microglia contribute to PND by producing proin ammatory cytokines including interleukin-1 (IL-1) and interleukin-6 (IL-6), as well as tumor necrosis factor-α (TNF-α) [7]. The upregulation of these proin ammatory factors predisposes individuals to cognitive impairment [8,9]. Therefore, understanding how to control microglial activation in the brain after surgery still requires further research.
In PND, microglia expressed NOD-like receptor family pyrin domain containing 3 (Nlrp3), and other components of the Nlrp3 in ammasome were elevated and activated [10]. The most researched class of NOD-like receptors is Nlrp3 (NLRs). NLRs are pattern-recognition receptors (PRRs) that can detect the presence of certain microbial components known as pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) [11]. Through proximity-induced self-cleavage, Nlrp3 activation activates caspase-1, resulting in the maturation of proin ammatory factors such as interleukin-1β (IL-1β) and interleukin-18 (IL-18) [12]. By modulating neuroin ammation, hippocampal IL-1β contributes signi cantly to surgically caused cognitive impairment. Pretreatment with IL-1 receptor antagonists or knockdown of IL-1 receptors in mice were effective in alleviating cognitive dysfunction induced by surgical trauma [13]. Recent research has shown that iso urane-induced cognitive impairment in elderly mice is associated with elevated Nlrp3 levels in the hippocampus, and that this damage may be repaired by inhibiting the Nlrp3-caspase-1-IL-1β pathway [10]. The mitochondrial reactive oxygen species (mtROS)/Nlrp3 in ammasome/IL-1β signaling pathway may provide an attractive target for disrupting the pathophysiology of PND [14].
Dexmedetomidine (Dex), a highly selective agonist of the α 2 receptor, is commonly used to give mild analgesia and sedation to surgical patients undergoing general anesthesia. A few preexisting metaanalyses have reported that Dex reduced postoperative delirium among patients who underwent cardiac and noncardiac surgery [15,16]. Dex inhibits microglia-mediated release of TNFα, interleukin 1β, and other factors that are essential for the proin ammatory cascade, and IL-1β and TNFα are related to the development of PND [17]. Furthermore, Dex inhibited the Nlrp3 in ammasome in different animal models, such as lung injury [18], acute kidney injury [19] and brain injury [20], which suggests that Dex may have a protective effect on PND patients. The mechanism by which Dex suppresses the Nlrp3 in ammasome in microglia, on the other hand, remains unknown. As a result, we investigated the underlying mechanisms by which Dex suppresses the Nlrp3 in ammasome via upstream regulatory molecules involved in Nlrp3 priming.

Materials And Methods
Cell culture and treatments

Cell viability
Cell viability was determined using a CCK-8 test kit (Beyotime Institute of Biotechnology, Nanjing, China). For treatment, BV-2 cells were seeded into each well of a 96-well microtiter plate, followed by the addition of 10 μL of CCK-8 solution and incubation at 37 °C for 4 hours. The absorbance of each well was then determined at 490 nm (OD490) using a microplate reader (BioTek Instruments, Vermont, USA). Three separate tests were conducted three times each.

ELISA
The cytokines IL-1β and TNF-α were determined in cell culture supernatants using an ELISA kit (R&D Systems, Minneapolis, Minnesota, USA) in accordance with the manufacturer's instructions.
Total RNA extraction and real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) Trizol reagent was used to extract total RNA from BV-2 cells (Life, Massachusetts, USA). To synthesis cDNA, we utilized a reverse transcriptase kit and an SYBR Premix Ex Taq kit (Takara, Tokyo, Japan) in a CFX96 Touch real-time PCR system (Bio-Rad, California, USA). The expression of β-actin served as a control to standardize the quantity of cDNA in the various samples. The comparable Ct method was used to assess real-time qPCR products according to the manufacturer's recommendations. The primer sequences for qRT-PCR were listed in Table 1.

ROS analysis
We measured ROS uorescence intensities using a ROS staining kit (Beyotime Institute of Biotechnology, Nanjing, China) according to the manufacturer's procedure. Brie y, cells were seeded in a 6-well plate and then washed three times with PBS after drug treatment. The cells were then incubated for 20 minutes at 37 °C in the dark with 2 mol/L DCFH-DA probe. After washing with PBS, the nuclei were counterstained with Hoechst 33342 (Sigma, St Louis, MO, USA). A uorescent microscope was used to determine the uorescence intensity of BV-2 cells.

BV-2 cell viability in the presence or absence of LPS or Dex
The CCK-8 method was used to determine the viability of BV-2 cells exposed to a range of LPS concentrations (0-800 ng/mL) over a range of time periods (6 h, 24 h, or 48 h). When activated BV-2 cells were subjected to doses higher than or equivalent to 100 ng/mL LPS for 24 hours, viability reduced signi cantly (p < 0.05, Fig. 1a). To evaluate the impact of Dex on BV-2 cell viability, we treated the cells with different doses of Dex (0, 0.1, 1, 5, 10, or 20 μM). The CCK-8 test revealed no change in viability between BV-2 cells treated with various Dex doses (p > 0.05, Fig. 1b).

Dex suppresses the in ammatory reactions produced by LPS in BV-2 cells
To evaluate the anti-in ammatory effect of Dex, the mRNA expression of Nlrp3, Caspase-1, interleukin-1β and the protein expression of Nlrp3, Caspase-1, and p65 were measured. In addition, the levels of IL-1β and TNF-α were determined in cell culture supernatants. The RT-qPCR results showed that Dex decreased Nlrp3 (Fig. 2a) and IL-1 (Fig. 2c) expression in LPS-induced BV-2 cells but did not affect caspase-1 expression (Fig. 2b). Western blot analysis revealed that LPS treatment enhanced Nlrp3 and p65 protein expression in BV-2 cells. However, Nlrp3 and p65 protein expression levels were decreased by 10 μM Dex in comparison to the LPS group (Fig. 2d). Furthermore, Caspase-1 protein expression was not enhanced in LPS-activated BV-2 cells, and 10 μM Dex had no effect on Caspase-1 expression in comparison to the LPS group (Fig. 2d). The ELISA results showed that Dex inhibited the expression of IL-1β and TNF-α in BV-2 cells stimulated by LPS (Fig. 2h and i).

Dex suppresses the activation of NF-κB by LPS in BV-2 cells
To determine whether Dex's inhibitory impact on the Nlrp3 in ammasome was due to the NF-κB signaling pathway, we employed the NF-κB inhibitor PDTC to disrupt the NF-κB pathway. As shown in Fig. 3, PDTC was e cient at inhibiting NF-κB p65 protein production as well as the in ammatory mediators IL-1β and TNF-α. This nding suggested that PDTC successfully blocked the NF-κB pathway. Moreover, when the NF-κB pathway was inhibited, the LPS+PDTC+Dex group demonstrated a reduction in Nlrp3 and the in ammatory mediators IL-1β and TNF-α when compared to the LPS+PDTC group. These results indicated that Dex acted on the Nlrp3 in ammasome through other pathways than the NF-κB pathway.

Dex's anti-in ammatory actions on LPS-induced BV-2 cells are mediated via the ROS-Nlrp3-IL-1β pathway
The impact of Dex on the signaling molecule ROS upstream of the Nlrp3 in ammasome was con rmed.
To assess Dex's impact on the ROS-NLRP3-IL-1β axis, the ROS-speci c inhibitor NAC was employed as a positive control ( Fig. 4a and b). The ndings indicated that NAC substantially decreased the amount of reactive oxygen species (ROS) in LPS-activated BV-2 cells, as well as the expression of the downstream protein Nlrp3, as shown by the ROS assay and Western blotting ( Fig. 4c-f). Dex had a similar impact to NAC on LPS-activated BV-2 cells. This nding suggested that Dex may have anti-in ammatory effects through the ROS-Nlrp3-IL-1β pathway.

Discussion
The current research established conclusively that Dex has anti-in ammatory properties in LPS-activated microglia. Dex inhibited the generation of TNF-α and IL-1β by LPS-activated BV-2 cells, as shown in our research. These results suggested that Dex exerted a signi cant anti-in ammatory impact, which may contribute to the reduction of postoperative tissue damage. Neuroin ammation may be generated in the brain when systemic in ammation is caused by surgery on peripheral tissues and organs [21,22]. A large number of studies have indicated that neuroin ammation, which is characterized by microglial activation, plays a vital role in PND [23]. Since Dex has anti-in ammatory effects, this property of Dex may mediate the reduction in PND. Additionally, previous research indicates that Dex may help prevent PND by decreasing the expression of the Nlrp3 in ammasome [24]. However, the exact mechanism is still unclear. The current study contributes to a better understanding of how Dex works as an anti-in ammatory.
Dex has been shown in a growing number of studies to inhibit the expression of the Nlrp3 in ammasome in a variety of disease models. It is currently recognized that microglial activation plays a vital role in PND [25]. Therefore, we adopted the widely used experimental model of BV-2 microglial activation by LPS [26,27]. According to a previous paper, we treated BV-2 cells with 0.1-20 µM Dex to examine cytotoxicity and found that Dex treatment did not induce cytotoxicity. In addition, we found that treatment with 10 µM Dex inhibited LPS-induced Nlrp3 and p65 activation in BV-2 cells. These results were consistent with those of previous study. LPS has been shown to cause proin ammatory and cytotoxic reactions in microglia [28].
Hu's research [29] shows that Nlrp3 in ammasome activation requires the participation of LPS and ATP. Moreover, LPS is involved in the initiation of Nlrp3 and does not cause an increase in caspase-1. We utilized LPS alone as a stimulant for BV-2 cells in this research and found enhanced expression of Nlrp3 and upstream signaling components. The LPS-induced BV-2 cell model was applied to measure Dex's mechanism of action in the Nlrp3 in ammasome's upstream signaling pathway.
Recent research revealed that NF-κB, an upstream factor of the Nlrp3 in ammasome, plays a key regulatory function in in ammasome activation [30]. NF-κB p65 is the main component of the NF-κB heterodimer, which is phosphorylated on Ser536 site in response to LPS and translocates to the nucleus to induce Nlrp3 and IL-1β production [31]. We obtained results similar with earlier research following LPS stimulation of BV-2 cells in the current study. Furthermore, we discovered that Dex inhibited NF-κB p65 and Nlrp3 protein expression. Yao et al demonstrated that Dex mitigated LPS-induced acute kidney injury (AKI) by inhibiting oxidative stress injury and NLRP3 in ammasome activation through the NF-κB pathway [32]. The NF-κB inhibitor PDTC can effectively block NF-κB in microglia, which has been well con rmed [33]. Interestingly, after blocking NF-κB p65 with PDTC in LPS-activated BV-2 cells, Dex could still reduce Nlrp3 and IL-1β protein expression. No previous study has shown a similar result. These data suggest that Dex can affect Nlrp3 expression through other upstream factors.
In terms of upstream factors affecting Nlrp3 expression, the ROS-Nlrp3-IL-1 signaling axis is critical. Mitochondrial dysfunction generates ROS, which trigger Nlrp3 oligomerization through the ROS-Nlrp3-IL-1β signaling axis, thus acting upstream of Nlrp3 activation [34]. NAC, an inhibitor of ROS, is commonly used to suppress the production of ROS and the Nlrp3 in ammasome. To clarify the effect of Dex on upstream Nlrp3 factors, we used NAC to block ROS in BV-2 cells treated with LPS. Our results showed that NAC substantially inhibited ROS generation and Nlrp3 in ammasome activation in BV-2 cells treated with LPS. These ndings corroborated earlier research [35]. Additionally, we found that Dex had a similar impact to NAC on LPS-activated BV-2 cells Dex substantially reduced the formation of LPS-induced ROS in BV-2 cells, suggesting that ROS suppression may be a process by which Dex suppresses Nlrp3 activation.
In summary, our study demonstrated that Dex inhibits Nlrp3 activation in LPS-induced BV-2 microglia through the NF-κB signaling pathway and the ROS-Nlrp3-IL-1β signaling axis (Fig. 5). When NF-κB p65 is inhibited, Dex can still suppress in ammatory mediators by reducing ROS and Nlrp3 protein expression. Dex blocks the ROS signaling pathway, which was consistent with the effects observed when we blocked the ROS signaling pathway using the speci c ROS inhibitor NAC. Dex may contribute to antineuroin ammation through its inhibitory effect on the Nlrp3 in ammasome's upstream signaling pathway. In conclusion, our data reveal a possible mechanism by which Dex reduces the expression of in ammatory mediators.

Declarations
Funding This study was supported by the Natural Science Foundation of Guangdong Province China (Grant No. 2017A030313514).
Con icts of Interest The authors declare that the publishing of this article does not involve any con ict of interest.
Availability of Data and Material The original data will be provided if required.
Code Availability Not applicable.  Figure 1 The