Dexmedetomidine Mitigated NLRP3-Mediated Neuroinflammation via the Ubiquitin-Autophagy Pathway to Improve Perioperative Neurocognitive Disorder in Mice

Abstract

Background: Surgery and anesthesia-induced perioperative neurocognitive disorder (PND) are closely related to NOD-like receptors (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome microglia inflammatory response. Inhibiting the occurrence of neuroinflammation is an important treatment method to improve postoperative delirium. Fewer NLRP3-targeting molecules are currently available in the clinic to reduce the incidence of postoperative delirium. Dexmedetomidine (DEX), an α2 adrenergic receptor agonist has been shown to have antioxidant and anti-inflammatory activities. The present study showed that DEX reduced the production of cleaved caspase1 (CASP1) and destroyed the NLRP3-PYD And CARD Domain Containing (PYCARD)-CASP1 complex assembly, thereby reducing the secretion of IL-1β interleukin beta (IL-1β). DEX promoted the autophagy process of microglia and reduced NLRP3 expression. More interestingly, it promoted the ubiquitination and degradation of NLRP3. Thus, this study demonstrated that DEX reduced NLRP3-mediated inflammation through the activation of the ubiquitin-autophagy pathway. This study provided a new mechanism for treating PND using DEX. Methods: C57BL/6 mice were pre-administered DEX 3 days in advance, and an abdominal exploration model was used to establish a perioperative neurocognitive disorder model. The anti-inflammatory effect of DEX was explored in vivo by detecting NLRP3-CASP1/IL-1β protein expression and behavioral testing. Primary microglia were stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in vitro, the expression of CASP1 and IL-1β was detected in the supernatant of cells, and the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) and sequestosome 1 (SQSTM1) was examined in the cytoplasm. Meanwhile, Co-immunoprecipitation (Co-IP) was used to detect NLRP3 protein ubiquitination so as to clarify the new mechanism underlying the anti-inflammatory effect of DEX. Results: Pre-administration of DEX reduced the protein expression of NLRP3, CASP1, and IL-1β in the hippocampus of mice induced by surgery and also improved the impairment of learning and memory ability. At the same time, DEX also effectively relieved the decrease in spine density of the hippocampal brain induced by surgery. DEX decreased the cleaved CASP1 expression, blocked the assembly of NLRP3-PYCARD-CASP1 complex, and also reduced the secretion of mature IL-1β in vitro. Mechanically, it accelerated the degradation of NLRP3 inflammasome via the autophagy-ubiquitin pathway and reduced the green fluorescent protein/red fluorescent protein MAP1LC3B ratio, which was comparable to the effect when using the autophagy activator rapamycin (Rapa). Furthermore, it increased the ubiquitination of NLRP3 after LPS plus ATP stimulated microglia. Conclusion: DEX attenuated the hippocampal brain inflammation by promoting NLRP3 inflammasome degradation via the autophagy-ubiquitin pathway, thus improving cognitive impairment in mice.

Keywords: NLRP3 inflammasome; dexmedetomidine; microglial; neuroinflammation; perioperative neurocognitive disorder.

FIGURE 1

NLRP3 activation in microglia was associated with surgery-induced inflammation. NLRP3 activation in microglia was associated with surgery-induced inflammation. (A): The expression levels of NLRP1, AIM2, NLRP2, NLRP3, and NLRC4 in the hippocampus were examined by Immunoblotting, and the statistical data are shown in (B) (means ± SEM, n = 3). (C): pro-IL1β/IL1β,pro-CASP1/CASP1/CASPASE1 protein expression in the hippocampus were measured by Immunoblotting and the quantitative analysis data shown in (D) (means ± SEM, n = 3). (E,F) Immunofluorescence staining of IBA-1, GFAP and NLRP3 in the hippocampal dentategyrus brain region. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding sham group, as determined by the Student’s t-test.

FIGURE 2

DEX mitigated surgically induced nerve damage and reversed cognitive function in mice. (A) Diagram of the experimental design, DEX was given once a day three days before the operation, and the first day after the operation was adapted to the environment of the behavioral test. The biological test was performed after the behavioral test. pro-IL1β/IL1β, pro-CASP1/CASP1 and PSD95 protein expression in the hippocampus were measured by Immunoblotting (B,C) and the quantitative analysis data shown in (D) (means ± SEM, n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group, as determined by the Student’s t-test. The expression of GFAP and IBA-1 were analyzed by immunochemistry in the hippocampal dentategyrus brain region (E–G), and the statistical data are shown in graph (F–H) (means ± SEM, n = 6). Scale bars = 100 μm. Quantification of the positive areas using ImageJ software. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group, as determined by the Student’s t-test. The hippocampus of the sections were examined by Golgi-Cox Staining (I), the pictures taken were analyzed with the NeuroJ plugin in ImageJ software to calculate the total length of dendrites and the density of synaptic spines (J,K, n = 6). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group, as determined by the Student’s t-test. Y-maze and new object recognition were tested of mice at the end of the experiment (means ± SEM, n = 10), the Y-maze trace of the mice were shown in (L), and the quantitative analysis data shown in (M–O). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group, as determined by the Student’s t-test.

FIGURE 3

DEX reduced microglia activation and attenuated neuronal cell apoptosis in vitro. DEX reduces microglia activation and attenuates neuronal cell apoptosis in vitro. The primary microglia viability and toxicity test were examined by the CCK-8 kit. Each group had three replicates, and the test was repeated 4 times independently (A). LPS-primed (100 ng/ml) primary microglia were treated with different doses of DEX for 5 h and then stimulated with ATP (5 mM) for 30 min. The expression of IBA-1 detected by Immunoblotting (B) and the statistical data shown in (C) (means ± SEM, n = 4). LPS-primed primary microglial cells were incubated with DEX for 5 h, then stimulated with ATP for 30 min.Microglia conditioned medium including without any treatment, treated with LPS (100 ng/ml, 5 h)plus ATP (5 mM, 30 min), and DEX (10 μM, 5 h) protected, MCM mixed with 2% B-27 Neurobasal medium (1:1) and incubated the cells for 24 h, primary neuronal cells were incubated with mix MCM separately, collected for Immunoblotting, immunofluorescence staining and cell flow analysis. Expression of apoptosis-related proteins BAX and Bcl-2 detected by Immunoblotting (D), the statistical data are shown in (E,F) (means ± SEM, n = 4). Apoptosis indicators were detected using a cell flow cytometer (G). Data analysis is shown in (H) (means ± SEM,n = 4). MAP-2 expression was stained by immunofluorescence and axon length was calculated by IMageJ software (I), data analysis in (J) (means ± SEM, n = 4). All the above data analysis were presented as the means ± SEM,*p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group by Student’s t-test.

FIGURE 4

DEX suppressed CASP1 and IL-1β maturation by interrupting NLRP3 inflammasome activation and assembly. DEX suppressed CASP1 and IL-1β maturation by interrupting NLRP3 inflammasome activation and assembly. Protein NLRP3 in the hippocampus brain region of different groups of mice was detected by Immunoblotting (A,B) is the corresponding statistical analysis (means ± SEM, n = 4). LPS-primed primary microglial cells were incubated with DEX for 5 h, and NLRP3 expression was detected via Immunoblotting (C), and the data shown in (D) (means ± SEM, n = 4). Primary microglial cells were treated as follows: MCC950 (0.01 μM) was administered 2 h in advance, LPS-primed primary microglial cells were incubated with DEX for 5 h and then ATP was gived for 30 min. The protein cell supernatant protein and cytosolic protein were collected for Immunoblotting to detect IL-1βand NLRP3 (E). The data is shown in (F,G) (means ± SEM, n = 4). The expression of the pro-IL-1β/IL-1β, pro-CASP1/CASP1 examined by Immunoblotting (H), the cells of the treatment is the same way as above. Protein expression analysis data were shown in (I,J) (means ± SEM, n = 4). The NLRP3-ASC interaction was analyzed by CO-IP and Immunoblotting (K). All the above data analysis were presented as the means ± SEM, p < 0.05 is defined as statistically different, and the ns means no significance, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group. The data analysis in (B,I,J) was one-way ANOVA, data analysis in (D,F,G) was two-way ANOVA. SN, supernatant; Ly, lysate.

FIGURE 5

DEX promotes NLRP3 degradation via autophagy pathway. DEX promoted NLRP3 degradation via the autophagy pathway. The cells were treated in the same way as before, and the NLRP3,ASC,pro-CAS1 were detected via Western blot (A,B, means ± SEM, n = 4). LPS-primed primary microglial were cultured with MG-132 (10 μM), 3-MA (5 mM) or BafA1 (100 nM) for 1 h and then treated with DEX (10 μM) for 5 h, followed by a 30-min incubation with 5 mM ATP. The protein expression of the Supernatants (SN) (IL-1β) and cell extracts (lysate) (NLRP3,pro-CASP1, and MAP1LC3B) were analyzed by Immunoblotting (C,E, means ± SEM, n = 4). The corresponding data analysis were shown in (D–F) (means ± SEM, n = 4). The protein expression of SQSTM1, ATG5, ATG7, and MAP1LC3B-II after LPS stimulation were analyzed by western blot (G,H, means ± SEM, n = 4). Cells used for experiments 4 days after RFP-GFP-MAP1LC3B adenovirus infection. The cells were treated in the same way as before. The relatively GFP-RFP ratio were counted (I) and the analysis data were shown in (J) (means ± SEM, n = 4). All the above data analysis were presented as the means ± SEM, p < 0.05 is defined as statistically different, and the ns means no significance,*p < 0.05, **p < 0.01, ***p < 0.001 compared with the corresponding group. Data analysis method was one-way ANOVA.

FIGURE 6

DEX promoting ubiquitination-mediated degradation of NLRP3. DEX promoted ubiquitination-mediated degradation of NLRP3. (A) Primary microglia cells were treated the same way as before. Cells were analyzed by NLRP3 and ubiquitin immunofluorescence. Scale bar = 20 μm. LPS-primed primary microglial were cultured with MG-132 (10 μM), 3-MA (5 mM) or, LY294002 (10 µM) for 1 h and then treated with DEX (10 μM) for 5 h. Immunoblotting analysis of Ub protein level in cell lysates immunoprecipitated with NLRP3 antibody (B,C).

FIGURE 7

Dexmedetomidine, an α2 adrenergic receptor agonist, alleviates the cognitive function via blocking the activation and assembly of NLRP3 inflammasome and accelerated the degradation of NLRP3 through the ubiquitin-autophagy pathway. Consequently, neuroinflammation level induced by surgery was reduced, thenPND of mice has been improved.