Maf1 Ameliorates Sepsis-Associated Encephalopathy by Suppressing the NF- k B/NLRP3 Inflammasome Signaling Pathway

Abstract

Background: The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome has been identified as an important mediator of blood-brain-barrier disruption in sepsis-associated encephalopathy (SAE). However, no information is available concerning the critical upstream regulators of SAE.

Methods: Lipopolysaccharide (LPS) was used to establish an in vitro model of blood-brain barrier (BBB) disruption and an in vivo model of SAE. Disruption of BBB integrity was assessed by measuring the expression levels of tight-junction proteins. NLRP3 inflammasome activation, pro-inflammatory cytokines levels, and neuroapoptosis were measured using biochemical assays. Finally, the FITC-dextran Transwell assay and Evan's blue dye assay were used to assess the effect of Maf1 on LPS-induced endothelial permeability in vitro and in vivo.

Results: We found that Maf1 significantly suppressed the brain inflammatory response and neuroapoptosis induced by LPS in vivo and in vitro. Notably, Maf1 downregulated activation of the NF-κB/p65-induced NLRP3 inflammasome and the expression of pro-inflammatory cytokines. In addition, we found that Maf1 and p65 directly bound to the NLRP3 gene promoter region and competitively regulated the function of NLRP3 in inflammations. Moreover, overexpression of NLRP3 reversed the effects of p65 on BBB integrity, apoptosis, and inflammation in response to LPS. Our study revealed novel role for Maf1 in regulating NF-κB-mediated inflammasome formation, which plays a prominent role in SAE.

Conclusions: Regulation of Maf1 might be a therapeutic strategy for SAE and other neurodegenerative diseases associated with inflammation.

Keywords: Maf1; NF-κB; NLRP3 inflammasome; blood–brain barrier disruption; sepsis-associated encephalopathy.

Figure 1

Maf1 alleviated BBB disruption, neuro-apoptosis, and inflammation induced by LPS. BMECs were transfected with a control vector (NC), Maf1 or shMaf1 plasmid, followed by LPS stimulation; they were then co-cultured with astrocytes to establish an in vitro BBB model. (A) The TEER values of monolayer cells at 0, 6, 12, and 24 h were measured using a bioelectric impedance analyzer. (B) Trans-endothelial permeability assays were performed to assess the in vitro BBB integrity by using the diffusion of 10 kDa FITC-dextran method. The relative permeability coefficient was determined after Transwell experiments were conducted. (C) Cell viability was analyzed using the CCK-8 assay. (D) Representative images of Annexin V/PI double staining, and (E) measurements of apoptosis. (F) The levels of Maf1, Claudin-5, Occludin, ZO-1, Bax, Bcl-2, and GSDMD were determined by western blotting. GAPDH served as a loading control. (G) Cell-free conditioned culture medium was collected and analyzed by ELISA for IL-1β and IL-18 levels. (H) Western blot analyses of pro-inflammatory cytokine (ASC, Caspas-1, IL-1β, andIL-18) and NLRP3expression levels. Results are expressed as the mean value ± SD of data obtained from three separate experiments. ***p< 0.001, compared with Control; ^## p < 0.01, compared with LPS + NC.

Figure 2

Maf1 suppressed inflammation in the LPS-induced in vitro BBB model through inactivation of the NLRP3 inflammasome. BMECs were transfected with the control vector (NC), Maf1 or a combination of Maf1and NLRP3, followed by LPS stimulation. The BMECs were then co-cultured with astrocytes to establish an in vitro BBB model. (A) The TEER values of monolayer cells at 0, 6, 12, and 24 h were measured using a bioelectric impedance analyzer. (B) Trans-endothelial permeability assays were performed to assess in vitro BBB integrity by using the diffusion of 10 kDa FITC-dextran method. The relative permeability coefficient was determined after the Transwell experiment. (C) Cell viability was analyzed using the CCK-8 assay. (D) Quantification of cell apoptosis. (E) Western blot results for the levels of Claudin-5, Occludin, ZO-1, Bax, Bcl-2, and GSDMD. (F) The cell-free conditioned culture medium was collected and analyzed IL-1β and IL-18 levels via ELISA (G) Western blot analyses for pro-inflammatory cytokine (ASC, Caspase-1, IL-1β, and IL-18) and NLRP3 expression levels. Results are expressed as the mean value ± SD of data obtained from three separate experiments. ***p < 0.001, compared with Control; ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with LPS + NC; ^$ p < 0.05 and ^$$ p < 0.01 compared with LPS + Maf-1.

Figure 3

Overexpression of p65 reversed the effects of Maf1 on in vitro BBB integrity, cell viability, and apoptosis in response to LPS. Cells were treated as shown in Figure 1 . (A) The cytoplasm and nucleus were separated. Western blot analyses of Maf1 and p65 expression in the cytoplasm and nucleus. (B) The co-location and expression levels of Maf1 and NLRP3 were determined by immunofluorescence assays. (C) Immunofluorescence staining of Maf1 and p65 is shown. BMECs were transfected with the control vector (NC), Maf1, or a combination of Maf1and p65, followed by LPS stimulation. The BMECs were then co-cultured with astrocytes to establish an in vitro BBB model. (D) The TEER of values of monolayer cells at 0, 6, 12, and 24 h were measured using a bioelectric impedance analyzer. (E) Trans-endothelial permeability assays were performed to assess in vitro BBB integrity by using the diffusion of 10 kDa FITC-dextran method. The relative permeability coefficient was determined after the Transwell experiment. (F) Cell viability was analyzed using the CCK-8 assay. (G) Quantification of cell apoptosis. (H) Western blot results for the levels of Claudin-5, Occludin, ZO-1, Bax, Bcl-2, and GSDMD. (I) The cell-free conditioned culture medium was collected and analyzed for IL-1β and IL-18 levels via ELISA (J) Western blot analyses of pro-inflammatory cytokine (ASC, Caspase-1, IL-1β andIL-18) and NLRP3 expression levels. Data are expressed as the mean value ± SD of data obtained from three separate experiments. ***p < 0.001, compared with Control; ^### p < 0.001, compared with LPS + NC; ^$ p < 0.05, ^$$ p < 0.01 and ^$$$ p < 0.05 compared with LPS+Maf-1.

Figure 4

Maf1 and p65 directly bound to the NLRP3 promoter. (A) The mutant binding site of the NLPR3promoter. (B, E) HEK293T cells were co-transfected with pcDNA4.0 or pcDNA4.0-Maf1/pcDNA4.0-p65 vectors and pGL-NLRP3-WT orpGL-NLRP3-MUT vectors, and the relative luciferase activities are shown. (C, F) ChIP assays were performed to determine the binding of Maf1 and p65 to the NLRP3 promoter in BMECs in vitro. (D, G) EMSA assays were performed to detect the direct binding of Maf1 and p65 to the NLRP3 promoter in BMECs in vitro. (H–K) ChIP assays were performed to explore howMaf-1 or p65 regulated the NLRP3 promoter in BMECs. Total chromatin from cell culture as the input, and normal rabbit IgG served as the negative control antibody. Results are expressed as the mean value ± SD of data obtained from three separate experiments, **p < 0.01, compared with NC.

Figure 5

Overexpression of NLRP3 reversed the effects of p65 on in vitro BBB integrity, apoptosis, and inflammation in response to LPS. BMECs were transfected with the control vector (NC), NLRP3 or a combination of shp65 and NLRP3, followed by LPS stimulation. The BMECs were then co-cultured with astrocytes to establish an in vitro BBB model. (A) The TEER values of monolayer cells at 0, 6, 12, and 24 h were measured using a bioelectric impedance analyzer. (B) Trans-endothelial permeability assays were performed to assess in vitro BBB integrity by using the diffusion of 10 kDa FITC-dextran method. The relative permeability coefficient was determined after the Transwell experiment. (C) Cell viability was analyzed using the CCK-8 assay. (D) Quantification of cell apoptosis. (E) Western blot results for the levels of Claudin-5, Occludin, ZO-1, Bax, Bcl-2, and GSDMD. (F) The cell-free conditioned culture medium was collected and analyzed for IL-1β and IL-18 levels via ELISA. (G) Western blot analysis of pro-inflammatory cytokine (ASC, caspase-1, IL-1β, andIL-18) levels. Results are expressed as the mean value ± SD of data obtained from three separate experiments. ***p < 0.001, compared with Control; ^### p < 0.001, compared with LPS+NC; ^$$ p < 0.01 and ^$$$ p < 0.001 compared with LPS + shp65.

Figure 6

Maf1 improved cognitive impairment and in vivo BBB integrity. (A) Analysis of the escape latency time to the platform in different groups. (B) The swim paths of mice in the MWM test. (C) The distance of crossings over the platform location in the probe trial. (D) Evan’s blue dye in the brain tissues. (E) The concentrations of Evan’s blue were measured. (F) The weights of the brains. (G) Immunofluorescence staining of Bax and Bcl-2 in brain tissues. (H) Western blot results for levels of Maf1, p65, Claudin-5, Occludin, ZO-1, Bax, Bcl-2, and NLRP3. (I) The survival rate of rats in the different groups was calculated. (J–L) Cerebral cortex tissue was collected and analyzed for S100B (J), IL-1β (K), and IL-18 (L) via ELISA. Results are expressed as the mean value ± SD of data obtained from three separate experiments. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with Sham; #, p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with SAE + NC.

Figure 7

Schematic diagram of the proposed molecular mechanisms. A schematic diagram depicting the molecular mechanism by which Maf1 inhibits blood–brain barrier disruption by competing with p65 to regulate NLRP3 expression. Maf1 protects the blood–brain barrier by directly targeting NLRP3 to decrease apoptosis, the secretion of pro-inflammatory cytokines, blood–brain barrier permeability, and nerve injury.