Uncoupling Protein 2 Deficiency Enhances NLRP3 Inflammasome Activation Following Hyperglycemia-Induced Exacerbation of Cerebral Ischemia and Reperfusion Damage In Vitro and In Vivo

Abstract

Mitochondrial uncoupling protein 2 (UCP2) deficiency exacerbates brain damage following cerebral ischemia/reperfusion (I/R). The Nod-like receptor protein-3 (NLRP3) inflammasome also plays a vital role in cerebral I/R damage. However, the effect of UCP2 on NLRP3 inflammasome-mediated hyperglycemia and I/R damage is not clear. In the present study, UCP2-knockout (UCP2^-/-) and wild-type (WT) mice were used to establish a model of middle cerebral artery occlusion (MCAO) and reperfusion under normo- and hyperglycemic conditions. HT22 cells were established as a model of oxygen-glucose deprivation and reoxygenation (OGD/R) with high glucose to mimic hyperglycemia and I/R in vitro. HT22 cells were treated with/without different concentrations of the UCP2-specific inhibitor genipin for different periods of time. The results showed that UCP2 deficiency significantly increased histopathological changes and apoptosis after cerebral I/R damage in hyperglycemic mice. Moreover, UCP2 deficiency enhanced NLRP3 inflammasome activation in neurons when cerebral I/R damage was exacerbated by hyperglycemia. Furthermore, UCP2 deficiency enhanced NLRP3 inflammasome activation and reactive oxygen species (ROS) production in HT22 cells under OGD/R and high-glucose conditions. UCP2 deficiency aggravated hyperglycemia-induced exacerbation of cerebral I/R damage. UCP2 deficiency also enhanced NLRP3 inflammasome activation and ROS production in neurons in vitro and in vivo. These findings suggest that UCP2 deficiency enhances NLRP3 inflammasome activation following hyperglycemia-induced exacerbation of cerebral I/R damage in vitro and in vivo. UCP2 may be a potential therapeutic target for hyperglycemia-induced exacerbation of cerebral I/R damage.

Keywords: Cerebral ischemia; Hyperglycemia; Nod-like receptor protein-3; Reactive oxygen species; Uncoupling protein 2.

Fig. 1

UCP2 deficiency exacerbated I/R-induced histopathological changes and apoptosis in hyperglycemic mice. a Representative images of TTC staining in each group. b Bar graph summarizing the percent infarction volume in WT and UCP2^−/− mice. c Assessment of neurological deficits. d HE staining and TUNEL staining. e Quantitative summary of pyknotic cells. f Bar graph summarizing the percentage of TUNEL-positive cells. ^#p < 0.05 vs. the WT group; *p < 0.05 vs. the NG group (n = 6 in each group)

Fig. 2

UCP2 deficiency enhanced NLRP3 inflammasome activation after cerebral I/R damage in hyperglycemic mice. a The expression of NLRP3, cleaved caspase 1, caspase 1, ASC, TXNIP, and IL-1β in the different groups of UCP2-/- and WT mice. b–g Semiquantitative analysis of NLRP3, cleaved caspase 1, caspase 1, ASC, TXNIP, and IL-1β protein expression. h, i The level of IL-1β and IL-18 in serum. ^@p < 0.05 vs. the sham group; ^#p < 0.05 vs. the WT group; ^*p < 0.05 vs. the NG group (n = 6 in each group)

Fig. 3

UCP2 deficiency promotes NLRP3 inflammasome activation in neurons under hyperglycemia-exacerbated cerebral I/R conditions. a Representative images showing double immunofluorescence staining for NLRP3 (red) and NeuN (green) in the cortical penumbra area. b Representative images of double immunofluorescence staining for cleaved caspase 1 (red) and NeuN (green) in the cortical penumbra area. c The percentage of NLRP3-positive cells in the cortical penumbra area. d The percentage of cleaved caspase 1-positive cells (%) in the cortical penumbra area. ^@p < 0.05 vs. the sham group; ^#p < 0.05 vs. the WT group; *p < 0.05 vs. the NG group (n = 6 in each group)

Fig. 4

UCP2 deficiency enhanced HT22 cell damage under OGD/R and high glucose conditions. a Light microscopy images showing morphological changes in HT22 cells treated with different concentrations of genipin (25 μM, 50 μM, 75 μM, and 100 μM). b Cell viability in HT22 cells at different concentrations was determined by CCK-8 assays. c, d The expression of UCP2 in the different groups at the indicated times was measured by western blotting. *p < 0.05 vs. the Control group. e Light microscopic images showing morphological changes in HT22 cells after different treatments. Scale bar = 20 μm. f Cell viability analysis of HT22 cells after different treatments. The experiments were repeated three times in triplicate for each condition. ^#p < 0.05 vs. Control; *p < 0.05 vs. HG; ^$p < 0.05 vs. NG + OGD/R; ^@p < 0.05 vs. HG + OGD/R; ^%p < 0.05 vs. NG + OGD/R + GE

Fig. 5

UCP2 deficiency enhanced NLRP3 inflammasome activation in HT22 cells under OGD/R and high glucose conditions. a Double immunostaining of NLRP3, ASC, Caspase 1, Cleaved Caspase 1, and IL-1β (red) with DAPI (blue)) was performed in the different groups. b Representative western blots showing NLRP3, ASC, Caspase 1, Cleaved Caspase 1, TXNIP, and IL-1β in the different groups. c–h Quantification of the average NLRP3, ASC, Caspase 1, Cleaved Caspase 1, TXNIP, and IL-1β expression normalized to β-actin. i The level of IL-1β in HT22 cell culture medium. The experiments were repeated three times in triplicate for each condition. ^#p < 0.05 vs. Control; *p < 0.05 vs. HG; ^$p < 0.05 vs. NG + OGD/R; ^@p < 0.05 vs. HG + OGD/R; ^%p < 0.05 vs. NG + OGD/R + GE

Fig. 6

UCP2 deficiency enhanced the production of ROS in HT22 cells under OGD/R and high glucose conditions. a DHE fluorescence. ROS were detected by the DHE probe (Red). b The presence of ROS was determined using DHE. c The MDA level in the different groups of HT22 cells. d SOD2 fluorescence. SOD (green color) was detected using antibodies. DAPI (blue color) labeled the nuclei. e Representative western blots showing SOD2 expression. f Quantification of the average SOD2 expression normalized to β-actin. The experiments were repeated three times in triplicate for each condition. ^#p < 0.05 vs. Control; *p < 0.05 vs. HG; ^$p < 0.05 vs. NG + OGD/R; ^@p < 0.05 vs. HG + OGD/R; ^%p < 0.05 vs. NG + OGD/R + GE