Sevoflurane Aggravates the Progress of Alzheimer's Disease Through NLRP3/Caspase-1/Gasdermin D Pathway

Abstract

Background: Alzheimer's disease (AD) is the most common form of dementia worldwide. Previous studies have reported that sevoflurane, a frequently used anesthetic, can induce cognitive impairment in preclinical and clinical settings. However, the mechanism underlying the development of this neurotoxicity is currently unclear. Methods: Seven-month-old APP/PS1 mice were placed in an anesthesia induction box containing 3% sevoflurane in 100% O₂ for 6 h, while BV2 cells were cultured with 4% sevoflurane for 6 h. Pyroptosis and tau protein expression in excised hippocampus tissues and cells were measured using Western blotting and immunofluorescence assay. Caspase-1 and NLRP3 were knocked out in BV2 microglia using CRISPR/Cas9 technology to determine whether they mediate the effects induced by sevoflurane. Results: Sevoflurane directly activated caspase-1 to induce pyroptosis in the mouse model of AD via NLRP3 and AIM2 activation. In addition, sevoflurane mediated cleavage of gasdermin D (GSDMD) but not gasdermin E (GSDME), promoted the biosynthesis of downstream interleukin-1β and interleukin-18, and increased β-amyloid (Aβ) deposition and tau phosphorylation. The nontoxic caspase-1 small-molecule inhibitor VX-765 significantly inhibited this activation process in microglia, while NLRP3 deletion suppressed sevoflurane-induced caspase-1 cleavage and subsequently pyroptosis, as well as tau pathology. Furthermore, silencing caspase-1 alleviated the sevoflurane-induced release of IL-1β and IL-18 and inhibited tau-related enzymes in microglia. Conclusion: This study is the first to report that clinical doses of sevoflurane aggravate the progression of AD via the NLRP3/caspase-1/GSDMD axis. Collectively, our findings elucidate the crucial mechanisms of NLRP3/caspase-1 in pyroptosis and tau pathogenesis induced by sevoflurane and suggest that VX-765 could represent a novel therapeutic intervention for treating AD.

Keywords: VX-765; gasdermin D; pyroptosis; sevoflurane; tau pathology.

FIGURE 1

Sevoflurane induces pyroptosis and activates tau pathology in APP/PS1 mice. (A) Representative immunoblot analysis of Aβ protein expression in control or sevoflurane-treated APP/PS1 mice using GAPDH as an internal control. (B) Immunoblot analysis of APP/PS1 mouse hippocampi stained for phosphorylated tau (p-tau), total tau, total PP2A subunit C, demethylated PP2A subunit C, p-CaMKII-α, total CaMKII-α, GSK-3β phosphorylated (p-GSK-3β), and total GSK-3β. (C) Quantification of kinases and phosphatases in B (n = 6). (D) Immunoblot analysis of NLRP3, AIM2, ASC, pro-caspase-1, cleaved caspase-1, and cleaved gasdermin D (GSDMD). (E) Quantification of proteins in D using GAPDH as an internal control (n = 6). (F) Immunoblot analysis and quantification of interleukin-1β and 18 in hippocampi after sevoflurane treatment. (G) Immunofluorescence staining of Iba1, cleaved-GSDMD, and DAPI. Scale bar: 100 μm. ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 2

Sevoflurane promotes caspase-1-dependent pyroptosis in BV2 microglia (A). Cleaved caspase-1 and caspase-3, IL-1β, and IL-18 levels were detected by Western blotting analysis in BV2 microglia exposed to 4% sevoflurane for 6 h (B). Western blotting analysis of caspase-1, caspase-3, IL-1β, and IL-18 induced by sevoflurane in BV2 cells pretreated with 100 ng/ml of lipopolysaccharide (LPS). (C) Pyroptosis-related proteins (gasdermin D (GSDMD) and gasdermin E (GSDME)) were detected by Western blotting analysis induced by sevoflurane in BV2 cells. (D) Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays of cell viability and LDH activity in BV2 cells. (E). FAM-FLICA caspase-1 assays of cleaved caspase-1 (green), propidium iodide (PI) (red), and DAPI (blue) in BV2 cells. Scale bar: 50 μm. Cleaved caspase-1 fluorescence intensity was calculated as the iod. (F). FAM-FLICA caspase-1 assays of cleaved caspase-1 (green) in BV2 cells. Scale bar: 50 μm. ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 3

Sevoflurane exposure induces caspase-1-dependent pyroptosis by activating the NLRP3-ASC pathway (A). Immunoblot analysis of NLRP1, AIM2, NLRP3, and NLPC4 expression in BV2 cells treated with 4% sevoflurane for 6 h or control. (B). Quantitative Western blotting analysis of NLRP1, AIM2, NLRP3, and NLPC4. (C). Iba1, ASC, and DAPI staining in BV2 cells imaged under a microscope. (D). Iba1 and ASC fluorescence intensity was calculated as the iod. ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 4

Tau-correlated kinases and phosphatases regulate the effects of sevoflurane in BV2 cells. (A) BV2 cells were treated with 4% sevoflurane for 6 h, and then p-tau and Aβ were detected by Western blotting. (B). Quantitation of p-tau and Aβ band intensity using ImageJ software. (C) Western blotting analysis of p-tau and Aβ induced by sevoflurane in BV2 cells pretreated with 100 ng/ml of lipopolysaccharide (LPS). (D). Quantitation of p-tau and Aβ band intensity using ImageJ software. ( E ) Expression of tau-correlated kinases and phosphatases (P-CaMKII, PP2A-D, and P-GSK-3β) as determined by Western blotting analysis. (F) Quantitation of P-CaMKII, PP2A-D and P-GSK band intensity using ImageJ software. ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 5

NLRP3 knockout alleviates sevoflurane-induced pyroptosis. (A) Western blotting analysis of cleaved gasdermin D (GSDMD), cleaved caspase-1, IL-1β, and IL-18 induced by sevoflurane in wild-type (WT) and NLRP3^−/− BV2 cells. Protein band intensity was quantified using ImageJ software. (B) Immunoblot analysis of p-tau, tau-correlated kinases, and phosphatases induced by sevoflurane in WT and NLRP3^−/− BV2 cells. (C) FAM-FLICA caspase-1 assay of cleaved caspase-1 (green) induced by sevoflurane in WT and NLRP3^−/− BV2 cells. Scale bar: 50 μm (D). Quantitation of cleaved caspase-1 fluorescence intensity as iod. (E). Lactate dehydrogenase (LDH) release and Cell Counting Kit-8 (CCK-8) assays of LDH activity and cell viability induced by sevoflurane in WT and NLRP3^−/− BV2 cells. ***p < 0.001, **p < 0.01, *p < 0.05.

FIGURE 6

Casp1 knockout alleviates sevoflurane-induced pyroptosis and Alzheimer’s disease (AD) development. (A) Western blotting analysis of cleaved gasdermin D (GSDMD) and caspase-1 in wild-type (WT) and caspase-1^−/− BV2 cells treated with 4% sevoflurane for 6 h. (B) Immunoblot analysis of, secreted IL-18, secreted IL-1β, induced by sevoflurane in WT and caspase-1^−/− BV2 cells. Protein band intensity was quantified using ImageJ software. (C) P-tau and tau-related phosphatases were detected by Western blotting analysis. (D) Lactate dehydrogenase (LDH) release and Cell Counting Kit-8 (CCK-8) assays of LDH activity and cell viability in WT and NLRP3^−/− BV2 cells. ***p < 0.001, **p < 0.01, *p < 0.05.