Effects of amyloid β (Aβ)42 and Gasdermin D on the progression of Alzheimer's disease in vitro and in vivo through the regulation of astrocyte pyroptosis

Abstract

Purpose: The study aimed to investigate whether astrocyte pyroptosis, and the subsequent neuroinflammatory response that exerts amyloid β (Aβ) neurotoxic effects, has an effect on endothelial cells, along with the underlying mechanisms.

Methods: In vivo, 5 μL of disease venom was injected into the lateral ventricle of APP/PS1 mice for treatment. Pyroptosis was induced by treating astrocytes with Aβ42 in vitro. Small interfering RNA (siRNA) was used to silence caspase-1 and Gasdermin D (GSDMD) mRNA expression. Cell viability was determined using a CCK-8 detection kit. Scanning electron microscopy (SEM), Annexin V/propidium iodide (PI) double staining, RT-qPCR, immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. The degree of pathological damage to the brain and aortic tissue was assessed by hematoxylin-eosin staining and immunohistochemistry.

Results: Aβ42 induced astrocyte pyroptosis dependent on the GSDMD/Gasdermin E (GSDME)/Caspase 11/NLRP3 pathway, releasing large amounts of inflammatory factors, such as TNF-α, IL-1α, IL-1β, and IL-18. Astrocyte pyroptosis caused endothelial cell dysfunction and release of large amounts of vasoconstrictors (ET and vWF). Knockdown of GSDMD reduced astrocyte pyroptosis in the cerebral cortex and hippocampal tissue, decreased the release of inflammatory factors IL-1 β and IL-18, reduced Aβ deposition and tau protein, increased the release of peripheral vasodilator substances (eNOS), and decreased the release of vasoconstrictor substances (ET, vWF), thereby reducing brain tissue damage and vascular injury in APP/PS1 mice.

Conclusion: Aβ42 induced astrocyte pyroptosis, while GSDMD knockout inhibited astrocyte pyroptosis, reduced the release of inflammatory factors, and alleviated brain tissue damage and vascular damage in APP/PS1 mice. Therefore, GSDMD is a novel therapeutic target for Alzheimer's disease.

Purpose: The study aimed to investigate whether astrocyte pyroptosis, and the subsequent neuroinflammatory response that exerts amyloid β (Aβ) neurotoxic effects, has an effect on endothelial cells, along with the underlying mechanisms.

Methods: In vivo, 5 μL of disease venom was injected into the lateral ventricle of APP/PS1 mice for treatment. Pyroptosis was induced by treating astrocytes with Aβ42 in vitro. Small interfering RNA (siRNA) was used to silence caspase-1 and Gasdermin D (GSDMD) mRNA expression. Cell viability was determined using a CCK-8 detection kit. Scanning electron microscopy (SEM), Annexin V/propidium iodide (PI) double staining, RT-qPCR, immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. The degree of pathological damage to the brain and aortic tissue was assessed by hematoxylin-eosin staining and immunohistochemistry.

Results: Aβ42 induced astrocyte pyroptosis dependent on the GSDMD/Gasdermin E (GSDME)/Caspase 11/NLRP3 pathway, releasing large amounts of inflammatory factors, such as TNF-α, IL-1α, IL-1β, and IL-18. Astrocyte pyroptosis caused endothelial cell dysfunction and release of large amounts of vasoconstrictors (ET and vWF). Knockdown of GSDMD reduced astrocyte pyroptosis in the cerebral cortex and hippocampal tissue, decreased the release of inflammatory factors IL-1 β and IL-18, reduced Aβ deposition and tau protein, increased the release of peripheral vasodilator substances (eNOS), and decreased the release of vasoconstrictor substances (ET, vWF), thereby reducing brain tissue damage and vascular injury in APP/PS1 mice.

Conclusion: Aβ42 induced astrocyte pyroptosis, while GSDMD knockout inhibited astrocyte pyroptosis, reduced the release of inflammatory factors, and alleviated brain tissue damage and vascular damage in APP/PS1 mice. Therefore, GSDMD is a novel therapeutic target for Alzheimer's disease.

Keywords: Alzheimer’s disease; GSDMD; amyloid β; astrocyte pyroptosis; endothelial cell.

Figure 1

Aβ42 promotes astrocyte proliferation. (A) CCK-8 assayed the cell proliferation ability of the control cells, 0.1 μmol Aβ42, or 1 μmol Aβ42-treated cells. (B) The effect of Aβ42 on astrocyte morphology was observed by scanning electron microscopy. (C) Real-time quantitative PCR was used to detect the relative expression of GFAP in the astrocytes treated with 1 μmol Aβ42. ^*p < 0.05, vs. control group (D) Detection of the GFAP expression in astrocytes by immunofluorescence.

Figure 2

Aβ42 induces astrocyte pyroptosis. (A) Astrocytes were stimulated with the 1 μmol Aβ42 for 48 h were harvested for PI staining and flow cytometry analysis. (B) Scanning electron microscopy was performed to observe astrocyte pyroptosis in the control and Aβ42 groups. (C) Real-time quantitative PCR was used to detect the expression of astrocyte pyroptosis-associated inflammasomes (NLRP1, NLRP3, NLRP4) in the control and Aβ42 groups. ^*p < 0.05 vs. control group. Abbreviations: Mv: microvilli; PS: pseudopods; CP: cell projection; Ch: cell hole; CO: cellular content.

Figure 3

siRNA-GSDMD inhibits astrocyte pyroptosis. (A) After the knockdown of GSDMD, astrocytes were stimulated with the 1 μmol Aβ42 for 48 h. All cells were harvested for PI staining, and flow cytometry analysis. ^*p < 0.05 vs. Aβ42 group. (B) The protein expression of GSDMD in the Aβ42 and siRNA-caspase 1/11 groups was detected by western blotting. (C) Concentrations of TNF-α, IL-1α, IL-1β and IL-18 were measured by enzyme-linked immunosorbent assay. ^*p < 0.05 vs. Aβ42 group, ^#p < 0.05 vs. Aβ42 group.

Figure 4

Real-time quantitative PCR was performed to detect the expression of eNOS, NO, ACE, ET and vWF in endothelial cells. Abbreviations: EC: endothelial cells; AS: astrocyte. ^*p < 0.05, vs. EC group, ^#p < 0.05, vs. EC + AS group, ^&p < 0.05, vs. EC + Aβ42 group, ^$p < 0.05, vs. EC + AS + Aβ42 group, ^@p < 0.05, vs. EC + AS + Aβ42 + scramble group.

Figure 5

Inhibition of astrocyte pyroptosis can alleviate brain tissue lesions in APP/PS1 mice. (A) H&E staining of brain tissues. (B) Immunohistochemical analysis and protein expression of each group of GFAP, Aβ, IL-1β, IL-18, tau, and tunnel.

Figure 6

Inhibition of astrocyte pyroptosis can alleviate vascular lesions in APP/PS1 mice. (A) H&E staining of aortic tissue. (B) Immunohistochemical analysis and protein expression of each group of eNOS, ET-1, and vwF.