Extracellular vesicles of ADSCs inhibit ischemic stroke-induced pyroptosis through Gbp3 regulation: A role for the NLRP3/GSDMD signaling pathway.

BACKGROUND

Mounting data indicates that extracellular vesicles (EVs) have the potential to improve the injury after a stroke. Pyroptosis is a recently identified kind of programmed cell death that initiates an inflammatory reaction. We aimed to ascertain the therapeutic implications and possible molecular processes of EVs obtained from adipose-derived stem cells (ADSCs) in inhibiting pyroptosis in ischemic stroke.

METHODS

The investigation employed transient middle cerebral artery occlusion (tMCAO) rat model and a BV2 of oxygen-glucose deprivation/reoxygenation (OGD/R) to ascertain ADSCs-EVs implications on inflammation and pyroptosis as assessed by neurological deficit scores, TTC staining, IHC, HE, CCK8, WB, ELISA, and immunofluorescence. RNA-Seq was performed on BV2 cells in the control, OGD/R, and OGD/R + ADSCs-EVs groups. Using sequencing data analysis, in the OGD/R group, we screened the upregulated genes regulated by EVs, overlapped with 74 pyroptosis-related genes, and identified Guanylate-binding protein 2 (Gbp2) and Guanylate-binding protein 3 (Gbp3) as key genes. Following the validation of the sequencing results in vivo and in vitro, Gbp3 was selected for further study. To test its regulatory effects on inflammation and pyroptosis, Gbp3 was knocked down and overexpressed in vitro.

RESULTS

The administration of ADSCs-EVs resulted in a significant reduction in neurological involvement scores and reduced infarct volume in rats with tMCAO. They were also protective against BV-2 cells after OGD/R. In vivo and in vitro, ADSCs-EVs inhibited inflammatory response and pyroptosis after stroke. The outcomes of the RNA-Seq data analysis manifested that the protective implications of EVs after stroke are mediated by the modulation of inflammation-related mechanisms. Moreover, treatment with EVs led to a significant reduction in Gbp3 expression in post-ischemic brain tissue and cells. When Gbp3 was knocked down, the expression of inflammatory molecules and proteins linked to pyroptosis had a significant decline. When Gbp3 was overexpressed, the opposite results were obtained.

CONCLUSIONS

ADSCs-EVs modulate the NLRP3/GSDMD signaling pathway via Gbp3 to attenuate the inflammatory response and reduce pyroptosis that occurs after stroke.