Advanced Glycation End-Products (AGEs) Promote Endothelial Cell Pyroptosis Under Cerebral Ischemia and Hypoxia via HIF-1α-RAGE-NLRP3.

This work mainly aimed to explore the role and mechanism of advanced glycation end-products (AGEs) in inducing cerebrovascular endothelial cell pyroptosis under oxygen glucose deprivation (OGD) condition. The mouse cerebral microvascular endothelial cells (BMECs and bEnd.3) were used as the objects to construct the OGD model in vitro. Then, cells were pretreated with AGE-modified human serum albumin (AGE-HSA). Thereafter, CCK-8 assay was conducted to detect cell viability, and flow cytometry (FCM) was performed to measure cell pyroptosis level. Meanwhile, the expression of inflammatory factors was detected by enzyme-linked immunosorbent assay (ELISA). The expression of HIF-α, NLRP3, and RAGE was detected by fluorescence staining. The opening status of cell membrane pore was observed under the electron microscope, and the expression levels of FL-GSDMD, NT-GSDMD, and caspase-1 were measured through Western Blot (WB) assay. Moreover, bEnd.3 cells were treated with siRAN-silenced NLRP3 and HIF-α inhibitor, so as to observe the effect of AGEs on cell pyroptosis level. In the mouse model, the middle cerebral artery occlusion (MCAO) model was constructed by the suture-occluded method. After intraperitoneal injection of AGEs, the pathological changes in mouse brain tissues were detected; the expression levels of NLRP3, ZO-1, and CD31 were determined by histochemical staining, and the levels of inflammatory factors and pyroptosis-related proteins were also detected. Under OGD condition, AGEs induced the pyroptosis of bEnd.3 cells, and the cell pyroptosis rate increased, higher than that of the OGD group. Meanwhile, the levels of inflammatory factors were up-regulated; the expression of HIF-α, NLRP3, and RAGE in cells increased; and the levels of NT-GSDMD and caspase-1 were markedly higher than those of the control and OGD groups. siRNA-NLRP3 or HIF-α inhibitor treatment suppressed pyroptosis and reduced the inflammatory factor levels. In mouse experiments, AGE injection aggravated brain injury in the MCAO mouse model, decreased the expression of ZO-1 and CD31, and elevated the levels of NLRP3 and inflammatory factors. Under cerebral ischemia condition, AGEs can induce endothelial cell pyroptosis via HIF-α-RAGE-NLRP3, thereby further aggravating brain injury.