Supplementary Material for: HMGB1 induced oxidative stress and Inflammation in endothelial cells exposed to Impinging Flow

Abstract Background: Oxidative stress and inflammation contribute to many aspects of the pathological processes involved in intracranial aneurysm (IA). However, the underlying mechanism for inducing oxidative stress and inflammation under impinging flow remains unclear. Accumulating evidence has shown that High mobility group box-1 (HMGB1) is associated with oxidative stress-related chronic diseases and inflammatory responses. Therefore, we aimed to investigate whether HMGB1 is involved in oxidative stress and inflammatory responses in endothelial cells (ECs) exposed to impinging flow. Methods: We used a modified T-chamber to simulate the in vitro situation of human umbilical vein endothelial cells (HUVECs) subjected to impinging flow at the arterial bifurcation in order to analyze the effect of wall shear stress (WSS) on the ECs. To investigate the role of HMGB1 in this process, we transfected ECs with shRNA before conducting impinging flow experiments. Intracellular reactive oxygen species (ROS) were measured by flow cytometry, and malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels were measured to assess oxidative stress. Inflammation was assessed by measuring the mRNA expression levels of IL-1β, IL-6 and IL-8 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We also examined the cellular localisation of HMGB1 by immunofluorescence. Results: Exposure of HUVECs to WSS can increase the level of oxidative stress and inflammatory response. WSS increased the expression of HMGB1 in ECs and promoted the translocation of HMGB1 from cytosol to cytoplasm. When we knocked down HMGB1, the level of oxidative stress and inflammatory response caused by WSS in ECs decreased, suggesting that HMGB1 can mediate the oxidative stress and inflammatory response in HUVECs exposed to WSS. Conclusions：HMGB1 induced oxidative stress and inflammatory response in ECs exposed to Impinging Flow.