GOLPH3 Contributes to Endothelial Cells Pyroptosis by Promoting Golgi Dispersal in Pulmonary Arterial Hypertension

Abstract

Background: Increased release of inflammatory cytokines caused by NLRP3 inflammasome activation-dependent endothelial cells pyroptosis is involved in the progression of pulmonary arterial hypertension (PAH). Disassembly of the trans-Golgi network (TGN) results in NLRP3 oligomerization and inflammasome activation, and Golgi structural proteins are the key factors to maintain Golgi homeostasis. We attempted to demonstrate the impact of Golgi structural proteins on activation of NLRP3 inflammasome and endothelial cells pyroptosis under pro-pulmonary hypertensive condition. Methods: Lung homogenates isolated from MCT-induced PAH rats and TNF-α-induced pulmonary artery endothelial cells (PAECs) lysates were obtained to access mRNA levels of Golgi matrix proteins by real-time PCR. Silence or overexpression approaches were used to determine potential involvement of selected Golgi matrix protein in regulating NLRP3 inflammasome activation. We utilized confocal scanning and co-immunoprecipitation to confirm interaction of TGN and NLRP3. To determine PAECs pyroptosis, we used western blot, LDH release, flow cytometry assay and Hoechst/PI double staining analysis. Findings: We found up-regulated GOLPH3 in PAH rats and TNF-α-induced PAECs. Silencing GOLPH3 in PAECs repressed TNF-α-induced NLRP3 inflammasome activation, then ameliorated GSDMD-mediated PAECs pyroptosis. And GOLPH3 enhanced dispersed TGN and its interaction with NLRP3 inflammasome. Interpretation: Our findings revealed that up-regulated GOLPH3 contributes to NLRP3 inflammasome activation thus causing PAECs pyroptosis through promoting Golgi dispersal in PAH, which indicate a novel pathophysiology mechanism and therapeutic target for PAH. Funding Information: This study was supported in part by the National Natural Science Foundation of China Projects 81930012 and 81730013, and Hunan Provincial Natural Science Foundation project 2019JJ50908. Declaration of Interests: There is no conflict of interest in this manuscript. Ethics Approval Statement: All animal protocols are based on the experimental animal welfare ethics guide drawn up by the Chinese Society of Laboratory Animals. This study was approved by the Animal Experimental Center of Central South University and the Animal Experimental Center of Hunan Normal University.