Galloflavin

Macrophage-mediated inflammation is closely linked to the pathogenesis of acute kidney injury (AKI) and the shift of macrophages to a pro-inflammatory phenotype being reliant on glycolytic metabolism. Galloflavin, a polyphenol derived from tea, functions as a lactate dehydrogenase A (LDHA) inhibitor, effectively obstructing glycolytic metabolic pathways. However, the specific immunometabolic regulatory functions of galloflavin in macrophages remain unclear. Here, we observed that galloflavin drives alleviation of glycolytic metabolism levels in lipopolysaccharide (LPS)-induced macrophages (RAW264.7 cells and human peripheral blood mononuclear cells-derived macrophages) through downregulation of LDHA expression, thereby inhibiting macrophage conversion to a pro-inflammatory phenotype and reducing the release of inflammatory cytokines. However, the overexpression of LDHA counteracts the effects of galloflavin in macrophages. In addition, in vivo experiments observed a protective effect of galloflavin against cecal ligation and puncture (CLP) and cisplatin-induced renal injury. The ability of galloflavin to inhibit glycolysis in renal macrophages, thereby regulating their phenotypic transition during AKI was further validated through the isolation of renal primary macrophages. This intervention ultimately ameliorated the inflammatory response and decelerated the progression of AKI. Collectively, galloflavin confers protection against AKI by suppressing glycolysis in macrophages through a LDHA-dependent mechanism, thereby positioning it as a potential therapeutic option for AKI in the future.

Maintaining barrier integrity, along with cell adhesion to the extracellular matrix and the subsequent process of cell spreading, are essential functions of endothelial cells, including human retinal endothelial cells (HRECs). Disruptions in these processes can lead to vision-threatening conditions like diabetic retinopathy. However, the bioenergetic mechanisms that regulate HREC barrier function and cell spreading remain incompletely understood. This study investigates the role of lower glycolytic components in modulating these critical functions of HRECs. In vitro, Electric Cell-Substrate Impedance Sensing (ECIS) technology was used to measure real-time changes in HREC barrier integrity (electrical resistance) and cell spreading (capacitance). Pharmacological inhibitors targeting lower glycolytic components were tested: heptelidic acid for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NG-52 for phosphoglycerate kinase (PGK), shikonin for pyruvate kinase M (PKM), galloflavin for lactate dehydrogenase (LDH), AZD3965 for lactate transporter (MCT-1), and MSDC-0160 for the mitochondrial pyruvate carrier (MPC). GAPDH knockdown was performed using siRNA, and cell viability was assessed via lactate dehydrogenase (LDH) release assays. For in vivo studies, wild-type C57BL/6J mice received intravitreal injections of heptelidic acid, while control mice received vehicle (DMSO). Retinal vascular permeability was assessed by fluorescein angiography (FA) and retinal albumin leakage. The most significant decrease in electrical resistance and increase in capacitance of HRECs were observed following the dose-dependent inhibition of GAPDH and the resulting reduction in 1,3-bisphosphoglycerate (1,3-BPG) and NADH by heptelidic acid. LDH level analysis at 24-48 hours post-treatment with heptelidic acid (1 and 10 μM) showed no significant difference compared to controls, indicating that the observed disruption of HREC functionality was not due to cell death. Supporting these findings, inhibition of downstream glycolytic steps that result in the accumulation of 1,3-BPG and NADH, such as treatment with NG-52 for PGK or shikonin for PKM, led to a significant increase in electrical resistance and a decrease in cell capacitance. Furthermore, GAPDH knockdown via siRNA also led to a significant decrease in cellular resistance in HRECs. In vivo, FA imaging demonstrated that intravitreal injection of heptelidic acid led to significant retinal vascular leakage, further supported by increased albumin extravasation in treated eyes. Conversely, pharmacological inhibition of other lower glycolytic components, including LDH, MCT, and MPC, did not significantly alter HREC barrier function or spreading behavior. This study highlights the distinct roles of lower glycolytic components in regulating HREC functionality. GAPDH and its downstream products (1,3-BPG and NADH) are shown to play a pivotal role in maintaining barrier integrity and promoting HREC adhesion and spreading. These findings guide the development of targeted interventions that modulate HREC bioenergetics to treat endothelial dysfunction in various retinal disorders, while minimizing potential adverse effects on healthy endothelial cells.