Modulation of oxidized low-density lipoprotein-affected macrophage efferocytosis by mitochondrial calcium uniporter in a murine model.

OBJECTIVE: Efferocytosis dysfunction contributes to the progression and rupture of atherosclerotic plaques. Efferocytosis is crucially modulated by intracytoplasmic Ca2+, and mitochondrial calcium uniporter (MCU) complex proteins serve as key channels for regulating Ca2+ concentration. Therefore, it was speculated that MCU may affect the development of atherosclerosis (AS) by regulating efferocytosis. In the present study, we aimed to investigate whether MCU could affect foam cell formation by regulating efferocytosis. METHODS: We stimulated primary macrophages (Møs) using oxidized low-density lipoprotein (ox-LDL) to mimic the atherosclerotic microenvironment and treated them with Ru360, an MCU-specific inhibitor, and UNC1062, an inhibitor of efferocytosis. Additionally, we conducted double staining to determine the Mø efferocytosis rate. We measured the expression of MCU complexes and efferocytosis-associated proteins using western blotting (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, we separately detected the Ca2+ level in the cytoplasm and mitochondria (MT) using Fluo-4 AM and Rhod-2 methods. We separately determined the reactive oxygen species (ROS) level in cytoplasm and MT using dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probing method and Mito-SOXTM superoxide indicator staining. Additionally, we conducted the enzyme-linked immunosorbent assay (ELISA) to detect the production of interleukin-6 (IL-6), interleukin-18 (IL-18), interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α). Oil Red O staining was performed to measure cytoplasmic lipid levels. RESULTS: Ru360 attenuated ox-LDL-induced efferocytosis dysfunction, and attenuated the upregulation of MCU and MCUR1 induced by ox-LDL, and meanwhile attenuated the downregulation of MCUb induced by ox-LDL. Ru360 attenuated the decrease of intracytoplasmic Ca2+ concentration induced by ox- LDL, Ru360 also attenuated the ROS production induced by ox- LDL, attenuated the release of IL-6, IL-18, IL-1ß, and TNF-α induced by ox- LDL, and attenuated the increase of intracytoplasmic lipid content induced by ox-LDL. UNC1062 attenuated the effects of Ru360 in reducing inflammatory cytokines and intracytoplasmic lipid content. CONCLUSIONS: In this study, we found that MCU inhibition modulated intracytoplasmic Ca2+ concentration, improved impaired Mø efferocytosis, and reduced ROS generation. Macrophage efferocytosis removed apoptotic cells and prevented the release of inflammatory factor and foam cell formation, and this can be a potential new therapeutic target for alleviating atherosclerosis.

Model-based temperature control for improving lactic acid production from glycerol.

To maximize the final lactic acid productivity and concentration, temperature control was optimized using a mathematical modelling approach. A kinetic model, including cell growth, product formation and substrate consumption equations, was proposed to describe the lactic acid production process by Escherichia coli AC-521 with glycerol as the substrate. By constructing four functions, the temperature effect was introduced on the fermentation process, where four parameters (X max, µ max, Y ps and ß) were observed to be significantly affected by the temperature. For the convenience of application, the temperature control strategies were simplified by dividing the whole fermentation process into several units. In each unit, the temperature was controlled constantly. Based on the model, the optimal temperature for each unit was determined to maximize the final lactate productivity. This temperature control strategy can be effectively applied in batch and fed-batch cultures, and the verified experimental evaluation showed a good correlation with the model data. Under improved temperature control conditions, a maximal lactic acid concentration of 90.4 g L-1 was obtained after 80 h of fed-batch fermentation, giving a productivity of 1.13 g L-1 h-1, which is 1.2 times more than that in the conventional constant temperature during the cultivation course.