The mammalian mitochondrial electron transport chain (ETC) includes complexes I‑IV, as well as the electron transporters ubiquinone and cytochrome c. There are two electron transport pathways in the ETC: Complex I/III/IV, with NADH as the substrate and complex II/III/IV, with succinic acid as the substrate. The electron flow is coupled with the generation of a proton gradient across the inner membrane and the energy accumulated in the proton gradient is used by complex V (ATP synthase) to produce ATP. The first part of this review briefly introduces the structure and function of complexes I‑IV and ATP synthase, including the specific electron transfer process in each complex. Some electrons are directly transferred to O2 to generate reactive oxygen species (ROS) in the ETC. The second part of this review discusses the sites of ROS generation in each ETC complex, including sites IF and IQ in complex I, site IIF in complex II and site IIIQo in complex III, and the physiological and pathological regulation of ROS. As signaling molecules, ROS play an important role in cell proliferation, hypoxia adaptation and cell fate determination, but excessive ROS can cause irreversible cell damage and even cell death. The occurrence and development of a number of diseases are closely related to ROS overproduction. Finally, proton leak and uncoupling proteins (UCPS) are discussed. Proton leak consists of basal proton leak and induced proton leak. Induced proton leak is precisely regulated and induced by UCPs. A total of five UCPs (UCP1‑5) have been identified in mammalian cells. UCP1 mainly plays a role in the maintenance of body temperature in a cold environment through non‑shivering thermogenesis. The core role of UCP2‑5 is to reduce oxidative stress under certain conditions, therefore exerting cytoprotective effects. All diseases involving oxidative stress are associated with UCPs.

The degree and duration of chemical hypoxia induced by sodium dithionite (Na₂S₂O₄) have not been reported. It is not yet clear how much reduction in the O₂ concentration (physical hypoxia) can lead to hypoxia in cultured cardiomyocytes. In this study, oxygen microelectrodes were used to measure changes in the O₂ concentration in media containing different concentrations of Na₂S₂O₄. Then, hypoxic effects of 0.8, 1.0, and 2.0 mM Na₂S₂O₄ or 1%, 3%, and 5% O₂ in cultured cardiomyocytes from neonatal rats were observed and compared. The results showed that the O₂ concentration failed to remain constant by Na₂S₂O₄ treatment during the 180-minute observation period. Only the 2.0 mM Na₂S₂O₄ group significantly increased the expression of hypoxia-inducible factor 1α (HIF-1α) and hypoxic responses. Notably, 3% O₂ only significantly increased the expression of HIF-1α in cardiomyocytes, while 1% O₂ not only increased the expression of HIF-1α but also increased the apoptotic rate in cardiomyocytes. These results suggest that Na₂S₂O₄ is not suitable for establishing a hypoxic model in cultured neonatal rat cardiomyocytes, and neonatal rat cardiomyocytes cultured at or below 1% O₂ induced significant hypoxic effects, which can be used as a starting O₂ concentration for establishing a hypoxic cell model.

Rapid adaptation to extreme hypoxia is a challenging problem, and there is no effective scheme to achieve rapid adaptation to extreme hypoxia. In this study, we found that withaferin A (WA) can significantly reduce myocardial damage, maintain cardiac function, and improve survival in rats in extremely hypoxic environments. Mechanistically, WA protects against extreme hypoxia by affecting BCL2-interacting protein 3 (BNIP3)-mediated mitophagy and the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)-mediated mitochondrial biogenesis pathway among mitochondrial quality control mechanisms. On the one hand, enhanced mitophagy eliminates hypoxia-damaged mitochondria and prevents the induction of apoptosis; on the other hand, enhanced mitochondrial biogenesis can supplement functional mitochondria and maintain mitochondrial respiration to ensure mitochondrial ATP production under acute extreme hypoxia. Our study shows that WA can be used as an effective drug to improve tolerance to extreme hypoxia.

Macrophages play crucial regulatory roles within the tumor immune microenvironment (TIME) and significantly influence tumor progression. GOLM1 has been identified as being closely associated with a variety of cancers. Nevertheless, the role of GOLM1 in macrophages on tumor progression remains unknown. This study aims to investigate the mechanism by which the GOLM1-ACLY axis in macrophages regulates tumor progression. The orthotopic hepatocellular carcinoma (HCC) inoculationand subcutaneous tumor implantation were used to establish mouse tumor models. The expression levels of the indicated proteins were assessed by immunohistochemistry and immunoblotting. Coimmunoprecipitation (Co-IP), mass spectrometry (MS), and immunofluorescence were employed to determine the interactions between proteins. Bulk RNA sequencing was used to explore the mRNA profiles in bone marrow-derived monocytes (BMDMs). Moreover, qPCR was used to quantify the mRNA levels. CUT&Tag sequence and qPCR assays were performed to assess the DNA sequences bound by H3K27ac. We identified that GOLM1 was observed to be markedly upregulated in macrophages from both liver/lung cancer patients and mouse tumor models. Macrophage deletion of GOLM1 suppressed tumor proliferation and provoked metabolic reprogramming in tumor cells. IP-MS analysis identified the Adenosine triphosphate citrate lyase (ACLY) protein as an interacting partner of GOLM1. Mechanistically, GOLM1 could bind to ACLY and impede the phosphorylation of ACLY by protein kinase A (PKA). The knockout of GOLM1 significantly increased the P-ACLY level in the nucleus of macrophage. Specifically, elevated P-ACLY significantly promoted monoacetylation at lysine 27 of histone H3 (H3K27ac). Combined analysis of CUT&Tag-seq and RNA-seq date further revealed a notable decrease of H3K27ac levels in the EFEMP1 gene, which promoted tumor proliferation via EGFR-MAPK/AKT signaling Genetic or pharmacological intervention via Acly-targeted siRNA or the ACLY inhibitor known as bempedoic acid (BA) abolished the tumor-suppressive effect induced by macrophage-specific GOLM1 deletion. Our findings reveal a novel mechanism involving the GOLM1-ACLY axis within macrophages that regulates tumor progression, which suggest a potential strategy for tumor intervention.