The present study found that the concentrations of eight metabolites were significantly changed in HUVECs 48 and 72 h after burn. Deoxycholate, glucose 1-phosphate, glucose 6-phosphate, mannose 6-phosphate, histidine, and 1-methyl-2-pyrrolidone were screened for decreased concentrations of metabolites after 48 h; the concentrations of sambucinol and flufenacet significantly increased. After 72 h, the concentrations of metabolites of azelate, 1-methyl-2-pyrrolidone, guanosine monophosphate, xanthosine monophosphate, cytidine, and flufenacet decreased significantly, while the concentration of dibutyl phthalate showed an increasing trend. We could judge the stage of disease progression based on the changes in the concentrations of these metabolites. For example, the concentration of flufenacet showed a tendency to increase first and then decrease 48 h after burn. Further in-depth understanding of the mechanism can be of great help in promoting repair and treatment after burns. With the rapid rise in metabolomics, the combination of metabolomics detection technology with computational biology and orthogonal experiments can help screen diabetes-related metabolites and infer metabolic pathways.12 The results showed that HUVECs affected five metabolic pathways, including starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, histidine metabolism, and galactose metabolism, within 48 h after burn. Among these, starch and sucrose metabolism and histidine metabolism had the greatest impact. However, purine metabolism and pyrimidine metabolism pathways were mainly affected in HUVECs 72 h after burn. We might further investigate through these four metabolic pathway maps. The main markers in starch and sucrose metabolism are D-glucose 1-phosphate and D-glucose 6-phosphate, whereas the markers in amino sugar and nucleotide sugar metabolism are D-glucose 1-phosphate and D-mannose 6-phosphate. The main significantly differential metabolic marker of histidine metabolism is L-histidine. Glucose, amino acids, and FAs are the three main substrates for energy and substance production in endothelial cells, as they have been the most extensively studied. While discussing the metabolism of endothelial cells after burn, many other factors may exert an influence. However, in this study, HUVECs were used for heat treatment and metabolomics measurement and analysis. One of the advantages of cell experiments is that the cells are reproducible and not easily affected by other factors.
Under normal physiological conditions, the endothelial cells in glucose metabolism do not shun all the glucose they absorb into oxidative phosphorylation for maximizing energy (ATP), but instead rely on glycolysis.13,14 In a study on rat coronary microvascular endothelial cells, approximately 98% of glucose was metabolized to lactate, whereas only 0.04% was oxidized during the entire cycle of tricarboxylic acid. Likewise, in HUVECs, the glycolytic flux was 200-fold higher than the glucose oxidative flux. However, it seems to contradict the hypothesis that endothelial cells can directly use high oxygen levels in the blood. Choosing glycolysis over oxidative phosphorylation reduces net ATP production per mole of glucose (approximately 20-fold lower), but may confer many benefits to ECs. First, high rates of glycolysis sustain the production of lactate, which functions as a pro-angiogenic signaling molecule.16–18 Second, a prerequisite for endothelial cell sprouting into an avascular hypoxic environment is glycolysis dependent, and the interstitial glucose level is not rate limiting, but the oxygen level is rate limiting.19,20 Among these, glucose–6--phosphate is a molecule generated after the phosphorylation of glucose (on the sixth carbon). It is also a common molecule in biological cells and is involved in biochemical pathways such as the pentose phosphate pathway and glycolysis. In glycolysis, this molecule is formed in the first step, which is catalyzed by hexokinase or other similar enzymes. In glycolysis, glucose-6-phosphate is catalyzed by phosphoglucose isomerase to form fructose-6-phosphate to continue the next step. D-Glucose 1-phosphate and D-glucose 6-phosphate can be converted into each other. The concentrations of these two metabolites were significantly reduced after burns and could be used as metabolic markers to study their metabolic pathways. In endothelial cells, lower glucose levels (1mM) led to increased mitochondrial respiration, while high glucose levels (25mM) had growth-inhibitory and respiration-reduced effects.14,21 This showed that hyperglycemia rapidly increased after burns, leading to cell growth inhibition. We can search for ways to promote endothelial cell growth from the major metabolites and the enzymes that produce them. In the human body, histidine can be obtained not only from the free and protein-bound histidine in the diet but also through the proteolysis of endogenous proteins and the hydrolysis of histidine-containing peptides in the diet. Not only does it play a role in protein synthesis, but it can also be converted into histamine or carnosine, and excess can be catabolized22. Histidine has unique roles in proton buffering, metal ion sequestration, scavenging reactive oxygen species and nitrogen, erythropoiesis, and histaminergic systems. Histidine (HIS) has been studied for treating various diseases and as a nutritional supplement to enhance muscle performance. Studies have shown that supplementation with HIS increases ammonia production in muscles and synthesis of alanine and glutamine. The turnover of proteins and HIS-containing peptides, among other things, results in decreased levels of glycine, methionine, and branched-chain amino acids (valine, leucine, and isoleucine).24,25 Histidine metabolism is a key pathway physiologically implicated in satiety, recognition memory, skin, neuroprotection, and allergic diseases. In this study, when endothelial cells were damaged by scalding, the histidine metabolic pathway was affected, and its representative marker histidine showed a downward trend. Whether we can increase histidine intake to treat endothelial cells is also an aspect of treatment research.
After heat treatment for 72 h in HUVECs, the main metabolic pathways affected were purine metabolism (inosine and GMP) and pyrimidine metabolism (cytidine). Purines and pyrimidines are important components of DNA and RNA and are closely associated with metabolic syndromes and disorders such as kidney disease, gout, and diabetic nephropathy.27 Purines also help regulate energy metabolism and signal transduction,and are structural components of some coenzymes. All cells require a balanced amount of purines to grow, proliferate, and survive. Under physiological conditions, the enzymes involved in purine metabolism maintain a balanced ratio between their synthesis and degradation in cells.28 Molecular inosine not only is an important secondary metabolite in purine metabolism, but also acts as a molecular messenger in cellular signaling pathways.29 Inosine was one of the first nucleobase modifications found in nucleic acids and identified in 1965 as tRNA La30, a component of the first sequence transfer RNA (tRNA). Inosine is a purine nucleoside that forms a screen by attaching hypoxanthine to the C1 carbon of ribose through its N9 nitrogen. The ability of inosine to increase the desirability and efficiency of nonenzymatic RNA replication has now been reported.31 This finding supports that inosine is an important component of nucleic acids.32,33 Therefore, the role of creatinine is particularly important. Purine nucleotides serve as energy sources, cofactors for metabolic enzymes, and signaling molecules. The molecule inosine is a central intermediate in purine biosynthesis and degradation pathways and also plays an important role in neuronal signaling. Within 72 h after burn, we found a significant decrease in creatinine, which is of great significance to our judgment and treatment.
Nucleotide metabolism is a key pathway for the production of purine and pyrimidine molecules for DNA replication, RNA synthesis, and cellular bioenergetics.34 Within complex metabolic pathways, pyrimidine biosynthesis is conserved in all organisms and is required to maintain basic cellular functions, namely DNA and RNA biosynthesis. In terms of digestion, we found dysregulated pyrimidine metabolism in gastric cancer (GC) and established a prognostic model for GC based on differentially expressed genes in pyrimidine metabolism. Pyrimidine is a six-membered heterocyclic compound that naturally occurs in nucleic acid components (uracil, thymine, and cytosine) and vitamin B1. It is a promising guiding molecule for synthesizing various substituted compounds for treating various diseases.37 Pyrimidine not only plays important roles as an organic reaction intermediates but also has a wide range of interesting biological activities, for example, antibacterial, antifungal, anticancer, anti-inflammatory, antiviral, and antiprotozoal activities.38 The screened cytidine showed a decreasing trend after burn. According to its corresponding pyrimidine metabolic pathway, further in-depth research can be conducted and more effective treatment methods can be found. We can further study the mechanism of faster recovery of endothelial cells after burn through the metabolites and enriched pathways screened in this study. Metabolomics analyzes not just simple biomarker identification tools but also techniques that influence the active drivers of biological processes. The metabolites we screened out can not only serve as a marker for screening burn time but also affect the entire metabolic pathway and body metabolism by increasing or decreasing the amount of metabolites or the corresponding enzymes. It is clear that the metabolome can influence cellular physiology by modulating other “omics”; the genome, epigenome, transcriptome, and proteome.39 The body seems to be an interlocking metabolic chain. Currently, metabolomics is increasingly used to diagnose diseases, understand disease mechanisms, identify new drug targets, customize drug treatments, and monitor treatment outcomes.40 The significantly different metabolites we screened in this study can provide a new angle and direction. However, further experiments or other cohorts are still needed in this HUVEC heat treatment experiment to validate our conclusions, quantitatively assess significantly altered metabolites, and explore the diversity of targeted metabolic pathways.