The results of the pain behavior analysis conducted in our study indicated that GIK decreased the pain behavior score, suggesting that GIK alleviated UCP in mice. Additionally, the results of the energy metabolism analysis showed that several compounds in the uterine tissue of mice treated with the combination of GIK and oxytocin, including L-citrulline, L-asparagine, ornithine, 3-phenyllactic acid, 2-phospho-D-glyceric acid, and 3-phosphoglyceric acid, increased compared to those in mice treated with oxytocin alone. Furthermore, the oxidative lipid metabolomics results indicated that the combination of GIK and oxytocin treatment decreased the levels of 19R-hydroxy PGF2α, while increasing the levels of docosahexaenoic acid (DHA),16,17-EpDPE and 8-HDoHE, and linoleic acid (9,10-EpOME and 12,13-EpOME) in the mouse uterine tissue. Conversely, the levels of linolenic acid (trans-EKODE-E-Ib) decreased in the same group compared to those in the group treated with oxytocin alone. The results of glycolysis product analysis indicated a decrease in ATP levels and an increase in lactate levels within the uterine tissue following oxytocin administration. However, the additional administration of GIK led to a recovery in ATP levels. This suggests that GIK improves uterine energy metabolism without increasing lactate accumulation. Overall, these results suggest that GIK increased the production of amino acids and DHA, while decreasing the production of prostaglandin F2α analogs. Additionally, GIK promoted glycolysis and affected the production of linoleic acid. To the best of our knowledge, this study is the first to investigate the principal metabolite responsible for UCP caused by oxytocin and how GIK relieves UCP from a metabolomics perspective.

Figure 4 illustrates the glycolysis process, which involves several intermediates, including glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate, among others. Compared to oxytocin only, the combination of GIK and oxytocin showed significant elevation in the levels of 2-phospho-D-glyceric acid and 3-phosphoglyceric acid, which are intermediates in glycolysis. Additionally, the results of glycolytic substance analysis indicated that compared to group O, group G exhibited an increase in ATP levels. These findings collectively suggest that, compared to using oxytocin alone, GIK promotes glycolysis and improves uterine energy supply.

Previous studies have reported that GIK has the potential to alleviate angina pectoris [12, 13] and skeletal muscle pain following ischemia [14].GIK provides glucose, and the insulin within it facilitates glucose uptake and utilization by cells, thus supplying energy to ischemic tissues. Research in late-term pregnant rats revealed an increase in glycogen levels in the uterine muscle layer, enhanced glycogen breakdown during labor to provide high energy, increased sensitivity of the muscle layer to insulin, and increased glucose uptake in the presence of insulin [18].This suggests that insulin and glucose play a crucial role in the energy supply to the laboring uterus. Furthermore, Anai et al. reported that pregnant women with LDH deficiency experience reduced ATP generation during uterine smooth muscle glycolysis, leading to increased UCP [19]. Disruptions in glycolytic metabolism can cause UCP and indicate that enhancing glycolysis may improve uterine energy metabolism and alleviate UCP. Based on these clues, the relief of UCP by GIK may be associated with its promotion of glycolysis and improvement of uterine energy metabolism. Morphological results of the uteri also suggest that group G exhibited more intact uterine muscle fibers and reduced interstitial edema, which may also be related to GIK’s improvement of uterine energy metabolism and mitigation of ischemic injury. However, this study did not find any differences in LDH levels among the three groups, indicating that the process by which GIK promotes glycolysis may occur upstream of lactate production. This warrants further research to provide direct evidence.

Our study found that the addition of GIK increased the levels of L-citrulline, L-asparagine, L-aspartic acid, and DL-3-phenyllactic acid in the uterine tissue of mice, compared to those in mice treated with oxytocin alone. These amino acids have been shown to improve tissue microcirculation [20,21,22,23,24].L-citrulline functions similarly to a nitric oxide (NO) supplement and can be converted to L-arginine in the body. L-arginine primarily acts through the arginine-NO pathway and generates citrulline and NO [20] in vivo. The release of NO can cause vasodilation, promote blood circulation, alleviate ischemic hypoxic injury [21], and reduce excessive oxygen consumption in muscles. Taking citrulline supplements can enhance athletic performance, increase exercise tolerance, and even alleviate post-exercise muscle soreness [22].The increase in L-citrulline levels in this study, after GIK administration, suggests that GIK may expand uterine blood vessels through the arginine-NO pathway, thereby increasing the tolerance of the uterus to ischemia and hypoxia. Ornithine can combine with carbamoyl phosphate to produce citrulline, thereby ameliorating uterine ischemia and hypoxia through the same pathway as that of citrulline. L-asparagine has peripheral vascular dilatation effects [23]. Xu et al. found that asparagine supplementation in mice significantly increased intracellular glycolysis intermediates (1,6 diphospho-fructose,6 phospho-fructose) and up-regulated the protein levels of key glycolysis enzymes (hexokinase 2, pyruvate kinase, phosphofructokinase ). They believed that asparagine could activate glycolysis, improving systemic metabolism [24]. Therefore, L-asparagine may improve uterine energy metabolism by dilating uterine blood vessels and promoting uterine glycolysis. Further, 3-phenyllactic acid is a derivative of 3,4-dihydroxyphenyllactic acid, a bioactive compound from the Chinese medicinal herb “Danshen.” It has pharmacological effects similar to those of salvianolic acid and can inhibit platelet aggregation and thrombosis, improve microcirculation, and has therapeutic effects on ischemic heart disease [25]. Therefore, 3-phenyllactic acid may also improve uterine microcirculation. In conclusion, these amino acids may play an important role in improving uterine microcirculation and promoting uterine energy metabolism.

Our analysis also revealed an increase in lactic acid levels, an end product of glycolysis, in both group G and group O compared to that in group C, thereby confirming that pressure on the uterine blood vessels during uterine contractions may lead to an increase in lactate levels due to uterine ischemia/hypoxia. However, there was no significant difference between group G and group C, indicating that although group G showed enhanced glycolysis, there was no significant lactic acid accumulation. Therefore, other metabolic pathways may be involved in lactic acid metabolism. As previously mentioned, compounds such as L-citrulline, ornithine, L-asparagine, and 3-phenyllactic-acid can improve microcirculation, which may accelerate blood flow in the uterus, and facilitate the removal and metabolism of lactic acid. However, further research is needed to clarify the specific mechanisms of this process.

Furthermore, we observed that mice in group O and group G exhibited changes in pain behavior after receiving oxytocin, with higher pain behavior scores than those in mice in group C, indicating successful creation of the UCP model. The mechanism of UCP induced by oxytocin involves the activation of G-protein-coupled oxytocin receptors in the uterus, leading to a significant increase in cellular Ca2+ concentration and prostaglandin synthesis. This results in the contraction of uterine smooth muscle, cell membrane damage, and UCP. Results of oxidative lipid metabolism analysis indicated that the expression of 10 arachidonic acids in group O was increased compared to that in group C. Among them, 5 S,15 S-DiHETE was shown to promote the contraction, migration, and proliferation of smooth muscle cells, and plays a crucial role in inducing inflammation and vasoconstriction [26]. Additionally, 6-trans-LTB4, 8-iso-PGF2α, and 15-keto-PGF2α were identified as powerful vasoconstrictors of contractile smooth muscle and pro-inflammatory substances, leading to intense contraction of uterine smooth muscle, ischemia and hypoxia, ultimately causing UCP. These results establish arachidonic acid to be the primary cause of UCP induced by oxytocin.

Linoleic acid is an essential fatty acid that plays a vital role in synthesizing phospholipids and maintaining the integrity of cell membranes. It also serves as a precursor of leukotrienes and prostaglandins [27]. Figure 7 shows that, compared to group O, group G had increased levels of linoleic acid (9, 10-epome, 12, 13-epome) and decreased levels of linolenic acid (trans-EKODE-E-Ib); therefore, the effect of changes in linoleic acid on UCP, after GIK administration, remains to be further studied. Linoleic acid is a precursor of arachidonic acid [27], which is known to induce uterine inflammation and contraction. However, this study found that the use of GIK, as opposed to oxytocin alone, reduced the production of 19R-hydroxy PGF2α (a derivant of PGF2α [28]) and effectively alleviated UCP. Furthermore, oleic acid can be converted into linolenic acid, which can then be converted into DHA substances. Compared to using only oxytocin, GIK could increase the production of DHA substances (16,17-EpDPE and 8-HDoHE). DHA has a beneficial effect on microcirculation and can reduce inflammatory responses [29, 30]. Therefore, the relief of UCP by GIK may be due to an increase in anti-inflammatory substances of DHA (16,17-EpDPE and 8-HDoHE) and a decrease in pro-inflammatory substances of 19R-hydroxy-PGF2α.

This study explore therapeutic strategies for UCP from the perspective of its pathogenesis and providing a new approach for its treatment. The GIK regimen, which encompasses glucose, potassium, and insulin, emerges as a cost-effective solution. Its deployment for myocardial safeguarding in clinical contexts over extensive periods substantiates its safety profile. For patients administered oxytocin to facilitate uterine involution in clinical settings, particularly those with intensified UCP such as individuals undergoing repeat delivery, the adjunct use of GIK alongside conventional analgesics may provide relief from UCP.

The study had some limitations. First, only energy metabolism and oxidized lipid metabolites were detected in this study; however, GIK may also act through other metabolic pathways. Second, uterine blood flow and oxygen levels were not monitored in this study because the animals were in an active state and could not be easily fixed for probing.