Discussion
Bovine endometritis has received wide attention, as it causes major economic losses to dairy farming. Therefore, accurate and rapid diagnosis of endometritis and effective control measures are crucial to the industry. Previous studies have used metabolomics techniques to identify serum markers of purulent vaginal secretions and subclinical endometritis (Pascottini et al., 2020). In this study, we used UPLC-QTOF-MS to generate and compare the uterine secretion metabolite profiles of healthy cows and those with clinical endometritis. The PCA results revealed differences between the uterine secretion metabolite profiles of the groups. Using OPLS-DA and t -test, we identified candidate cow biomarkers and pathways linked with the endometritis disease status.
Specifically, several metabolic differences were observed in uterine secretion samples collected from endometritis cows compared to healthy cows. There were significant differences in protein and lipid metabolism between the healthy cows and those with clinical endometritis. As the endometritis developed, fewer metabolites of carbohydrate and energy metabolism were detected in the clinical endometritis cow uterine secretions, possibly because of consumption of these metabolites by the pathogen.
The potential biomarkers involved in amino acid metabolism are glutamine, L-glutamate, arginine, and phenylalanine. Glutamine is a nonessential amino acid but has many important physiological functions, such as regulating the metabolism and immune state of the body, protecting the structure and function of the intestinal barrier, and improving the antioxidant capacity of the body (Yeon-Kyung et al., 2018). Under the action of glutamine synthetase, glutamic acid is converted into glutamine, which can be metabolized into glutamic acid. Glutamic acid is a glycogenic amino acid converted into α-ketoglutarate by glutamate dehydrogenase, which is then converted into malic acid to participate in gluconeogenesis to provide energy for the body (Castell et al., 1994). However, some studies showed that L-glutamic acid is the most important excitatory neurotransmitter in the central nervous system, and a large amount of glutamic acid can cause excitotoxicity, leading to swelling, apoptosis, and necrosis of neurons (Kamel et al., 2018; Kim et al., 2018). Additionally, glutamate can reduce the ratio of Bcl-2 to Bax levels and induce apoptosis (Yamagata et al., 2008). Arginine plays an essential role in hormone secretion, endothelial function, and immunity (Sakari et al., 2015). Under the action of nitric oxide synthase, arginine is converted into citrulline and nitric oxide. Alternatively, it decomposes into urea and ornithine. Ornithine produces proline under the action of pyrroline-5-carboxylic acid reductase. Proline can be used as a free radical scavenger or converted into glutamic acid (Yoshimi et al., 2016). However, high doses of arginine can reduce the synthesis of polyamines and inhibit the synthesis of nucleic acids and proteins. Arginine is the precursor of nitric oxide (NO). After activation of nitric oxide synthase, arginine is converted into NO with the participation of NADPH. Excessive NO release can cause excessive telangiectasis, local inflammatory diffusion, and tissue oxygen utilization reduction, resulting in tissue damage (Jun et al., 2010; Sakuma et al., 1988). Under normal physiological conditions, phenylalanine is hydroxylated to tyrosine, and a transamination reaction produces a small amount of phenyl pyruvic acid. However, under pathological conditions, the conversion of phenylalanine to tyrosine is limited, and instead, it reacts with ketoglutarate to produce phenyl pyruvic acid to result in the accumulation of phenylpyruvate in vivo . Excessive accumulation of phenylpyruvate typically causes damage to the body.
Pyruvate metabolism in cows with clinical endometritis was also altered; the lactic acid and succinic acid contents were increased. The increase in lactate in the uterine secretions of cows with clinical endometritis may result from mitochondrial dysfunction and obstruction of cell respiration due to inflammation in the uterus. To meet energy requirements, anaerobic glycolysis increases, leading to lactic acid accumulation (Okorie et al., 2011). Succinic acid, a metabolite of innate immune signaling, can increase the production of IL-1β via the transcription factor hypoxia-inducible factor-1α and amplify the inflammatory effect (Tannahill et al., 2013). The increase in the succinic acid content indicates the development of inflammation.
The potential biomarkers involved in lipid metabolism are linoleic acid, arachidonic acid, lysophosphatidyl cholines, and palmitic acid. Arachidonic acid and linoleic acid are ω-6 polyunsaturated fatty acids, which are biomarkers of various cancers (Ip et al., 1999). Leukotriene, the metabolite of arachidonic acid, mediates inflammatory reactions (Fordhutchinson, 1985). Palmitic acid is the main component of saturated fatty acids in palm oil and has been shown to be associated with metabolic syndrome, cardiovascular disease, neurodegenerative disease, and inflammation (Ebbesson et al., 2015; Warensjö et al., 2005). The levels of linoleic acid, arachidonic acid, and palmitic acid in the clinical endometritis group were higher than those in the healthy group, indicating uterine inflammation.
Glycerin phospholipids are not only important components of the cell membrane but also are closely related to inflammation (Yuan et al., 2019). Lysophosphatidyl cholines (lysoPCs), as chemotactic mediators, can participate in the inflammatory process by modifying immune cell activation by specific G protein-coupled receptors (Gregor et al., 1998). When the lysoPC content is excessive, the cell membrane is damaged and further leads to disordered phospholipid metabolism. Some studies have suggested that some PCs are important biomarkers of inflammation because their levels reliably match the degree of inflammation (Song et al., 2016). LysoPC is a type of bioactive lysophospholipid which is a key signal molecule in cell and tissue metabolism and participates in plasma membrane formation, cell growth and death, and inflammatory reactions. LysoPC is also a new class of inflammatory lipids, which can combine and share metabolic pathways and regulatory mechanisms with thromboxanes, leukotrienes, and prostaglandins (Hideo et al., 2013; Seastou et al., 2013). In uterine secretions, lysoPC 16:0, lysoPC 18:2, lysoPC 17:0, and lysoPC 18:1 were identified as potential regulators of glycophospholipid metabolism. Furthermore, in dairy cows with clinical endometritis, defects in glycerin phospholipid metabolism, fatty acid metabolism, linoleic acid metabolism, and α-linolenic acid metabolism were observed.
We observed an increase in small peptides in uterine secretion samples from cows with clinical endometritis. The increase in small peptides (e.g., Val-Val-Val, Trp-Glu, Phe-His, Tyr-Leu, Trp-Ser, Gln-Phe, and others) may be due to the activities of proteolytic enzymes of endogenous or bacterial origin, or both (Moussaoui et al., 2002; Wedholm et al., 2008). Thus, the uterine secretion peptide profiles may serve as interesting metabolite biomarkers for diagnosing bovine endometritis; however, further experimental validation is necessary.
Compared with healthy cows, the levels of coumaric acid and benzoic acid, both metabolites of phenylalanine metabolism, were decreased in clinical endometritis uterine secretions. Benzoic acid, a precursor of hippurate, can inhibit the growth of coliforms (Knarreborg et al., 2002). Coumaric acid exerts essential antioxidant, cardio-protective, anti-malarial, anti-mutagenic, anti-platelet, anti-inflammatory, and immune-modulatory effects (Jiao et al., 2018; Pei et al., 2016; Pragasam et al., 2013). Notably, the uterine secretions of healthy dairy cows contain large amounts of benzoic acid and coumaric acid, protecting the uterus from invasion by Escherichia coli , and inducing anti-inflammatory effects. We also observed a significant decrease in equol content. Equol is the metabolite obtained from soybean isoflavones under the action of the intestinal microflora, which has hormone-like effects and antioxidant and immune regulatory functions (Sugiyama et al., 2013). Compared with healthy cows, the content of equol in the uterine secretion of cows with clinical endometritis is lower, indicating that the reproductive tract is affected.
Apart from confirming some known bovine endometritis biomarkers (e.g., cholesterol, salivary amylase, cortisol, uric acid, adenosine deaminase, and acetylcholinesterase), we identified new metabolites such as lactic acid, succinic acid, glutamine, palmitic acid, and benzoic acid, which varied considerably between the healthy and endometritis groups. This study improves the understanding of the pathobiology of endometritis and provides tools for diagnosing endometritis.
However, one limitation of this study was the relatively small sample size. Further experimental validation of our findings is needed prior to the application of this method to farm conditions. Different microbial infections of the uterus may significantly impact uterine secretion metabolism. A more comprehensive multi-omics analysis can further reveal the pathological and biological reactions of endometritis.