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.