Abstract

   High temperature induces heat stress, adversely affecting the growth
   and lactation performance of cows. Research has shown the protective
   effect of taurine against hepatotoxicity both in vivo and in vitro.
   This study aimed to investigate the effect of taurine on the
   metabolomics of mammary epithelial cells of dairy cows under
   high-temperature conditions. Mammary epithelial cells were exposed to
   0 mmol/L (HS, control), 8 mmol/L (HT-8), and 32 mmol/L (HT-32) of
   taurine, then incubated at 42°C for 6 h. Metabolomics analysis was
   conducted using Liquid Chromatograph Mass Spectrometer (LC–MS).
   Compared with the HS group, 2,873 and 3,243 metabolites were detected
   in the HT-8 group in positive and negative ion modes. Among these, 108
   and 97 metabolites were significantly upregulated in positive and
   negative ion modes, while 60 and 166 metabolites were downregulated.
   Notably, 15 different metabolites such as palmitic acid, adenine and
   hypoxanthine were screened out in the HT-8 group. Compared with the HS
   group, 2,873 and 3,243 metabolites were, respectively, detected in the
   HT-32 group in the positive and negative ion modes. Among those
   metabolites, 206 metabolites were significantly up-regulated, while 206
   metabolites were significantly downregulated in the positive mode. On
   the other hand, 497 metabolites were significantly upregulated in the
   negative mode, while 517 metabolites were reported to be downregulated.
   Noteworthy, 30 distinct metabolites, such as palmitic acid,
   phytosphingosine, hypoxanthine, nonanoic acid, and octanoic acid, were
   screened out in the HT-32 group. KEGG enrichment analysis showed that
   these metabolites were mainly involved in lipid metabolism, purine
   metabolism and other biological processes. Overall, our study indicates
   that taurine supplementation alters the metabolites primarily
   associated with purine metabolism, lipid metabolism and other pathways
   to alleviate heat stress in bovine mammary epithelial cells.

   Keywords: metabolites, cattle, heat stress, metabolic pathways, taurine

1. Introduction

   In livestock production, stress can be induced by factors such as
   environment, nutrition, management, pathogens and disease ([33]1).
   Climate change-induced increases in diurnal temperatures and global
   warming have attracted increasing attention. Heat stress (HS) happens
   when an animal is not able to dissipate the heat load generated by the
   body’s metabolism and the environment, thereby losing the body’s
   thermal homeostasis. The cows under HS conditions reduce the body heat
   load by reducing dry matter intake (DMI), resulting in insufficient
   energy and nutrients being available to maintain normal production
   ([34]2–[35]4). HS reduces daily rumination time and milk production in
   lactating high-producing dairy cows ([36]5, [37]6). Furthermore, HS
   poses a significant threat to dairy farming, disrupting cows’
   productivity, reproductive performance, and overall health ([38]7,
   [39]8). In addition, animal management has become a huge challenge due
   to the increase in the number of production animals and the increased
   metabolic activity due to high temperatures ([40]9). Therefore, HS
   caused to huge economic loss on dairy production in the world.

   The structural and functional integrity of the mammary tissue is
   considered a crucial factor in the performance of lactation in cows
   ([41]10). Research has shown that environmental and management factors
   can affect mammary gland function at both molecular and cellular levels
   ([42]11). For example, high temperatures can inhibit the proliferation
   of mammary epithelial cells, causing the occurrence of various diseases
   such as mastitis, and affecting the yield and milk quality of cows
   ([43]12–[44]14). HS led to a variety of protein chaperone genes
   up-regulated and interfere with cytoskeletal and cell transport
   function in bovine mammary epithelial cells in vitro ([45]15).

   Taurine, as a sulfur-containing non-protein amino acid, is one of the
   most abundant free amino acids in mammalian tissues ([46]16). It is
   involved in many biological processes, including anti-inflammatory,
   antioxidant activities, bile acid binding, membrane stabilization,
   osmoregulation, regulation of cellular calcium flux, and
   immunomodulation ([47]17, [48]18). It has been shown to be protective
   in stress models and toxic situations such as high temperatures,
   endotoxin excitation, and high stocking densities ([49]19). Previous
   studies have shown that taurine supplementation promotes milk fat and
   protein synthesis and alleviates oxidative stress and inflammation in
   bovine mammary epithelial cells ([50]20, [51]21). Moreover, taurine can
   reverse the decreases in the activity of superoxide dismutase and
   glutathione peroxidase induced by HS, then alleviate cellular oxidative
   stress, and thus protect bovine mammary epithelial cells against HS
   ([52]22). Furthermore, the protective effect of taurine on mammary
   glands has been achieved by attenuating mammary gland epithelial
   integrity damage and inflammatory response under HS conditions
   ([53]23).

   The metabolites of the organism, especially the macromolecules, can
   directly reflect the changes in life activities in the organism
   ([54]24). Metabolomics is an important method that allows for a
   comprehensive analysis of metabolites in the organism ([55]25). It can
   be used to explore the dynamic response of living system metabolites to
   changes in endogenous or exogenous factors in a quantitative way
   ([56]26). Previous research has demonstrated that HS results in an
   elevated concentration of amino acids in the mammary epithelial cells
   of dairy cows, thereby enhancing the transportation of amino acids and
   stimulating the activity of the mTOR signaling pathway ([57]27).
   Additionally, 16 metabolites have been identified in the plasma of
   dairy cows treated with N-Carbamylglutamate (NCG), and found that NCG
   could relieve HS, resulting in an improvement in milk production under
   HS ([58]28). Furthermore, another study using analyzing metabolomics
   proved the protective effect in the yeast chromium by reducing rectal
   temperature, decreasing serum insulin concentration, and increasing
   serum glucose and plasma nicotinamide concentration to improve the
   lactation performance of dairy cows under heat stress ([59]29).

   It has been found that heat stress in dairy cows is associated with
   many metabolic pathways, such as glucose metabolism, amino acid
   metabolism, and nucleotide metabolism ([60]30, [61]31). However, the
   direct impact of taurine on the metabolism of bovine mammary epithelial
   cells under HS remains underexplored. This study aims to address this
   gap by employing liquid chromatography-mass spectrometry (LC-MS) to
   investigate the metabolite profile of bovine mammary epithelial cells
   supplemented with taurine under high-temperature conditions. By
   shedding light on the molecular mechanisms underlying taurine’s
   protective effects, this research seeks to provide a theoretical basis
   for developing targeted interventions to mitigate the adverse effects
   of HS in dairy cows.

2. Materials and methods

2.1. Cells and reagents

   The bovine mammary epithelial cells were preserved by our laboratory
   (Rongchang, Chongqing); fetal bovine serum and DMEM/F-12 medium were
   purchased from Gibco; taurine (Taurine, 99% biotech grade) was
   purchased from Macklin, and methanol (chromatographically pure,
   HPLC-grade) were purchased from Sigma.

2.2. Cell culture and treatment

   The mammary epithelial cells were cultured in DMEM/F-12 medium
   containing 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL
   streptomycin at 37°C, 5% CO[2] in an incubator, and then cultured in
   10 cm dishes at a density of 6 × 10^6 cells/mL until cells reached
   80–90% confluence, and then replaced with culture medium containing
   0 mmol/L (HS, control group), 8 mmol/L (HT-8) and 32 mmol/L (HT-32) of
   taurine, respectively. After cells were cultured at 37°C for 6 h, they
   were then transferred to 42°C for 6 h-incubation based on similar work
   by Salama et al. ([62]32–[63]34). All of the treatments were conducted
   in six biological replicates (n = 6).

2.3. Samples research topic

   After the hyperthermia treatment, the medium was discarded and washed
   twice with pre-cooled (4°C) PBS. Afterwards, the dish containing the
   cells was exposed to liquid nitrogen for 10 s and then placed on ice.
   750 μL of methanol–water (Methanol/Water = 8:2, V/V) was added to each
   dish, and the cells were scraped off with a cell scraper and
   transferred into Eppendorf tubes, while the remaining cells were
   scraped off with another 750 μL of methanol–water and put into the same
   tube. The collected samples were stored at −80°C for 30 min (mins) and
   then pulverized by ultrasound. The samples were centrifuged at 14,000 g
   for 15 min at 4°C. The supernatant was transferred in a 1.5 mL
   Eppendorf tube and stored at −80°C for measurement.

2.4. Detection of metabolites

   LC-MS/MS analysis was performed using UHPLC equipment (UHPLC3000,
   Dionex, Sunnyvale, CA, Germany). Detection conditions were presented as
   follows.

2.4.1. Chromatographic conditions

   Column: UPLC Hypersil Gold C18 column (2.1 Hypers, particle size 1.91,
   Thermo Fisher Science, United States) and Q-Exactive Orbitrapp (Thermo
   Fisher Science, United States), flow rate 0.2 mL/min, column
   temperature 35°C, injection volume 2 μL; mobile phases: solvent A
   (0.1%formic acid), solvent B (methanol containing 0.1% formic acid),
   solvent C (0.1%NH3), and D (methanol containing 0.1%NH3); gradient
   elution program: positive ions were 0–10 min, 5%B and 95%A; 10–12 min,
   5%A and 95% B; 12–13 min, 5%A and 95%B; 13.1–14 min 95%A and 5%B;
   negative ions were 0–2.5 min, 95%C and 5%D; 2.5–16.5 min, 95%D and 5%C;
   16.5–19 min, 95%D and 5%C; 19–20 min, 95%C and 5%D.

2.4.2. Mass spectrometry conditions

   MS/MS spectra were acquired in an information-dependent acquisition
   (IDA) mode using Q-ExactiveOrbitRAP under the control of the
   acquisition software (X Cup, Thermo Fisher Science, United States);
   HASI source operating parameters: sheath and auxiliary gas flow rates
   of 40 and 10 arb, respectively, capillary temperature of 320°C, full
   mass scan range m/z 70–1,050 with a resolution of 70,000; the MS/MS
   scan mode was set to a data-dependent MS2 (dd-MS2) scan with a
   resolution of 35,000, high collisional dissociation, and a spray
   voltage of 3.5 kV (positive-ion mode)/−2.5 kV (negative-ion mode) in
   NCE mode.

2.5. Data analysis

2.5.1. Data processing

   The raw data from the mass spectrometry downgauge was analyzed with
   Compound Discoverer 3.2 (Thermo Fisher, United States) for peak
   processing and peak integration. The measured mass spectral primary and
   mass spectral secondary information were matched with the mzCloud,
   Chemspider, and mzVault databases to analyze the metabolites to which
   they could be matched.

2.5.2. Analysis of differential metabolites

   Multivariate statistical analysis was performed using SIMCA 14.1
   (Umetrics, Sweden) software. The unsupervised principal component
   analysis (PCA) was used to categorize the ions detected in the positive
   and negative modes, and then the data were analyzed using Orthogonal
   Partial Least Squares Discriminant Analysis (OPLS-DA). The model was
   verified by the permutation test to find out the projected importance
   values (VIPs) of the variables. The metabolites were screened for
   differences between treatment and the control. The metabolite analysis
   was performed using the online software MetaboAnalyst 5.0. Kyoto
   Encyclopedia of Genes and Genomes (KEGG) database is a comprehensive
   database that integrates genomic, chemical, and systemic functional
   information to reveal the genetic material and chemical blueprint of
   life phenomena. The samples that met the criteria were compared with
   the KEGG database to identify the metabolites and explain their
   biological functions and physicochemical properties. For unidentified
   compounds, KEGG annotations can be used to determine their identity.
   Using the MBROLE 2.0 online website, identified differential
   metabolites were mapped to relevant metabolic pathways to better
   understand their functions and roles in biological systems.

   p values were obtained by univariate analysis of the multiplicity of
   differences (Fold-Change) and T-statistical test, combined with VIP
   values (Variable Important for the Projection) obtained by multivariate
   statistical analysis OPLS-DA to screen for differential metabolites.
   Differential metabolites were required to fulfill the following
   conditions simultaneously: (1) |Log2 Fold Change| ≥ 1; (2) P-value ≤
   0.05; (3) VIP ≥ 1.

3. Results

3.1. Multivariate statistical analysis

   In order to find the differential metabolites between the treatment and
   control groups, multivariate statistical analysis of cell lysates was
   performed. The stability of the model was indicated by QC samples.
   [64]Figure 1 showed the plots of PCA scores for the degree of
   dispersion between the groups of samples with different concentrations
   of taurine and the control group in the positive and negative ion mode,
   respectively. Almost all of the samples were within the ellipse
   representing the 95% confidence interval, and all treatment groups were
   separated from the blank group in the horizontal coordinate t [1]
   (first principal component), and the QC samples were clustered
   together, which indicated that the established system was stable and
   reliable. Further, taurine had a significant effect on the metabolism
   patterns of mammary epithelial cells of cows under high-temperature
   conditions.

Figure 1.

   [65]Figure 1
   [66]Open in a new tab

   PCA score plots of different samples. (A) PCA score plots of three
   groups in cationic mode (positive ion). (B) PCA score plots of three
   groups in anionic mode (negative ion). Green represents the quality
   control group (QC), Red represents control group (HS), blue represents
   8 mmol/L taurine group (HT-8), yellow represents 32 mmol/L taurine
   group (HT-32), (n = 6) the same as below.

   To verify the reliability of the model and to identify the potential
   biological markers affecting cellular metabolic patterns, orthogonal
   partial least squares discriminant analysis (OPLS-DA) was carried out
   between the HS, HT-8 and HT-32 groups in positive and negative ion
   modes, respectively. The parameters of the model, R^2X and R^2Y,
   represented the explanatory rate of the constructed model for the X and
   Y matrices, respectively. The value of Q^2 indicated the predictive
   ability of the model, and their sizes directly reflected the
   reliability of the model. The OPLS-DA score plots between different
   groups in positive and negative ion mode were shown in [67]Figures
   2A–[68]D, and their related model parameters, R^2 and Q^2 values, were
   shown in [69]Table 1. The R^2 values of each group were greater than
   0.5, which showed that the models of the different concentrations of
   taurine treatment groups were stable; the Q^2 values of HT-8 and HT-32
   groups in positive ion mode were 0.823, 0.836, respectively, and the
   Q^2 values of HT-8 and HT-32 group in negative ion mode were 0.780 and
   0.863, respectively, indicating that the model predictability was good.

Figure 2.

   [70]Figure 2
   [71]Open in a new tab

   OPLS-DA model and verification diagram of different samples. (A)OPLS-DA
   scores plot of HT-8 vs. HS in cationic mode. (B) OPLS-DA scores plot of
   HT-8 vs. HS in anionic mode. (C) OPLS-DA scores plot of HT-32 vs. HS in
   cationic mode. (D) OPLS-DA scores plot of HT-32 vs. HS in anionic mode.
   (E–H) OPLS-DA model verification diagram of panel (A–D).

Table 1.

   OPLS-DA model parameters with different concentrations of taurine.
                     Positive                Negative
             HT-8 vs. HS HT-32 vs. HS HT-8 vs. HS HT-32 vs. HS
   R^2X(cum)    0.674       0.534        0.603       0.526
   R^2Y(cum)    0.999       0.976        0.999       0.997
   Q^2(cum)     0.823       0.836        0.780       0.863
   [72]Open in a new tab

   In order to decide whether the data derived from the OPLS-DA analysis
   were analytically significant, the data were subjected to a permutation
   test (Permutation test). As shown in [73]Figures 2E–[74]H, the slope of
   the fitted line of the various treatment groups was greater than 0, and
   the next step in the analysis could be carried out.

3.2. Effects of taurine on the metabolite profile of the mammary epithelial
cells

   Metabolomic analysis was conducted to reveal the changes in metabolic
   profiles of the mammary epithelial cells following taurine treatment
   under high temperatures. Compared to the control group, a total of
   2,873 metabolites were detected in the positive ion mode when treated
   with 8 mmol/L taurine ([75]Figure 3A), of which 108 metabolites were
   significantly up-regulated, 60 metabolites were significantly
   down-regulated, and the remaining 2,705 metabolites did not show
   significantly different. On the other hand, a total of 3,243
   metabolites were detected in the negative ion mode, among which 97
   metabolites were significantly up-regulated, 166 metabolites were
   significantly down-regulated, and the remaining 2,980 metabolites were
   not significantly different ([76]Figure 3B). In 32 mmol/L taurine
   group, 2,873 metabolites were found in the positive ion mode, of which
   206 metabolites were significantly up-regulated, 206 metabolites were
   significantly down-regulated and the other 2,461 metabolites did not
   show significant changes ([77]Figure 3C). In addition, a total of 3,243
   metabolites were detected in negative ion mode, of which 497
   metabolites were significantly up-regulated, 517 metabolites were
   significantly down-regulated, and the remaining 2,229 metabolites
   showed no significant difference ([78]Figure 3D).

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Volcano diagram of differential expressed metabolites between different
   samples. (A) Volcano diagram of HT-8 vs. HS in cationic mode. (B)
   Volcano diagram of HT-8 vs. HS in anionic mode. (C) Volcano diagram of
   HT-32 vs. HS in cationic mode. (D) Volcano diagram of HT-32 vs. HS in
   anionic mode. Red dots represent metabolites with significant and
   upregulated differences, green dots represent metabolites with
   significant and downregulated differences, and black represents
   metabolites with non-significant differences.

3.3. Metabolite clustering analysis

   Heatmaps constructed from the peak areas of the differential
   metabolites showed the combined differentiation between treatments. The
   clustering of metabolite contents among groups could be clearly
   observed by horizontal comparison. As shown in [81]Figure 4, the HT-8
   and HT-32 groups had similar metabolite expression patterns in the
   positive ion mode, whereas the HT-8 and HS groups had similar
   metabolite expression patterns in the negative ion mode.

Figure 4.

   [82]Figure 4
   [83]Open in a new tab

   Cluster diagram of differentially expressed metabolites. (A) Cluster
   diagram of three groups in cationic mode (positive ion). (B) Cluster
   diagram of three groups in anionic mode (negative ion). Red dots
   represent up-regulated metabolites and green dots represent
   down-regulated metabolites. The vertical axis indicates clustering of
   all groups and the horizontal axis indicates clustering of all
   metabolites, with shorter cluster branches indicating higher
   similarity.

3.4. Analysis of differential metabolites

   Based on the OPLS-DA assay, potential metabolic markers were screened
   using a “multi-criteria” technique. Differential metabolites between
   treatment and the control group were screened under the conditions of
   |log2 Fold Change| ≥ 1, VIP value > 1, and P-value ≤ 0.05. As shown in
   [84]Table 2, a total of 15 differential metabolites were screened in
   the HT-8 group compared with the control group, of which
   N,N-Dimethyldecylamine N-oxide, Norethindrone, Bis(2-ethylhexyl)
   phthalate, Taurine, Bacteriopheophytin, L-Glutathione oxidized and
   PC(18:0/24:0) for seven upregulated metabolites, G Benzylpenicillin,
   Creatinine, 3-Phosphoglyceric acid, Guanosine 5′-diphosphate,
   Hypoxanthine, Adenosine 5′-monophosphate, α-D-Glucose-1,6-bisphosphate,
   Palmitic Acid as 8 downregulated metabolites.

Table 2.

   Differential metabolites of HS vs. HT-8 group.
   Name Molecular formula Log[2] FC P-value VIP Up/down
   N,N-Dimethyldecylamine N-oxide C12 H27 N O 7.54 0.000609 1.63657 Up
   Norethindrone C20 H26 O2 2.98 0.009498 1.62568 Up
   Bis(2-ethylhexyl) phthalate C24 H38 O4 2.51 0.022253 1.39681 Up
   Taurine C2 H7 N O3 S 2.15 0.000803 2.09867 Up
   Bacteriopheophytin C55 H76 N4 O6 1.7 0.015532 1.34182 Up
   L-Glutathione oxidized C20 H32 N6 O12 S2 1.5 0.036793 1.44644 Up
   PC (18:0/24:0) C50 H100 N O8 P 1.45 0.048642 1.29947 Up
   G Benzylpenicillin C16 H18 N2 O4 S −1.17 0.041552 1.42848 Down
   Creatinine C4 H7 N3 O −1.21 0.0084 1.82303 Down
   3-Phosphoglyceric acid C3 H7 O7 P −1.36 0.042315 1.71386 Down
   Guanosine 5′-diphosphate C10 H15 N5 O11 P2 −1.36 0.044289 1.63198 Down
   Hypoxanthine C5 H4 N4 O −1.45 0.001688 1.77526 Down
   Adenosine 5′-monophosphate C10 H14 N5 O7 P −1.66 0.048445 1.23457 Down
   α-D-Glucose-1,6-bisphosphate C6 H14 O12 P2 −1.83 0.039922 1.5497 Down
   Palmitic Acid C16 H32 O2 −4.92 0.002485 1.51669 Down
   [85]Open in a new tab

   As shown in [86]Table 3, a total of 30 differential metabolites were
   screened in the HT-32 group compared with the control group, including
   13 upregulated metabolites such as Phenol, Norethindrone, Nonanoic
   acid, Decanoic acid, and Guanine, and ADP, L-Glutathione (reduced),
   UDP, GDP, GTP, ATP, Hypoxanthine, Phytosphingosine,
   2-Amino-1,3-octadecanediol, Palmitic Acid and 17 other downregulated
   metabolites.

Table 3.

   Differential metabolites of HS vs. HT-32 group.
   Name Molecular formula Log[2] FC P-value VIP Up/down
   Phenol C6 H6 O 7.89 5.97E−05 1.13794 Up
   N,N-Dimethyldecylamine N-oxide C12 H27 N O 7.34 0.000522 1.18492 Up
   Taurine C2 H7 N O3 S 6.05 1.21E−07 1.58637 Up
   Norethindrone C20 H26 O2 6.02 1.75E−06 1.19654 Up
   Nonanoic acid C9 H18 O2 4.55 0.000363 1.39485 Up
   Decanoic acid C10 H20 O2 3.49 2.02E−06 1.39083 Up
   Bacteriopheophytin C55 H76 N4 O6 3.07 3.48E−06 1.48380 Up
   PC (18:0/24:0) C50 H100 N O8 P 2.6 0.004076 1.09031 Up
   Adipic acid C6 H10 O4 1.99 0.037432 1.14083 Up
   Valeric acid C5 H10 O2 1.45 4.69E−05 1.41050 Up
   Caprylic acid C8 H16 O2 1.33 0.001663 1.39478 Up
   Mangostin C24 H26 O6 1.18 0.000708 1.29905 Up
   Guanine C5 H5 N5 O 1.09 0.000243 1.22641 Up
   Adenosine diphosphate (ADP) C10 H15 N5 O10 P2 −1 0.039933 1.13877 Down
   chloroacetic acid C2 H2 Cl2 O2 −1 0.000828 1.44780 Down
   L-Histidine C6 H9 N3 O2 −1.11 0.014266 1.58664 Down
   Phosphoenolpyruvic acid C3 H5 O6 P −1.13 0.038488 1.23543 Down
   Isoniazid C6 H7 N3 O −1.14 0.036489 0.90460 Down
   DL-Arginine C6 H14 N4 O2 −1.2 0.036092 1.36733 Down
   L-Glutathione (reduced) C10 H17 N3 O6 s −1.25 0.026896 1.29385 Down
   Uridine 5′-diphosphate (UDP) C9 H14 N2 O12 P2 −1.35 0.0296 1.30708 Down
   Guanosine 5′-diphosphate (GDP) C10 H15 N5 O11 P2 −1.41 0.018383 1.25923
   Down
   Guanosine triphosphate (GTP) C10 H16 N5 O14 P3 −1.43 0.006164 1.16313
   Down
   Adenosine triphosphate (ATP) C10 H16 N5 O13 P3 −1.45 0.004106 1.10366
   Down
   2,3-Bisphospho-D-glyceric acid C3 H8 O10 P2 −1.49 0.007239 1.27946 Down
   Creatinine C4 H7 N3 O −1.75 4.95E−05 1.07949 Down
   Hypoxanthine C5 H4 N4 O −1.98 0.000226 1.65350 Down
   Phytosphingosine C18 H39 N O3 −2.4 0.015633 1.25813 Down
   2-Amino-1,3-octadecanediol C18 H39 N O2 −4.85 0.001825 1.23619 Down
   Palmitic acid C16 H32 O2 −5.5 0.000499 1.75619 Down
   [87]Open in a new tab

   Further analysis of these differential metabolites, as shown in
   [88]Figure 5, revealed that 8 mmol/L and 32 mmol/L taurine resulted in
   the upregulation of decanoic acid, valeric acid, octanoic acid, and
   oxidized glutathione, and the downregulation of ADP, ATP, hypoxanthine,
   phytosphingosine, 2-amino-1,3-octadecanediol (sphingolipid analogs),
   reduced glutathione, and palmitic acid, compared to the control.

Figure 5.

   [89]Figure 5
   [90]Open in a new tab

   A plot of the peak values of several major metabolites between
   treatments. (A-L) indicate peak plots of Arabinosylhypoxanthine, ADP,
   ATP, Phytosphingosine, 2-Amino-1,3-octadecanediol, Palmitic acid,
   Nonanoic acid, Decanoic acid, Caprylic acid, Valeric acid,
   L-Glutathione (reduced) and L-Glutathione (oxidized) between
   treatments, respectively. Values are means ± SEM (n = 3). Asterisks (*,
   ** and ***) represent significant differences with p < 0.05, p < 0.01
   and p < 0.001 respectively.

3.5. Analysis of differential metabolite metabolic pathways

   In order to identify potential biomarkers of KEGG identification, the
   online software of MetaboAnalyst 5.0 was utilized to analyze KEGG. KEGG
   pathway enrichment analysis was performed by the MBROLE.2.0·online
   website. [91]Figure 6 showed differentially expressed metabolites
   through the KEGG pathway analysis. [92]Figures 6A,[93]B showed the top
   20 KEGG metabolic pathways enriched by differential metabolites between
   the HT-8 vs. HS and HT-32 vs. HS groups, respectively. After being
   treated with 8 mmol/L taurine, purine metabolism, glutathione
   metabolism, fatty acid biosynthesis and metabolism, linoleic acid
   metabolism, and mTOR signaling pathway were significantly changed
   ([94]Figure 6A). The changes in purine metabolism, fatty acid
   metabolism, taurine and hypo-taurine metabolism, mTOR signaling
   pathway, and metabolic pathway were significant after 32 mmol/L taurine
   (HT-32) treatment ([95]Figure 6B). In conclusion, taurine affected the
   metabolic pathways: purine metabolism, amino acid metabolism, glucose
   metabolism, fatty acid metabolism and biosynthesis in bovine mammary
   epithelial cells under high temperature condition.

Figure 6.

   [96]Figure 6
   [97]Open in a new tab

   The KEGG pathway analysis of differentially expressed metabolites. (A)
   The KEGG pathway of HT-8 vs. HS. (B) The KEGG pathway of HT-32 vs. HS.
   The size of the solid circle in the figure indicated the number of
   differential metabolites enriched by the pathway, and the darkness of
   the color of the circle represents the size of the p-value.

4. Discussion

   The aim of this study was to assess the impact of taurine
   supplementation on the metabolite composition of bovine mammary
   epithelial cells subjected to high-temperature stress. In this
   experiment, employing liquid chromatography-mass spectrometry (LC-MS),
   a total of 15 differential metabolites were identified in the mammary
   epithelial cells of dairy cows by supplementation of 8 mmol/L taurine
   under high temperature, and 30 differential metabolites were identified
   with the addition of 32 mmol/L taurine, compared with the control
   group. The KEGG enrichment analysis revealed that these metabolites
   mainly regulated the pathways of purine metabolism, fatty acid
   metabolism, fatty acid biosynthesis and sphingolipid metabolism.

   Fatty acids and their derivatives are widespread in all living
   organisms, both as energy providers and as part of the composition of
   biological membranes ([98]35). They can regulate gene expression via
   activating transcription factors and cell membrane receptor signaling
   ([99]36). They also affect their own metabolism as well as the
   metabolism of other compounds by influencing gene transcription
   ([100]37). Under high temperature condition, cells need to regulate
   membrane fluidity to maintain normal physiological function, as the
   amount of saturated fatty acids in the membrane affects the fluid
   properties of the membrane. Therefore, cells increase the synthesis of
   unsaturated fatty acids to regulate membrane properties in response to
   HS ([101]38). The synthesis of short- and medium-chain unsaturated
   fatty acids are fatty acid groups consisting of a carbon chain of 3–12
   carbon atoms, which have a small molecular mass and a strong ability to
   penetrate cell membranes. Short-chain fatty acids include formic acid,
   acetic acid, and valeric acid, while medium-chain fatty acids include
   caproic acid, octanoic acid, nonanoic acid, decanoic acid, etc.
   ([102]39, [103]40). The medium chain fatty acids include hexanoic acid,
   octanoic acid, nonanoic acid and decanoic acid. Studies have shown that
   pathways related to lipid metabolisms, such as fatty acid biosynthesis
   and amino acid metabolism, are inhibited under high temperature
   conditions ([104]41). In the present study, 32 mmol/L taurine
   significantly upregulated the expression of short-chain fatty acids,
   such as caproic acid, valeric acid, nonanoic acid, and decanoic acid in
   the mammary epithelial cells of dairy cows under high temperature
   conditions. Therefore, it can be hypothesized that taurine could
   protect the cells by regulating the expression of short and medium
   chain fatty acids in the fatty acid synthesis pathway, thereby
   increasing the expression of heat shock proteins and enhancing the
   tolerance to heat stress.

   Palmitic acid (PA) is the most abundant saturated free fatty acid in
   the blood and one of the main substrates for fat synthesis ([105]42).
   PA can regulate cellular metabolism via the activation of
   phosphatidylinositol 3-kinase (PI3K) ([106]43). Excessive PA leads to
   endoplasmic reticulum stress, mitochondrial damage, and apoptosis
   ([107]44–[108]46). Excessive PA also induces programmed necrosis
   (necrotic apoptosis) in endothelial cells by initiating enhanced
   autophagy ([109]47). Besides, PA has been reported to stimulate
   hepatocyte apoptosis by inducing oxidative stress through intracellular
   generation of reactive oxygen species ([110]48) and apoptosis of bovine
   mammary epithelial cells by inducing extreme endoplasmic reticulum
   stress ([111]49). In this study, we found that 8 mmol/L and 32 mmol/L
   of taurine downregulated the expression of palmitic acid in bovine
   mammary epithelial cells under high temperature conditions. Therefore,
   taurine may activate cellular autophagy pathways, and alleviate
   endoplasmic reticulum stress, mitochondrial damage, and apoptosis
   induced by palmitic acid. Moreover, taurine can reduce cellular
   autophagy by inhibiting the PI3K pathway and inhibiting the mTOR
   pathway through down-regulation of adenosine monophosphate (AMP)
   expression. It also reduces the production of intracellular reactive
   oxygen species under high temperature conditions, thereby alleviating
   oxidative stress and to some extent relieving the damage of mammary
   epithelial cells in dairy cows under high temperature conditions.

   Sphingolipids are one of the major lipid classes in eukaryotes and play
   a crucial role in cells as structural components of membrane lipid
   bilayers and signaling molecules ([112]50, [113]51). They are related
   to important physiological and pathological processes, including cell
   adhesion, recognition of viral and bacterial toxins, skin barrier
   formation, neural function, apoptosis, and glucose metabolism ([114]52,
   [115]53). Phytosphingosine (PHS) is a sphingolipid found in plants and
   animals and is unique since it has an extra hydroxyl group compared to
   other long-chain bases ([116]54). 2-Amino-1,3-octadecanediol
   (2-Amino-1,3-octadecanediol), a sphingosine analog that inhibits
   protein kinase C, is used in the treatment of skin diseases and cancer
   ([117]55). Studies have shown that sphingolipid metabolites associated
   with PHS, as well as sphingosine 1-phosphate, can cause programmed cell
   death ([118]56). They can also directly or indirectly interfere with
   mitochondria and induce apoptosis ([119]57) and promote apoptosis by
   disrupting mitochondrial autophagy ([120]54, [121]58). Furthermore, PHS
   causes abnormal nuclear morphology, micronuclei and DNA damage,
   inhibits cell proliferation by damaging DNA, and activates the
   ATM/P53/p21 pathway, resulting in cell cycle arrest in S phase
   ([122]59). In this experiment, we found that the metabolites of PHS and
   its analog (2-Amino-1,3-octadecanediol) in the mammary epithelial cells
   of dairy cows under high temperature conditions were significantly
   downregulated by 32 mmol/L taurine. It means that taurine might
   alleviate apoptosis under high temperature conditions by
   down-regulating sphingomyelin metabolism.

   Purine nucleotides, essential components for cell proliferation,
   provide cells with energy, and help cells against the adverse effects
   of the external environment ([123]60, [124]61). When an animal is
   subjected to HS, it will release some endogenous substances, such as
   catalase and cytokines, which can affect the purine metabolic pathway
   and lead to changes in the activity of the relevant enzymes ([125]62).
   Heat stress may also lead to a change in purine metabolism to produce
   excessive free radicals and oxidative stress, which may adversely
   affect the cells ([126]63). Hypoxanthine is an important product of the
   nucleotide degradation pathway and can be used as a substrate for ATP
   synthesis ([127]64, [128]65). It can induce apoptosis by regulating the
   expression of proteins associated with apoptosis ([129]66), which
   induces cell death and ROS production. Studies have shown that HS first
   affects the animal’s feed intake and nutrient absorption, and then
   affects the body’s metabolism. During heat production, a large number
   of free radicals are generated, causing oxidative stress in the body
   ([130]67, [131]68). In this experiment, 8 and 32 mmol/L taurine
   inhibited purine metabolism by significantly down-regulating the
   production of ATP, ADP, UTP, UDP and hypoxanthine in mammary epithelial
   cells of cows at high temperature. By reducing the levels of these
   metabolites, taurine attenuates high temperature-induced oxidative
   stress and free radical release. Thus, it attenuates the damage and
   apoptosis of mammary epithelial cells in dairy cows under high
   temperature conditions.

   The results of this study emphasize the vital role of taurine in
   alleviating HS in dairy cows through various metabolic pathways. These
   effects not only help maintain the health and productivity of dairy
   herds but also have substantial implications for improving the economic
   sustainability of dairy farming operations. Therefore, ensuring
   sufficient taurine intake through careful nutritional management and
   feeding practices is essential for managing HS and optimizing overall
   herd health and performance.

   In summary, the effect of taurine on the metabolome of mammary
   epithelial cells of dairy cows under high temperature conditions is a
   complicated biological process, and the potential biomarkers are mainly
   involved in the regulation of purine metabolism, lipid metabolism,
   sphingolipid metabolism, amino acid metabolism, which constitutes a
   complicated regulatory network. However, this study primarily utilized
   metabolomics analysis to pinpoint potential biomarkers indicating
   taurine’s capacity to mitigate HS, it did not proceed to validate the
   precise mechanisms underlying the action of the selected metabolites.
   To strengthen the findings, future research could incorporate cellular
   function experiments, such as assessing cell proliferation and
   apoptosis, to validate the metabolomics results and delve further into
   taurine’s specific effects on mammary epithelial cells.

5. Conclusion

   The LC-MS technique and multivariate statistical analysis were utilized
   to screen the significantly different metabolites of the mammary
   epithelial cells of dairy cows under high-temperature conditions after
   taurine treatment with different concentrations of taurine. These
   metabolites are mainly involved in the pathways of purine metabolism,
   lipid metabolism, and sphingomyelin metabolism, which accumulates data
   for the in-depth study of the mitigation of heat stress in dairy cows
   by taurine.

Data availability statement

   The original contributions presented in the study are included in the
   article/supplementary material, further inquiries can be directed to
   the corresponding author.

Ethics statement

   The animal study was approved by the Ethics Committee/Institutional
   Review Board. The study was conducted in accordance with the local
   legislation and institutional requirements.

Author contributions

   FL: Data curation, Writing – original draft, Formal analysis,
   Methodology. LL: Methodology, Writing – review & editing,
   Visualization. ZL: Writing – review & editing, Data curation. GZ:
   Writing – review & editing. FZ: Methodology, Writing – review &
   editing. LW: Funding acquisition, Writing – review & editing,
   Supervision.

Acknowledgments