Subacute Ruminal Acidosis as a Potential Factor that Induces Endometrium Injury in Sheep

The relative changes of LPS and TNFα concentrations in rumen fluid, serum, and endometrial tissue supernatant during high-concentrate-feed-induced SARA in sheep were explored. The pH trend in rumen fluid is shown in Figure 1A. Compared with the average pH of the low-concentrate (LC) group (6.6 ± 0.29), that of the high-concentrate (HC) group rumen fluid (6.2 ± 0.09) was significantly lower. In the HC group, the rumen fluid pH value remained in the 5.2–5.8 range for more than 3 h. In addition, the LPS and TNFα concentrations in the rumen fluid, serum, and endometrial tissue supernatant of the HC group were significantly higher than those in the LC group (p < 0.05; Figure 1B,C).

Through hematoxylin-eosin staining and qRT-PCR, we investigated whether the increase of LPS concentration in the endometrium of sheep during SARA would induce the inflammatory response. Hematoxylin-eosin staining revealed a large number of inflammatory cells in the endometrium of the HC group (Figure 2A). Further, the transcription levels of TLR4, NFκB, and TNFα in the endometrial tissue of the HC group increased (p < 0.05; Figure 2B).

We investigated the expression sites of claudin-1 and occludin in the uterus and the effect of SARA on the expression of claudin-1 and occludin proteins in the endometrial epithelium. Immunohistochemical staining showed that claudin-1 and occludin were positively expressed in the endometrial epithelium (Figure 3). In the HC group, the relative protein expression level of claudin-1 in the endometrial epithelial tissue of the HC group increased while that of occludin decreased (p < 0.05; Figure 4A,B).

The primary endometrial epithelial cells cultured in vitro were confirmed via cellular immunofluorescence staining, and cytokeratin 18 was found to be positively expressed in the cytoplasm of the cells (Figure 5).

To explore the effect of elevated LPS in the endometrial epithelium on epithelial cells during SARA, LPS at final concentrations of 0, 10, 50, and 100 ng/mL induced endometrial epithelial cells, and TLR4, NFκB, and TNFα mRNA relative expression levels were determined using qRT-PCR. The results showed that LPS at a concentration of 100 ng/mL increased the transcription levels of TLR4, NFκB, and TNFα (Figure 6A–C). In addition, LPS concentrations of 0, 10, 50, and 100 ng/mL resulted in increased claudin-1 relative protein expression and decreased occludin protein expression (p < 0.05; Figure 6D–F).

In order to explore the regulatory effects of E₂ and P₄ on sheep endometrial epithelial claudin-1 and occludin, epithelial cells were treated with E₂ and P₄ at final concentrations of 0, 10⁻⁹, 10⁻⁸, and 10⁻⁷ M. We found that E₂ at a concentration of 10⁻⁷ M inhibited claudin-1 protein expression, while concentrations of 10⁻⁹, 10⁻⁸, and 10⁻⁷ M promoted occludin protein expression (p < 0.05; Figure 7A,B). Meanwhile, P₄ concentrations of 10⁻⁸ and 10⁻⁷ M promoted claudin-1 protein expression and those of 10⁻⁹, 10⁻⁸, and 10⁻⁷ M inhibited occludin protein expression at (p < 0.05; Figure 7C,D).

We explored the differences in the expression of ERα, ERβ, and PGR in sheep uterine samples during the luteal phase and the effect of LPS on the transcription levels of ERα, ERβ, and PGR in sheep endometrial epithelium during SARA. ERα, ERβ, and PGR were positively expressed in the endometrial epithelium (p < 0.05; Figure 8). The transcription levels of the E₂ receptors ERα and ERβ and the P₄ receptor PGR in the endometrial epithelial tissue of the HC group were significantly lower than those of the LC group (p < 0.05; Figure 9A). LPS at final concentrations of 0, 10, 50, and 100 ng/mL induced endometrial epithelial cells. Furthermore, concentrations of 10, 50, and 100 ng/mL reduced ERα and PGR mRNA expression levels (p < 0.05; Figure 9B,D), while those of 50 and 100 ng/mL inhibited ERβ transcription (p < 0.05; Figure 9C).

We investigated the effects of E₂ and P₄ on endometrial epithelial claudin-1 and occludin, and LPS on their functions. Treatment with ICI 182,780 at a concentration of 10⁻⁶ M mitigated the effect of 10⁻⁷ M E₂ on claudin-1 and occludin (p < 0.05; Figure 10A,B). Similarly, 100 ng/mL LPS hindered the effect of 10⁻⁷ M E₂ on claudin-1 and occludin proteins (p < 0.05; Figure 10C,D). Meanwhile, treatment with RU 486, a 10⁻⁶ M P₄ receptor inhibitor, mitigated the effects of P4 on claudin-1 and occludin (p < 0.05; Figure 11A,B). LPS at a concentration of 100 ng/mL also inhibited the impact of 10⁻⁷ M P₄ on claudin-1 and occludin proteins (Figure 11C,D).

All experimental procedures were approved by the Animal Care and Use Committee of the College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu, China. The experiment was carried out during the period of anestrus in the sheep. Sexually mature but never-mated grass-fed healthy female sheep purchased from local farmers (n = 16; 7 ± 1 months old; 25 ± 5 kg) were pre-fed with corn flour and alfalfa (2:8) for one week before the start of the experiment. They were randomized into two groups: the low-concentrate (LC) group (n = 8), wherein the ratio of corn flour to alfalfa was adjusted to 7:3, and the high-concentrate (HC) group (n = 8), wherein the ratio remained unchanged. Rumen fluid was extracted from the rumen at 1 h intervals for 10 h on days 5, 6, and 7 of the 10th week of the experiment. The rumen fluid was collected by a gastric tube sheep rumen fluid sampler (MDW16; Chengdu Huazhi Kaiwu Technology Co., Ltd., Chengdu, China). In order to avoid the influence of saliva on the pH of the rumen fluid, the rumen fluid collected for the first time was discarded, and the subsequent samples were filtered through four layers of gauze. The pH of the rumen fluid was measured immediately using a pH meter (Sartorius, Goettingen, Germany). Serum and rumen fluid were collected after 10 weeks, and the uteri of luteal stage sheep (LC, n = 5; HC, n = 4) were collected after killing for subsequent experiments.

Recently collected sheep endometrial tissue (0.5 g; LC: n = 5, HC: n = 4) was weighed and homogenized at 4 °C with 100 μL of phosphate buffer (PBS). The tissue homogenate, serum, and rumen fluid were centrifuged at 3000 rpm at 4 °C for 20 min to collect the supernatant. Absorbance was measured at 450 nm according to the manufacturer’s instructions, and a standard curve was constructed to determine the relative concentrations of LPS (YFXESh00066; Yifeixue Bio Tech, Nanjing, China) and TNFα (YFXESh00022; Yifeixue Bio Tech).

Uterine tissues were fixed in 4% (v/v) paraformaldehyde with 0.1 M PBS (pH 7.4) and embedded in paraffin. Sections of 4 μm thickness were mounted onto gelatin/poly L-lysine-coated glass slides and dried in an incubator at 60 °C for 2 h. Paraffin sections were submerged in hematoxylin dyeing solution (D10393↗; Bioss, Beijing, China) for 2 min, 1% Acid Alcohol Fast Differentiation Solution (C0163M; Beyotime, Haimen, China) for 3 s, and eosin (S0159; Bioss) for 1 min. The sections were then processed through gradient alcohol and xylene dehydration. Sections were observed and photographed using an Olympus-DP73 optical microscope (Olympus, Tokyo, Japan).

Uterine tissues were fixed in 4% (v/v) paraformaldehyde with 0.1 M PBS (pH 7.4) and embedded in paraffin. Sections of 4 μm thickness were mounted onto gelatin/poly L-lysine-coated glass slides and dried in an incubator at 60 °C for 2 h. They were then processed through xylene dehydration and gradient alcohol dewaxing. An immunohistochemical streptavidin-peroxidase conjugate kit was used (SP−0023 and SP−0024; Bioss) following the manufacturer’s instructions. The sections were incubated overnight at 4 °C with antibodies (diluted 1:100) against claudin-1 (28674–1-AP; Proteintech Group, Wuhan, China), occludin (66378–1-Ig; Proteintech Group), ERα (ab−66102; Abcam), ERβ (ab−3576; Abcam), and PGR (bs−23376R; Bioss), after which they were washed with PBS. Sections were incubated with streptavidin-peroxidase (sp−0023 and sp−0024; Bioss) at 37 °C for 15–20 min and then washed with PBS. Next, the sections were incubated with HRP-labeled streptavidin (sp−0023 and sp−0024; Bioss) for 15–20 min at 37 °C, and a positive signal was observed using DAB (D10294↗; Bioss). The paraffin sections were washed and immersed in hematoxylin dyeing solution (D10393↗; Bioss) for 10–20 s, after which the dye was removed with water. The sections were then processed through gradient alcohol and xylene dehydration. For the negative control, PBS was used instead of primary antibodies for incubation, following the procedure described above. Sections were observed and photographed using an Olympus-DP73 optical microscope (Olympus).

Total RNA was extracted from endometrial tissue and cultured endometrial epithelial cells using TRIzol reagent (Solarbio, Beijing, China) and was reverse-transcribed using the Prime Script RT Reagent Kit with a cDNA Eraser (Takara Bio, Dalian, China). mRNA levels were determined via qRT-PCR using the LightCycler 480 Real-Time Detection System (Roche, Basel, Switzerland). The reaction mixtures consisted of 10 μL of 2 × SYBR Green II PCR Mix (Takara Bio), 25 μmol/L each of forward and reverse primers, 2 μL of template, and distilled water up to a final volume of 20 μL. The reaction conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 10 s. A melting curve was obtained from 65 °C to 95 °C, increasing at intervals of 0.5 °C every 5 s. β-actin was used as an internal control. Four replicates were used for each sample to ensure the accuracy of the relative expression of target genes. The primers used were designed based on the mRNA sequence data available in GenBank; TLR4 [50], NFκB, TNF-α, Erα [51,52], ERβ, PGR [53], β-actin [52], and the sequences are listed in Table 1. The relative expression of target genes was calculated using the 2^−ΔΔCT method [54].

Cells and tissue samples were lysed using a radioimmunoprecipitation assay buffer (Solarbio) containing 1:100 (v/v) phenylmethylsulfonyl fluoride (Solarbio) and were then centrifuged at 12,000 rpm for 5 min at 4 °C to obtain protein samples. The protein concentration was determined using a Bicinchoninic Acid Protein Assay Kit (Solarbio). Protein (40 μL) was mixed with 4 × loading buffer (Solarbio) and separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto polyvinylidene fluoride membranes (Millipore, Atlanta, GA, USA) and washed with Tris-buffered saline with Tween 20 (TBST; Solarbio) at room temperature for 30 min. The membranes were blocked with 5% skim milk in TBST at room temperature for 30 min and incubated with antibodies against claudin-1 (28674–1-AP; Proteintech Group) and occludin (66378–1-Ig; Proteintech Group) at 4 °C for 8 h, with β-actin (diluted 1:3000; bs−0061R; Bioss) as an internal reference. The membranes were then washed and incubated with HRP-conjugated AffiniPure goat anti-rabbit IgG (H + L) (diluted 1:4500; SA00001–2; Proteintech) or HRP-conjugated AffiniPure goat anti-mouse IgG (H + L) (diluted 1:4500; SA00001–1; Proteintech). Immunoreactivity was observed using enhanced chemiluminescence (Abnova, Taipei, Taiwan), and the signal was quantified using ImageJ 10.0 software (National Institute of Health, Bethesda, MD, USA).

To further investigate the effect of elevated LPS concentrations during SARA on the endometrial epithelium TJs, the sheep uterus of the LC group was collected and primary endometrial epithelial cells were cultured [55]. Antibiotics (50 IU/mL penicillin and 50 IU/mL streptomycin; Solarbio) at 37 °C in PBS and 75% alcohol were used to sequentially wash the endometrial tissue thrice, and the endometrial epithelium was cut with 0.2% collagenase type 4 (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 10 min. Centrifugation was performed at 1200 rpm for 5 min to remove the supernatant, followed by washing with PBS thrice. The cells were then cultured in Dulbecco’s modified Eagle medium (DMEM)/F12 complete medium suspension containing 100 IU/mL penicillin (Solarbio), 100 IU/mL streptomycin (Solarbio), and 10% fetal bovine serum at 37 °C with 95% O₂ and 5% CO₂ in a humidified incubator. The medium was changed every 2 d, and the cells were split upon reaching 80–90% confluency and cultured in six-well plates (Corning, Glendale, AR, USA), which contained 2 mL of medium. During treatment, DMEM/F12 was added to the original culture for 12 h and was used to treat according to the test needs for 24 h, after which the supernatant was discarded, and the cells were washed with PBS precooled at 4 °C three times and stored at −80 °C until use.

Cytokeratin is a component of the cytoskeleton, mainly present in all epithelial cells and some non-epithelial cells, and is used to maintain the shape of cells and resist external stress. CK18 and its type II pair CK8 are probably the most common products of the intermediate fiber gene family. For example, in some highly specialized parenchyma epithelial cells and in some endocrine cells and normal liver cells, CK8 and CK18 are the only keratin proteins present [56,57]. Therefore, primary cultured endometrial epithelial cells were identified by CK18 immunofluorescence staining. The culture medium was discarded when the cell density in the six-well plate (Corning) reached 70–80% confluency. The cells were washed with prechilled PBS at 4 °C, fixed with 4% (v/v) paraformaldehyde, washed again with PBS, and then treated with 0.1% Triton x−100 (Solarbio) at room temperature for 15 min, followed by a final wash with PBS. The cells were incubated with 5% goat serum at 37 °C for 30 min and incubated overnight at 4 °C with antibodies against cytokeratin 18 (diluted 1:300). The following day, the cells were washed with PBS and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (H + L) antibody (ab150077; Abcam) for 45 min, after which the nuclei were counterstained with 1 μg/mL 4′,6-diamidino−2-phenylindole. Images were acquired using a Revolve Omega fluorescence microscope (Apex Bio, Houston, TX, USA).

Statistical analyses were performed using SPSS 21.0 (IBM Corporation, Armonk, NY, USA). Quantitative data are presented as the mean ± standard error of the mean. All data were tested for normality and homoscedasticity and subjected to one-way analysis of variance followed by Duncan’s multiple range test. Statistical significance was set at p < 0.05.

The datasets produced and/or analyzed during the current study are available from the corresponding author upon reasonable request.