Protective Effect of Clostridium butyricum on Escherichia coli-Induced Endometritis in Mice via Ameliorating Endometrial Barrier and Inhibiting Inflammatory Response.

Endometritis impairs female fertility by disrupting endometrial function, generating an unfavorable environment for embryos in the female reproductive tract and interfering with follicle function and oocyte health ( 1 , 2 ). Currently, endometritis represents a considerable worldwide economic problem for the animal husbandry industry, as it is highly correlated with reduced reproductive activity ( 3 ). The principal pathogenic bacterium reported to cause endometritis include Escherichia coli , Staphylococcus aureus , and Streptococcus spp. ( 4 ). During mating and birth, the endometrium is exposed to bacteria, which may lead to endometritis and eventually cause infertility. In endometritis, the uterus releases large amounts of inflammatory mediators after stimulation of neutrophils. Previous studies have shown that pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin (IL)-1β are involved in the development of bacterial endometritis ( 4 , 5 ). It has been determined that inhibition of these pro-inflammatory cytokines may reduce bacterial endometritis. To our knowledge, the epithelial barrier integrity of the endometrium is impaired and tight junction protein (TJP) content in uterine tissue is significantly reduced during endometritis ( 6 ). Pathogenic microorganisms activate the innate immune response of the body by binding to Toll-like receptors (TLRs) and secreting inflammation-associated factors in infected sites ( 7 ). Lipopolysaccharide (LPS) is an immunostimulatory component of the cell wall in Gram-negative bacteria such as E. coli , which can activate the TLR4/NF-κB signaling pathway and cause a strong inflammatory response ( 8 , 9 ). The innate immune defense system of the endometrium is composed primarily of three parts: TLRs, acute phase proteins, and antimicrobial peptides ( 1 ). Therefore, E. coli induces the production of cytokines and inflammatory mediators by activating TLRs on the endometrium, promoting the infiltration of neutrophils and macrophages and leading to endometritis ( 10 ). HADCs are a class of enzymes that remove acetyl groups from histone lysine residues, cause chromatin condensation, and reduce the expression of certain genes ( 11 ). These enzymes play an important regulatory role in anti-inflammatory functions and modulate the activity of the transcription factor NF-κB in different cell types ( 12 , 13 ). Antibiotics are widely used for the treatment of endometritis in clinical practice, but their misuse often leads to antibiotic resistance ( 14 ). Thus, there is an urgent need for new therapeutic agents to treat bacterial endometritis and overcome bacterial resistance ( 15 ). Probiotics influence the interaction between pathogenic microorganisms and the physiological functions of the host ( 16 ). Clostridium butyricum is a Gram-positive, rod-shaped, spore-forming obligate anaerobic bacterium that can ferment a variety of carbohydrates into butyric acid. It is also a common animal gut symbiotic bacterium and is frequently found in the environment. Previous studies have demonstrated that C. butyricum exhibits excellent protection against colitis, acute pancreatitis, bacterial vaginosis, and other inflammatory conditions ( 17 – 20 ). However, until recently, the protective effect of C. butyricum against E. coli -induced endometritis remained unknown, especially its underlying molecular mechanisms. Thus, in this trial, we evaluated the protective effect of C. butyricum on E. coli -induced endometritis in mice and investigated its potential mechanism of action.

The effects of C. butyricum on E. coli -induced histopathological changes were detected by hematoxylin and eosin (H&E) staining. As shown in Fig. 1A through F , the control group showed normal uterine tissue structure with no evident histopathological changes. Compared with the control group, the E. coli group exhibited more severe and distinct pathological changes in their uterine tissues, and diffuse infiltration of macrophages and neutrophils was observed within the endometrial glands. Furthermore, we observed stromal edema, distorted architecture, and stroma alteration in the E. coli group. However, the E. coli -induced pathological changes were markedly ameliorated by pretreatment with C. butyricum in a dose-dependent manner. Notably, the effect was more prominent at the highest dose of C. butyricum in our study, which was closely matched with the control group. In addition, compared to the damage in the E. coli group, a histological analysis showed normal morphology in the C. butyricum group. Histopathologic sections of uterine tissues (hematoxylin and eosin [H&E], ×400 magnification). (A) Control group treated with phosphate-buffered saline. (B) Group treated with Clostridium butyricum . (C) Group pretreated with Escherichia coli . (D) Group pretreated with C. butyricum (1 × 10 6 CFU/mL) and E. coli . (E) Group pretreated with C. butyricum (1 × 10 7 CFU/mL) and E. coli . (F) Group pretreated with C. butyricum (1 × 10 8 CFU/mL) and E. coli . Myeloperoxidase (MPO) is an enzyme that reflects the levels of inflammation and oxidative stress. Malondialdehyde (MDA) content is used to assess the antioxidative activity of C. butyricum on oxidative stress in E. coli -induced mice. In our research, E. coli stimulation led to significantly increased MPO activity and increased MDA levels in the E. coli group compared with the control group ( Fig. 2 ). However, treatment with C. butyricum reduced the levels of MPO activity and MDA content compared with those in the E. coli group ( Fig. 2 ). Effects of C. butyricum on myeloperoxidase (MPO) activity and malondialdehyde (MDA) content. Values are shown as the mean ± standard error of the mean (SEM). #, P < 0.01 versus control group. *, P < 0.05; **, P < 0.01 versus E. coli group. TNF-α and IL-1β play important roles in inflammatory diseases. To evaluate the effects of C. butyricum on E. coli -induced endometritis, the levels of inflammatory cytokines TNF-α and IL-1β were determined by enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 3 , E. coli stimulation induced significant increases in TNF-α and IL-1β production relative to the control group. Compared with those in the E. coli group, the levels of TNF-α and IL-1β expression were markedly reduced in a dose-dependent manner by treatment with C. butyricum . In addition, there were no significant differences between the C. butyricum group and the control group. The results showed that C. butyricum decreased E. coli -induced endometritis by downregulating the proinflammatory cytokines TNF-α and IL-1β to directly inhibit the inflammatory response. Effects of C. butyricum on tumor necrosis factor α (TNF-α) and interleukin (IL)-1β levels. Values are shown as the mean ± SEM. #, P < 0.01 versus control group. *, P < 0.05; **, P < 0.01 versus E. coli group. It has been confirmed that ZO-1, claudin-3, and occludin are important epithelial TJPs for preventing harmful substances from breaching the mucosa epithelial cells. Therefore, we furthered evaluated the effect of C. butyricum on TJP expression in the E. coli -induced endometritis model, and determined the levels of ZO-1, claudin-3, and occludin using Western blotting. As shown in Fig. 4 , compared with levels in the control group, ZO-1, claudin-3, and occludin expression were strongly significantly inhibited by E. coli treatment. However, pre-treatment with C. butyricum markedly enhanced the levels of ZO-1, claudin-3, and occludin in a dose-dependent manner. Effects of C. butyricum on ZO-1, claudin-3, and occludin expression. Values are shown as the mean ± SEM. #, P < 0.01 versus control group. *, P < 0.05; **, P < 0.01 versus E. coli group. TLR4/NF-κB plays a pivotal role in the development of inflammation. To confirm whether C. butyricum affects TLR4/NF-κB signaling pathway activation, we investigated the degrees of TLR4, IκB, and p65 protein expression by Western blotting. The data showed that the phosphorylation levels of NF-κB p65, and IκB and the levels of TLR4 were dramatically increased in the E. coli group compared with the control group ( Fig. 5 ). However, these changes were markedly downregulated in the C. butyricum administration groups compared with the E. coli group ( Fig. 5 ). Effects of C. butyricum on the expression of Toll-like receptor 4 (TLR4) and NF-κB activation. Values are shown as the mean ± SEM. #, P < 0.01 versus control group, *, P < 0.05; **, P < 0.01 versus E. coli group. The histone H3 acetylation (Ac-H3) levels in uterine tissues were measured by Western blotting. As shown in Fig. 6 , E. coli induced the downregulation of Ac-H3 protein expression in the uterus compared to that in uterine tissues without E. coli stimulation. This effect was completely prevented by C. butyricum pre-treatment in a dose-dependent manner. This result indicated that C. butyricum protected against E. coli -induced endometritis by inhibiting HDAC. Effects of C. butyricum on histone deacetylase (HDAC) expression. Values are shown as the mean ± SEM. #, P < 0.01 versus control group. *, P < 0.05; **, P < 0.01 versus E.coli group. First, we tested the effects of heat-killed C. butyricum and spent culture supernatants (SCS) from C. butyricum on TNF-α and IL-1β expression in E. coli -induced endometritis. In this study, TNF-α and IL-1β levels were reduced significantly by SCS from C. butyricum compared with those in the E. coli -treated group ( Fig. 7 ). Pretreatment with heat-killed C. butyricum did not alter TNF-α and IL-1β levels in E. coli -induced endometritis. We next evaluated the effects of inactivated C. butyricum and SCS from C. butyricum on histopathological changes in uterine tissues. In this study, pretreatment with heat-killed C. butyricum did not affect histopathological changes in uterine tissues. However, SCS from C. butyricum could inhibit E-coli-induced histopathological changes in uterine tissues ( Fig. 7 ). Effects of inactivation and spent culture supernatants (SCS) from C. butyricum on inflammatory response to endometritis. Values are shown as the mean ± SEM. #, P < 0.01 versus control group. *, P < 0.05; **, P < 0.01 versus E.coli group. To investigate the molecular mechanism that C. butyricum protects against E. coli -induced endometritis in mice, we examined butyrate levels in both the blood and uterus by gas chromatography-mass spectrometry (GC-MS). As shown in Fig. 8 , butyrate content in the blood and uterus was markedly elevated after pretreatment with C. butyricum compared with that in the control group. Thus, we concluded that butyrate was likely the active ingredient responsible for the inhibitory effect of C. butyricum on endometritis. Gas chromatography-mass spectrometry analyses of the blood and uterine tissues in C. butyricum treatment mice. Values are shown as the mean ± SEM. #, P < 0.01 versus control group.

Endometritis is considered a critical infertility element and causes enormous economic losses in the dairy industry ( 21 ). Endometritis often occurs during the postpartum period: in this period, the microbial community in the uterus fluctuates, with cycles of infection, elimination, and reinfection with bacteria ( 1 ). The bacteria most commonly isolated from animals with endometritis are E. coli , Trueperella pyogenes , and Prevotella and Bacteroides spp. ( 22 ). E. coli is the main pathogen which causes the clinical signs of uterine disease ( 23 ). Previous studies have established the endometritis model by infusing E. coli into the uteri of mice and cattle ( 24 , 25 ). Therefore, we used a mouse model of E. coli -induced endometritis to investigate the effect and mechanism of endometritis prevention in cows. Meanwhile, the treatment of endometritis attracts much discussion because animals have reduced fertility even after successful treatment. In addition, endometritis not only causes pain and suffering but also perturbs ovarian function and oocyte health ( 1 ). Therefore, we suggest that prevention is better than a cure for endometritis. Probiotics, as beneficial active microbial preparations for humans and animals, are increasingly used in the treatment of diseases and have achieved some efficacy. Our investigation revealed the protective role of C. butyricum on E. coli -induced endometritis in an experimental mice model. C. butyricum is a Gram-positive anaerobic bacterium that efficiently generates butyrate and has been shown not to produce cytotoxins ( 26 ). Additionally, it has good resistance to stomach acid and temperature ( 27 ). Several studies have demonstrated the protective effects of C. butyricum in colitis, immune response, intestinal barrier, etc. ( 17 , 28 , 29 ). Notably, many functions of gut microbiota in ameliorating disease occur via metabolites such as short-chain fatty acids (SCFAs), including acetic acid and butyric acid ( 30 ). Recently, accumulating evidence has shown that SCFAs have anti-inflammatory effects and regulate different processes, including hormone secretion, immune responses, and cell differentiation ( 31 , 32 ). Previous studies have demonstrated that SCFAs have anti-inflammatory effects such as lowering NF-κB transactivation and inhibiting TNF-α, IL-6, and myeloperoxidase activity ( 33 – 35 ). Several studies have also shown that SCFAs may regulate the integrity of the tight junction barrier and decrease intestinal permeability and risk of injury. Butyric acid, which can be produced by fermentation of C. butyricum and accounts for 85% of all intestinal SCFAs ( 36 ), has been shown to inhibit TLR4-mediated signaling and reduce HDAC levels ( 37 ). This prompted us to ask whether C. butyricum or its metabolite, butyric acid, had similar protective effects against endometritis. In the present study, we observed significant uterus pathological alterations, such as inflammatory cell infiltration, endometrial epithelial shedding, and serious structural damage, in the E. coli -induced endometritis group without C. butyricum treatment. Moreover, three different viable C. butyricum doses all mitigated uterus injury compared to E. coli alone. MPO activity is a marker of macrophage and neutrophil influx into parenchymal tissue ( 38 ). MPO levels significantly increased after E. coli injection compared with those in the control group. However, pretreatment with C. butyricum inhibited MPO activity in a dose-dependent manner in E. coli -induced endometritis mice. The MPO results also showed the anti-inflammatory activity of C. butyricum by suppressing the infiltration of inflammatory cells. In addition, E. coli significantly enhanced reactive oxygen species (ROS) levels in endometritis, contributing to the inflammatory reaction ( 39 ). MDA, a biomarker of oxidative stress ( 40 ), was dramatically increased in the E. coli group in our study, and C. butyricum significantly attenuated E. coli -induced MDA levels. In response to E. coli treatments, the production of pro-inflammatory cytokines was clearly upregulated. Pro-inflammatory cytokines such as TNF-α and IL-1β have been found in endometritis. TNF-α is the main endogenous cytokine which stimulates the production of cellular adhesion molecules, and activation of neutrophils impairs vascular endothelial cells ( 41 , 42 ). IL-1β, a subtype of IL-1, is a cytokine released early in the inflammatory response by monocytes, endothelial cells, and macrophages ( 43 ). It has been demonstrated that IL-1β is involved in the clearance of invading pathogens and stimulation of neutrophil activation ( 44 ). Consistent with results from previous studies, TNF-α and IL-1β concentrations were markedly elevated in the E. coli -induced endometritis group in this study. However, pretreatment with C. butyricum suppressed TNF-α and IL-1β expression in a dose-dependent manner. Therefore, we conclude that C. butyricum decreased inflammatory responses by reducing the release of pro-inflammatory cytokines TNF-α and IL-1β. In this context, to further evaluate the potential molecular mechanism by which C. butyricum suppresses the production of pro-inflammatory factors, we hypothesized that C. butyricum could alleviate endometritis by suppressing the TLR4/NF-κB signaling pathway and HDAC levels. E. coli contains LPS in its cytoderm, which induces an inflammatory response by triggering TLR4-mediated signal transduction pathways, including NF-κB, mitogen-activated protein kinase, and PI3K/Akt ( 8 , 45 ). As a pattern recognition receptor, TLR4 is one of the primary inflammatory signaling proteins and is crucial to activating NF-κB ( 46 ). Evidence has shown that NF-κB is an important transcription factor that plays an imperative role in inducing various pro-inflammatory chemokines and cytokines, such as IL-6, IL-1β, TNF-α, and Cox-2 ( 47 , 48 ). It is a widespread heterodimeric transcription factor, and the activation of NF-κB p65 accompanies the degradation of inhibitory protein IκBα in the proteasome. Next, the activated NF-κB p65 moves into the nucleus after phosphorylation binds to specific sites and initiates the transcription of pro-inflammatory function by regulating downstream gene expression ( 49 ). As described in Fig. 5 , C. butyricum pretreatment not only effectively inhibits IκBα degradation but also reduces NF-κB p65 gene expression. There have been few reports about augmented HDAC activity in endometritis. HDACs are related to the removal of epsilon amino groups from the lysine residue in histones, causing the agglomeration of DNA chromatin ( 50 ). Previous studies have demonstrated that inhibition of HDAC increases histone acetylation, leading to a more open structure of chromatin and promoting DNA transcription ( 51 , 52 ). Among these HDACs, HDACs 1, 2, and 3 are known for their roles in promoting pathology and inflammation ( 53 , 54 ). Therefore, inhibition of HDAC activity would be a novel therapeutic measure against endometritis. In our study, E. coli significantly decreased Ac-H3 levels, but pretreatment with C. butyricum elevated Ac-H3 levels. Our results suggest that C. butyricum ameliorates E. coli -induced endometritis by inhibiting the TLR4/NF-κB signaling pathway and the HDAC level. The vulva, vagina, cervix, and cervical mucus plug supply physical barriers to fight infection during gestation ( 1 ). The next line of endometrial defense is the columnar epithelium, which has tight junctions between cells. Recent studies have shown that tight junctions play an important role in the pathogenesis of endometritis ( 55 , 56 ). These structures are complexes composing of cytosolic and transmembrane proteins that restrict the diffusion of solutes through adjacent epithelial cells ( 56 , 57 ). Inflammatory stimuli and barrier damage lead to downregulation of the tight junctions, which is a progression of endometrial epithelial pathology ( 58 ). Furthermore, accumulating evidence has shown that maintaining tight junction complexes can prevent uterus injury ( 56 ). ZO-1, claudin-3, and occludin proteins are key components of the intact intercellular barrier at tight junctions ( 59 ). Our reports showed that there was an obvious reduction in TJPs (ZO-1, claudin-3, and occludin) in the uterus exposed to E. coli , and treatment with C. butyricum had a dramatic protective effect on the TJPs. C. butyricum is a vital probiotic to inhibit the growth of pathogenic species, and the possible mechanisms for its protective effect against endometritis include competition for nutrients and the production of SCFAs. In the present study, we investigated whether SCS from C. butyricum and heat-killed C. butyricum have similar protective effects against endometritis to that of activated C. butyricum . Also, SCS from C. butyricum inhibited the production of pro-inflammatory factors and activation of TLR4/NF-κB signaling pathways. However, the inactivated C. butyricum was not significantly discrepant with the E. coli group. These results suggest that the anti-inflammatory effects of C. butyricum may be due to the production of SCFAs, such as butyric acid and propionic acid. Interestingly, we found that both uterus and blood butyric acid levels were significantly increased after C. butyricum treatment compared to those in the control group. Other reports have definitively shown that butyrate, as a function of HDAC inhibition, has anti-inflammatory effects by lowering NF-κB transactivation and upregulating TJP expression ( 60 – 63 ). Therefore, our results are consistent with those from previous studies. For these reasons, we propose butyrate as a treatment to ameliorate E. coli -induced endometritis. In conclusion, these data suggest that C. butyricum exerted a protective effect against E. coli -induced endometritis in mice. These effects include suppression of inflammatory cytokine production, inhibition of ROS production and neutrophil infiltration, and activation of endometrial protection barriers. In particular, we elucidate the molecular mechanisms for the beneficial effects of C. butyricum , which downregulates TLR4/NF-κB signal pathways and decreases HDAC activity. Furthermore, the study suggests that butyric acid, a metabolite of C. butyricum , may be involved in the protection of C. butyricum against endometritis induced by E. coli treatment. Therefore, C. butyricum and its metabolite butyric acid could be prospective therapeutic agents for the prevention and management of endometritis in dairy cattle. Based on these encouraging results in mice, further investigations should be carried out to evaluate the protective effects of C. butyricum against endometritis in cattle.

All animal procedures were performed in strict accordance with the legislation for care and use of laboratory animals of the People’s Republic of China and approved by the Animal Ethics Committee of Jilin University. Escherichia coli O111:K58 (CVCC1450) was obtained from the China Institute of Veterinary Drug Center (Beijing, China) and cultured in nutrient Luria-Bertani (LB) broth (Sigma-Aldrich L3022, Burlington, MA, USA) overnight at 37°C with shaking. Clostridium butyricum (ATCC 19398) was obtained from the China General Microbiological Culture Collection Center and cultured in sterile Reinforced Clostridial Medium at 37°C under anaerobic conditions until the logarithmic phase of growth with a bacterial density of 0.5 at A 600 . Primary antibodies for ZO-1, occludin, claudin 3, TLR4, β-actin, and phosphorylated and nonphosphorylated forms of NF-κB were purchased from Cell Signaling Technologies, Inc. (Beverly, MA, USA). Horseradish peroxidase (HRP) secondary antibody and the primary antibody of H3, Ac-H3, were purchased from GeneTex (Irvine, CA, USA). Specific pathogen-free, female BALB/c mice (6 to 8 weeks old, 22 to 25 g) were provided from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The animals were provided with 11 to 13 h light and dark cycles under specific pathogen-free conditions with a room temperature of approximately 25 ± 5°C. Mice were raised for 1 week with free access to water and food to adapt to the feeding environment before the experiment. The inactivation and SCS of C. butyricum were performed according to a modified method ( 29 , 64 , 65 ). In summary, the culture broth of C. butyricum was centrifuged at 12,000 rpm for 10 min. Then, the bacteria were suspended in sterile phosphate-buffered saline (PBS) and heat-killed at 121°C for 30 min. Next, C. butyricum was collected, washed 3 times with PBS, and suspended in sterile saline at a final concentration of 1 × 10 8 CFU/mL for subsequent treatments. SCS from C. butyricum were obtained by centrifugation of the bacterial solution at 8,000 × g for 30 min and then filtrated through a sterile filter with a pore size of 0.22 μm and stored at low temperature. All-female BALB/c mice were stochastically divided into the following 6 experimental groups, with 8 mice in each group. (i) The control group was composed of healthy mice that did not receive any treatment. (ii) For the E. coli group, inocula containing E. coli 1 × 10 5 CFU/mL were prepared with sterile normal saline before the experiment. Mice then received a 50-μL injection of E. coli into each side of the uterus horn and no drug treatment. (iii) For the C. butyricum treatment groups, E. coli -infected mice received an intragastric dose of C. butyricum with different contents: low- (1 × 10 6 CFU/mL), medium- (1 × 10 7 CFU/mL), and high-doses (1 × 10 8 CFU/mL). Mice were then treated with 200 μL C. butyricum once a day for 1 week before E. coli infection. (iv) For the individual C. butyricum group, mice received a 1-week oral administration of C. butyricum (1 × 10 8 CFU/mL) and no other treatment. To investigate the mechanism by which C. butyricum plays a protective role in endometritis, the female mice were divided into 4 groups ( n  = 8) at random: control group, E. coli group, C. butyricum SCS (200 μL of supernatant from 1 × 10 8 CFU C. butyricum /day) + E. coli group, and inactivated C. butyricum (1 × 10 8 CFU at 200 μL/day) + E. coli group. The mice were euthanized with sodium pentobarbital after 24 h of treatment with E. coli and the uterine samples were collected and stored at −80°C until analysis. After the mice were sacrificed, the uterus tissues were gathered and washed 2 to 3 times with PBS. After immersed in 4% paraformaldehyde solution for 24 h, the tissues were embedded in paraffin, cut into 5-μm thick sections, and later stained with hematoxylin and eosin (H&E) reagent. After staining, the pathological tissue sections were subsequently observed under an optical microscope (Olympus, Japan). Photos were taken after typical spots had been found. Uterus tissues were homogenized with reaction buffer (weight:volume ratio of 1:19) using a glass homogenizer and centrifuged at 13,000 rpm to collect the supernatant. The MPO activity and MDA content in samples was determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance peak (optical density, OD) was determined at a wavelength of 460 nm by a microplate reader and defined as MPO activity = (ODtest − ODcontrol)/(11.3 × weight [g]). MDA content in uterine tissues was measured according to the manufacturer’s instructions. The effects of C. butyricum on the levels of cytokines in uterus tissues were measured by ELISA. Briefly, uterine tissues were collected from each group, homogenized in PBS on ice, and then centrifuged at 12,000 rpm for 10 min. The levels of TNF-α and IL-1β in the supernatants were determined by ELISA (BioLegend, San Diego, CA, USA) according to the manufacturer’s directions. The absorbance was measured at 450 nm using a microplate reader and the concentrations of TNF-α and IL-1β were determined using the standard curve generated from the known values. Samples of uterine tissues were collected, and the total proteins were extracted by a total protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommended protocol. The concentration of the protein was measured with a bicinchoninic acid protein test kit (Thermo Fisher Scientific). Next, protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST) for 3 h at room temperature. Subsequently, the membranes were treated with specific primary antibodies (1:1,000 to 1:2,000 dilutions) at 4°C overnight, washed 3 times for 15 min each with TBST and secondary HRP-conjugated anti-mouse (1:10,000) or anti-rabbit (1:5,000) IgG antibodies and incubated at room temperature for 2 h. After 3 washes times with TBST, the blots were detected using an enhanced chemiluminescence reagent kit (Beyotime, China) and visualized with the Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA). All data are presented as the ratio of the target protein to the internal control (β-actin). At the end of the 7-day-experimental period (1 × 10 8 CFU C. butyricum /mL), mice were euthanized, and uterus and blood samples were collected. Blood was kept on ice for 30 min and centrifuged at 3,500 rpm for 10 min at 4°C, and serum was obtained for analysis. The samples obtained for measurement of butyric acid (a short-chain fatty acid) were immediately frozen at −20°C and stored at −80°C until quantification. Butyric acid measurement was determined following a modified protocol, as previously described. First, a standard curve sample was prepared. Next, the SCFA-containing ether layers were obtained and pooled for GC-MS analysis using an Agilent 7890 gas chromatography system coupled with an Agilent 7000D mass spectrometer. The mass spectrometry data were acquired in MRM mode with a solvent delay of 3 min. All values are presented as the mean ± standard error of the mean. The images were generated by using GraphPad Prism 8.0 software (La Jolla, CA, USA) and data analysis was performed using SPSS 20.0 (SPSS Inc., Chicago, IL). Each group included three technical or biological replicates, and differences between the mean values of normally distributed data were measured by one-way analysis of variance followed by Tukey’s multiple-comparisons test. The results were considered statistically significant at P < 0.05 and very significant at P  < 0.01. The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher.