https://orcid.org/0000-0002-8904-7758
Abstract
Omega-3 polyunsaturated fatty acids (n-3 PUFAs), essential fatty acids for humans and animals, have been reported to play a beneficial role in a variety of inflammatory diseases. In this study, we investigated the inhibitory effects and potential molecular mechanisms of n-3 PUFAs on the inflammatory response in lipopolysaccharide (LPS)-stimulated mammary alveolar cell line (MAC-T). Results showed that n-3 PUFAs could abate LPS-induced secretions of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β in MAC-T cells through the nuclear transcription factor kappa B (NF-κB) signal pathway. Meanwhile, n-3 PUFA intervention attenuated histopathologic changes of mammary glands, the white blood cell number decrease, and the alkaline phosphatase level decrease in the serum of mice challenged by LPS. Furthermore, n-3 PUFA intervention improved the ecological structure of the flora in terms of the structural disorder of the non-significant dominant flora induced by LPS in mice. Collectively, both in vitro and in vivo experiments revealed that n-3 PUFAs have a positive effect on LPS-induced inflammatory response, which was possibly mediated by the NF-κB signaling pathway and the intestinal microbiota.
Introduction
Mastitis causes serious hazards to bovine health, and it occurs as a result of mammary gland inflammation, often accidentally due to the contagion of environmental pathogenic microorganisms and physiological trauma, or related metabolic pathway changes [1]. Pathological changes in mammary gland tissues of diseased bovines may cause severe reductions in the quality and quantity of milk, as well as the reproductive capacity. Part of this problem is reflected in a significant increase of somatic cells in milk, and most notably the number of leukocytes [2,3]. Mammary epithelial cells (MECs) exert sentinel as well as effector functions of immune defense of the mammary gland [4]. They are the first line of defense against bacterial pathogen invasion as a physical barrier. On the other hand, pathogens that invade the mammary glands could induce MECs to express a large number of cytokines, chemokines, and factors, directly fighting off pathogens [4]. For instance, stimulation by lipopolysaccharide (LPS) causes MECs to produce cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β [5]. In addition, MECs are equipped with innate immunity receptors, such as toll-like receptor (TLR) 4 and TLR2, which allow them to sense several microbe-associated molecular patterns [6]. Escherichia coli acts as a kind of common mastitis pathogen in dairy cows, and its main pathogenic component LPS infusion in the mammary gland has been proved to elicit an immune response [7]. TLR4, as a receptor of LPS, involves in the activation of nuclear transcription factor kappa B (NF-κB). The signal transduction cascade of NF-κB is activated in response to infection, inflammation, and other stress conditions and causes rapid changes in the gene expression in the cell [8].
Antibiotics have been used as conventional drugs against mastitis [9]. However, the increasing number of multidrug-resistant pathogens has become a great and global public health threat [10]. Under such circumstances, developing more effective and safer drugs against mastitis is critical. Omega-3 polyunsaturated fatty acids (PUFAs), called ω-3 PUFAs or n-3 PUFAs, are essential fatty acids for humans. They include eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) [11]. These n-3 PUFAs play a beneficial role in a variety of inflammatory diseases, including atherosclerosis, asthma, and arthritis [12,13]. In addition, n-3 PUFAs have many other properties like anti-cardiovascular disease and anticancer [14]. Although n-3 PUFAs have become a hot research topic in the field of treatment of inflammatory disorders in the clinic, whether the early n-3 PUFA intervention has beneficial effects on bovine mastitis is not known. According to previous reports, dysbiosis of intestinal microbiota may be one cause of mastitis [15], and n-3 PUFAs as a dietary intervention alleviated circadian gut microbiota dysregulation in mice caused by a high-fat diet for the prevention of hyperlipidemia [16,17]. Previous studies showed that n-3 PUFAs provided multiple health benefits for different chronic degenerative diseases such as cardiovascular diseases [18] and rheumatoid arthritis [19] by improving the intestinal flora [20]. In this study, we aimed to evaluate the anti-inflammatory effect of n-3 PUFAs on LPS-stimulated bovine mammary gland epithelial cells and LPS-challenged mammary glands of mice. The findings of this study may have potential applications for the development of effective strategies to prevent and treat bovine mastitis.
Materials and Methods
Cell culture and treatment
The immortalized mammary alveolar cell line expressing SV-40 large T antigen (MAC-T) was a gift from Prof. Mark D. Hanigan (Virginia Polytechnic Institute and State University, Blacksburg, USA) and grown in complete medium containing Dulbecco's modified Eagle’s medium/F12 (Life Technology, Burlington, USA), 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, USA), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Gibco BRL), at 37°C at a humidified 5% CO2 environment. MAC-T cells grown to ∼80% confluence in culture dishes were treated with n-3 PUFAs (Yuwang Pharmaceutical Co. Ltd, Dezhou, China) at a series of concentrations from 0 to 1000 µg/ml for 24 h. Inflammatory responses of MAC-T cells were induced by LPS (Sigma-Aldrich, St Louis, USA) at a concentration of 10 µg/ml for 6 h according to our previous publication [21]. To explore the protective effect of n-3 PUFAs on LPS-induced inflammatory response of MAC-T cells, cells were firstly cultured in complete medium with n-3 PUFAs for 18 h. Then, LPS was added into the medium and cultured for another 6 h to induce the inflammatory response.
CCK-8 assay
Cell viability was assayed by using a Cell Counting Kit-8 (CCK-8; TransGene, Shanghai, China). In brief, 5 × 103 MAC-T cells were seeded in 96-well plates with 100 µl medium. After 6 h of incubation, cells were treated with 100 µl of n-3 PUFAs at different concentrations (0‒1000 µg/ml) for 24 h. Then, the medium was substituted with 100 µl fresh medium containing 10 µl of CCK-8 solution and incubated for another 1 h. The absorbance of each well was measured by using a microplate reader at a wavelength of 450 nm, and five parallel wells were set for each sample.
Enzyme-linked immunosorbent assay
After cells were treated with n-3 PUFAs and/or LPS as described above, the medium in each well of 6-well plates was replaced by 2 ml fresh complete medium. Then, cells were cultured for another 24 h, and supernatants were collected from cell culture. Cell debris was further removed by centrifugation at 200 g for 3 min. Cytokines TNF-α, IL-6, and IL-1β in supernatants were measured by using corresponding enzyme-linked immunoassay kits (Kanglang Biological Technology Co, Ltd, Shanghai, China) according to the manufacturer’s instructions.
Western blot analysis
Proteins from each sample were extracted by using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea) according to the manufacturer’s instruction. Total protein concentration was determined using Bradford Easy Protein Quantitative Kit (TransGene). Equal amounts of proteins were separated by 10%‒15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). Membranes were blocked for 2 h with 5% bovine serum albumin (BSA) (Sigma-Aldrich) in TBST and then incubated with antibodies against MyD88, p-IκB-α, IκB-α, p-p65, p65, and p50 (Santa Cruz Biotech, Santa Cruz, USA) or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; TransGene) at 4°C overnight. Subsequently, the membranes were washed with Tris-buffered saline with 0.5% Tween20 (TBST) for three times (5 min each), followed by incubation with their corresponding horseradish peroxidase–conjugated secondary antibodies (TransGene) at room temperature for 2 h. Immunoreactive protein bands were detected using an enhanced chemiluminescence solution (Beyotime, Shanghai, China).
Immunofluorescence analysis
Cells from each sample were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min. Subsequently, cells were blocked with 5% BSA in phosphate buffered saline (PBS) for 1 h and incubated with anti-p-p65 antibody at 4°C overnight. After being washed with PBS, samples were incubated with Alexa Fluor‐conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, USA) for 1 h at room temperature. After cells were washed with PBS, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for 10 min. Finally, immunofluorescence was then visualized under an immunofluorescence microscope (Zeiss, Jena, Germany).
Luciferase assays
MAC-T cells were grown until about 80% confluence in 48‐well culture plates and transiently co‐transfected with NF‐κB luciferase plasmids (Promega, Madison, USA) and Renilla luciferase plasmids (Promega) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). After 12 h of transfection, cells were treated with n-3 PUFAs and/or LPS as described above. Finally, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instruction.
Animal experiments
Six-week-old female Kunming strain mice were obtained from the Experimental Animal Center of Fourth Military Medical University (Xi’an, China). All animals were given ad libitum access to food and water and housed under a conventional 12-h/12-h light/dark cycle. One-week acclimatization was provided prior to further treatments. The experimental procedure was approved by the Animal Care and Use Committee of Northwest A&F University, and animals were treated in accordance with the animal welfare and ethics guidelines.
Mice were randomly divided into four groups, and they were designated as the LPS group, n-3 PUFA intervention group, normal control group, and n-3 PUFA control group. In the LPS group, a mouse model of mastitis was established by an inoculation of 50 μl of 0.2 mg/ml LPS into the L4 and R4 abdominal mammary glands according to the method described in our previous publications [22,23]. At 24 h after LPS inoculation, blood samples were collected from the orbital sinus vein, and mammary tissues were extracted from the mice that were anesthetized and sacrificed through cervical dislocation. In the n-3 PUFA intervention group, mice were administered with n-3 PUFAs (240 mg/kg/day) by gavage a month before LPS treatment. The mice administered with an equal volume of solvent of n-3 PUFAs, soybean oil, were used as the normal control. In the n-3 PUFA control group, mice were administered with n-3 PUFAs and without any other treatment.
Histopathologic examination of mammary gland tissues
Mammary tissue samples were extracted and fixed in 4% paraformaldehyde for 72 h, dehydrated with gradient alcohol, embedded in paraffin, and then stained with hematoxylin and eosin (H&E). Histopathologic changes of H&E-stained sections were examined under a light microscope.
Biochemical analysis of blood and serum
White blood cells (WBCs) were counted by using a Neubauer improved cell counting chamber. The serum biochemical parameters alkaline phosphatase (ALP), C-reactive protein (CRP), and procalcitonin (PCT) were measured with an auto-analyzer (Hitachi 7180, Tokyo, Japan).
Gut bacterial DNA extraction and 16S rDNA sequencing
Genomic DNA was extracted from intestinal contents and amplified to target the V3-V4 hyper-variable regions of 16S ribosomal DNA (rDNA) gene by using the 341F (5′-CCTACGGGRSGCAGCAG-3ʹ) and 806R (5′-GGACTACVVGGGTATCTAATC-3ʹ) primer set. The differences of intestinal flora among groups were analyzed by high-throughput sequencing of the 16S rDNA V3–V4 region with the Illumina HiSeq 250 platform (Ruiyi Biotechnologies Inc., Shanghai, China).
Both alpha and beta diversity metrics of the microbiome were generated using the QIIME package with default parameters. We used observed species diversity and Chao1 diversity indices for the comparison of bacterial operational taxonomic unit (OTU) richness and the Shannon diversity index for the comparison of bacterial OTU diversity of the gut microbiota. Principal coordinate analysis (PCoA) was performed on weighted UniFrac distances of the relative abundances (normalized for each sample) of OTUs to visualize whether there is segregation of gut microbiota structure among the three groups. To test the differences in the microbial communities of the gut, we performed analyses of similarity (ANOSIM) to calculate R and P values using the phylogeny-based weighted UniFrac distance metric. The linear discriminant analysis effect size (LEfSe) algorithm was used to analyze the differential abundance of gut microbiota among groups. The correlation network between blood and serum parameters and the intestinal microbiota was visualized with R Cytoscape software.
Statistical analysis
Statistical analyses were performed using Statistical Package for Social Sciences and GraphPad Prism 6.0. All data were expressed as mean±standard deviation (SD). The Kruskal–Wallis test with Bonferroni post hoc test was used to analyze nonparametric data. One-way analysis of variance (ANOVA) with least significant difference (LSD) t-tests was used to analyze parametric data. A value of P<0.05 indicated statistical significance.
Results
Effects of n-3 PUFAs on cell viability
To screen an appropriate dose of n-3 PUFAs for its potential anti-inflammatory function, the cytotoxicity of n-3 PUFAs on MAC-T cells was evaluated by CCK-8 assay. Results showed that cell viability was not affected by n-3 PUFAs at the concentration range of 0‒100 µg/ml (Fig. 1A). However, when the concentration exceeded 100 µg/ml, the viability of MAC-T cells gradually decreased with the increase of concentration of n-3 PUFAs up to 1000 µg/ml (Fig. 1B). In order to more representatively reflect the effect of n-3 PUFAs on LPS-stimulated MAC-T cells, n-3 PUFAs at a concentration of 50 µg/ml were used in the subsequent experiments. There was no significant difference among all the groups (Fig. 1C). These results indicated that both LPS and n-3 PUFAs did not affect the viability of MAC-T cells under this condition.
Effects of n-3 PUFAs on cell viability of MAC-T cells (A,B) Cells were treated with a series of concentrations (0-1000µg/ml) of n-3 PUFAs for 24 h, and cell viabilities were detected by CCK-8 assay. (C) Cells were cultured with 50µg/ml n-3 PUFAs in the absence or presence of 10µg/ml LPS for 24 h. Data were presented as the mean±SD (n=5). Statistical analysis was performed by one-way ANOVA with LSD t-test. **P<0.01 vs 0µg/ml.
n-3 PUFAs alleviated LPS-induced secretions of pro-inflammatory cytokines in MAC-T cells
Pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β are indicators of inflammatory response. To determine whether n-3 PUFAs have anti-inflammatory functions, the levels of TNF-α, IL-6, and IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 2, compared with the control group, LPS enhanced the secretion levels of TNF-α (Fig. 2A), IL-6 (Fig. 2B), and IL-1β (Fig. 2C), but their increases induced by LPS were inhibited by n-3 PUFA pretreatment. No significant difference was observed between the control group and n-3 PUFA group (Fig. 2A–C)
Effects of n-3 PUFAs on secretions of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the LPS-induced MAC-T cells Cells were pretreated with n-3 PUFAs before LPS stimulation as described in the ‘Material and Methods’ section, and untreated cells were used as the control. The levels of TNF-α (A), IL-6 (B), and IL-1β (C) secreted in the supernatant were measured by ELISA. Data are presented as the mean±SD of three independent experiments. Statistical analysis was performed by one-way ANOVA with LSD t-test. *P<0.05, **P<0.01.
n-3 PUFAs suppressed the activation of NF-κB signaling pathway induced by LPS
The NF-κB signaling pathway is well-known to mediate the process of inflammatory response. The protein levels of several key molecules of the NF-κB signaling pathway, including MyD88, p50, p-IκB-α, and p-p65 were detected. Results of western blot analysis showed that all of these protein expression levels were higher in the LPS group than that in the control group, and n-3 PUFA pretreatment significantly inhibited LPS-induced phosphorylation of p65 and IκB-α (Fig. 3A). Moreover, immunostaining images identically showed that n-3 PUFAs efficiently inhibited the translocation of phosphorylated p65, the subunit of NF-κB, from the cytoplasm to the nucleus in LPS-induced MAC-T cells (Fig. 3B). To further confirm this, we performed dual‐luciferase reporter assays to examine the activation of NF-κB. Results showed that n-3 PUFAs significantly inhibited the activity of the NF-κB luciferase reporter gene expression induced by LPS (Fig. 3C). These results suggested that n-3 PUFA pretreatment was able to inhibit the NF-κB activation in MAC-T cells under inflammatory conditions.
LPS-induced activation of NF-κB signaling pathway was inhibited by n-3 PUFAs (A) Representative images of western blots and quantitative data of MyD88, p-IκB-α, IκB-α, p-p65, p65, p50, and GAPDH protein expression in MAC-T cells. GAPDH was used as the loading control. (B) Immunostaining images of p-p65 (green) and nuclear staining with DAPI (blue) in MAC-T cells (scale bar: 20 μm). (C) The activity of the NF‐κB luciferase reporter gene expression was examined by the Dual-Luciferase Reporter Assay System, and the activity of firefly luciferase was normalized to that of Renilla luciferase. Statistical analysis was performed by one-way ANOVA with LSD t-test. *P<0.05, **P<0.01.
n-3 PUFAs relieved inflammatory responses of mice challenged by LPS
The effects of n-3 PUFAs on LPS-induced histopathological changes were detected by H&E staining. Mice in the normal control group and n-3 PUFA group displayed normal structures of mammary glands with large luminal area, limited stroma, and integrated alveoli (Fig. 4A). Mice in the LPS group showed severe pathologic changes. For instance, mammary alveolus obviously got thick, and a mass of inflammatory cells infiltrated into the lumen (Fig. 4A). However, n-3 PUFA intervention could significantly reduce the histologic changes of mammary glands caused by LPS (Fig. 4A).
n-3 PUFAs relieved inflammatory response of mice induced by LPS (A) Effect of n-3 PUFAs on the pathological changes of mammary tissue induced by LPS. Representative images of HE staining sections of mammary tissue of mice in the control group, LPS group, n-3 PUFA+LPS group, and n-3 PUFA group. (B) Effect of n-3 PUFAs on the levels of biochemical parameters ALP, WBC, PTC, and CRP in the blood or serum of mice. (C) n-3 PUFAs inhibited LPS-induced activation of NF-κB signaling pathway. Protein levels were measured by western blot analysis (left), and quantitative analysis of each protein was normalized to the internal control. Statistical analysis was performed by one-way ANOVA with LSD t-test. *P<0.05, **P<0.01.
In addition, the effects of n-3 PUFAs on LPS-induced changes of the number of WBC in blood and the levels of biochemical factors ALP, PCT, and CRP in serum were explored. The number of WBC in the blood of mice was significantly decreased in the LPS group compared with that in the control group, whereas n-3 PUFA intervention prevented the decrease of WBC number induced by LPS. The treatment of n-3 PUFAs without LPS challenge did not affect the number of WBC in the blood of mice (Fig. 4B). In addition, LPS treatment significantly reduced the serum level of ALP compared with the control, whereas n-3 PUFA intervention alleviated the LPS-induced decrease of ALP (Fig. 4B). However, serum levels of PCT and CRP among all experimental groups were not significantly altered (Fig. 4B).
We further analyzed the expressions of key molecules of the NF-κB signaling pathway in the animal study. Results showed that LPS injection induced the activation NF-κB pathway, as indicated by increased protein expression levels of MyD88, p-IκB-α/IκB-α, p-p65/p65, and p50 compared with the control, whereas n-3 PUFA intervention alleviated LPS-induced increases of these protein expressions (Fig. 4C). All these data indicated that n-3 PUFAs had a beneficial effect on the mastitis of mice induced by LPS through mediating the activation of the NF-κB signaling pathway.
Overall structural changes of the gut microbiota in response to LPS and n-3 PUFA intervention
To investigate the effects of n-3 PUFAs on the microbiota structure of mice, we performed a high-throughput sequencing of the 16S rDNA V3–V4 region with the Illumina HiSeq 250 platform for intestinal contents collected from mice, which produced 544,419 clean reads from 15 samples. The degrees of OTUs shared among the three groups were summarized in the Venn diagram (Fig. 5A), which showed that 478 OTUs were common to three groups, while some OTUs were still unique to each group (45 for the control group, 29 for the LPS group, and 69 for the n-3 PUFA intervention group), revealing a more OTU diversity in the n-3 PUFA intervention group.
Gut microbiota diversity analysis (A) Venn diagram summarizing the number of OTUs shared between different groups. (B) Good’s coverage was used to assess sequencing depth. (C) Observed species index (left) and Chao1 index (middle) were used to measure richness. Shannon index (right) was used to estimate diversity; NS indicates that the inter-group differences were not statistically significant. (D) ANOSIM analysis of similarity. R-values >0 indicate significant differences in the inter-group population compared with the intra-group population. P-values <0.05 indicate a significantly different level between the groups. PCoA of the gut microbiota. The percentage of variation explained by PCoA1 and PCoA2 are indicated on the axis. (E) Distances between the samples were based on similarity in OTU composition (OTU similarity: 97%). A greater distance implies lower similarity, whereas similar OTUs will cluster together. A heatmap based on weighted UniFrac distances of the 15 samples. Rows and columns represent the 15 samples. Dissimilarity between the samples was indicated by a color gradient from blue (similar) to yellow to red (dissimilar). Statistical analyses were performed by the Kruskal–Wallis test with Bonferroni post hoc test.
The alpha diversity analysis was used to evaluate the species distribution of a single sample. Based on OTU richness, Good’s coverage was over 99.60% (Fig. 5B), suggesting that the vast majority of taxonomic units were detected in the samples. The observed species and Chao1 value and Shannon value (Fig. 5C) were not significantly different among all groups, which suggested that gut microbiota abundance and diversity were comparable in all samples.
The beta-diversity analysis of ANOSIM based on weighted UniFrac ranks confirmed that statistically different bacterial community structures existed among the three groups (R=0.252, P=0.035; Fig. 5D, left). Based on principal component analysis (PCA)1 (36.01% of variance explained, P=0.298) and PCA2 (29.84% of variance explained, P=0.031) analyses of PCA on weighted UniFrac distances, all samples were grouped into three distinct clusters (Fig. 5D, right). Moreover, the comparison of heatmaps based on weighted UniFrac distances showed that lower distances were observed in the n-3 PUFA intervention or control groups when compared with the LPS group (Fig. 5E). These analyses revealed that the gut microbial communities were different among the three groups and that the pattern of microbiota in the n-3 PUFA intervention group was relatively more similar to that in the control group, while the LPS group showed a greater difference with the other two groups.
n-3 PUFAs recovered the changes of gut bacterial phenotypes of mice challenged by LPS
To shed light on the bacterial phenotypes that contribute to the differences in gut microbiota communities, LEfSe measurements were performed. The data demonstrated 19 gut bacterial clades with significant differences, and Rikenellaceae and Deferribateraceae were the predominant genera in the LPS group, which were modified by n-3 PUFA intervention (Fig. 6A). In addition, LEfSe analysis revealed 35 OTUs at the phylum (1 OTUs), class (6 OTUs), order (5 OTUs), family (7 OTUs), and genus levels (16 OTUs), which showed significant differences among the groups. Among the significantly different OTUs, Alistipes and Odoribacterwere were the two most abundant bacteria in the LPS group, while Bacteroides and Ruminococcus were the most abundant in the n-3 PUFA intervention group at the genus level (Fig. 6B).
Analysis of the structure of the gut microbiota (A) Taxonomic cladogram generated by LEfSe analysis illustrating significant changes in the gut microbiota among three groups (n=5). (B) The result of the LEfSe analysis. LEfSe was used to analyze total bacteria that contribute to the differences at the phylum, class, order, family, and genus levels (LDA score >2).
Furthermore, the top 20 dominant genera from each group were analyzed and presented in a pie chart. Prevotella, Alloprevotella, and Bacteroides were the top three dominant bacterial genera in all three groups, suggesting that the LPS challenge did not affect the main flora bacterial composition patterns of mice, regardless of whether the mice were administrated by n-3 PUFAs before LPS treatment (Fig. 7A). Interestingly, we found that the secondary main flora bacterial composition patterns of the control group (from the 4th to 7th dominant genera) and n-3 PUFA intervention group (from the 6th to 9th dominant genera) were similar, and they were Lactobacillus, Parasutterella, and Barnesiella. However, the secondary main flora bacterial composition of the LPS group was different from that of the other two groups (Fig. 7A). At the genus level, we found that LPS decreased abundances of Lactobacillus, Parasutterella, Barnesiella, and Bifidobacterium and increased abundances of Odoribacter and Alistipes of gut microbiota when compared with the control group, while n-3 PUFA intervention alleviated this abundance changes induced by LPS (Fig. 7B). These data suggested that n-3 PUFA intervention could modify the dysbacteriosis caused by LPS stimulus and restore the homeostasis of the intestinal flora microecology.
Impact of n-3 PUFA on gut microbiota composition (A) Dominant genera (top 20) assigned to control, LPS, and n-3 PUFA+LPS groups. The area of different sectors in the pie chart represents the relative abundance of the various genera. (B) The histogram showing the relative abundance of six key bacterial genera Lactobacillus, Parasutterella, Barnesiella, Bifidobacterium, Odoribacter, and Alistipes in each group. Statistical analysis was performed by one-way ANOVA with LSD t-test. *P<0.05, **P<0.01. (C) Heat map describing the correlation of the abundances of key bacterial genera and inflammation-related blood and serum parameters. The correlation heatmap was produced using the R software package, and Spearman’s rank correlation was used to test the associations between the differential genera and biochemical indexes. *P<0.05, **P<0.01.
Correlation between overall microbiota structure and inflammatory parameters
To elucidate whether the changes of gut microbiota of mice challenged with LPS are correlated with inflammatory indices, the relationships between the 11 differential microflora genera and the two types of biochemical factors WBC and ALP were analyzed using the Spearman’s correlation coefficient. It was found that the control and n-3 PUFA intervention groups that were enriched in Lactobacillus and Barnesiella exhibited positive relationships with the levels of ALP and WBC. Conversely, the LPS-induced abundance increases of Odoribacter and Alistipes showed negative correlations with the levels of WBC and ALP, respectively. Specifically, ALP and WBC levels were negatively correlated with the abundance of Anaerotruncus and Clostridium XIVb. The WBC level was significantly positively correlated with abundances of Bifidobacterium, Allobaculum, and Ruminococcus but significantly negatively correlated with abundances of Parabacteroides and Mucispirillum (Fig. 7C).
Discussion
Mastitis is one of the most frequent diseases of dairy cows, and it has a huge impact on public health, milk processing characteristics, milk quality, animal health, and farm profitability [24–26]. The treatment of mastitis is expensive, and no satisfactory control effect can be obtained [27]. In most cases, large amounts of antibacterial drugs were used for the treatment and prevention of mastitis [28]. Some studies have shown that antimicrobial treatment cannot improve the outcomes of mastitis caused by E. coli [29]. Therefore, the development of new antibiotic alternatives has become a new strategy for the treatment for mastitis. The n-3 PUFAs, as essential fatty acids for humans, play a beneficial role in a variety of inflammatory human diseases [30], including diabetes, atherosclerosis, asthma, and arthritis [13], indicating their potential anti-mastitis effects. LPS-induced mammary epithelial inflammatory response model is widely used to study the mechanism of drugs on anti-mastitis. In our previous studies, we established an inflammatory cell model of LPS-induced MAC-T [21]. In this study, we demonstrated the anti-inflammatory effects of n-3 PUFAs through both in vitro and in vivo experiments.
As we all know, inflammatory cytokines play an important role in host defense against invading pathogenic microorganisms [31]. Among these inflammatory cytokines, TNF-α, IL-6, and IL-1β are considered to be important inflammatory mediators involved in the occurrence and development of mastitis [32]. In order to explore whether n-3 PUFAs affect the secretion of inflammatory cytokines, we measured the secretion levels of TNF-α, IL-6, and IL-1β and found that n-3 PUFAs can reduce the secretion increases of TNF-α, IL-6, and IL-1β induced by LPS in MAC-T cells. Therefore, early intervention with n-3 PUFAs may be a potential alternative therapy to prevent mastitis, which is different from the administration of antibiotics after the onset of infection and the development of drug resistance, because n-3 PUFAs are generally considered to be a nutrient that can be safely used as feed additive. Our results indicate that n-3 PUFAs can reduce the inflammatory responses induced by LPS by reducing the production of these pro-inflammatory cytokines.
LPS could activate the MyD88-dependent pathways [33] and NF-κB signaling pathway [34] to induce inflammation. NF-κB is a key nuclear transcription factor that plays an extremely important role in regulating the production of pro-inflammatory cytokines. In the basal state, the NF-κB is kept inactive in the cytoplasm through binding to the inhibitory protein IκB-α. Once NF-κB is stimulated by an inducer such as LPS, the IκB-α protein becomes phosphorylated and degraded [35], and then, NF-κB p65 and p50 subunits are released and transferred to the nucleus to promote the transcription of inflammatory genes [36]. So we further investigated whether the inhibition of inflammatory response by n-3 PUFAs is through NF-κB signaling pathway. Our results demonstrated that n-3 PUFAs inhibited the phosphorylation of p65 and IκBα and the translocation and activation of NF-κB in MAC-T cells stimulated by LPS.
LPS-induced mouse mastitis model has been used as a practical method for studying bovine mastitis [37]. Striking divergence in mastitis-associated intestinal microbiota exists between diseased cows and mice, and they shared few mastitis-associated bacterial organismal or functional markers, but the mouse model of mastitis has the ability to recapitulate key physiological and immunological features of bovine mastitis [15]. We further studied the effect of n-3 PUFA intervention on LPS-induced mastitis in a mouse model and found that LPS caused serious damages to mammary tissues in histopathological examination, such as mammary lobule damages and inflammatory cell infiltration, and administration of n-3 PUFAs improved these pathological changes.
CRP, PCT, and WBC number, as markers of acute appendicitis [38], were used to measure the occurrence of LPS-induced mastitis. Although CRP and PCT did not show any significant difference between the groups, WBC count results showed that the number of WBC in the blood of mice was significantly reduced after LPS treatment, and the intervention with n-3 PUFAs corrected this reduction. Studies have shown that there is a correlation between serum ALP and inflammation in the general population [39], and our results also showed a trend similar to that of WBC counts. This study may provide a new idea to explore the clinical examination of mastitis serological indicators, but their specific mechanism is worthy of our continuous study.
It has been reported that n-3 PUFAs mediate multiple biological processes such as neurobehavioural development related to cognitive, anxiety, and social behaviors [40] and intestinal immunity and inflammation [12] by affecting the gut microbiome. It was reported that intestinal microbiota from mastitis cows could induce mastitis in germ-free mice through cow-to-mouse fecal transplantations, and it was concluded that the dysbiosis of intestinal microbiota may be one cause of mastitis [15]. In our study, we found that LPS injection could induce inflammatory reactions in mammary glands of mice, which prompted us to explore whether LPS-induced mastitis is accompanied by gut microbiota composition changes. Interestingly, we found that LPS challenges did not affect the main flora bacterial composition patterns of mice, but LPS did affect the secondary main flora bacterial composition patterns, and the intervention of n-3 PUFAs made the structure of the genus toward the normal control group, indicating a restoration of the microecology of the intestinal flora. Lactobacillus and Bifidobacterium are known as two kinds of probiotics [41] that can regulate intestinal bacteria and balance intestinal microbes and communities, adjust the intestinal transit time, and then improve the intestinal symptoms. In the intestinal immune system, probiotics can protect the host from infection, inflammatory processes, and other immune-related diseases [42]. The relationships among the interaction of these genera, n-3 PUFAs, and mastitis may be the focus of our next research work. This study indicates that n-3 PUFAs have the effect of improving the ecological structure of the flora in terms of the structural disorder of the non-significant dominant flora induced by LPS.
In summary, our results demonstrated that LPS-induced inflammatory responses of bovine MECs could be beneficially attenuated by n-3 PUFA pretreatment through inhibiting the secretion of pro-inflammatory cytokines and the activation of NF-κB signaling pathway. The n-3 PUFA intervention could effectively alleviate the pathological changes of mammary glands and hematological parameters in mice through inhibiting the activation of NF-κB signaling pathway and amending the structural changes of the intestinal microbiota of mice. These findings suggest that n-3 PUFAs may be used to prevent and treat mastitis.
Funding
The work was supported by the grant from the key research and development program of Shaanxi province (No. 2019NY-088).
Conflict of Interest
The authors declare that they have no conflict of interest.
