Introduction

Dairy cow mastitis is an inflammatory disease worldwide and causes severe economic losses in the dairy industry due to decreased milk production, higher veterinary care costs, and increased culling of dairy cows [1, 2]. Escherichia coli is one of the main pathogens in dairy cow mastitis and is widely present in bovine feces, humid soil, and composts [3]. Recent studies revealed that E. coli often causes acute inflammatory responses and might contribute to extensive mammary tissue damage [4]. There are currently no medications or other prophylactic methods effective against this disease, and common treatments are antibiotic treatment [5]. However, misuse of antibiotics inevitably leads to multi-antibiotic resistance and antibiotic residue, which causes threats to human and animal health globally [6]. Therefore, there is an urgent need to find effective and safe alternative antimicrobial agents for conventional antibiotics.

Lactobacillus plantarum is one of the most widely used probiotics with great beneficial effects on human and animal health [7]. Existing studies have shown that L. plantarum can produce lactic acid and various metabolites during colonization, which can effectively abrogate pathogenic bacteria growth and modulate immune functions [8]. In addition, some metabolites of L. plantarum may have anti-inflammatory properties in addition to their antimicrobial effects; this feature provides its therapeutic potential for various inflammatory diseases [9]. Fernsndez et al. found that oral administration of L. salivarius PS2 positively affected the prevention of infectious mastitis in late pregnancy [10]. Frola et al. have stated that intramammary infusion of L. plantarum CRL 1716 was an effective way of treating dairy cow mastitis [11]. Previous studies performed by our research team showed that L. plantarum 17–5 could attenuate E. coli-induced inflammatory responses in bovine mammary epithelial cells [12]. However, the effect of intramammary infusion of L. plantarum 17–5 on mice mastitis and its mechanism of action remains unclear. Here, our study establishes the murine model of mastitis using E. coli. The aim is to determine whether L. plantarum 17–5 has prevention effects on mastitis in vivo and provide a basis for developing and utilizing microecological agents.

Materials and Methods

Bacteria and the Culture Conditions

The Lactobacillus plantarum 17–5 strain (ATCC 8014, provided by American Type Culture Collection, Manassas, VA, USA) was cultivated statically in de Man, Rogosa, and Sharpe (MRS) broth (Aobox, Beijing, China) at 37 ℃ under microaerobic conditions. Escherichia coli O111:K58 (CVCC1450, provided by China Constitute of Veterinary Drug Centre, Beijing, China) was grown overnight in Luria Broth (LB) medium (Aobox, Beijing, China) at 37 ℃ with shaking. The number of colony-forming units (CFUs) was counted after three generations.

Animals and Experiment Design

SPF-grade male and female Kunming mice (8 weeks old) were purchased from Liaoning Changsheng Biotechnology Corporation (Benxi, China). Females and males were placed in the same microisolator cage at a ratio of 2:1 until the females were pregnant, and water and food were provided ad libitum. Animal assays were approved by the Animal Ethics Committee of Hebei Agricultural University (protocol number 2020044). The mouse mastitis model was established by referring to previous studies [13, 14]. Briefly, after ether anesthesia, the tip of the L4 and R4 abdominal mammary glands was carefully snipped, and bacteria or PBS was injected into the mammary ducts 7 days after delivery. The lactating mice were randomly divided into six groups (n = 8): the control group (PBS), the E. coli group (107 CFU/100 µL), L. plantarum (105, 106, and 107 CFU/100 µL) + E. coli and the L. plantarum group (107 CFU/100 µL). The L. plantarum or PBS was injected into each side of the nipple for 3 h prior to adding E. coli and then injected with E. coli using the same method. At 24 h after the last injection, mice were sacrificed, and the mammary gland tissues were collected and stored at − 80 ℃ until further analysis.

Histopathological Evaluation

The mammary tissues of the mice were observed for general condition and scored using a clinical scoring system ranging from 1 to 5, with higher scores indicating greater tissue damage. Specifically, 1 represents no damage, 2 represents slight redness, 3 represents slight redness and minor bleeding, 4 represents moderate redness and bleeding, and 5 represents severe redness and bleeding. Subsequently, tissue samples were fixed in 4% paraformaldehyde solution, dehydrated with gradient ethanol, and then embedded in paraffin.

The paraffin-embedded tissue sections were cut into 5 µm thickness, stained with hematoxylin and eosin (HE), and examined under an optical microscope. The same histological score (1 to 5) previously described was used for evaluating the degrees of tissue damage (necrosis and neutrophil and macrophage infiltration). The higher the score, the more serious the injury.

Immunofluorescence Staining

Paraffin sections were dewaxed with water, antigen repaired with sodium citrate, and blocked with 5% BSA (Solarbio, Beijing, China). Then, slides were incubated with primary antibody against claudin-3 (1:500; Bioss, Beijing, China) overnight at 4 ℃, then in FITC-labeled secondary antibody (1:200; Solarbio, Beijing, China) for 1 h at room temperature. After counterstaining with DAPI (Solarbio, Beijing, China), the fluorescence was observed under a fluorescence microscope.

MPO Activity Determination

The mammary tissues were homogenized, and the homogenates were centrifuged at 2500 rpm for 10 min at 4 ℃ to obtain supernatants. The activity of MPO in mammary tissue homogenates was assayed using MPO Detection Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol.

qRT–PCR Analysis

Total RNA was extracted from mammary tissue using the Ultrapure RNA extraction kit (CWBio, Beijing, China). The concentration and purity of RNA samples were measured by NanoDrop-2000 (Thermo Scientific, DE, USA), and the integrity was detected by agarose gel electrophoresis. Then, RNA was reversely transcribed into cDNA using a reverse transcription kit (US Everbright Inc, CA, USA), and quantitative real-time PCR was performed according to the following procedures: 300 s at 95 ℃ followed by 45 cycles of 5 s at 95 ℃, 30 s at 57 ℃ and 15 s at 72 ℃. The efficiency of the amplification was evaluated by establishing the standard curve. Gene relative expression levels were calculated using the 2−ΔΔCt method and normalized to the expression of GAPDH and β-actin. Primer sequences were listed in Table 1.

Table 1 The primer sequences used for qRT-PCR
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Western Blot Analysis

Total protein from mammary tissue was extracted using RIPA lysis buffer (Solarbio, Beijing, China), and its concentration was quantified by BCA protein assay kit (Solarbio, Beijing, China). A total of 30 µg of protein from each sample were separated by 10% SDS-PAGE gels, electrotransferred onto nitrocellulose membranes (Beyotime, Shanghai, China) and then blocked with 5% skim milk. The membranes were incubated with primary antibodies against claudin-3 (1:1000), occludin (1:1000), NF-κB p65 (1:1000), NF-κB phospho-p65 (1:1000), phospho-IκBα (1:500), and β-actin (1:1000) from Bioss Biotech Limited Company (Beijing, China) and antibodies against p38 (1:1000), phospho-p38 (1:1000), ERK (1:1000), phospho-ERK (1:2000), JNK (1:1000), phospho-JNK (1:1000), and IκBα (1:1000) from Cell Signaling Technology (MA, USA). After incubation with a secondary antibody (1:2000; Zhongshan Golden Bridge, Beijing, China), the NBT/BCIP color development kit (Solarbio, Beijing, China) was used to visualize the stainings, and ImageJ software (ImageJ Software Inc., MD, USA) was used for densitometric analyses of western blot bands.

Statistical Analysis

All data in this study was shown as means ± standard error of the mean (SEM). Comparisons between multiple independent groups were performed by one-way ANOVA and Tukey’s or Dunnett’s T3 tests. P values < 0.05 were considered significantly different.

Results

Effect of L. plantarum 17–5 on Histopathological Changes in Mice Mammary Tissue

No visible redness, swelling, or bleeding were seen in the mammary tissues in the control group, high dose of L. plantarum pretreatment group, and the L. plantarum group, and the tissue injury scores were significantly lower (P < 0.05) in three doses of L. plantarum pretreatment groups compared with the E. coli group (Fig. 1). Obvious inflammatory changes were observed in the mammary tissues of the E. coli group with infiltration of neutrophils and macrophages in the mammary acini, ducts, and connective tissue. However, these histopathological changes were ameliorated in the L. plantarum pretreatment group, with a significant reduction (P < 0.05) in histological scores among medium and high doses of L. plantarum pretreatment groups (Fig. 2).

Fig. 1
figure 1

Effect of L. plantarum 17–5 on the histopathological impairment in the mice mammary tissue. A The control group. B The E. coli group. C–E 105, 106, and 107 CFU/100 µL L. plantarum + E. coli group. F The L. plantarum group. The injury score from each group ranged from 1 to 5 with higher scores indicating greater tissue damage. Data shown as means ± SEM (n = 5) and different letters indicate significance at P < 0.05. The same as the following figures

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Fig. 2
figure 2

Effect of L. plantarum 17–5 on the histopathological changes in the mice mammary tissue (H&E 100 ×). A The control group. B The E. coli group. C–E 105, 106, and 107 CFU/100 µL L. plantarum + E. coli group. F The L. plantarum group. Scale bars: 100 µm. The histological score from each group ranged from 1 to 5 with higher scores indicating greater tissue damage. Data were the mean ± SEM (n = 5)

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Effect of L. plantarum 17–5 on the MPO Activity in the Mammary Glands

As shown in Fig. 3, the MPO activity in the E. coli group increased significantly (P < 0.05) compared with the control group. Pretreatment with different doses of L. plantarum 17–5 significantly (P < 0.05) reduced these increases.

Fig. 3
figure 3

MPO activity in mammary tissue from the control group, the E. coli group, and pretreatment with 105, 106, and 107 CFU/100 µL L. plantarum (LP) and 107 CFU/100 µL L. plantarum group. Data were expressed as means ± SEM (n = 5)

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Effect of L. plantarum 17–5 on Tight Junction Proteins in the Mammary Glands

Immunofluorescence staining for the claudin-3 was performed in mammary gland sections (Fig. 4A). In control and L. Plantarum groups, claudin-3 was localized to the cell membrane at cell–cell contacts and showed a complete and continuous structure. In the E. coli group, the claudin-3 positive signals were intermittent and markedly weaker than the above groups showing that the tight junctions were disrupted. Pretreatment with L. plantarum alleviated the E. coli-induced damage in tight junction proteins.

Fig. 4
figure 4

Effects of L. plantarum 17–5 on the structure and protein expression in the tight junction proteins. A Representative images of the FITC albumin staining in each group. Green shows the claudin-3 signal and blue shows the DAPI signal. (a) The control group. (b) The E. coli group. (c-e) 105, 106, and 107 CFU/100 µL L. plantarum + E. coli group. (f) The L. plantarum group. Scale bars: 100 µm. B Representative western blots showed expression of claudin-3 and occludin in each group. Data were expressed as means ± SEM from three independent experiments

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To further evaluate the effect of L. plantarum 17–5 on tight junction protein level, we examined the levels of claudin-3 and occludin by western blot (Fig. 4B). As expected, the protein levels of claudin-3 and occludin in the E. coli group were significantly (P < 0.05) lower than those in the control group. However, the reduction of claudin-3 and occludin levels was alleviated in the L. plantarum pretreatment group.

Effect of L. plantarum 17–5 on the mRNA Expression of Inflammatory Cytokines in the Mammary Glands

The results in Fig. 5 showed that the expression levels of IL1β, IL6, and TNFα in the E. coli group were significantly (P < 0.05) enhanced. However, these E. coli-induced expression alterations were partially inhibited (P < 0.05) by pretreatment with L. plantarum 17–5.

Fig. 5
figure 5

The mRNA expression levels of IL1β (A), IL6 (B), and TNFα (C) in the mammary tissue from each group. Data were expressed as means ± SEM (n = 3)

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Protein Expression of the NF-κB and MAPK Signaling Pathways in the Mammary Glands

The western blot analysis of NF-κB and MAPK signaling pathway protein expression is shown in Figs. 6 and 7. The results showed that compared with the control group, the phosphorylation levels of p65, IκBα, p38, ERK, and JNK increased significantly (P < 0.05) after E. coli stimulation. However, the L. Plantarum pretreatment group suppressed these increases to varying degrees.

Fig. 6
figure 6

Effects of L. plantarum 17–5 on NF-κB signaling pathway in the mammary tissue. Data were expressed as means ± SEM from three independent experiments

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Fig. 7
figure 7

Effects of L. plantarum 17–5 on MAPK signaling pathway in the mammary tissue. Data were expressed as means ± SEM from three independent experiments

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Discussion

E. coli is the most common environmental pathogen causing dairy cow mastitis in dairy herds [3]. Coliform mastitis is often characterized by a severe local and systemic inflammatory response, which causes huge economic losses for dairy farmers due to reduced milk production and premature culling [15]. Lactobacillus plantarum has been continuously studied as a potential novel anti-inflammatory agent. The current studies show that L. plantarum can produce organic acids and bacteriocins, inhibit the growth of different pathogens, and exert an anti-inflammatory effect during the proliferation process [16]. At present, the management of dairy cow mastitis is predominantly accomplished through intramammary infusion [17, 18]. Although some scholars have expressed concerns about the intramammary infusion of active probiotics [19], more and more studies have shown that intramammary injection of Lactococcus not induces inflammation but enhances the expression of immune proteins in the mammary glands of healthy cows [20, 21]. Thus, this study explores the preventive effect of intramammary infusion of L. plantarum 17–5 on mice mastitis and sets the L. plantarum group to verify the safety of this method.

Mastitis is characterized by the destruction of the acinar structure and neutrophil infiltration in mammary tissue, accompanied by the secretion of pro-inflammatory factors [22, 23]. We next performed the histological evaluation of mice mammary glands to evaluate the effect of L. plantarum 17–5 on histological changes in mice mammary tissue. The results showed that the mammary gland tissue in the E. coli group had obvious redness, swelling and bleeding, and massive infiltration of inflammatory cells in the mammary tissue. However, these characteristics were significantly attenuated in the L. plantarum pretreatment group. This indicated that L. plantarum 17–5 might protect against inflammation and was consistent with the report by Chen et al. that Lactobacillus plantarum can alleviate the inflammatory response of LPS-induced murine mastitis [7]. Notably, there were no obvious pathological changes in the mammary gland tissue in the L. plantarum 17–5 group, indicating that Lactobacillus plantarum 17–5 does not cause an inflammatory response in mice mammary tissue; this is coincident with previously reported results [20].

The blood–milk barrier is an important physical barrier in organisms, which maintains normal lactation function and is an important barrier against pathogen invasion [24, 25]. The integrity of the blood–milk barrier primarily depends on mammary epithelial tight junctions (TJs) [26]. There are studies indicating that inflammation can disrupt the integrity of TJs and increase its permeability [27, 28]. To investigate the effect of Lactobacillus plantarum 17–5 on TJs in mice mammary tissue, we focused on changes in the transmembrane protein family claudin-3 and occludin closely related to TJs. Immunofluorescence staining showed that the claudin-3 signal in the E. coli group was significantly weakened, and the tight junction structure was disrupted. In contrast, the claudin-3 signal in the L. plantarum 17–5 pretreatment group appeared stronger, and the tight junction structure was improved to some extent. Subsequently, we further detected the protein levels of claudin-3 and occludin in mice mammary tissue by western blot. As expected, claudin-3 and occludin levels were lower in the E. coli group and higher in the L. plantarum 17–5 pretreatment group, this suggests that the loss of aforementioned proteins led to the decrease in claudin-3 and occludin levels seen in the E. coli group. Similar findings were yielded by Zheng et al. [29].

MPO plays an important role in the process of inflammatory cells resisting microbial infection and is an important indicator for assessing neutrophil infiltration and damage in tissues [30]. In the present study, MPO activity was significantly higher in the E. coli group, indicating that inflammatory cells clustered around the injection site; this also validates the histopathological changes in mammary gland sections. Pretreatment with L. plantarum 17–5 could decrease the elevation of MPO activity, further ameliorating the aggregation of inflammatory cells and inflammatory injury in mice mammary tissue; this corresponds to previous reports [31]. Moreover, some pro-inflammatory cytokines such as IL1β, IL6, and TNFα are involved in the induction, amplification, and regulation of other inflammatory factors and play an important role in the development of inflammation and pathological processes [32,33,34]. Previous studies have shown that L. plantarum can reduce the secretion of IL1β, IL6, and TNFα in the mammary tissue [7]. Our results also indicated that L. plantarum 17–5 could inhibit the expression of the above cytokines and alleviate the inflammatory process in E. coli-induced mastitis.

To further clarify the mechanism of L. plantarum anti-inflammatory, we next detected the NF-κB and MAPK signaling pathways. NF-κB is a transcription factor with various biological activities involved in cell differentiation, inflammation, and immunomodulation [35, 36]. NF-κB normally exists in the cytoplasm in the inactive state; when stimulated by upstream signals, IκBα is rapidly degraded, and NF-κB is released into the nucleus to regulate downstream genes. Simultaneously, this effect is accompanied by increases in NF-κB and IκB phosphorylation [37]. In addition, the MAPK signaling pathways, which include p38 MAPK, ERK1/2, and JNK, are regulated by diverse transduction cascades [38]. It regulates inflammatory genes via phosphorylation of ERK, JNK, and p38 [39, 40]. In this study, we demonstrated that E. coli activated the NF-κB and MAPK signaling pathways in mice mammary tissue. However, pretreatment with L. plantarum 17–5 inhibited the phosphorylation levels of key proteins in these pathways. We speculate that the anti-inflammatory effect of L. plantarum 17–5 may involve inhibiting the NF-κB and MAPK signaling pathways.

Conclusion

In summary, our study indicated that pretreatment with L. plantarum 17–5 could alleviate inflammatory damage to the mammary tissue, decrease the expression of pro-inflammatory genes, and inhibit the activation of the NF-κB and MAPK signaling pathways in mice mammary tissue. Therefore, we believe that L. plantarum 17–5 has protective effects against E. coli-induced mastitis in mice and may be useful as a potential therapeutic agent for mastitis. Finally, a more comprehensive model evaluation should be conducted in vivo to advance their clinical applications further.