Lentinan was obtained from Shanghai Yuanye Biotechnology Co., Ltd., and dissolved with DMSO to prepare a final concentration of 100 mg/ml. When lentinan is used, it is diluted to the experimental concentrations (DMSO<0.1%). LPS was purchased from Sigma Chemical CO (St. Louis, United States). A mouse myeloperoxidase (MPO) ELISA kit was obtained from MultiSciences (Lianke) Biotech Co., Ltd (Zhejiang, China).

Mice were purchased from the Laboratory Animal Center of Zhejiang University (Hangzhou, China). Ninety BALB/c female mice (8 week old) were used in this experiment. Food and water were available ad libitum. The mice were kept in separate cages for a 12 h dark light cycle under controlled temperature (24°C ± 1°C) and 60% humidity for 1 week before the research. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee in Zhejiang University.

The mice were randomly classified into six groups, each comprising fifteen mice: Control group, LPS group, lentinan (5, 10, and 20 mg/kg) + LPS groups, and dexamethasone group (5 mg/kg). The mastitis model was carried out as described previously by us (Xingxing et al., 2018). In brief, 100 μl of LPS (1 mg/ml) was infused into two abdominal mammary glands (R4 and L4) in mice under anesthesia with pentobarbital. Mice received an intraperitoneal injection (ip) of different lentinan concentrations (5, 10, and 20 mg/kg) or dexamethasone after 1 h of LPS or saline ip treatment. After 24 h, the mice were sacrificed by CO₂ inhalation at the same time. The mouse mammary tissues were collected and stored at −80°C until being analyzed.

The mouse mammary gland tissues were excised and fixed in 10% formalin for subsequent histopathological analysis. In brief, tissues were dehydrated with different concentrations of alcohol, paraffin-embedded sections were prepared at a 4-µm thickness, and hematoxylin and eosin (H and E) staining was then performed to observe the morphology changes with an optical microscope (Olympus, Japan).

MPO activity in mammary gland tissue was detected in tissue homogenates prepared as described above using the ELISA kit following the instruction book of the producer. In addition, mammary tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then incubated with the MPO antibody (Servicebio, China). Immunopositive cells were counted, and positive cells in mammary gland tissue sections were quantified to the tissue area.

Epithelial cells from the mammary gland tissue of lactating mice were cultured as described previously (Wu et al., 2017). The mouse mammary epithelial cells (mMECs) were cultured in DMEM containing 10% FBS, 100 U/mL penicillin–streptomycin, and 10 μg/ml insulin in a 5% CO₂ incubator. The cells were pretreated with different concentrations of lentinan (5, 10, and 20 μg/ml) or dexamethasone for 1 h before LPS challenge.

The cells were fixed with paraformaldehyde at room temperature for 15 min and then washed three times with PBS in a twelve-well plate. Next, the cells were sealed with 10% normal goat serum at room temperature for 1 , followed by incubation with the primary antibody CK-18 at 4°C for 12 h. The cells were then incubated with the fluorescent-labeled secondary antibody (Bioss, China) for 45 min at room temperature and washed three times in PBS. Finally, DAPI was used to stain the cell nuclei, which were then observed using a laser scanning confocal microscope (Leica, Germany).

The mMEC viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) experiment. Cells (1 × 10⁵ cell/mL) were cultured in 96-well plates for 6 hours. The cells were stimulated with lentinan (5, 10, and 20 μg/ml) for 24 h. Next, the MTT (5 mg/ml) agent was added in mMECs for 4 h, and 100 μl of DMSO per well was added. The optical density (OD) at 570 nm was read with a microplate reader (Thermo, United States).

Immunofluorescence staining assay of the mouse mammary tissue and mMECs was carried out. Briefly, the mouse mammary tissues or cells were fixed in 10% formalin, and tissues were embedded in paraffin. The tissue or cell slice was permeated with PBS appending Triton X-100 (0.3%, Sigma, United States) and 10% BSA. The tissue or cell slice was hatched for 12 h at 4°C with a special antibody for Wnt3a and β-catenin (Servicebio, China) and a Cy3 secondary antibody. Then, Wnt3a and β-catenin proteins were determined and immobilized using mounting media supplemented with DAPI. Finally, all of the slices were observed with fluorescence microscopy.

The cytokine secretion after LPS challenge was assessed with the ELISA kit in the mouse mammary tissues and mMECs. The tissues were homogenized in ice-cold PBS and then centrifugation at 10,000 rpm, 4°C for 15 min. Harvested tissue and cell supernate to detect the production of cytokines (IL-1β and TNF-α) using the ELISA kit following the instruction book of the producer. Finally, the optical density (OD) at 450 nm is read with a microplate reader.

The total protein of mMECs was obtained by the lysis solution containing the phosphatase repressor. The BCA kit was used to determine the protein concentration. Then, samples with the same amount of protein were applied to 10% SDS-PAGE gel electrophoresis and then transferred to the PVDF membrane. After being placed in the 5% skim milk, the membrane was washed three times and incubated with the corresponding primary antibody at 4°C for 12 h. Next, the membrane was incubated with the secondary antibody at room temperature for 1 hour. The protein band density was detected using a chemiluminescence system.

SPSS software was used for data analysis. Statistical data were expressed as the mean ± S.E.M. of three individual experiments. Data were analyzed by Student’s t-test or one-way analysis of variance (ANOVA). p ≤ 0.05 was deemed a statistically significant difference.

The histological analysis was used to evaluate the mouse mammary tissue damage. Histological analysis showed that the mammary tissue of the control group was intact without histopathological changes (Figure 1A). In the LPS group, the injury of mouse mammary tissue was obvious, and inflammatory cell infiltration was extensive and hyperemia (Figure 1B). However, the extensive inflammatory cell infiltration and hyperemia were relieved, and the tissue structure was relatively complete in lentinan or dexamethasone treatment (Figures 1C–F). Moreover, in order to further verify the degree of tissue damage, the histopathological changes of the mouse mammary gland were evaluated according to the number of infiltrated inflammatory cells. As described in the literature, the pathological grade was scored according to 0–5 (Xingxing et al., 2018). As expected, the result was consistent with pathological sections (Figure 1G).

MPO is an enzyme in neutrophils, and its activity is related to neutrophil infiltration (Lin et al., 2020). As displayed in Figure 2A, compared with the control group, the MPO activity was obviously enhanced in LPS challenge. Lentinan treatment reduced MPO activity in a dose-dependent manner, especially at high concentration. In order to further verify the effect of Lentinan on MPO activity, the immunofluorescence technique was performed. As expected, LPS-enhanced MPO activity was decreased by lentinan or dexamethasone treatment (Figure 2B).

The secretion of proinflammatory factors in mouse mammary tissues was detected using ELISA kits. The result of ELISA assay displayed that LPS markedly promoted the production of TNF-α and IL-1β. In contrast, lentinan or dexamethasone treatment substantially decreased the levels of TNF-α and IL-1β (Figure 3). These abovementioned results suggested that lentinan significantly inhibited the secretion of proinflammatory factors in LPS-induced mastitis at a concentration of 20 mg/kg. Thus, this concentration of lentinan was used to study the protective mechanism of LPS-induced mastitis.

The Wnt/β-catenin signaling pathway is an evolutionarily conserved mechanism that is fundamentally vital for inflammation-related diseases (Guan et al., 2021). We evaluated whether lentinan alleviated the LPS-induced inflammatory response by suppressing the Wnt/β-catenin pathway. The result of immunofluorescence assay displayed that LPS treatment significantly enhanced the activation of the Wnt/β-catenin signaling pathway that was reduced by lentinan treatment (Figure 4).

CK-18 is commonly used to identify the integrity of epithelial cells. Thus, mMECs were pretreated with the blue fluorescent pigment to identify the cell nucleus and CK-18 labeled with the green fluorescent pigment to show the cell integrity. The result is displayed in Figure 5.

First, the potential cytotoxicity of lentinan on mMECs was detected by MTT experiment. As shown in Figure 6A, the cell viability was not affected by lentinan treatment. To investigate the effects of lentinan on inflammatory response in LPS-stimulated mMECs, the TNF-α and IL-1β protein levels were detected using the ELISA kit. As displayed in Figure 6B, LPS increased TNF-α and IL-1β secretion that was inhibited by lentinan or dexamethasone administration.

The activation of the Wnt/β-catenin signaling pathway in LPS-stimulated mMECs was also determined by immunofluorescence assay. The result showed that LPS challenge greatly enhanced the activation of the Wnt/β-catenin pathway, but that was reduced by lentinan treatment or dexamethasone administration (Figures 7A,B). In order to confirm the effect of lentinan on the Wnt/β-catenin pathway activation, western blot was performed in mMECs. Consistent with the results in Figure 7C, the downstream factors in the Wnt/β-catenin pathway, Wnt3α, and β-catenin were activated upon LPS challenge, while lentinan or dexamethasone administration downregulated the Wnt3α and β-catenin protein expression.