Chemicals
LTA from S. aureus was purchased from Sigma-Aldrich Inc. (St. Louis, MO, US). S. aureus (ATCC29213) was purchased from Micro biologics Inc. (St. cloud, MN, US). Mouse IL-1α, IL-1β and TNF-α enzyme-linked immunosorbent assay (ELISA) kit was purchased from Cusabio Biotech (Wuhan, Hubei, China). Trizol was purchased from Invitrogen (Carlsbad, CA, US). The reverse transcriptase synthesis kit was purchased from Takara Biotech (Dalian, Liaoning, China). SYBR Premix Ex TaqTM II was purchased from Sheng gong Biotech (Shanghai, China). Bicinchoninic acid (BCA) protein assay kit was purchased from BioChain Institute Inc (Silicon Valley, CA, US). Polyvinylidene fluoride (PVDF) membrane was purchased from Merck KGaA (Darmstadt Germany). NLRP3 rabbit monoclonal antibody (mAb) was purchased from Abcam (Cambridge, UK). Caspase-1, P65, P-P65, IκBα, P-IκBα, P38, P-P38, Jnk, P-Jnk, Erk, P-Erk and β-actin rabbit mAb and HRP-conjugated goat anti-rabbit antibodies were purchased from Cell Signaling Technology (CST; Danvers, MA, US).
Experimental design
The schematic of experimental design is shown in Fig. 1.
Characterization of inflammation induced by LTA
Eighty SPF BALB/c female mice and ten male mice (60 days old) were purchased from the Center of Experimental Animals of Linyi University and the protocol was approved by the Animal Care and Ethics Committee of the Linyi University. The mice were fed separately in an air-conditioned room maintained at a temperature of 24 ± 1 °C for 2 weeks to adapt to the environment. The diet was prepared consulting the standard of AIN-93 purified diets for laboratory rodent in Animal Nutrition and Feed Science Laboratory of Linyi University. Pregnancy is determined by vaginal plugs. Pregnant mice were randomly divided into two groups: Control group and Experimental group. Control group (CG, n = 40) were infected with 0.1 mL physiological saline from milk ducts; Experimental group (EG; n = 40) were infected with 0.1 mL LTA (5 µg). At each time point (0, 12, 24, 48 and 72 h), twenty mammary glands (CG = 10, EG = 10; R4, L4) were collected.
Characterization of LTA treatment on the activation of NLRP3/NF-κB and NLRP3/MAPK signaling pathway induced by S. aureus
Forty SPF pregnant mice were randomly divided into four group: Control group (CG, n = 10) were infected with 0.1 mL physiological saline from milk ducts; Model group (MG, n = 10) was infected with 0.1 mL S. aureus (105 CFU/mL); LTA control group (LCG, n = 10) was infected with 0.1 mL LTA (5 µg) for 72 h; LTA treatment group (LTG, n = 10) was infected with 0.1 mL LTA (5 µg) for 72 h and then infected with 0.1 mL S. aureus (105 CFU/mL). 12 h after stimulus, mammary gland (R4, L4) in each group were collected.
Histopathologic examination
The tissues were collected and fixed in 10% formaldehyde solution, and then embedded in paraffin wax. The mammary tissue sections were stained with hematoxylin eosin (H&E) staining and the pathological changes were observed under a microscope (Olympus DX45, Japan).
Immunohistochemistry analysis
The mammary tissues were collected and fixed in 10% form-aldehyde solution, and then embedded in paraffin wax. For the immunohistochemistry staining, the mammary section was routinely processed. Tissue sections were deparaffinised, rehydrated and then antigen retrieval was applied by microwave heat for 10 minutes at medium voltage in a 10 mM citrate buffer, pH 6.0. After cooling at room temperature, the sections were incubated in 3% hydrogen peroxide (H2O2) for 30 minutes to block endogenous peroxidase. Nonspecific staining was eliminated by 10 minutes incubation with normal goat serum at the room temperature. Excess normal serum was removed and slides were then incubated with primary antibody (1:100) at 4˚C overnight. After washing the slides, the sections were incubated with biotinylated secondary antibody for 15 minutes and replaced in the streptavidin, horseradish peroxidase (HRP) conjugate for 15 minutes at the room temperature. The colour was developed with DAB in PBS for 5 minutes. Slides were counterstained with Harris haematoxylin, dehydrated and mounted with Entellan. IPP6.0 software was used to analyze the optical density of immunohistochemical photos. Four 400-fold photos were selected from each section for optical density analysis.
Western blot analysis
Total protein was extracted by using a total protein extraction kit and the protein concentrations were determined by BCA protein assay kit. 50 µg (5 µL) proteins were loaded into a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel for electrophoresis and transferred to PVDF membranes. The membranes were blocked in 5% nonfat milk for 1 h to 2 h and incubated overnight with the primary monoclonal antibodies, including Caspase-1, NF-κB, MAPK and β-actin. Then, all of the membranes were subsequently incubated with HRP-conjugated goat anti-rabbit antibodies. The blots were washed with PBS-T and processed with supersignal west pico chemiluminescent substrate.
qRT-PCR analysis
The total RNA of tissue was extracted according to the manufacturer's instructions by using Trizol reagent. The quantity and quality of the extracted RNA was evaluated with a Nanodrop 2000 spectrophotometer (Thermo, USA). The ratio of absorption (OD 260 nm/OD 280 nm) was between 1.8 and 2.2, and the RNA integrity was verified with electrophoresis using a 1% agarose gel. Then, RNA was converted to cDNA using a reverse transcriptase synthesis kit. qRT-PCR primers were designed using “Primer 3 web” (http://bioinfo.ut.ee/primer3/) to detect the genes encoding IL-1α forward (5′-TCTCAGATTCACAACTGTTCGTG-3′) and reverse (5′-AGAAAATGAGGTCGGTCTCACTA-3′); IL-1β forward (5′-ACCTGTGTCTTTCCCGTGG-3′) and reverse (5′-TCATCTCGGAGCCTGTAGTG-3′); TNF-α forward (5′-TGCTGTCCCTGTATGCCTCT-3′) and reverse (5′-GGCAGCCTTGTCCCTTG-3′); β-actin forward (5′-TGCTGTCCCTGTATGCCTCT-3′) and reverse (5′-TTTGATGTCACGCACGATTT-3′). The PCR reaction system contained 10 µL of SYBR Green PCR mix, 0.8 µL of each primer (both 10 µmol/L), and 2 µL of cDNA template in a final volume of 20 µL per reaction. The cycling parameters for real-time PCR were 95 °C for 2 min, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s using a CFX connect real-time PCR system (BIO-RAD, US). The real-time quantitative PCR reaction was performed in triplicate for each sample, and the mean value was used to calculate the mRNA expression levels. The fold change (n-fold) for gene expression was calculated using the relative quantification method as previously described [22].
ELISA analysis
Mammary tissue samples were weighed and grinded with RIPA lysis buffer until homogenized. Then the homogenate was centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected in − 20 °C until measurement. The expression of IL-1α, IL-1β and TNF-α were measured by ELISA and according to the manufacturer.
Statistical analysis
All data is shown as the mean ± S.E.M, and experiments were independently repeated at least 3 times. One-way ANOVA and Dunnett's test were used for statistical analyses. Values of p < 0.05 were considered statistically significant.