Animals and LPS-induced mouse mastitis model
BALB/c mice, 6-8 weeks old (18 female and 9 male), were divided into three groups, and each group contained two female and one male raised in a cage. After suckling mice born on the 10th day, 18 female were randomly divided into three groups for next experiments. Group Ⅰ was used as control. Group Ⅱ was treated with LPS group. Group Ⅲ treated with LPS and NSC23766 group. The method of establishment of mastitis mouse model refers to our previous reports . In brief, the teat duct of GroupⅡwas infused with LPS (200 μg/mL). Group Ⅲ mice were intraperitoneally injected with NSC23766 (5 mg/kg, MedChemExpress) 1h before LPS (200 μg/mL) treatment. Group Ⅰ mice received an equal volume of PBS treatment. At 24 h after the LPS perfusion, mice were killed by CO2 inhalation. The mammary glands were collected and stored at -80 °C for later analysis. All animal experiments were in line with Jilin University experimental animal management regulations, and the People's Republic of China Animal Protection Law.
Cell culture and treatments
The mouse mammary epithelial cell line (EpH4-Ev) were cultured in DMEM medium F12 (Hyclone) supplemented with 10 % fetal bovine serum (Clark) at 37°C with 5 % CO2. Cells plated into a 6-pore plate and pre-treated with NSC23766 (50, 100 and 200 μM) for 30 min. After that the cells were treated with LPS (5μg/mL) for 12 h, and collected for qRT-PCR and western blot analysis.
The collected mammary tissues were fixed in 10 % formalin for 48-72 h. Then fixed mammary glands were dehydrated with graded alcohol, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Finally, the histopathologic changes were observed by light microscopy.
Myeloperoxidase (MPO) Assay
In order to detect the activity of MPO in the mammary glands, the collected mammary glands were homogenized, and detected by MPO kit (Jiancheng Bioengineering Institute of Nanjing). The specific experimental procedures were carried out according to the instruction.
qRT- PCR Analysis
Total RNA of mammary glands was extracted with Trizol (Invitrogen, USA), and complementary DNA (cDNA) was reversed transcription by the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, San Jose, CA). Primers were acquired from Sangon Biotech Co. Ltd (Shanghai, China, Table 1). The genes expressions of mammary glands were analyzed by using a 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.
Total protein in mammary tissue was extracted with the T-PER™ Tissue Protein Extraction Reagent (ThermoFisher, product number: 78510), which contains a propreitary detergent in 25 mM bicine and 150 mM sodium chloride. Protein concentration was determined by BCA protein assay kit. The samples were separated by 10 % SDS polyacrylamide gel, and then proteins were transferred to PVDF membranes. Membranes were blocked in 5 % bovine serum albumin for 2 h. Whereafter, the membranes were probed with primary antibody (1: 1000 dilution in TBST) overnight at 4 °C, washed thrice in TBST with gentle mixing, and then incubated with peroxidase-conjugated secondary antibody (1: 20000 dilution in TBST) for 3 h. After thrice washing in TBST, the samples were visualized with the ECLPlus Western Blotting Detection System (GE Healthcare, Chalfont St Giles, UK). Finally, the western blot bands were quantified by the software of ImageJ. The primary antibodies of ERK, P38, P65, Ikb, NLRP3 and GADPH were purchased from Boster bioengineering co. LTD (China).The primary antibodies of p-ERK (Thr202+Tyr204), p-p38 (Tyr182), p-p65 (ser536), p-iκb (S32/S36) were purchased from Bioworld Technology, Inc. (USA).
The data was analyzed by using GraphPad Prism 5.0 software. The western blot band intensities were quantified by the software of ImageJ. Difference among groups was analyzed by one-way analysis of variance (ANOVA) and the Tukey multiple comparison test. Values were expressed as the means the ± standard deviation (SD). The significance threshold was set at 0.05.