Assay the concentration of AIF-1 in milk
The milk samples were obtained from 56 healthy midlactation Holstein-Friesian cows, 75 cows suffering from clinical mastitis and 23 cows recovering from clinical mastitis after antibiotic treatment from one dairy farm in Sichuan province of China on the basis of a clinical examination. The concentrations of AIF-1 in milk samples were determined using a commercial bovine AIF-1 assay kit (No.YX-010907B, Pengshida, Sichuan, China). All procedures were performed according to the manufacture’s guideline. The optical absorbance at 450 nm was measured.
Synthesis of bovine AIF-1 protein
Spleen samples of bovines were harvested from a local slaughterhouse (Chengdu, Sichuan, China). Total RNA was extracted, cDNA was synthesized via reverse transcription and the entire open reading frame of bovine AIF-1 gene was amplified by PCR as previously described [18] using specific primers (sense: 5′-ATGAGCGAAACTAGGGATTTAC-3′, antisense: 5′-TCAGGGCAACTCAGAG
ATAGC-3′) designed based on the nucleotide sequence of bovine AIF-1 (GenBank accession No. NM 173985). The PCR fragments were purified using agarose gel electrophoresis and cloned into pMD19-T vector (TaKaRa, Dalian, China), then transformed into competent E.coli DH5α cells (Novagen, Germany), and applied to DNA sequencing. Thereafter, bovine AIF-1 open reading frame was cloned into pET-32a plasmid at NdeI and XhoI sites. The recombinant plasmids (pET32a-AIF1) were also identified by DNA sequencing and transformed into competent BL21 (DE3) cells (Novagen, Germany) to express recombinant bovine AIF-1. The cells were cultured overnight, then fusion protein was induced by 0.8 mmol/L isopropyl-1-thio-β-D-galactopyranoside (IPTG, Sigma, USA) and recombinant bovine AIF-1 was purified using HisTrap™ HP (No. 17-5247-01, GE Healthcare, USA) according to the product description.
Cell culture and treatment
Bovine mammary gland epithelial cell line (MAC-T) was a generous gift from Professor Jinfeng Miao (Nanjing Agricultural University, China). The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Fremont, CA, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37 ℃ in the humidified atmosphere of 5% CO2. 0, 0.1, 1, 10 nmol/L recombinant bovine AIF-1 protein was added into medium and incubated for 48 hours.
Cell viability and apoptosis
Cell Counting Kit-8 (CCK8, No.1706, Bioswamp, China) was applied to assess bovine mammary gland epithelial cell viability and proliferation. In detail, 3 × 103 cells was inoculated in 96 well cell culture plate overnight and exposed to 0, 0.1, 1 or 10 nmol/L bovine AIF-1 for 48 hours, then CCK8 solution was added for 4 hours and the optical absorbance was estimated at 450 nm. The data were shown as the percentage of viable cell count with AIF-1 treatment compared with that in control group. For cell apoptosis assay, the cells were stained with annexin V-FITC/PI (No. 1065, Beyotime, Nanjing, China). The cell percentage at different apoptotic stages was analyzed by flow cytometer (Beckman Coulter FC500, Brea, CA).
Nitric oxide and reactive oxygen species (ROS) generation, inflammatory cytokines and lactate dehydrogenase (LDH) release
At the same time, nitric oxide and reactive oxygen species (ROS) generation by the cells was measured with nitric oxide quantitation kit (A-013-2, Jiancheng, Nanjing, China) and cellular reactive oxygen species detection assay kit (No.PAB180052, Bioswamp, China), respectively. In addition, the supernatants were collected. The levels of TNF-α (No.HM10001, Bioswamp, China), interleukin 6 (IL6, No.HM10205, Bioswamp, China), monocyte chemoattractant protein 1 (MCP-1, No.HM10894, Bioswamp, China) and LDH (No. A020-1, Jiancheng, Nanjing, China) released from epithelial cells were also detected with the corresponding kits.
Mitochondrial membrane potential (ΔΨm) and intracellular ATP content
Using fluorescent probe JC-1 (No.180068, Beyotime, Nanjing, China), mitochondrial membrane potential was estimated [19]. In brief, the cells were incubated with 5 mg/L JC-1 for 20 min at 37 °C, then harvested by centrifugation for 5 min at 400 g, washed thoroughly and suspended in 0.5 mL PBS. JC-1 monomer and aggregate signals were detected by flow cytometry analysis and the ratio of aggregate/monomer fluorescent intensity was calculated to determine mitochondrial membrane potential. Additionally, the concentration of intracellular ATP was detected with an ATP assay kit (No. A095, Jiancheng, Nanjing, China). The cells were lysed in 200 µL ATP assay buffer and the level of intracellular ATP was measured by chemiluminescence using ATP as standard (West Pico kit, Pierce, Loughborough, UK) and normalized to protein concentrations.
Western blot
The expression of cleaved caspase-3 and phosphorylation of IκBα in bovine mammary epithelial cells were assayed by western blot. In detail, total cellular were dissolved in lysis buffer containing protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Then the proteins were applied to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Whatman, London, UK). The membranes were blocked with 5% w/v bovine serum albumin in Tris-HCl buffer containing 0.1% Tween 20. After that, the proteins on the membranes were immunologically reacted with the specific primary antibodies against cleaved caspase 3 (Asp175, No. 9661, 1:1000, Cell Signaling Techology, Danvers, MA, USA), p-IκBα (Ser32, No. 2859, 1:1000, Cell Signaling Techology, Danvers, MA, USA), GAPDH (No. PAB36269, 1:1000, Bioswamp, China). Thereafter, the corresponding horseradish peroxidase conjugated secondary antibodies were added. Finally, specific immune complexes were assessed by chemiluminescence (West Pico kit, Pierce, Loughborough, UK).
Statistical analysis
The concentrations of bovine AIF-1 in milks were expressed as mean ± standard deviation. Statistical analyses were performed with SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL) and statistical differences between AIF-1 treatment groups and control group were determined by one-way ANOVA. A value of P < 0.05 was considered as statistical significance.