Gentamycin

Curcumin (purity ≥ 95.0%) was purchased from Shanghai Yuanye Biotech (Shanghai, China). Carboxymethylcellulose sodium was obtained from Sigma-Aldrich (Saint Louis, MO, USA). Neomycin, streptomycin, penicillin, vancomycin, metronidazole, bacitracin, ciprofloxacin, ceftazidime and gentamycin were purchased from Sangon Biotech (Shanghai, China). TRIzol® Reagent was from Invitrogen (Carlsbad, CA, USA). High-Capacity cDNA Reverse- Transcription Kits were purchased from Applied Biosystems (Foster City, CA, USA). SYBR Green PCR Master Mix was obtained from Promega (Fitchburg, MI, USA). Proteinase inhibitor cocktail and phosphorylase inhibitor were purchased from Roche (Basel, Switzerland). Bicinchoninic acid (BCA) Protein Assay Kits were purchased from Genstar Technologies (Beijing, China). Antibodies against Akt (9272), pAkt (9271) were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against FGF15 (ab229630) was obtained from Abcam. The antibody against Tubulin (CW0098) was from Cwbiotech (Beijing, China).

The phagocytosis assay was performed using the mouse macrophage cell line RAW 264.7 (ATCC catalogue number TIB-71) and peritoneal macrophages as previously described⁵³ . Peritoneal macrophages were collected from 7-week-old female C57BL/6J mice on the 5th day after intraperitoneal injection of 1 ml 5% thioglycollate broth (BD Difco, catalogue number 211716)⁵⁴ . Briefly, 5 × 10⁵ cells were infected with bacteria at a multiplicity of infection of 50 for 1 h. The cells were then washed three times with 1× PBS and treated with 500 μl Dulbecco’s modified eagle medium (Servicebio, catalogue number G4515) containing 300 μg ml⁻¹ gentamycin (Sangon Biotech, catalogue number A506614) or amikacin (Solarbio, catalogue number A9660) for 1 h to eliminate the extracellular bacteria. After washing three times with 1× PBS, the cells were treated with 1 ml of 1% TritonX-100 (Sangon Biotech, catalogue number A110694) for 10 min to release the intracellular bacteria. Then intracellular bacteria were counted by serial dilution agar plating method.

For infections, RAW264.7 macrophages were seeded into 24-well plates at 2 × 10⁵ cells/well or into 6-well plates at 1 × 10⁶ cells/well and maintained for 24 h prior to infection. Parallelly, Brucella M5 was inoculated in TSB and grown in the exponential phase. The bacterial suspension was diluted in DMEM supplemented with 1% FBS and added to each well at a multiplicity of infection (MOI) of 100. The infected cells were then centrifuged at 400× g for 5 min at room temperature and incubated for 1 h at 37 °C in a 5% CO₂ atmosphere. After that, the cells were washed three times with phosphate-buffered saline (PBS; Gibco) and treated with 50 μg/mL of gentamycin (#A100304; Sangon, Shanghai, China) for 1 h to kill the uninternalized bacteria. Subsequently, the cells were washed three times with PBS and incubated with a medium containing 25 μg/mL of gentamycin for the desired time points.

H9c2 cells were maintained in DMEM supplemented with 10% FBS, 2% l-glutamine, 10% sodium bicarbonate, 10% sodium pyruvate, 5% Hepes, 1% penicillin/streptomycin and 1% gentamycin (Sangon Biotech Co., Ltd., Shanghai, China) in an incubator (37°C, 95% air + 5% CO₂). The cells were cultured for 1–2 days under these normal conditions, until they reached 90% confluence prior to anoxic treatment. EGCG (0, 5, 10, 15 and 20 µM) and Zn²⁺ (0, 5, 10, 15 and 20 µM) were added to the myocytes 30 min prior to reoxygenation. Hypoxic stress was implemented by incubating cultured H9c2 cells in serum- and glucose-free DMEM. The culture dish was placed in an airtight incubator at 37°C under an atmosphere of 95% N₂ and 5% CO₂ for 3 h followed by reoxygenation for 1 h in normal (37°C, 95% air + 5% CO₂) conditions with 10% FBS-DMEM. EGCG and Zn²⁺ at various concentrations were added 30 min prior to the reoxygenation. Myocytes not exposed to H/R served as normoxic controls. At the end of the H/R treatment, myocytes were examined for viability and apoptosis by Hoechst 33258 (Beyotime Institute of Biotechnology, Haimen, China) staining and western blot analysis.