Minocycline

FK506 (Cat#F-4900) was purchased from LC Laboratories (New Boston Street Woburn, Massachusetts, USA). Purity was ≥99%. Minocycline (M9511) was purchased from Sigma Aldrich (St Louis, MO, USA). Purity was ≥99%. FK506 and Minocycline combined stock solution (0.5 mg/mL) was prepared by dissolving the compounds in saline (0.9% NaCl) containing 1.25% PEG40 Castor Oil (Solarbio Life Sciences, Beijing, China, Cat#C9510) and 2% ethanol (Beijing Chemical Works, Beijing, China). For the calculation of best dosage for our study, we conducted a pilot study where we injected different cocktail concentration combinations of FK506 and Minocycline (2.5 mg:15 mg, 5 mg:25 mg and 10 mg:40 mg). Animals treated with 5 mg FK506 and 25 mg Minocycline in combinatory therapy were found most effective. Animals with 2.5 mg:15 mg did not prolong survival, but with no side effects. On the other hand, 10 mg:40 mg also did not prolong survival, and they had severe weight loss and some other side effects observed in kidney tissues. Animals in group B were injected i.p with 0.12 mg of FK506 (5 mg/kg) and 0.60 mg of Minocycline (25 mg/kg) dissolved in 100 mL of the vehicle solution mentioned above [4 (link),22 (link),74 ,75 (link),76 (link),77 (link)]. The stock solution was stored frozen in a light protected bottle.

The employed strains, A. Baumannii strains A1 (minocycline-sensitive strain), A163 (minocycline-mediated strain), and A156 (minocycline-resistant strain), were clinically isolated from Zhangjiagang people’s hospital, Xuyi hospital, and Affiliated Hospital of Yangzhou University, respectively and preserved in the pathogenic microbiology laboratory of Yangzhou University while the strains were grown in Mueller–Hinton (MH) broth or on MH agar. Metformin were obtained from Macklin (Shanghai,China), minocycline were obtained form Solarbio (Beijing, China), other antibiotics were obtained from Sangon Biotech (Shanghai, China).

The 1,2-dichloroethane was purchased from Sinopharm Chemical Reagent Co., Ltd. (Ningbo, China). The BCA protein assay kit, multicolor prestained protein marker, and ECL plus kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies for claudin 5 and occludin were obtained from Abcam (Cambridge, UK). Antibodies against glial fibrillary acidic protein (GFAP) and soluble calcium-binding protein 100B (S100B) were the products of ABMART (Shanghai, China). Antibodies against ionized calcium-binding adapter molecule1 (Iba-1), interleukin 6 (IL-6), cluster of differentiation 11b (CD11b), and nuclear factor-κB (NF-κB, p-p65) were obtained from ImmunoWay (Texas, USA). Antibodies for tumor necrosis factor α (TNF-α) and β-actin were purchased from Elabscience (Wuhan, China). Antibodies for arginase-1 (Arg-1), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), MMP-9, inducible nitric oxide synthase (iNOS), Toll-like receptor 4 (TLR4), and MyD88 were obtained from Wanleibio (Shenyang, China). RIPA lysis buffer and the PAGE Gel Kit were obtained from EpiZyme (Shanghai, China). Minocycline was purchased from Solarbio (Beijing, China). DL-Fluorocitric acid barium salt and melatonin were obtained from Sigma-Aldrich (St. Louis, MO, USA).