February 18, 2025
COX

Whereas expression of intact caspase-9 remained relatively constant following exposure of rats to SM, expression of activated cleaved caspase-9 increased; this was observed at 6 and 24 h post treatment at all doses of SM tested

Whereas expression of intact caspase-9 remained relatively constant following exposure of rats to SM, expression of activated cleaved caspase-9 increased; this was observed at 6 and 24 h post treatment at all doses of SM tested. MMP-9 was prominent in bronchial epithelium. In contrast, expression of the anti-oxidant hemeoxygenase, and the anti-inflammatory collectin, surfactant protein-D, decreased in the lung after SM exposure. These data demonstrate that SM-induced oxidative stress and injury are associated with the generation of cytotoxic inflammatory proteins which may contribute to the pathogenic response to this vesicant. for 10 min and SB 216763 the supernatant collected and stored at ?80 C until analyses. Immediately following lavage, the left lobe, along with the right cranial, medial, caudal, and accessory lobes of the lung were removed, snap frozen in liquid nitrogen and stored in ?80 C. For histologic evaluation, the trachea, bronchus and lung lobes were removed and fixed in formalin. Sections (5 m) were prepared, stained with hematoxylin Lamp3 and eosin, and examined by light microscopy. Images were acquired using DP controller software (Ver. SB 216763 3.3.1.292) from Olympus Corporation (Center Valley, PA). The extent of inflammatory changes including macrophage and neutrophil localization, alterations in alveolar epithelial barriers and edema were assessed blindly by a veterinary pathologist (Sherritta Ridgely, D.V.M, Ph.D). Preparation of tissue lysates Frozen tissue samples (20C200 mg) were pulverized twice using an ice cold 316 stainless steel tissue pulverizer (Cole-Parmer, Vernon Hills, IL). The tissue was then lysed (330 mg/ml) in ice cold buffer (pH 7.4) consisting of 20 mM HEPES, 150 mM sodium chloride, 1 mM EGTA, 1.5 mM magnesium chloride, 10% glycerol and 1% Triton X-100 and protease inhibitors (1 mM PMSF, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM lactacystin and 5% protease inhibitor cocktail). Samples were then centrifuged at 14,000for 10 min at 4 C. Supernatants containing tissue lysates were frozen at ?80 C until analyses. Protein measurement Total protein content in cell-free BAL and tissue lysates was quantified using a BCA protein assay kit (Pierce Biotechnologies Inc., Rockford, IL) following the manufacturers directions with bovine serum albumin as the standard. All samples were assayed SB 216763 in triplicate. Antibodies Rabbit monoclonal anti-cleaved caspase-3, polyclonal anti-caspase-9, polyclonal anti-poly-ADP-ribose polymerase (PARP)-1, and polyclonal anti-LC3B antibody, and horse radish peroxidase (HRP)-conjugated secondary goat anti-rabbit and horse anti-mouse antibodies were purchased from Cell Signaling Technology (Beverly, MA). Rabbit monoclonal anti-MMP-9, and polyclonal anti-COX-2 antibodies were from Abcam Inc. SB 216763 (Cambridge, MA). Mouse monoclonal anti-surfactant protein-D (SP-D) and rabbit polyclonal anti-inducible nitric oxide synthase (iNOS) antibodies were from Millipore Corp. (Billerica, MA). Rabbit polyclonal anti-hemeoxygenase (HO)-1, rabbit polyclonal Cu/Zn-superoxide dismutase (SOD) and Mn-SOD antibodies were from Assay Designs (Ann Arbor, MI). Primary goat polyclonal anti-TNF and secondary goat anti-rabbit antibodies were purchased from Santa SB 216763 Cruz Biotechnology (Santa Cruz, CA). Western blot analysis Proteins (10C100 g) were fractionated on 15% SDS polyacrylamide or 4C12% Novex Bis-Tris gels (Invitrogen, San Diego, CA) and then transferred onto nitrocellulose membranes. Nonspecific binding was blocked by incubation of the blots with 5% non-fat dry milk in Tris-buffered saline/Tween-20 (20 mM Tris Base, pH 7.6, 137 mM sodium chloride, and 0.1% Tween 20) for 1 h at room temperature (RT). The blots were incubated overnight at 4 C with primary antibodies (1:500 to 1 1:1000) in Tris-buffered saline/Tween-20, washed three times, and then incubated for 1 h at RT with HRP-conjugated secondary antibody (1:2000C1:20,000), diluted in Tris-buffered saline/Tween-20. Immunoreactive bands were visualized using an ECL detection system (GE Healthcare Biosciences, Piscataway, NJ). In some experiments membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL) and reprobed with different antibodies. Representative gels were analyzed by densitometry using.