Figure 6. Senescence-associated leukotriene synthesis promotes pulmonary fibrosis.

WT C57BL/6J mice received a single intratracheal injection of PBS (vehicle control) or bleomycin (Bleo, 1.9 U/kg). Mice received, from days 7–14,vehicle (Veh) or 50 mg/kg/day ABT-263 by gavage. (A) Level of p16^INK4a and p21^WAF1 mRNA levels normalized to tubulin mRNA. (B) Activation of cytosolic phospholipase A2 (cPLA2) was determined by calculating the ratio of the level of expression of phosphorylated cPLA2 to the level of expression of total cPLA2. Representative pictures of Western blot of lung lysates collected day 14 after the bleomycin injury for the expression of the phosphorylated form of cPLA2, cPLA2-S505P, and nonphosphorylated form cPLA2. n = 3 lysates per group. (C) Level of expression of mRNA of gene encoding enzymes of the leukotriene (ALOX5 and LTC4S) and the prostaglandins (PTGDS, PTGS2, and PTGES) biosynthesis pathways was measured by qPCR normalized to tubulin mRNA, in samples collected 14 days after bleomycin injury. Data are presented as dot plot graphs or heatmap. (D) Lipids were extracted from broncho-alveolar lavage fluid (BALF) collected 14 days after bleomycin injury and treatment with vehicle or ABT-263. BALF lipid content was analyzed for cysteinyl leukotrienes and for PGE2 by ELISA. (E) Representative pictures of Picrosirius red staining of lungs collected 21 days after bleomycin injury and treatment with vehicle or ABT-263. Original magnification, ×100. (F) Hydroxyproline levels obtained using the right lung lobes of mice, 21 days after bleomycin injury and treatment with vehicle or ABT-263. Unless stated otherwise, lung and BALF from at least 5 PBS-treated mice, 5 bleomycin + vehicle-treated mice, and 3 bleomycin + ABT-263 mice were analyzed. Statistical analyses were performed using 1-way ANOVA test. *P ≤ 0.05; **P ≤ 0.01.