Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear translocation

Cells were pre-treated with nimbolide for 24 h and oxidative stress was induced with LPS (1？μg/ml). After 24 h of incubation, the nitrite levels were measured in cell culture supernatants of (a) RAW 264.7 and (b) differentiated THP-1 cells by Griess assay. Whole cell protein was extracted and subjected to western blot and measured the expression of nitrotyrosine and iNOS in both (c) RAW 264.7 and (d) differentiated THP-1 cells. After 30？min of LPS stimulation, intracellular and mitochondrial ROS (mROS) levels were measured by (e, g) DCFDA and (f, h) MitoSOX Red staining in both RAW 264.7 and differentiated THP-1 cells, respectively. The fluorescent images were captured at ×200 magnification from different groups. (i) RAW264.7 cells were pre-treated with nimbolide (1？μM) and oxidative stress was induced by LPS (1？μg/ml). After 12 h of LPS exposure, cells were incubated with Pa and macrophage bactericidal activity was calculated by counting Pa colonies on nutrient agar plates. Western blot was performed to determine the Nrf-2, HO-1, and SOD1 expression in both (j) RAW 264.7 and (k) differentiated THP-1 cells from whole cell lysate. Data represented as mean？±？SEM (n？=？3 independent experiments). *P？<？0.05, ***P？<？0.001, and ****P？<？0.0001 is significantly different from the normal control (NC) group; ###P？<？0.001 and ####P？<？0.0001 are significantly different from the LPS grou