Figure 5. HO-1 inhibitor SnPP attenuated NF-κB activation and macrophage biomarker expression against celastrol in LPS-stimulated RAW264.7 cells. (A) Restoration of NF-κB nuclear translocation against celastrol in LPS-stimulated macrophages. After drug treatment, the cells were stained for NF-κB p65 subunit, whereas the cell nuclei were stained with DAPI. The cells were imaged under a Zeiss fluorescence microscopy. Representative images were shown. Cela, celastrol (0.75 μM). Scale bar, 20 μm. (B) Specific up-regulation of iNOS expression against celastrol in LPS-stimulated macrophages. After 24 h treatment, the cellular proteins were analyzed by Western blotting with specific antibodies and GAPDH (as loading control). The blots (n = 3) were quantified by a densitometric method. Representative blots were shown. ***, p < 0.001. (C) Attenuation of celastrol inhibition on macrophage M1 and M2 biomarkers in LPS-stimulated RAW264.7 cells. After drug treatment, the expression of selected macrophage M1 and M2 biomarkers was quantified by qRT-PCR technique using Qiagen primers. C0.75, celastrol (0.75 μM); *, p < 0.05; **, p < 0.01; ***, p < 0.001. (D) Schematic illustration of the potential mechanisms. Celastrol activates Nrf2/HO-1 pathway while inhibits MAP kinases (e.g., ERK1/2, p38, JNK) and NF-κB pathway.