Research Paper Volume 9, Issue 10 pp 2069—2082

Natural product celastrol suppressed macrophage M1 polarization against inflammation in diet-induced obese mice via regulating Nrf2/HO-1, MAP kinase and NF-κB pathways

Figure 4. Effects of celastrol on the activation of MAP kinases, NF-κB and Nrf2/HO-1 pathways in RAW264.7 cells. (A) Western blot analysis of MAP kinase activation in LPS-stimulated RAW264.7 cells. After treatment with LPS alone or in combination with celastrol, the cellular proteins were analyzed by Western blotting with specific antibodies. Representative blots were shown. The blots (n = 3) were quantified by a densitometric method. C0.25, celastrol (0.25 μM); C0.5, celastrol (0.5 μM); C0.75, celastrol (0.75 μM); **, p < 0.01; ***, p < 0.001. (B) Nuclear translocation of NF-κB p65 subunit. After treatment with 1 μg/ml LPS alone or in combination with 1 μM celastrol, the nuclear and cytoplasmic proteins were prepared and analyzed by Western blotting with specific antibodies. Lamin B and GAPDH were used as loading control. Representative blots were shown. (C) Nuclear translocation of Nrf2. After treatment with celastrol, the nuclear and cytoplasmic proteins were prepared and analyzed by Western blotting with specific antibodies. Lamin B and GAPDH were used as loading control. Representative blots were shown. (D) Induction of HO-1 expression. After 24 h treatment with celastrol, the cellular proteins were analyzed by Western blotting with specific antibodies and GAPDH (as loading control). Representative blots were shown. The blots (n = 3) were quantified by a densitometric method. *, p < 0.05; ***, p < 0.001.