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. 2013 Dec 10;171(1):237–252. doi: 10.1111/bph.12440

Figure 8.

Figure 8

Andrographolide activates p38 MAPK phosphorylation to stimulate the Nrf2–HO-1 signalling pathway for anti-HCV activity. (A) Andrographolide activated p38 MAPK phosphorylation in a time-dependent manner. Ava5 cells were treated with 10 μM andrographolide at the indicated time points (0–120 min), and total protein was assayed by Western blotting with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK and anti-GAPDH (loading control) antibodies. Band intensity was quantified by densitometric scanning and presented as fold value relative to the time point of 0 min (defined as 1) following normalization against the GAPDH protein level. (B) Restoration of andrographolide-reduced HCV protein synthesis by p38 MAPK inhibitor. Ava5 cells were co-treated with 10 μM andrographolide and each MAPK molecule inhibitor against the ERK (PD98059), p38 (SB203580) or JNK inhibitor (SP600125) at the indicated concentration for 3 days. The cell lysates were assayed by Western blotting with anti-NS5B, anti-Nrf2, anti-HO-1 or anti-GAPDH antibody. Equal loading of cell lysates was confirmed by the GAPDH protein level. (C) The andrographolide-induced Nrf2 transactivation was restored to control levels by the p38 MAPK inhibitor SB203580. The cell lysates of p2xARE-Luc-transfected Ava5 cells were analysed for luciferase activity under the experimental conditions described earlier. Each transfection mixture contained 0.1 μg pSEAP reporter plasmid as a control of transfection efficiency. Nrf2-mediated ARE transactivation was assayed for luciferase activity and presented as fold activation relative to drug-untreated cells (defined as 1) following normalization against SEAP activity. (D) The p38 MAPK inhibitor suppressed the andrographolide-induced Nrf2 transactivation in a concentration-dependent manner. The cell lysates of p2xARE-Luc-transfected Ava5 cells were assayed for luciferase activity in the presence of 10 μM andrographolide with increasing concentrations of the p38 MAPK inhibitor SB203580 (0–15 μM) for 3 days. The luciferase activity was presented as fold change relative to andrographolide/SB203580-untreated cells (defined as 1) following normalization against SEAP activity. The experiments were performed in triplicate, and error bars indicate mean ± SD. *P < 0.05, **P < 0.01.