Ingredients from Litsea garrettii as Potential Preventive Agents against Oxidative Insult and Inflammatory Response

Figure 4

MPP activates Nrf2 signaling pathway in human lung epithelial Beas-2B cells. (a) MPP had no cytotoxicity up to 50 μM. Cells were treated with indicated doses of MPP for 48 h, and then cell viability was determined using MTT assay. (b) MPP induced the ARE-dependent luciferase activity in a dose-dependent manner. Cells were transfected with ARE-firefly luciferase and TK-Renilla luciferase plasmids and then treated with the indicated doses of MPP or SF (5.0 μM) for 16 h. (c) MPP dose dependently induced the protein levels of Nrf2 and its downstream genes. Cells were treated with or without indicated doses of MPP or SF (5.0 μM) for 16 h, and then the total cell lysates were subjected to immunoblot analysis. (d and e) MPP induced the nuclear translocation of Nrf2. For (d), cells were treated with or without MPP (25 μM) or SF (5 μM) for 8 h and then subjected to indirect fluorescence staining. For (e), cells were treated with SF (5 μM) and SF (5 μM) and indicated doses of MPP for 8 h, and then the nuclear extracts were collected and subjected to immunoblot analysis. (f) MPP increased the half-life of Nrf2. Cells were left untreated or treated with MPP (25 μM) for 4 h. Cycloheximide (50 μM) was added to block protein synthesis. Cells were harvested at the indicated time points, and then total cell lysates were subjected to immunoblot analysis. (g) MPP induced Nrf2, NQO1, and GCLM but had no effect on Keap1 in the time course study. Cells were treated with MPP (25 μM) for the indicated times, and then the protein levels were measured by immunoblot analysis. (h) MPP induced Nrf2, NQO1, and GCLM in a dose-dependent manner but had no effect on Keap1. Cells were treated with indicated doses of MPP for 16 h, and then the protein levels were measured by immunoblot analysis. Values were presented as mean ± SD (). , , , treated versus control; C: control.

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