Effects of ursolic acid on interleukin-33 expression in HaCaT cells induced by interferon-γ

Objective To evaluate effects of ursolic acid (UA)on interleukin-33 (IL-33)expression in HaCaT cells induced by interferon-γ (IFN-γ), and to explore their mechanism. Methods Some HaCaT cells were treated with UA at different concentrations (0, 0.1, 1, 5, 10, 20, 40 and 80 μmol/L)for 24, 48 and 72 hours separately. Then, methyl thiazolyl tetrazolium (MTT)assay was conducted to evaluate cell proliferative activity. A cell model of inflammation was established by culture of HaCaT cells with the presence of 200 μg/L IFN-γ. Some HaCaT cells were classified into several groups to be treated with IFN-γ (200 μg/L)and UA (10 and 15 μmol/L)alone or in combination (firstly treated with IFN-γ followed by UA treatment), and those receiving no treatment served as the blank control group. Reverse transcription PCR (RT-PCR)was performed to detect mRNA expressions of IL-6 and IL-33, and Western-blot analysis to measure IL-33 protein expression after 12-hour culture. The expressions of extracelluar signal-regulated kinase 1/2 (ERK1/2)and phosphorylated ERK1/2 (p-ERK1/2)were also measured by Western-blot analysis after 5-and 60-minute treatments with IFN-γ and UA alone or in combination. Results MTT assay showed that the treatments with 5-20 μmol/L UA for 24 hours had no effects on cell proliferative activity, while 40-80 μmol/L UA could significantly inhibit it at 24, 48 and 72 hours (all P 0.05), while IL-33 protein expression was significantly lower in the IFN-γ+ 15-μmol/L UA group than in the IFN-γ+ 10-μmol/L UA group (P < 0.05). The p-ERK1/2 protein expression significantly increased in HaCaT cells treated with IFN-γ for 5 and 60 minutes compared with the blank control group, but significantly decreased in the IFN-γ+ 15-μmol/L UA group compared with the IFN-γ group (0.458±0.053 vs. 0.941±0.042 at 5 minutes, 0.302±0.054 vs. 0.509±0.032 at 60 minutes, both P < 0.05). However, no significant differences were observed in the total ERK1/2 protein expression between the IFN-γ+ 15-μmol/L UA group and IFN-γ group at 5 or 60 minutes. Conclusion UA can suppress IL-33 expression in HaCaT cells induced by IFN-γ, likely by regulating expressions of the ERK signaling pathway-related proteins. Key words: Ursolic acid; Interferon-gamma; Interleukin-1; Interleukin-6; Extracellular signal-regulated MAP kinases; HaCaT cells; Interleukin-33