Abstract:

Hu Hua, Song Xiangfeng, Sun Min, Fu Dandan, Li Min, Tian Zhongwei Deparment of Dermatology, First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453100, Henan, China （Hu H, Sun M, Fu DD, Li M, Tian ZW）; School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang 453100, Henan, China （Song XF） Corresponding author: Tian Zhongwei, Email: zhonwt@xxmu.edu.cn 【Abstract】 Objective To evaluate effects of ursolic acid （UA） on interleukin?33 （IL?33） 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 s of IL?6 and IL?33, and Western?blot analysis to measure IL?33 protein after 12?hour culture. The s 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）. Thus, 10 and 15 μmol/L were chosen as the concentrations of UA for further study. After the treatment with 200 μg/L IFN?γ, there was a significant increase in the s of IL?33 mRNA （0.812 ± 0.036 vs. 0.412 ± 0.021）, IL?6 mRNA （0.947 ± 0.091 vs. 0.595 ± 0.030） and IL?33 protein （1.317 ± 0.119 vs. 0.147 ± 0.036） in HaCaT cells compared with the blank control group （all P < 0.05）. Compared with the IFN?γ group, the IFN?γ + 10?μmol/L UA group and IFN?γ + 15?μmol/L UA group both showed significantly decreased s of IL?33 mRNA （0.447 ± 0.042 and 0.438 ± 0.028 respectively, both P < 0.05）, IL?6 mRNA （0.437 ± 0.099 and 0.350 ± 0.075 respectively, both P < 0.05） and IL?33 protein （0.923 ± 0.058 and 0.564 ± 0.113 respectively, both P < 0.05）. There were no significant differences in IL?33 mRNA between the IFN?γ + 10? or 15?μmol/L UA group and blank control group （P > 0.05）, while IL?33 protein 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 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 between the IFN?γ + 15?μmol/L UA group and IFN?γ group at 5 or 60 minutes. Conclusion UA can suppress IL?33 in HaCaT cells induced by IFN?γ, likely by regulating s of the ERK signaling pathway?related proteins.