熊果酸对干扰素γ刺激HaCaT细胞产生白细胞介素33的影响

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

目的：探讨熊果酸对干扰素γ（IFN？γ）刺激HaCaT细胞产生白细胞介素33（IL？33）的影响及其机制。方法不同浓度熊果酸（0、0.1、1、5、10、20、40、80μmol/L）分别刺激HaCaT细胞24、48、72 h，MTT法检测其对细胞增殖的影响。将HaCaT细胞与200μg/L IFN？γ共培养建成炎性细胞模型，再与10和15μmol/L熊果酸共培养，以IFN？γ诱导的HaCaT炎性细胞模型为对照，探讨熊果酸的抗炎作用，RT？PCR法检测IL？6和IL？33 mRNA的表达，Western印迹法检测IL？33、p？ERK1/2和ERK1/2蛋白的表达。结果 MTT法检测显示，5~20μmol/L熊果酸作用24 h对细胞活力影响不大，而40~80μmol/L熊果酸在各个时间点均可明显抑制HaCaT细胞增殖（P〈0.05），故后续实验采用10和15μmol/L浓度。HaCaT细胞经IFN？γ刺激后，IL？33 mRNA（0.812±0.036）、IL？6 mRNA（0.947±0.091）表达量和IL？33蛋白表达（1.317±0.119）均显著高于空白对照组（分别为0.412±0.021、0.595±0.030和0.147±0.036，均P〈0.05）；而IFN？γ＋10μmol/L熊果酸组（分别为0.447±0.042、0.437±0.099和0.923±0.058）和IFN？γ＋15μmol/L熊果酸组（分别为0.438±0.028、0.350±0.075和0.564±0.113）又较IFN？γ组（分别为0.812±0.036、0.947±0.091和1.317±0.119）显著降低（均P〈0.05），且这两个熊果酸组IL？33 mRNA水平与空白对照组相比差异无统计学意义（P〉0.05），而IFN？γ＋15μmol/L熊果酸组IL？33蛋白水平显著低于IFN？γ＋10μmol/L熊果酸组（P〈0.05）。HaCaT细胞分别经IFN？γ刺激5、60 min后，p？ERK1/2蛋白表达均明显高于空白对照组。IFN？γ＋15μmol/L熊果酸5 min组和60 min组p？ERK1/2蛋白的相对表达量（0.458±0.053、0.302±0.054）分别低于IFN？γ5 min组（0.941±0.042）和60 min组（0.509±0.032），差异均有统计学意义（P〈0.05），而总ERK1/2蛋白表达量不变。结论熊果酸能够降低IFN？γ刺激的HaCaT细胞IL？33的表达量，其机制可能与调节ERK信号通路相关蛋白的表达有关。

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 80μmol/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 200μg/L IFN？γ. Some HaCaT cells were classified into several groups to be treated with IFN？γ（200μg/L）and UA（10 and 15μmol/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＆nbsp;after 5？and 60？minute treatments with IFN？γand UA alone or in combination. Results MTT assay showed that the treatments with 5-20μmol/L UA for 24 hours had no effects on cell proliferative activity, while 40-80μmol/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 200μg/L IFN？γ, there was a significant increase in the expressions 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？γgroup, the IFN？γ＋10？μmol/L UA group and IFN？γ＋15？μmol/L UA group both showed significantly decreased expressions 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 expression between the IFN？γ＋10？or 15？μmol/L UA group and blank control group（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？γgroup（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？γgroup 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.