Effects Of Ursolic Acid On Interleukin-33 Expression In HaCaT Cells Induced By Interferon-γ

BackgroundPsoriasis is a chronic skin disease that includes an infiltration of immune cells in the dermis and epidermis.It is considered a fact that the immune system plays an important role in the pathogenesis of psoriasis.Recently the profound success of anti-cytokine therapy has shown the importance of the key cytokines IL-23,TNF,and IL-17 in this process.IL-33,a newly found important psoriatic cytokine gives us new insight to explore the mechanism of Psoriasis.Ursolic acid(UA),a natural pentacyclic triterpenoid carboxylic acid,its anti-inflammatory effects and the molecular mechanism of action of UA for the anti-inflammatory activity are also studied.The influence of UA on the release of IL-33 is unclear.Much more work need to be done to explore anti-inflammation effect and the detailed mechanism of anti-inflammatory activity of UA in Psoriasis.ObjectiveTo evaluate effects of ursolic acid(UA)on interleukin-33(IL-33)expression in HaCaT cells induced by interferon-γ(IFN-γ),and to explore its mechanism.MethodsSome cultured 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 cellular proliferative activity.A cell model of inflammation was established by using 200 μg/L IFN-γ.Some HaCaT cells were classified into several groups to be treated with IFN-γ(200 μg/L)as well as UA(10 and 15 μmol/L)alone or in combination(firstly treated with IFN-γfollowed 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 in these cells after 12-hour culture,and 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.Results1.MTT assay showed that the treatments with 5~20 μmol/L UA for 24 hours had no effects on cellular 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 UA were chosen for further study.2.After the treatment with 200 μg/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 m RNA(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).What’s more,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).3.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 total ERK1/2 protein expression between the IFN-γ + 15-μmol/L UA group and IFN-γ group at 5 or 60 minutes.Conclusions1.The effect of UA on the proliferation of HaCaT cells was time and concentration dependent.2.UA can suppress IL-33 expression in HaCaT cells induced by IFN-γ,and it has the anti-inflammatory effect.3.The anti-inflammatory effect of the UA may be regulate expressions of the ERK signaling pathway-related proteins.