紫檀芪对不同波长紫外线照射后HaCaT细胞内Nrf2与Bach1的平衡关系影响研究

Effect of pterostilbene on balance between Nrf2 and Bach1 in HaCaT cells under ultraviolet irradiation at different wavelengths

目的:探讨紫檀芪是否影响不同波长紫外线照射后HaCaT细胞内Nrf2与Bach1的平衡关系。方法:用不同浓度紫檀芪(0、2.44、4.88、9.75、19.5、39、78、156μmol/L)处理HaCaT细胞后,采用MTS法检测不同浓度紫檀芪对细胞增殖的影响,从而筛选出适宜的浓度进行后续实验;Western-blot检测紫檀芪对Nrf2和Bach1表达的调控;紫外比色法检测细胞内蛋白羰基含量;TBA法检测细胞内MDA含量。结果:MTS结果显示,当紫檀芪浓度为4.88μmol/L时对细胞增殖无影响;Western-blot结果显示UVA可引起Nrf2胞质蛋白减少(q=5.64,P<0.05),胞核蛋白增多(q=16.38,P<0.01),UVB不引起Nrf2胞质蛋白减少(q=1.86,P>0.05),可引起胞核蛋白的含量升高(q=17.24,P<0.01),添加紫檀芪再进行UVA/UVB照射,Nrf2胞质蛋白明显减少(q_(UVA)=8.27,qUVB=6.90,P<0.05),胞核蛋白明显增加(q_(UVA)=17.07,qUVB=36.30,P<0.01)。同时,UVA、UVB均可引起Bach1胞质蛋白增多(q_(UVA)=34.81,qUVB=29.25,P<0.01),胞核蛋白减少(q_(UVA)=27.48,qUVB=20.68,P<0.01),添加紫檀芪再进行UVA/UVB照射,Bach1胞质蛋白明显增多(q_(UVA)=66.48,qUVB=210.20,P<0.01),胞核蛋白明显减少(q_(UVA)=88.52,qUVB=22.25,P<0.01)。紫外比色法结果显示,与UVA组相比,UVA+紫檀芪组蛋白羰基的含量显著减少(q=10.44,P<0.01);与UVB组相比,UVB+紫檀芪组蛋白羰基的含量显著减少(q=7.03,P<0.01)。TBA法结果显示,与UVA组相比,UVA+紫檀芪组MDA的含量显著减少(q=11.22,P<0.01);与UVB组相比,UVB+紫檀芪组MDA的含量显著减少(q=17.49,P<0.01)。结论:紫檀芪通过影响核内Nrf2与Bach1的平衡关系发挥抗氧化作用。

Objective：To explore whether pterostilbene（PTS）has the ability to affect the balance between Nrf2 and Bach1 in HaCaT cell when it was exposed under different wavelengths of ultraviolet irradiation （UVA or UVB） or not. Methods：HaCaT cell was treated with different concentrations of PTS（0, 2.44, 4.88, 9.75, 19.5, 39, 78, 156 μmol/L）and [3-（4,5-dimethylthiazol2yl）-5-（3carboxymethoxyphenyl）-2-（4-sulfophenyl） -2H-tetrazolium, inner salt] （MTS） assay was utilized to test the effect of PTS on cell proliferation, which was in order to screen the most suitable concentration of PTS for followup study. Protein levels of Nrf2 and Bach1 were detected by Westernblot. Protein carbonyl content was tested by ultraviolet colorimetric method. Malondialdehycle （MDA） content was tested by TBA method. Results：Based on the results of MTS assay, the most suitable concentration of PTS is 4.88 μmol/L. Westernblot results showed that UVA can cause Nrf2 cytoplasmic proteins reduced （q=5.64, P〈0.05） and nuclear protein increased （q=16.38, P〈0.01）, UVB did not induce Nrf2 cytoplasmic protein to decrease （q=1.86, P〉0.05）, while can increase the content of nuclear proteins （q=17.24, P〈0.01）. When PTS was added and exposed under UVA/UVB irradiation, Nrf2 protein in the cytoplasm was significantly reduced （qUVA=8.27, qUVB=6.90, P〈0.05）, nucleus protein increased significantly （qUVA=17.07, qUVB=36.30, P〈0.01）. At the same time, UVA and UVB could induce Bach1 protein in the cytoplasm increased （qUVA=34.81, qUVB=29.25, P〈0.01）, nuclear protein （qUVA=27.48, qUVB=20.68, P〈0.01） reduced. When added PTS and UVA/UVB irradiation, Bach1 protein in the cytoplasm increased significantly （qUVA=66.48, qUVB=210.20, P〈0.01）, and nuclear protein was significantly reduced （qUVA=88.52, qUVB=22.25, P〈0.01）. The results of UV colorimetric analysis showed that compared with the UVA group, the content of the carbonyl group in the UVA＋PTS decreased significantly （q=10.44, P〈0.01）. Compared with the UVB group, the content of the carbonyl group in the UVB＋PTS group decreased significantly （q=7.03, P〈0.01）. The results of TBA showed that compared with the UVA group, the content of MDA in the UVA＋PTS group decreased significantly （q=11.22, P〈0.01）. Compared with the UVB group, the content of MDA in the UVB＋PTS group decreased significantly （q=17.49, P〈0.01）.Conclusion：PTS might protect HaCaT cell against ultravioletinduced damage through mediating the balance of Nrf2 and Bach1 in nucleus.