摘要
目的探讨转录因子Nrf2/Bach1在枸杞多糖(LBP)防御紫外线(UV)致Ha Ca T细胞光损伤的作用。方法体外培养的Ha Ca T细胞经300μg/m L LBP预处理24 h后,分别接受30 J/cm^2UVA及300 m J/cm^2UVB照射,孵育24h。以噻唑蓝(MTT法)检测细胞增殖活性;尼罗红荧光染色法检测过氧化脂质含量;采用单细胞凝胶电泳法检测DNA损伤;RT-q PCR法检测Nrf2和Bach1 mRNA及下游II相解毒酶基因的表达,Western blot法检测Nrf2及Bach1蛋白在细胞内分布及表达情况。结果强度为30 J/cm^2UVA及300 m J/cm^2UVB均可造成Ha Ca T细胞增殖能力下降,过氧化脂质含量及DNA荧光强度、迁移距离增加,与空白组相比,差异均有统计学意义(P<0.05);300μg/m L LBP预处理可显著提高细胞增殖活性,降低过氧化脂质水平,减轻DNA链断裂损伤,且增加Nrf2核蛋白及Bach1质蛋白的量,促进Nrf2 mRNA及下游II相解毒酶SOD,CAT,NQO1,GCLC,GCLM mRNA的表达,抑制Bach1mRNA的表达(均P<0.05)。结论枸杞多糖可有效减轻UV诱导的HaCaT细胞氧化损伤,其机制可能是通过激活Nrf2/Bach1转位及下游II相解毒酶基因的表达,发挥光防护作用。
Objective To evaluate the role of transcriptional factors of Nrf2 and Bach1 in the prevention of LBP against UV-induced damage in HaCaT cells.Methods The HaCaT cells cultured in vitro were pretreated with 300 μg/mL of LBP for 24 hours, and then were irradiated with 30 J/cm2UVA and 300 mJ/cm2UVB respectively.The cell viability was inspected by the MTT assay.Lipid peroxide levels were detected by the Nile red staining.Then the DNA strand breaks were evaluated through the comet assay.We employed real time PCR to detect the expressions of Nrf2 Bach1 mRNA, and the expressions of phase II enzyme genes.Western blot was carried out to measure distribution and expression of Nrf2 and Bach1.Results The intensity of 30 J/cm2UVA and 300 mJ/cm2UVB decreased the cell proliferation activity, increased the content of lipid peroxide, the DNA fluorescence intensity, and the migration distance.Compared with the blank group, the difference was significant（P〈0.05）.Pretreated HaCaT cells with LBP and irradiated by UVA/UVB, we found that it can significantly improve the cell proliferation activity, and reduce lipid peroxide content and DNA damage（P〈0.05）.Besides, report to blank group, LBP promoted protein expression of Nrf2 in nucleus and Bach1 in cytoplasms.Also, it can obviously up-regulate the expression of Nrf2, SOD, CAT, NQO1, GCLC, GCLM mRNA, inhibiting the expression of Bach1 mRNA（P〈0.05）.Conclusion LBP can effectively attenuate the oxidative damage induced by UV in HaCaT cells.It＇s mechanism may activate the translocation of Nrf2/Bach1 and increase the expression of phase II enzyme genes, acting as photoprotection.
出处
《中国皮肤性病学杂志》
CSCD
北大核心
2017年第11期1169-1174,共6页
The Chinese Journal of Dermatovenereology
基金
广东省建设中医药强省科研课题(20141221)
2014年广东省医学科研基金(A2014592)
2015年广州市科技计划项目(1563000503)
2015年广东省自然科学基金项目(2015A030313765)
2016年广州科技计划项目(201604020093)
广东省科技计划项目(2014A020212570)
广州市医药卫生科技项目(20141A011069)
