3种中药对中波紫外线辐射HaCaT细胞的干预及其机制

Effect and regulatory mechanisms of three traditional Chinese medicines on HaCaT cells irradiated by UVB

目的观察中波紫外线(UVB)辐射后HaCaT细胞光产物胸腺嘧啶二聚体(cyclobutanepyrimidine dimers,CPDs)的生成和清除情况及3种中药活性成分:黄芩(baikal skullcap root,BSR)、川芎(szechwan lovge rhizome,SLR)和表没食子儿茶素没食子酸酯[(-)-epigallocatechin-3-gallate,EGCG]对该过程的干预情况并探讨上述药物对相关调控分子的作用机制。方法以含10%小牛血清的RMPI1640培养基培养永生化人角质形成细胞株HaCaT细胞,根据实验设计定时定量照射培养细胞并预先或UVB辐射后加入相关药物进行干预处理。免疫组织化学法检测CPDs;RT-PCR法检测受试各组细胞p53和PCNA基因mRNA表达;Western blot法检测受试各组细胞p53和PCNA蛋白表达。结果HaCaT细胞损伤程度随照光剂量加大而加重;30mJ/cm2UVB照射后细胞CPDs生成量在辐射后0.5h左右达到高峰,同时细胞也开始清除CPDs,辐射后4h内清除速率较快,4h后清除速率逐渐降低,照光前加入黄芩溶液,可以减少CPDs生成,照光后即刻加入川芎和EGCG溶液,可加快CPDs清除(P<0.05);UVB照射后可明显增加HaCaT细胞中p53和PCNA mRNA及蛋白表达水平,药物可在不同程度上下调上述基因及蛋白表达。结论UVB辐射对HaCaT细胞的损伤程度呈剂量依赖性并导致DNA损伤而产生光产物CPDs,细胞自身有清除CPDs的能力,所试3种中药均可减少光产物水平;这种光保护作用可能与受试中药影响p53和PCNA基因mR-NA和蛋白表达有关。

Aim To investigate the production and removal of cyclobutane pyrimidine dimers （CPDs） after UVB irradiation and the effect of baikal skullcap root, szechwan lovge rhizome and epigallocatechingallate （EGCG） on the irradiation damage in HaCaT cells, and finally, to clarify the probable regulatory mechanisms. Methods HaCaT cells were cultured in RMPI-1640 medium with 10% fetal bovine serum. The time and dosage of UVB irradiation were conducted according to the experimental design. Tested medicines were added into the medium before or after UVB irradiation. The production and removal of CPDs were examined by immunohistochemical method; the p53 and PCNA mRNA expressions were examined by RT-PCR; the p53 and PCNA protein expressions were examined by Western blot. Results The damage of HaCaT cells was of UVB irradiation. CPDs appeared in HaCaT cells and immediately after irradiation reached the peak in 0. 5 h and they were removed rapidly during the first 4 h and slowly thereafter. The production of CPDs was decreased when HaCaT cells were preincubated with baikal skullcap root solution before UVB irradiation and the removal of CPDs was accelerated when HaCaT cells were coincubated with szechwan lovge rhizome and EGCG solution after UVB irradiation （ P 〈 0. 05 ） ; UVB irradiation could induce mRNA and protein expressions of p53 and PCNA in cultured HaCaT cells and mRNA and protein levels of p53 and PCNA were decreased after tested medicine intervention in UVB irradiated group. Conclusions The photodamage of HaCaT cells induced by UVB irradiation was dose dependent, which induced the production of CPDs. DNA lesions could be repaired by cell itself; three traditional Chinese medicines could decrease the level of CPDs; inhibiting expressions of mRNA and proteins of p53 and PCNA may be part of mechanisms related to these photoprotecting effects of UVB irradiation.