双环醇拮抗DDT引起的细胞间隙连接通讯功能抑制研究

目的研究治肝炎药物双环醇对促癌剂滴滴涕(DDT)引起细胞间隙连接通讯(GJIC)功能抑制的拮抗作用及作用机制。方法划痕标记染料示踪技术直接观察DDT引起的大鼠肝上皮WB-F344细胞GJIC功能抑制并分析双环醇的作用。利用Western blot方法检测间隙连接蛋白43(Cx43)、磷酸化Cx43、E-cadherin及β-Catenin的表达和活性。细胞免疫荧光技术考察WB-F344细胞Cx43蛋白亚细胞定位、间隙连接的形成及E-cadherin和β-Catenin在细胞内的表达。结果 DDT能剂量依赖性的抑制WB-F344细胞GJIC功能,20μM浓度时小分子荧光染料Luciferyellow CH仅能从伤沿细胞向后传递1-2列细胞。双环醇能部分恢复DDT引起的GJIC功能抑制,且具有一定剂量依赖关系,其作用机制与抑制DDT引起的磷酸化Cx43蛋白表达量升高,进而恢复DDT损伤的间隙连接形成有关。DDT和双环醇对与Cx43蛋白功能密切相关的E-cadherin及β-Catenin的表达、活性及细胞内定位均无明显影响。结论双环醇能通过影响Cx43蛋白的磷酸化水平,部分恢复环境促癌剂DDT引起的WB-F344细胞间隙连接的形成,改善GJIC功能。对GJIC的功能抑制是多种促癌剂诱发肿瘤发生的重要原因,前期研究显示双环醇具预防二甲基亚硝胺/苯巴比妥诱发肝癌发生的作用,本文研究结果进一步提示,双环醇在预防杀虫剂DDT(一种主要的环境致癌物)诱发的肿瘤方面亦具有一定潜能。

Effects of Bicyclol on the Inhibition of Gap Junctional Intercellular Communication by DDT in WB-F344 Cells

Objective To determine the effects of bicyclol on dichloro diphenyl trichloroethane（DDT）-induced inhibition of gap junctional intercellular communication in WB-F344 rat liver epithelial cells and the mechanisms involved in the process. Methods The scrape loading/dye transfer（SL/DT） technique was used to assay the inhibition of GJIC induced by DDT and the preventive effects of bicyclol. The expression of total-Cx43, phosphor-Cx43, E-cadherin and jS-catenin were assessed by Western blot. Immunofluorescence analysis was used to determine the subcellular distribution of Cx43, E-cadherin and /3-Catenin. Results Two hours treatment of DDT could inhibit GJIC of WB-F344 cells in a dose-dependent manner. The cells were pre-incubated with bicyclol for 22h and this was followed by treatment with DDT for 2h, bicyclol could prevent the inhibition of GJIC induced by DDT. The reduction of high phosphor-Cx43 expression induced by DDT and the restoration of gap junction maybe contribute to the mechanisms of bicyclol. Both DDT and bicyclol showed no significant effect on expression, activity and cellular distribution of E-cadherin and ~8-catenin. Conclusion Bicyclol prevented the DDT-induced GJIC inhibition via regulating the expression ofphosphor-Cx43 and restoring the gap junction. The inhibition of GJIC is a known cellular event associated with tumor promotion. Previous results showed that bicyclol could prevent the hepatocarcinogenesis induced by diethylnitrosamine/phenobarbital. This paper suggested that bicyclol maybe also have the potential prevention to DDT induced tumorgenesis, which is a pesticide and strongly persists in the environment.