叶绿酸对氧化损伤的防护作用及其机制

The protective effect of chlorophyllin against oxidative damage and its mechanism

目的 观察叶绿酸能否通过诱导血红素加氧酶-1（HO-1）表达来发挥抗氧化功能以保护细胞并探讨其相关机制。方法利用流式细胞术检测叶绿酸对氧化损伤的细胞保护作用，电子自旋共振波谱直接测定自由基水平；逆转录PCR、Western blot、免疫荧光激光共聚焦显微技术、酶活性检测验证叶绿酸对HO-1的诱导表达；Western blot检测是否涉及信号通路P13K／Akt的激活。结果 叶绿酸通过清除人脐静脉血管内皮细胞中由H202损伤产生的过量自由基而起到保护作用。叶绿酸能以剂量和时间依赖的方式诱导HO-1的表达。叶绿酸诱导HO-1表达涉及信号通路P13K／Akt的激活。P13K抑制剂LY294002可以剂量依赖方式明显抑制叶绿酸对P13K／Akt的激活和HO-1的诱导表达。结论 叶绿酸具有清除自由基的细胞保护作用，HO-1的诱导表达在其中发挥了关键性作用。P13K／Akt信号通路的激活在抗氧化酶HO-1诱导表达过程中是必需的。

Objective To investigate whether chlorophyllin could protect human umbilical vein endothelial cell （HUVEC） against oxidative damage by inducing the expression of heme oxygenase-1 （HO-1） and to explore the underlying mechanism. Methods The cellular protection of chlorophyllin against oxidative damage was detected by cell-survival assay with flow cytometry. The level of free radicals was detected directly by electron spin resonance spectra. The induced expression of HO-1 was shown by RT-PCR, Western blot, immunofluorescence confocal laser microscopy and enzymatic activity test. Whether the activation of PI3K/Akt pathway was involved was detected by Western blot. Results Chlorophyllin could protect HUVEC against oxidative damage caused by H202 via scavenging the excessive free radicals. Chlorophyllin treatment could induce expression of HO-1 in a dose- and time-dependent manner. The activation of PI3K/Akt pathway was required in the induction of HO-1. LY294002, the specific inhibitor of PI3K, could suppress the activation of PI3K/Akt and the induced expression of HO-1 in a dose-dependent manner. Conclusions Chlorophyllin shows cellular protection against oxidative damage by counteracting the excessive free radicals. Up-regulation of HO-1 expression plays a pivotal role in the protection of chlorophyllin, while the activation of PI3K/Akt signaling pathway is required in the induction of HO-1.