滋补肝肾方调控B16黑素细胞氧化损伤的分子机制

Molecular mechanism of Zibu Ganshen Formula on regulating the oxidative damage of B16 melanocytes

目的:探讨滋补肝肾方组方配伍抑制B16黑素细胞氧化损伤的分子机制。方法:研究共分6组:正常对照组、模型组、祛风组、养血组、补肝肾组、全方组。采用流式细胞仪检测细胞凋亡率,Hoechst 33342观察凋亡细胞的核变化,激光共聚焦显微镜观察细胞线粒体膜电位,底物荧光法检测Caspase-3/9的活性,Western Blot法测定细胞凋亡B淋巴细胞瘤-2基因(Bcl-2)、细胞色素c(Cyt-c)蛋白的表达。结果:(1)与正常对照组比较,模型组凋亡率、Caspase-3/9活性显著增高(P<0.01),线粒体膜电位、Bcl-2蛋白表达显著降低(P<0.01),Cyt-c蛋白表达量升高(P<0.01)。(2)与模型组比较,滋补肝肾含药血清各组凋亡率显著降低(P<0.01,P<0.05),线粒体膜电位降低得到改善,Caspase-3/9活性降低(P<0.01),可使Cyt-c蛋白表达量降低,Bcl-2蛋白表达量升高(P<0.01)。结论:线粒体信号转导通路可能是滋补肝肾含药血清干预氧化应激诱导B16黑素细胞凋亡的调控机制之一,全方组抑制氧化应激效果最好,体现了中医治疗白癜风、综合作用的优势。

Objective:To explore the molecular mechanism of the compatibility of nourishing liver and kidney prescription to inhibit the oxidative damage of B16 melanocytes.Methods:The study was divided into 6 groups:normal control group,model group,Qufeng group,Yangxue group,Buganshen group and Zibu Ganshen Formula group.The apoptosis rate was detected by flow cytometry,the nuclear changes of apoptotic cells were observed by Hoechst 33342,the mitochondrial membrane potential was observed by laser confocal microscope,the activity of Caspase-3/9 was detected by substrate fluorescence method,and Western Blot method was used.The expression of Bcl-2 and Cyt-c protein was measured.Results:(1)Compared with the normal control group,the apoptosis rate and Caspase-3/9 activity in the model group increased significantly(P<0.01),mitochondrial membrane potential and Bcl-2 protein expression decreased significantly(P<0.01),and Cyt-c protein expression increased(P<0.01).(2)Compared with the model group,the apoptosis rate and Caspase-3/9 activity of nourishing liver and kidney medicated serum decreased significantly(P<0.01,P<0.05),mitochondrial membrane potential reduction was improved,and the expression of Cyt-c protein decreased and the expression of Bcl-2 protein increased(P<0.01).Conclusion:Mitochondrial signal transduction pathway may be one of the regulatory mechanisms of nourishing liver and kidney medicated serum to interfere with oxidative stress-induced apoptosis of B16melanocytes,and the whole formula group has the best effect of inhibiting oxidative stress,which shows that the advantages of traditional Chinese medicine in the treatment of vitiligo and its comprehensive function have been gained.