滋补肝肾方全方及拆方对黑素细胞氧化损伤致凋亡的影响

Effect of nourishing liver and kidney prescription and its component prescriptions on apoptosis induced by oxidative injury of melanocytes

目的观察滋补肝肾方中滋补肝肾药、活血药、祛风药及全方对氧化损伤后的黑素细胞活性、凋亡率、线粒体膜电位及释放凋亡蛋白的调节作用,探讨滋补肝肾方的作用机制。方法以B16黑素细胞为研究对象,用过氧化氢刺激造成黑素细胞氧化损伤模型。根据组方规律将滋补肝肾方中的药物拆分为滋补肝肾组、活血组、祛风组,采用冻干粉溶液作用细胞。MTT比色法观察各组黑素细胞的生长抑制;流式细胞仪技术分析DNA含量,确定凋亡细胞所占比例;线粒体膜电位检测试剂盒(JC-1法)检测线粒体膜电位变化;Western blot法检测凋亡蛋白。结果滋补肝肾方全方组(200 μg/mL、100 μg/mL)、活血组(100 μg/mL)可升高氧化损伤后细胞活性,以全方组作用最为明显。全方组细胞凋亡率低,在保护黑素细胞免受氧化损伤方面比其他组效果更明显。与对照组相比,50 μg/mL全方组、200μg/mL滋补肝宵组、50μg/mL滋补肝肾组Bcl-2蛋白表达升高(P<0.05),说明全方组、滋补肝肾组抗凋亡作用明显。Capase 9、Capase 3、CytC模型组与各药物组蛋白表达差异无统计学意义(P>0.05)。25 μg/mL祛风组、12.5μg/mL祛风组AIF蛋白与模型组相比差异有统计学意义(P<0.05)。50μg/mL全方组、50μg/mL滋补肝肾组、50μg/mL活血组、25μg/mL祛风组、12.5μg/mL祛风组Bax蛋白与模型组比较差异有统计学意义(P<0.05)。除全方组、滋补肝肾组外,其余各药物组Smac蛋白表达与模型组比较差异有统计学意义(P<0.05)。结论滋补肝肾方及其拆方可抑制黑素细胞氧化损伤,可能通过线粒体通路发生作用,提示白癜风临床治疗当补血活血、滋补肝肾、祛风共用。

Objective To explore the regulation of drugs for nourishing the liver and kidney,activating blood circulation,eliminating wind as well as the whole prescription for melanocyte activity and apoptosis rate,mitochondrial membrane potential and release of apoptosis protein induced by oxidative damage and discuss the action mechanism.Methods B16 melanocytes were used as the subject and oxidative damage model of melanocytes induced by hydrogen peroxide stimulation,then protective effect on melanocytes were observed.The prescription for nourishing liver and kidney were divided intothe following groups:group of nourishing liver and kidney,group of activating blood,group of eliminating wind,and freeze-dried powder solution was adopted to act on cells.MTT colorimetry was used to observe the growth inhibition of melanocytes in each group;flow cytometry was used to analyze DNA content and determine the proportion of apoptotic cells.Mitochondrial membrane potential detection kit(JC-1 method)was used to detect mitochondrial membrane potential changes.Western blot method was used to detect apoptosis protein.Results In the experiments,the whole prescription group of nourishing liver and kidney(200μg/m L、100μg/mL)and the group of activating blood(100μg/mL)can increase the cell activityafter oxidative damage,the whole prescription group is the most obviously seen.The apoptosis rate of cells in the whole prescription group was the lowest,it is more effective than other groups in protecting melanocytes from oxidative damage.Compared with the control group,there was a significant difference between the 50μg/mL whole prescription group,200μg/mL group of nourishing liver and kidney,the 50μg/mL group of nourishing liver and kidney group,and the protein expression of Bcl-2 were increased(P<0.05)and it indicated that the anti-apoptosis effect was more obvious in whole prescription group and group of nourishing liver and kidney.Capase 9,capase3,and CytC models groups had notsignificant statistical difference with other groups in protein expression.AIF protein in 25μg/mL group of eliminating wind,12.5μg/mL group of eliminating wind had statistical significancewith the model group(P<0.05).50μg/mL whole prescription group,50μg/mL group of nourishing liver and kidney group,50μg/mL group of activating blood,25μg/mL group of dispelling wind,12.5μg/mL group of dispelling wind had statistical significance in Bax protein compared with the model group.Except the whole prescription group and group of nourishing kidney and liver,there was statistical significance in Smac protein expression compared between the drug groups and model group(P<0.05).Conclusion The nourishing liver and kidney recipe and its component recipes can inhibit melanocyte oxidative damage,which may occur through mitochondrial pathway,suggesting that clinical treatment of vitiligo should be combined with enriching blood and promoting blood circulation,nourishing liver and kidney,and dispelling pathogenic wind.