[1]陈小艳,惠 坤,马科党.咖啡酸衍生物WSY6对H2O2诱导的黑素细胞氧化损伤保护作用及潜在分子机制研究[J].陕西医学杂志,2021,50(10):1199-1203，1213.[doi:DOI:10.3969/j.issn.1000-7377.2021.10.006]
　CHEN Xiaoyan,HUI Kun,MA Kedang.Protective effect of caffeic acid derivative WSY6 on H2O2-induced oxidative damage of melanocytes and its potential molecular mechanism[J].,2021,50(10):1199-1203，1213.[doi:DOI:10.3969/j.issn.1000-7377.2021.10.006]
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咖啡酸衍生物WSY6对H2O2诱导的黑素细胞氧化损伤保护作用及潜在分子机制研究
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《陕西医学杂志》[ISSN:1000-7377/CN:61-1281/TN]

卷:
50
期数:
2021年10期
页码:
1199-1203，1213
栏目:
基础研究
出版日期:
2021-10-05

文章信息/Info

Title:
Protective effect of caffeic acid derivative WSY6 on H2O2-induced oxidative damage of melanocytes and its potential molecular mechanism
作者:
陈小艳1; 惠 坤2; 马科党2
(1.延安大学咸阳医院皮肤科,陕西 咸阳 712000; 2.陕西省中医医院皮肤科,陕西 西安 710003)
Author(s):
CHEN Xiaoyan; HUI Kun; MA Kedang
(Department of Dermatology,Xianyang Hospital,Yan'an University,Xianyang 712000,China)
关键词:
咖啡酸衍生物WSY6;  H2O2;  黑素细胞氧化损伤;  白癜风;  保护作用;  分子机制
Keywords:
affeic acid derivative WSY6;  H2O2;  Melanocyte oxidative damage;  Vitiligo;  Protective effect;  Molecular mechanism
分类号:
R 96
DOI:
DOI:10.3969/j.issn.1000-7377.2021.10.006
文献标志码:
A
摘要:
目的:观察应用咖啡酸衍生物WSY6对H2O2诱导的黑素细胞氧化损伤的保护作用及潜在分子机制研究。方法: 将人表皮黑素细胞进行体外培养,设为6.25 μmol/LWSY6组、12.5 μmol/LWSY6组、25 μmol/LWSY6组(分别采用6.25、12.5、25 μmol/LWSY6预处理1 h后,采用1 mmol/L H2O2处理1 h)、H2O2组(1 mmol/L H2O2处理)、空白组(不做任何处理),持续培养1 d后,对黑素细胞存活率及LDH释放量进行测定。将部分黑素细胞分为H2O2组(直接用1 mmol/L H2O2处理1 h)和抑制剂组(用p38抑制剂预处理1 h,再用1 mmol/L H2O2处理1 h),处理完成后持续培养1 d,对LDH的释放量进行培养。用25 μmol/L WSY6对部分黑素细胞进行预处理1、2、4 h后,再用H2O2处理1 h,对细胞活性氧(ROS)水平进行检测; 分别采用6.25、12.5、25 μmol/LWSY6预处理部分黑素细胞1 h后,再用H2O2处理1 h,并观察c-Jun氨基末端激酶(p-JNK)、细胞外调节蛋白激酶(p-ERK)、磷酸化丝裂原活化的蛋白激酶(p-p38 MAPK)、半胱氨酸天冬氨酸蛋白酶3(caspase-3)、caspase-9、细胞色素C(Cyto-C)的表达。结果:①1 d后,加入100 μmol/L WSY6后细胞存活率与空白组比较显著降低(P<0.05); ②H2O2组细胞存活性明显低于空白组,而0.39～50 μmol/L WSY6预处理后,细胞存活率明显高于H2O2组,且浓度越高细胞存活率越高,差异有统计学意义(均P<0.05); ③6.25～25 μmol/L WSY6细胞LDH释放量均较空白组显著升高,而较H2O2组显著降低(均P<0.05),且WSY6浓度越低则降低越明显(r=-0.949,P<0.01),p38抑制剂组和H2O2组黑素细胞LDH释放量比较差异不具有统计学意义(均P>0.05); ④不同时间处理25 μmol/L WSY6与H2O2组比较显著降低(均P<0.05),黑素细胞内ROS含量随WSY6预处理时间越长而越低(r=-0.892,P<0.05); ⑤黑素细胞仅少量表达caspase-3、Cyto-C及caspase-9,而采用H2O2处理后,caspase-3、Cyto-C及caspase-9表达含量均明显升高(均P<0.05),但经WSY6分别对caspase-3、Cyto-C及caspase-9处理后,caspase-3、Cyto-C及caspase-9表达均降低(P<0.05)。P-p38、p-ERK和p-JNK均采用H2O2处理后,其表达水平均高于空白组(均P<0.05); 对WSY6预处理后,H2O2WSY6浓度明显低于p-p38的表达,H2O2 WSY6浓度越低则H2O2WSY6表达越低,而p-JNK、p-ERK的表达无改变; 而WSY6浓度升高后p38 MAPK下游产物p-p53的表达则降低(P<0.05),联合WSY6或单独H2O2处理JNK、ERK及p38总蛋白水平均无明显变化(均P>0.05)。结论: H2O2诱导的黑素细胞氧化应激损伤采用咖啡酸衍生物WSY6具有明显的保护作用,其途径可能是通过p38 MAPK发挥。
Abstract:
Objective:To observe the protective effect of the caffeic acid derivative WSY6 on melanocyte oxidative damage induced by H2O2 and its potential molecular mechanism.Methods:Human epidermal melanocytes were cultured in vitro and set as 6.25 μmol/L WSY6 group,12.5 μmol/L WSY6 group,25 μmol/L WSY6 group(after pretreatment with 6.25,12.5,25 μmol/L WSY6 for 1 hour,1 mmol/L H2O2 was used for 1 hour),H2O2 group(1 mmol/L H2O2 treatment),and blank group(no treatment).After continuous culture for 1 day,the survival rate of melanocytes and the release of LDH were determined.Part of melanocytes were divided into H2O2 group(directly treated with 1 mmol/L H2O2 for 1 hour)and inhibitor group(pretreated with p38 inhibitor for 1 hour,and then treated with 1 mmol/L H2O2 for 1 hour).After the treatment was completed,the LDH release was determined by continuous culture for 1 day.Part of melanocytes were pretreated with 25 μmol/L WSY6 for 1,2,4 hours,and then treated with H2O2 for 1 hour to detect cellular reactive oxygen species(ROS)levels.Some black cells were pretreated with 6.25,12.5,and 25 μmol/L WSY6,respectively.After 1 hour,the cells were treated with H2O2 for 1 hour,and the expression of c-Jun N-terminal kinase(p-JNK),extracellular regulatory protein kinase(p-ERK),phosphorylated mitogen-activated protein kinase(p-p38 MAPK),caspase-3,caspase-9,Cyto-C were observed.Results:①After 1 day,the cell survival rate after adding 100 μmol/L WSY6 was significantly lower than that of the blank group(P<0.05).②The cell viability of the H2O2 group was significantly lower than that of the blank group,and after 0.39-50 μmol/L WSY6 pretreatment,the cell survival rate was significantly higher than that of the H2O2 group,and the higher the concentration,the higher the cell survival rate,and the difference was significant(P<0.05).③The LDH release of 6.25-25 μmol/L WSY6 cells was significantly higher than that of the blank group,but was significantly lower than that of the H2O2 group(all P<0.05),and the lower the concentration of WSY6,the more obvious the decrease(r=-0.949,P<0.01),but there was no statistically significant difference in the LDH release of melanocytes between the p38 inhibitor group and the H2O2 group(P>0.05).④Treatment of 25 μmol/L WSY6 and H2O2 group at different time significantly decreased(all P<0.05),and the content of ROS in melanocytes decreased with the longer the pretreatment time of WSY6(r=-0.892,P<0.05).⑤Melanocytes expressed only a small amount of caspase-3,Cyto-C and caspase-9,and after treated with H2O2,the expression levels of caspase-3,Cyto-C and caspase-9 were all significantly increased.However,the expression of caspase-3,Cyto-C and caspase-9 all decreased after WSY6 treatment(all P<0.05).After p-p38,p-ERK and p-JNK were treated with H2O2,their expression levels were higher than those of the blank group.After pretreatment with WSY6,the concentration of H2O2WSY6 was significantly lower than that of p-p38.The lower the concentration of H2O2WSY6,the higher the expression of H2O2WSY6.The expression of p-JNK and p-ERK remained unchanged.However,the expression of p38 MAPK downstream product p-p53 decreased when the concentration of WSY6 increased.The total protein levels of JNK,ERK and p38 did not change significantly when combined with WSY6 or H2O2 alone.Conclusion:Caffeic acid derivative WSY6 can protect melanocytes from oxidative stress injury induced by H2O2,which may be mediated by p38 MAPK.

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备注/Memo

备注/Memo:
基金项目:陕西省中医药管理局科研项目(15-JC013)
更新日期/Last Update: 2021-10-08