异鼠李素对H_(2)O_(2)诱导HaCaT细胞氧化应激损伤的保护作用

Protective effect of isorhamnetin on oxidative stress injury of HaCaT cells induced by H_(2)O_(2)

目的探讨异鼠李素对过氧化氢(H_(2)O_(2))诱导人正常皮肤细胞(HaCaT)氧化损伤的保护作用。方法体外培养HaCaT细胞。使用不同浓度的H_(2)O_(2)(300、600、900、1200μmol/L)处理HaCaT细胞12 h后,CCK-8法检测细胞增殖活力,试剂盒检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,筛选合适的H_(2)O_(2)浓度建立氧化应激模型。使用不同浓度异鼠李素预处理HaCaT细胞12 h,CCK-8法检测细胞存活率,确定异鼠李素安全浓度,用于后续实验。使用安全浓度的异鼠李素预处理HaCaT细胞12 h,600μmol/L H_(2)O_(2)干预HaCaT细胞12 h,进行细胞增殖活力检测、SOD活性检测和MDA含量测定。结果随着H_(2)O_(2)浓度升高,细胞存活率逐渐下降、细胞中SOD活性逐渐下降和MDA含量逐渐升高。其中,600、900、1200μmol/L H_(2)O_(2)组细胞存活率与对照组相比差异均具有统计学意义(P<0.05);H_(2)O_(2)组(300、600、900、1200μmol/L)的SOD活性和MDA含量与对照组比较差异均具有统计学意义(P<0.05),选择600μmol/L H_(2)O_(2)建立HaCaT细胞氧化应激模式。20、40、60、80和100μmol/L异鼠李素处理HaCaT细胞12 h,对细胞无毒性作用,选择20、40和60μmol/L异鼠李素进行后续实验。与H_(2)O_(2)组比较,40、60μmol/L异鼠李素组的细胞增殖活性明显增加[(72.21±5.11)%、(76.08±4.91)%,P<0.05];20、40、60μmol/L异鼠李素SOD活性增高(19.81±0.38、20.52±0.52、15.45±3.13,P<0.05)和MDA含量下降(35.94±0.31、22.04±0.26、19.26±1.36,P<0.05)。结论异鼠李素对H_(2)O_(2)诱导的HaCaT细胞氧化应激损伤具有保护作用,提示异鼠李素可能是治疗白癜风的潜在药物成分。

Objective To investigate the protective effect of isorhamnetin on oxidative stress injury of HaCaT cells induced by H_(2)O_(2).Methods HaCaT cells were cultured in vitro and treated with different concentrations of H_(2)O_(2)(300,600,900,1200μmol/L)for 12 h.Cell proliferation activity was detected by cell counting kit-8(CCK-8)assay;SOD activity was detected by superoxide dismutase(SOD)kit and malondialdehyde(MDA)content was detected by MDA assay.The oxidative stress model was established by the selection of suitable H_(2)O_(2) concentration.HaCaT cells were pretreated with isorhamnetin at different concentrations for 12 h,and cell survival rate was detected by CCK-8 method to determine the safe concentration of isorhamnetin for subsequent experiments.HaCaT cells were pretreated with safe concentration of isorhamnetin for 12 h,and H_(2)O_(2) was used to interfere with HaCaT cells for 12 h.Cell proliferation activity,SOD activity and MDA content were detected.Results With the increase of H_(2)O_(2) concentration,the cell survival rate decreased gradually,the SOD activity decreased gradually and MDA content increased gradually.Compared with the control group,the survival rate of 600,900 and 1200μmol/L H_(2)O_(2) groups was statistically significant(P<0.05);The SOD activity and MDA content of H_(2)O_(2) groups(300,600,900,1200μmol/L)were significantly different from those of the control group(P<0.05).The oxidative stress model of HaCaT cells was established by 600μmol/L H_(2)O_(2).HaCaT cells treated with 20,40,60,80 and 100μmol/L isorhamnetin for 12 h showed no cytotoxic effect.20,40 and 60μmol/L isorhamnetin was selected for subsequent experiments.Compared with H_(2)O_(2) groups,the cell proliferation activity in 40 and 60μmol/L isornetin groups was significantly increased[(72.21±5.11)%,(76.08±4.91)%,P<0.05],SOD activity increased(19.81±0.38,20.52±0.52,15.45±3.13,P<0.05)and MDA content decreased(35.94±0.31,22.04±0.26,19.26±1.36,P<0.05).Conclusions The flavonoid isorhamnetin has a protective effect on oxidative stress injury induced by H_(2)O_(2) in HaCaT cells,suggesting that isorhamnetin may be a potential drug component in the treatment of vitiligo.