槲皮素对5-氟尿嘧啶诱导的人永生化角质形成细胞损伤的保护作用及其机制  

Protective effect of quercetin against 5-fluorouracil-induced damage in human immortalized keratinocytes and its mechanism

作　　者：李佳欣 王一 吴婷婷 郝诗睿 付晓 LI Jiaxin;WANG Yi;WU Tingting;HAO Shirui;FU Xiao(Second Outpatient Department of Traditional Chinese Medicine,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121000,China;Key Laboratory of Surgery of Liaoning Province,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121000,China;Department of Emergency,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121000,China;Department of Anesthesiology,First Affiliated Hospital,Jinzhou Medical University,Jinzhou 121000,China)

机构地区：[1]锦州医科大学附属第一医院中医科二门诊,辽宁锦州121000 [2]锦州医科大学附属第一医院辽宁省外科学重点实验室,辽宁锦州121000 [3]锦州医科大学附属第一医院急诊科,辽宁锦州121000 [4]锦州医科大学附属第一医院麻醉科,辽宁锦州121000

出　　处：《吉林大学学报(医学版)》2025年第2期428-436,共9页Journal of Jilin University:Medicine Edition

基　　金：国家中医药管理局第五批全国中医临床优秀人才研修项目(国中医药人教函[2022] 1号);辽宁省卫健委第一批优秀中医临床人才培养项目(辽中医药函[2021] 1号);辽宁省科技厅自然科学基金项目(2019-ZD-0809)。

摘　　要：目的:探讨槲皮素对5-氟尿嘧啶(5-FU)诱导的人永生化角质形成细胞(HACAT)损伤的保护作用,并阐明其作用机制。方法:将HACAT细胞分为对照组(正常培养细胞)、5-FU组(7.5 mg·L^(-1) 5-FU作用于HACAT细胞24 h)和低、中及高剂量槲皮素组(25,50和75μmol·L^(-1)的槲皮素与7.5 mg·L^(-1) 5-FU共同作用于HACAT细胞24 h)。采用细胞计数试剂盒8 (CCK-8)法检测不同剂量(0、10、25、50、75和100μmol·L^(-1))槲皮素处理后各组HACAT细胞存活率,活性氧(ROS)荧光探针法检测各组HACAT细胞中ROS水平,荧光素标记的AnnexinⅤ-FITC/PI双染法检测HACAT细胞凋亡情况,Western blotting法检测各组HACAT细胞中B细胞淋巴瘤2 (Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解型天冬氨酸特异性的半胱氨酸蛋白水解酶3 (Cleaved Caspase-3)、环氧合酶2(COX-2)、白细胞介素1β (IL-1β)和白细胞介素6 (IL-6)蛋白表达水平。结果:CCK-8法检测,与0μmol·L^(-1)槲皮素组比较,10、25、50和75μmol·L^(-1)槲皮素组HACAT细胞存活率无明显变化,差异无统计学意义(P>0.05),而100μmol·L^(-1)槲皮素组HACAT细胞存活率明显降低(P<0.05);与5-FU组比较,低、中和高剂量槲皮素组细胞存活率明显升高(P<0.05)。ROS荧光探针法检测,与5-FU组比较,低、中和高剂量槲皮素组HACAT细胞中ROS水平明显降低(P<0.05)。Annexin V-FITC/PI双染法检测,与5-FU组比较,低、中和高剂量槲皮素组HACAT细胞凋亡率明显降低(P<0.05)。Western blotting法检测,与5-FU组比较,中和高剂量槲皮素组HACAT细胞中Bcl-2蛋白表达水平明显升高(P<0.05);低、中和高剂量槲皮素组HACAT细胞中Bax和Cleaved Caspase-3蛋白表达水平明显降低(P<0.05);中和高剂量槲皮素组HACAT细胞中COX-2蛋白表达水平明显降低(P<0.05);中剂量槲皮素组HACAT细胞中IL-1β和IL-6蛋白表达水平明显降低(P<0.05)。结论:槲皮素对5-FU诱导的HACAT细胞损伤具有保护作用,其机制可能与减少ROS和COX-2等炎症因子Objective:To investigate the protective effect of quercetin against 5-fluorouracil(5-FU)-induced damage in the human immortalized keratinocytes(HACAT),and to elucidate its possible mechanism.Methods:The HACAT cells were divided into control group(normal cultured cells),5-FU group(treated with 7.5 mg·L^(-1)5-FU for 24 h),and low,medium,and high doses of quercetin groups(HACAT cells treated with 25,50,and 75μmol·L^(-1) quercetin combined with 7.5 mg·L^(-1)5-FU for 24 h).Cell counting kit-8(CCK-8)assay was used to detect the survival rates of HACAT cells treated with different doses(0,10,25,50,75 and 100μmol·L^(-1))of quercetin in various groups.The fluorescent probe of reactive oxygen species(ROS)was used to detect ROS levels in the HACAT cells in various groups.AnnexinⅤ-FITC/PI double staining was used to detect the apoptosis of HACAT cells in various groups.Western blotting method was used to detect the expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Cleaved Caspase-3,cyclooxygenase-2(COX-2),interleukin-1β(IL-1β),and interleukin-6(IL-6)in the HACAT cells in various groups.Results:The CCK-8 assay results showed that compared with 0µmol·L^(-1) quercetin group,the survival rates of HACAT cells in 10,25,50 and 75μmol·L^(-1) quercetin groups showed no significant differences(P>0.05),while the survival rates of HACAT cells in 100μmol·L^(-1) quercetin group was significantly decreased(P<0.05).Compared with 5-FU group,the survival rates of the HACAT cells in low,medium and high doses of quercetin group were significantly increased(P<0.05).Compared with 5-FU group,the ROS levels in low,medium,and high doses of quercetin groups were significantly decreased(P<0.05).AnnexinⅤ-FITC/PI double staining assay showed that compared with 5-FU group,the apoptotic rates in low,medium and high doses of quercetin groups were significantly decreased(P<0.05).The Western blotting results showed that compared with 5-FU group,the expression levels of Bcl-2 protein in medium and high doses of qu

关 键 词：槲皮素 手足综合征 人永生化角质形成细胞 细胞凋亡 环氧合酶2 

分 类 号：R275.9[医药卫生—中西医结合]