枸杞糖肽对放射诱导人角质形成细胞氧化应激损伤的保护作用及其机制  

Protective effect of Lycium barbanun glycopeptide on human keratinocytes against radiation-induced oxidative stress damage and its mechanism

作　　者：江思婧 李静 刘畅 牟雁东[2] JIANG Sijing;LI Jing;LIU Chang;MU Yandong(School of Stomatology,Southwest Medical University,Luzhou 646000,Sichuan Province,China;不详)

机构地区：[1]西南医科大学口腔医学院,四川泸州646000 [2]四川省人民医院口腔科,四川成都610072

出　　处：《中国生物制品学杂志》2023年第11期1324-1328,1334,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(82071168);四川省科技厅基金(2021YFS0009)。

摘　　要：目的探讨枸杞糖肽(Lycium barbanun glycopeptide,LbGP)对放射诱导人角质形成细胞HaCaT损伤的保护作用及其机制。方法采用直线加速器(速率6 Gy/min)分别按4、8、12、16、20、24、28 Gy剂量对HaCaT细胞进行照射,CCK-8法检测细胞活性。以0、0.05、0.1、0.5、0.8、1.0、1.5、3 mg/mL的LbGP作用HaCaT细胞4h,进行12 Gy剂量放射,CCK-8法检测细胞活性。将HaCaT细胞分为空白对照组(不加LbGP不放射)、放射组(12 Gy剂量照射)、LbGP+放射组(0.8 mg/mL LbGP作用24 h,12 Gy剂量照射),照射1 h后,流式细胞术检测活性氧(reactive oxygen species,ROS)产生量,WST-8法检测超氧化物歧化酶(superoxide dismutase,SOD)活性,Western blot法检测细胞中核因子E2相关因子2(nuclear factor-E2 related factor 2,Nrf2)、p-Nrf2、NADPH氧化还原酶1(NADPH quinone oxidoreductase 1,NQO1)、血红素加氧酶-1(heme oxygenase-1,HO-1)蛋白表达水平;分别于放射1、3、5 h后,qRT-PCR法检测Nrf2、HO-1、NQO1基因mRNA的转录水平。结果12 Gy放射辐射量可诱导约50%细胞死亡,0.8 mg/mL LbGP对放射细胞活性保护作用最佳。放射1 h后,与空白对照组比较,放射组HaCaT细胞内ROS含量显著增加(F=2.55,P<0.001),SOD活力显著降低(F=1.23,P<0.01),NQO1、Nrf2蛋白含量差异无统计学意义(F分别为1.78和1.00,P均>0.05),HO-1蛋白含量明显增加(F=1.37,P<0.05),p-Nrf2蛋白含量显著下降(F=2.75,P<0.01);与放射组比较,LbGP+放射组HaCaT细胞内ROS含量明显降低(F=3.61,P<0.001),SOD活性明显升高(F=1.23,P<0.05),Nrf2,p-Nrf2、HO-1及NQO1蛋白含量均显著上升(F分别为4.00、2.25、6.25及1.27,P均<0.05)。放射1、3、5 h,与放射组比较,LbGP+放射组Nrf2、HO-1、NQO1基因mRNA转录水平均明显升高(F=0.20~36.00,P均<0.05)。结论LbGP可减轻放射对HaCaT细胞的氧化应激损伤,保护细胞活性,该作用可能是通过激活Nrf2及提高其下游抗氧化酶SOD、HO-1、NQO1水平而发挥作用的。Objective To investigate the protective effect of Lycium barbanun glycopeptide(LbGP)on human keratinocyte HaCaT cells against radiation-induced cytotoxicity and its mechanism.Methods HaCaT cells were exposed to radiation with linear accelerator(rate 6 Gy/min)at doses of 4,8,12,16,20,24 and 28 Gy respectively,and the cell activity was detected by CCK-8 assay.HaCaT cells were treated with LbGP(0,0.05,0.1,0.5,0.8,1.0,1.5 and 3 mg/mL)for 4 h,exposed to radiation of 12 Gy,and detected for the cell viability by CCK-8 assay.HaCaT cells were divided into control group(without LbGP and radiation),radiation group(radiation of 12 Gy)and LbGP+radiation group(0.8 mg/mL LbGP for 24 h,radiation of 12 Gy).After irradiation for 1 h,the reactive oxygen species(ROS)production was measured by flow cytometry,the superoxide dismutase(SOD)activity was determined by WST-8 assay and the expression of nuclear factor-E2 related factor 2(Nrf2),p-Nrf2,NADPH quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)were detected by Western blot;The mRNA transcription levels of Nrf2,HO-1 and NQO1 were detected by qRT-PCR at 1,3 and 5 h after irradiation.Results Radiation of12 Gy induced about 50%cell death,and 0.8 mg/mL LbGP showed the best protective effect on the activity of irradiated cells.After irradiation for 1 h,compared with the control group,the content of ROS in HaCaT cells increased significantly in radiation group(F=2.55,P<0.001),the activity of SOD decreased significantly(F=1.23,P<0.01),the contents of NQO1 and Nrf2 protein had no significant difference(F=1.78 and 1.00,respectively,P>0.05),the content of HO-1protein increased significantly(F=1.37,P<0.05),and the content of p-Nrf2 protein decreased significantly(F=2.75,P<0.01);Compared with the radiation group,the content of ROS in HaCaT cells of LbGP+radiation group decreased significantly(F=3.61,P<0.001),the activity of SOD increased significantly(F=1.23,P<0.05),and the contents of Nrf2,p-Nrf2,HO-1 and NQO1 protein increased significantly(F=4.00,2.25,6.25 and 1.27,respectively,P<0.

关 键 词：枸杞糖肽 人角质形成细胞 放射 氧化应激 细胞损伤 

分 类 号：R78[医药卫生—口腔医学]