摘要
目的探讨海藻多糖对H_2O_2诱导人胚肺成纤维细胞MRC-5氧化损伤的影响及机制。方法将体外培养的人胚肺成纤维细胞MRC-5随机分为空白组、H_2O_2组、海藻多糖组、海藻多糖+H_2O_2组、H_2O_2+海藻多糖组。空白组正常培养,H_2O_2组给予H_2O_2处理,海藻多糖组给予海藻多糖处理,海藻多糖+H_2O_2组先给予海藻多糖干预1 h、再给予H_2O_2处理,H_2O_2+海藻多糖组先给予H_2O_2干预1 h、再给予海藻多糖处理。各组H_2O_2浓度均为600μmol/L,海藻多糖浓度均为0.312 5 mg/m L。各组均于干预0、24、48、72 h时采用MTT法检测细胞增殖抑制率;处理24 h时检测丙二醛(MDA)、超氧化物歧化酶(SOD)、活性氧(ROS)表达,采用免疫荧光法检测核转录因子E2相关因子2(Nrf2)蛋白表达,采用RT-PCR法检测Nrf2、Kelch样ECH联合蛋白1(Keap1)、NADP(H)醌氧化还原酶1(NQO1)、血红素氧合酶1(HO-1)、CGLC mRNA表达。结果培养24、48、72 h时,H_2O_2组、海藻多糖+H_2O_2组、H_2O_2+海藻多糖组细胞增殖抑制率均高于空白组及海藻多糖组,海藻多糖+H_2O_2组、H_2O_2+海藻多糖组均低于H_2O_2组,组间比较P均<0.05。与空白组及海藻多糖组比较,H_2O_2组、海藻多糖+H_2O_2组、H_2O_2+海藻多糖组MDA、ROS表达增加,SOD表达降低;与H_2O_2组比较,海藻多糖+H_2O_2组、H_2O_2+海藻多糖组MDA、ROS表达降低,SOD表达增加,组间比较P均<0.05。与空白组及海藻多糖组比较,H_2O_2组、海藻多糖+H_2O_2组、H_2O_2+海藻多糖组Nrf2 mRNA和蛋白相对表达量均降低,海藻多糖+H_2O_2组、H_2O_2+海藻多糖组均较H_2O_2组升高,组间比较P均<0.05。与空白组及海藻多糖组比较,H_2O_2组、海藻多糖+H_2O_2组、H_2O_2+海藻多糖组Keap1 mRNA相对表达量均升高,NQO1、CGLC、HO-1 mRNA相对表达量均降低;与H_2O_2组比较,海藻多糖+H_2O_2组、H_2O_2+海藻多糖组Keap1 mRNA相对表达量均降低,NQO1、CGLC、HO-1 mRNA相对表达量均升高;组间比较P均<0.05。结论海藻多糖可抑制H_2O_2诱导人胚肺成纤维细胞MRC-5的氧化应激损伤,上调Nrf2表达、激活Nrf2/Keap1/抗氧化反应序列元件信号通路可能是其作用机制。
Objective To investigate the effects of seaweed polysaccharides on H2O2-induced oxidative damage of human embryonic lung fibroblasts MRC-5 and its mechanism. Methods MRC-5 cells cultured in vitro were randomly divided into the blank group,H2O2 group,seaweed polysaccharide group,seaweed polysaccharide ＋ H2O2 group,and H2O2＋ seaweed polysaccharide group. Cells in the blank group were cultured normally,cells in the H2O2 group were treated with H2O2,cells in the seaweed polysaccharide group were treated with seaweed polysaccharide,cells in the seaweed polysaccharide ＋ H2O2 group were treated with seaweed polysaccharide for 1 h,then followed by H2O2,and cells in the H2O2＋seaweed polysaccharide group were treated with H2O2 for 1 h,then followed by H2O2. The concentration of H2O2 in each group was 600 umol/L,and the concentration of seaweed polysaccharide was 0. 3125 mg/m L. At 0,24,48 and 72 h after treatment,the cell proliferation inhibitory rate in each group was determined by MTT assay. The levels of malondialdehyde（ MDA）,superoxide dismutase（ SOD）,and reactive oxygen species（ ROS） were measured at 24 h. NF-E2-related factor2（ Nrf2） protein was detected by immunofluorescence assay. The expression levels of Nrf2,Kelch-like ECH-associated protein 1（ Keap1）,NADP（ H） quinone oxidoreductase 1（ NQO1）,heme oxygenase 1（ HO-1）,and CGLC mRNA were detected by RT-PCR. Results The cell inhibition rates of the H2O2 group,seaweed polysaccharide ＋ H2O2 group,and H2O2＋ seaweed polysaccharide group were higher than those of the blank group and seaweed polysaccharide group at 24,48 and 72 h,and those of the seaweed polysaccharide ＋ H2O2 group and H2O2＋ seaweed polysaccharide group were lower than that of the H2O2group（ all P〈0. 05）. Compared with the blank group and the seaweed polysaccharide group,the levels of MDA and ROS in the H2O2 group,the seaweed polysaccharide ＋ H2O2 group,and the H2O2＋ seaweed polysaccharide group increased,and the SOD level decreased. Compared with the H2O2 group,the MDA and ROS levels decreased and the SOD level increased in the seaweed polysaccharide ＋ H2O2 group and the H2O2＋ seaweed polysaccharide group（ all P〈0. 05）. Compared with the blank group and the seaweed polysaccharide group,the expression of Nrf2 mRNA and protein in the H2O2 group,the seaweed polysaccharide ＋ H2O2 group,and the H2O2＋ seaweed polysaccharide group decreased,and the seaweed polysaccharide ＋ H2O2 group and H2O2＋ seaweed polysaccharide group were higher than the H2O2group（ all P〈0. 05）. Compared with the blank group and the seaweed polysaccharide group,the relative expression of Keap1 mRNA in the H2O2 group,the seaweed polysaccharide ＋ H2O2 group,and the H2O2＋ seaweed polysaccharide group increased,and the relative expression of NQO1,CGLC,and HO-1 mRNA decreased. Compared with the H2O2 group,the relative expression of Keap1 mRNA in the polysaccharide ＋ H2O2 group and H2O2＋ seaweed polysaccharide group decreased, and the relative expression of NQO1, CGLC, and HO-1 mRNA increased（ all P〈0. 05）.Conclusion Seaweed polysaccharide could inhibit the oxidative stress injury of MRC-5 induced by H2O2 through up-regulating the expression of Nrf2 and activating Nrf2-Keap1-ARE signaling pathway.
作者
刘骅漫
刘学
贾新华
张心月
张伟
LIU Huaman LIU Xue JIA Xinhua ZHANG Xinyue ZHANG Wei(Shandong University of Traditional Chinese Medicine, Jinan 250014, China)
出处
《山东医药》
CAS
北大核心
2017年第32期17-20,共4页
Shandong Medical Journal
基金
国家自然科学基金资助项目(81273704)
“泰山学者”建设工程(ts20110819)
