摘要
目的探讨欧亚旋覆花总黄酮(IBFTF)对衰老人皮肤成纤维细胞lncRNA TERRA的影响及其机制。方法采用高效液相色谱法(HPLC)分析IBFTF的成分及含量。将人皮肤成纤维细胞分为对照组、IBFTF组(使用IBFTF处理)、衰老模型组[使用D-半乳糖(D-gal)诱导建立衰老模型]与衰老模型+IBFTF组(衰老模型建立后使用IBFTF处理)。采用CCK-8法检测各组细胞存活率;β-半乳糖苷酶(SA-β-Gal)染色观察细胞蓝染情况,计算阳性细胞比例;酶标仪测定各组超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)等氧化应激指标水平;qRT-PCR检测各组lncRNA TERRA、Telomere、人端粒酶反转录酶(hTERT)mRNA的表达;Western blotting检测各组hTERT及衰老相关蛋白p53、p16的表达。结果IBFTF中槲皮素、异鼠李素、山奈酚和芦丁的含量分别为6.447、2.044、1.272、0.781 mg/g。CCK-8法及SA-β-Gal染色结果显示,IBFTF可增高衰老模型组细胞存活率,降低SA-β-Gal阳性细胞比例(P<0.05)。酶标仪检测结果显示,IBFTF可增加衰老细胞SOD、GSH-Px的活性,降低衰老细胞MDA含量(P<0.05)。qRT-PCR检测结果显示,IBFTF可降低衰老细胞lncRNA TERRA的表达水平,增高hTERT mRNA的表达水平,延长端粒长度(P<0.05)。Western blotting检测结果显示,与衰老模型组比较,衰老模型+IBFTF组p53、p16蛋白表达水平降低,hTERT蛋白表达水平升高(P<0.05)。结论IBFTF可降低D-gal诱导的衰老人皮肤成纤维细胞lncRNA TERRA的表达,发挥抗衰老作用,其机制可能与恢复端粒酶活性、延长端粒长度有关。
Objective To investigate the effect of Inula britannica flower total flavonoids(IBFTF)on the expression of lncRNA TERRA in the senile skin fibroblasts and its mechanism.Methods High performance liquid chromatography(HPLC)was used to analyze the composition and content of IBFTF.The human skin fibroblasts cell line was divided into control group,IBFTF group(cells were treated with IBFTF alone),cell senescence model group[cell senescence model was induced by D-galactose(D-gal)]and cell senescence model+IBFTF group(cell senescence model was treated with IBFTF).The cell viability was detected by CCK-8.The senescence-associatedβ-galactosidase(SA-β-Gal)staining was used to detect the positive rate of human skin fibroblasts cells,and the contents of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)were detected by enzyme microplate reader.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression of lncRNA TERRA,hTERT mRNA and the relative length of telomere in the cells.The protein expression of agingrelated proteins such as p53,p16 and human telomerase reverse transcriptase(hTERT)were detected by Western blotting.Results The contents of quercetin,isorhamnetin,kaempferol and rutin in IBFTF were 6.447,2.044,1.272,0.781 mg/g,respectively.CCK-8 and SA-β-Gal staining showed that IBFTF increased the viability of cells in senescence model group,and reduced the proportion of SA-β-Gal positive cells(P<0.05).Enzyme labeling results showed that IBFTF increased the activity of SOD and GSH-Px,and reduced the MDA level of senescent cells(P<0.05).qRT-PCR showed that IBFTF decreased the expression of lncRNA TERRA,increased the expression of hTERT mRNA and prolonged the telomere length in senescent cells(P<0.05).Western blotting results showed that,compared with cell senescence model group,the protein expressions of p53,p16 decreased,and the hTERT increased in cell senescence model+IBFTF group(P<0.05).Conclusions IBFTF can decrease the expression of lncRNA TERRA in D-ga
作者
周茂强
况杰
胡翰林
果磊
Zhou Mao-Qiang;Kuang Jie;Hu Han-Lin;Guo Lei(Department of Plastic and Burn Surgery,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2020年第11期1117-1124,共8页
Medical Journal of Chinese People's Liberation Army
基金
重庆市科卫联合中医药科研项目(2019ZY023405)。
