没食子酸激活Nrf-2信号通路降低高糖诱导NIH-3T3细胞氧化应激与炎症反应  

Gallic Acid Activates Nrf-2 Signaling Pathway to Alleviate High Glucose-Induced Oxidative Stress and Inflammation of NIH-3T3 Cells

作　　者：郑时旭 陈丽 丁雅容 周忠志[2] 谢晨磊 雷晓明 ZHENG Shixu;CHEN Li;DING Yarong;ZHOU Zhongzhi;XIE Chenlei;LEI Xiaoming(Medical School,Hunan University of Chinese Medicine,Changsha 410208;The First Hospital of Hunan University of Chinese Medicine,Changsha 410007;Key Laboratory of Vascular Biology and Translational Medicine,Changsha 410208)

机构地区：[1]湖南中医药大学医学院,长沙410208 [2]湖南中医药大学第一附属医院,长沙410007 [3]血管生物学与转化医学湖南省重点实验室,长沙410208

出　　处：《中药药理与临床》2024年第2期78-84,共7页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金(编号:81973862、82074447);湖南省大学生创新创业课题(编号:S202210541142);湖南中医药大学研究生创新课题(编号:2022CX176、2022CX177)。

摘　　要：目的:研究没食子酸激活核因子E2相关因子-2(Nrf-2)通路减轻高糖诱导的NIH-3T3细胞炎症反应与氧化应激,恢复NIH-3T3细胞功能的效应机制。方法:不同浓度葡萄糖及没食子酸干预24 h后,以CCK-8法检测NIH-3T3细胞增殖活力,以确定最佳高糖损伤造模浓度及没食子酸干预浓度;以CCK-8法检测没食子酸对高糖诱导下NIH-3T3细胞增殖活力;以DCFH-DA荧光探针检测NIH-3T3细胞活性氧(ROS)含量;免疫荧光法检测Nrf-2入核;RT-PCR和Western blot法检测Nrf-2及核因子κ-B(NF-κB)信号通路相关蛋白及mRNA表达;Western blot法检测Ⅰ型胶原蛋白(COL-Ⅰ)表达。结果:选用100 mmol/L葡萄糖为造模浓度,选用5μmol/L为没食子酸干预浓度。与空白对照组相比,模型对照组细胞增殖活性显著降低(P<0.01),Nrf-2、KEAP-1、HO-1、IκB蛋白下调(P<0.01),P65、p-P65表达上调(P<0.05或P<0.01),p-P65/P65比值增大(P<0.01);Keap1、Nrf2、Nqo1、P65、Ii6、Tnfa mRNA上调(P<0.05),Ho1、Sod、Iκb mRNA下调(P<0.05,P<0.01);细胞中ROS水平升高;Nrf-2表达、核易位减少(P<0.05或P<0.01),细胞COL-Ⅰ表达下调(P<0.01);与模型对照组相比,没食子酸干预可恢复细胞增殖活性(P<0.01);Nrf-2、HO-1、IκB蛋白上调(P<0.05或P<0.01),p-P65下调、p-P65/P65(P<0.01)降低;Nrf2、Ho1、Sod、Nqo1、Cat、Gst、Iκb mRNA上调(P<0.05或P<0.01),Keap1、P65、Ii6、Tnfa mRNA下调(P<0.05或P<0.01);细胞中ROS水平下调,Nrf-2表达、核易位增多(P<0.05或P<0.01);细胞COL-Ⅰ表达上调(P<0.01)。结论:没食子酸可通过激活Nrf-2信号通路降低NIH-3T3细胞ROS水平,减轻氧化应激并抑制炎症反应,上调COL-Ⅰ蛋白表达,从而恢复高糖损伤NIH-3T3细胞功能。Objective: To investigate the role of gallic acid (GA) in activating the nuclear factor erythroid 2-related factor 2 (Nrf-2)pathway to reduce inflammation and oxidative stress induced by high glucose in NIH-3T3 cells and restore cell function.Methods: The proliferation activity of NIH-3T3 cells exposed to different concentrations of glucose and treated with GA for24 h was examined by the Cell-Counting Kit-8 (CCK-8) method, and thus the optimal glucose concentration for modelingand GA concentration for intervention were determined. The proliferation viability of NIH-3T3 cells exposed to highglucose and treated with GA was detected by the CCK-8 method. The DCFH-DA fluorescent probe was used to measure thecontent of reactive oxygen species (ROS) in NIH-3T3 cells. Immunofluorescence was used to detect the nucleartranslocation of Nrf-2. Western blotting and RT-PCR were employed to determine the protein and mRNA levels,respectively, of the proteins in the Nrf-2 and nuclear factor-kappa B (NF-κB) signaling pathways. In addition, the expressionof collagen type I (COL-I) was measured by Western blotting. Results: The cells were modeled with 100 mmol/L glucoseand treated with 5 μmol/L GA. Compared with the normal control group, the model group showed decreased cellproliferation (P<0.01), down-regulated protein and mRNA levels of proteins in the Nrf-2 signaling pathway, up-regulatedprotein and mRNA levels of proteins in the NF-κB signaling pathway (P<0.05 or P<0.01), increased ROS content,decreased Nrf-2 expression and nuclear translocation (P<0.05 or P<0.01), and attenuated COL-I synthesis (P<0.01).Compared with the model group, GA intervention restored cell proliferation (P<0.01), up-regulated the expression ofproteins in the Nrf-2 pathway, down-regulated the expression of proteins in the NF-κB signaling pathway (P<0.05 orP<0.01), reduced ROS content, increased the Nrf-2 expression and nuclear translocation (P<0.05 or P<0.01), and enhancedCOL-I synthesis (P<0.01). Conclusion: GA can scavenge ROS and reduce oxidative s

关 键 词：没食子酸 糖尿病创面 高糖损伤 氧化应激 炎症反应 

分 类 号：R285.5[医药卫生—中药学]