摘要
目的:基于核因子2(Nrf2)/血红素加氧酶1(HO-1)信号通路研究夜关门提取物对谷氨酸诱导小鼠海马细胞HT22损伤的保护作用及机制。方法:采用谷氨酸(5 mmol/L)建立HT22细胞损伤模型后,以水溶性维生素E为阳性对照(50μmol/L),采用MTT法检测0(空白对照)、25、50、100μg/mL夜关门的石油醚、二氯甲烷、乙酸乙酯提取物预处理12 h后对谷氨酸诱导损伤细胞增殖的影响。以水溶性维生素E为阳性对照(50μmol/L),采用2′,7′-二氯荧光黄双乙酸盐探针(DCFH-DA)法检测0(空白对照)、25、50、100μg/mL夜关门二氯甲烷提取物预处理12 h后对谷氨酸诱导损伤细胞中活性氧(ROS)水平的影响;以HO-1激动剂钴-原卟啉为阳性对照,采用Western blotting法检测0(空白对照)、25、50、100μg/mL夜关门二氯甲烷提取物处理24 h后对细胞中HO-1蛋白表达的影响;分别采用Western blotting法(药物分别处理0.5、1、1.5 h)和免疫荧光染色法(药物处理1 h)检测100μg/mL夜关门二氯甲烷提取物处理后对细胞核内外Nrf2蛋白表达的影响。采用小干扰RNA(si-RNA)转染技术进行HO-1基因沉默后,考察100μg/mL夜关门二氯甲烷提取物对谷氨酸诱导损伤细胞的存活率以及细胞中ROS水平的影响。结果:与空白对照比较,50、100μg/mL夜关门二氯甲烷提取物均可显著升高细胞的存活率(P<0.05),并降低细胞中ROS水平(P<0.05);25、50、100μg/mL夜关门二氯甲烷提取物均可显著升高细胞中HO-1蛋白表达水平(P<0.05),100μg/mL夜关门二氯甲烷提取物可显著降低细胞质中Nrf2蛋白水平并升高细胞核中Nrf2蛋白水平(P<0.05)。HO-1基因沉默后,夜关门二氯甲烷提取物对谷氨酸诱导损伤细胞的促增殖以及降低ROS水平的作用被逆转(P<0.05)。结论:夜关门二氯甲烷提取物可通过激活Nrf2信号通路,诱导HO-1蛋白的表达,从而发挥对谷氨酸诱导损伤HT22细胞的保护作用。
OBJECTIVE:To study the protective effects of Lespedeza cuneata extract on glutamate-induced hippocampal cells HT22 injury of mice and its possible mechanism based on Nrf2/HO-1 signaling pathway.METHODS:Using glutamate(5 mmol/L)to extablish the injury model of HT22 cells.Using water soluble vitamin E as positive control(50μmol/L),MTT assay was used to detect the effects of 0(blank control),25,50,100μg/mL petroleum ether extract,dichloromethane extract,ethyl acetate extract of L.cuneata on the proliferation of glutamate-induced injury cellsafter pretreated for 12 h.Using water soluble vitamin E as positive control(50μmol/L),DCFH-DA assay was used to detect the effects of 0(blank control),25,50,100μg/mL L.cuneata dichloromethane extract on the level of active oxygen(ROS)in glutamate-induced injury cells after pretreated with 12 h.Using HO-1 agonist CoPP as positive control,Western blotting method was used to detect the effects of 0(blank control),25,50,100μg/mL L.cuneata dichloromethane extract on the protein expression of HO-1 after treated for 24 h.Western blotting method(treated for 0.5,1,1.5 h)and immunofluorescence staining(treated for 1 h)were used to detect the effects of 100μg/mL L.cuneata dichloromethane extract on protein expression of Nrf2 inside and outside the nucleus.After HO-1 gene was silenced by small interfering RNA(Si RNA)transfection technology,the effects of 100μg/mL L.cuneata dichloromethane extract on the survival rates of glutamate-induced injury cells and the level of ROS were detected.RESULTS:Compared with blank control,50,100μg/mL L.cuneata dichloromethane extract could significantly improve the survival rate of glutamate-induced injury cells(P<0.05),while reduced the level of ROS(P<0.05).25,50,100μg/mL L.cuneata dichloromethane extract could increase the protein expression of HO-1 in cells(P<0.05),while 100μg/mL L.cuneata dichloromethane extract could significantly decrease the protein level of Nrf2 in cytoplasm and increase that in nucleus(P<0.05).After HO-1 gene silencing,the
作者
郭丰
黄山
李斌
GUO Feng;HUANG Shan;LI Bin(Dept.of Pharmacy,Baicheng Medical College,Jilin Baicheng 137000,China;Dept.of Pharmacy,Qingdao University of Science and Technology,Shandong Qingdao 266000,China)
出处
《中国药房》
CAS
北大核心
2020年第11期1303-1308,共6页
China Pharmacy
基金
国家自然科学基金资助项目(No.81760779,No.31500288,No.81360686)。
