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基于Nrf2/HO-1信号通路的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研究
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| 篇名： | 基于Nrf2/HO-1信号通路的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研究 |
| TITLE： | Study on Protective Effects and Mechanism of Lespedeza cuneata Extracts on Glutamate-induced Hippocampal Cells HT 22 Injury of Mice Based on Nrf 2/HO-1 Signaling Pathway |
| 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（5mmol/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12h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12h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Westernblotting法检测0（空白对照）、25、50、100µg/mL夜关门二氯甲烷提取物处理24h后对细胞中HO-1蛋白表达的影响；分别采用Westernblotting法（药物分别处理0.5、1、1.5h）和免疫荧光染色法（药物处理1h）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。 |
| ABSTRACT： | OBJECTIVE：To study the protective effects of Lespedeza cunea ta extract on glutamate-induced hippocampal cells HT22 injury of mice and its possible mechanism based on Nrf 2/HO-1 signaling pathway. METHODS ：Using glutamate （5 mmol/L） to extablish the injury model of HT 22 cells. Using water soluble vitamin E as positive control （50 µmol/L），MTT assay was used to detect the effects of 0（blank control ），25，50，100 µg/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 （50 µmol/L），DCFH-DA assay was used to detect the effects of 0（blank control ），25，50，100 µg/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 ），25，50，100 µg/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 100 µg/mL L. cuneata dichloromethane extract on protein expression of Nrf 2 inside and outside the nucleus. After HO-1 gene was silenced by small interfering RNA （Si RNA ）transfection technology ，the effects of 100 µg/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， 100 µg/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， 100 µg/mL L. cuneata dichloromethane extract could increase the protein expression of HO- 1 in cells（P＜0.05），while 100 com µg/mL L. cuneata dichloromethane extract could significantly decrease the protein le vel of Nrf 2 in cytoplasm and increasethat in nucleus （P＜0.05）. After HO-1 gene silencing ，the effects of L. cuneata dichloromethane extract on the proliferation promotion of glutamate-induced injury cells and the reduction of ROS level were reversed （P＜0.05）. CONCLUSIONS ：L. cuneata dichloromethane extract can protect HT 22 cells against injury induced by glutamate through activating Nrf 2 pathway，inducing HO- 1 expression. |
| 期刊： | 2020年第31卷第11期 |
| 作者： | 郭丰，黄山，李斌 |
| AUTHORS： | GUO Feng，HUANG Shan，LI Bin |
| 关键字： | 夜关门；谷氨酸；血红素加氧酶 1；核因子2；小鼠；海马细胞HT22；机制 |
| KEYWORDS： | Lespedeza cuneata ；Glutamate；HO-1；Nrf2；Mice；Hippocampal cells HT 22；Mechanism |
| 阅读数： | 821 次 |
| 本月下载数： | 9 次 |
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