| 吴平平,叶丽,邵雅婕,等.知柏地黄汤通过调节ARE信号通路激活细胞抗氧化反应治疗阴虚火旺证的机制研究[J].浙江中医药大学学报,2020,44(4):316-323. |
| 知柏地黄汤通过调节ARE信号通路激活细胞抗氧化反应治疗阴虚火旺证的机制研究 |
| Zhibai Dihuang Decoction Inhibits Oxidative Stress Induced by “Hyperactivity of Fire Due to Yin Deficiency Syndrome” through ARE Pathway |
| DOI：10.16466/j.issn1005-5509.2020.04.002 |
| 中文关键词: 知柏地黄汤 干姜附子肉桂汤 氧化应激 阴虚火旺证 ARE通路 代谢组学 |
| 英文关键词: Zhibai Dihuang decoction Ganjiang Fuzi Rougui decoction oxidative stress hyperactivity of fire due to Yin deficiency syndrome ARE pathway metabolomics |
| 基金项目:国家自然科学基金项目(81673857)；国家重点基础研究发展计划(973计划)项目(2014CB543000) |
|
| 摘要点击次数: 2692 |
| 全文下载次数: 1370 |
| 中文摘要: |
| [目的]通过研究知柏地黄汤对细胞抗氧化反应元件(antioxidant response element，ARE)信号通路的调节及其对大鼠血清代谢物的干预作用，探讨其治疗阴虚火旺证的可能机制。[方法]将人肝癌细胞系HepG2细胞分为对照组、知柏地黄汤低、中、高浓度组，分别以0、1、20和100mg·mL-1的知柏地黄汤干预24h，通过双萤光素酶报告基因实验筛选知柏地黄汤显著调控的信号通路；荧光定量PCR检测所筛选通路相关基因的表达，并利用磷钼酸比色法及氧化酶法检测细胞内三磷酸腺苷(adenosine triphosphate，ATP)含量、细胞耗氧率(oxygen consumption rate，OCR)、超氧化物歧化酶(superoxide dismutase，SOD)活性的变化。45只SD大鼠随机分成3组，空白组以0.9%氯化钠溶液灌胃21d，干姜组先以10g·kg-1干姜附子肉桂汤灌胃14d、再以0.9%氯化钠溶液灌胃7d，干知组先以10g·kg-1干姜附子肉桂汤灌胃14d、再以知柏地黄汤灌胃7d，灌胃体积为10mL/(kg·d)，以液相色谱-质谱联用(liquid chromatography-mass spectrometry，LC-MS)技术检测血清中代谢物的改变。[结果]双萤光素酶报告基因实验显示，知柏地黄汤对ARE元件调控最为显著，荧光定量PCR结果显示知柏地黄汤可以促进核因子红细胞2相关因子2(nuclear factor erythroid-2-related factor 2，Nrf2)、小肌腱膜纤维肉瘤(musculoaponeurotic fibrosarcoma，Maf)的转录(P＜0.001，P＜0.05)，同时抑制Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1，Keap1)的转录(P＜0.001)。与对照组比较，知柏地黄汤组细胞ATP含量(P＜0.05)、OCR(P＜0.001)及SOD活性(P＜0.05)均升高。动物实验证实，知柏地黄汤灌胃治疗可以改善阴虚火旺模型大鼠胆碱、甘胆酸及棕榈酰左旋肉碱(palmitoyl-L-carnitine，PALC)的紊乱，其中胆碱及PALC含量差异有统计学意义(P＜0.001)。[结论]知柏地黄汤能够通过ARE信号通路，抑制机体的氧化应激反应，进而改善阴虚火旺引起的代谢紊乱，这可能是知柏地黄汤治疗阴虚火旺证的作用机制之一。 |
| 英文摘要: |
| [Objective] To demonstrate the regulation of Zhibai Dihuang decoction on the antioxidant response element(ARE) signaling pathway in vitro, and its influence on serum metabolites of rats model, then to clarify the mechanism of Zhibai Dihuang decoction in the treatment of hyperactivity of fire due to Yin deficiency syndrome. [Methods] HepG2 cells were divided into four groups, control group, Zhibai Dihuang decoction low concentration group, Zhibai Dihuang decoction medium concentration group and Zhibai Dihuang decoction high concentration group, each group was treated by 0, 1, 20 and 100mg·mL-1 Zhibai Dihuang decoction. The dual luciferase reporter gene assay was used to screen the involved pathways, the expression of the ARE pathway related genes were quantified by Real-time PCR. Intracellular adenosine triphosphate(ATP) content, cell oxygen consumption rate(OCR) and superoxide dismutase(SOD) activity were measured with phosphomolybdic acid colorimetric kit or oxidase assay kit. Forty five SD rats were separated into 3 groups at random, blank group was gavaged with 0.9% sodium chloride solution for 21 days; Ganjiang group was treated by 0.9% sodium chloride solution for 7 days after gavaged with 10g·kg-1 Ganjiang Fuzi Rougui decoction for 14 days; Ganzhi group was treated by Zhibai Dihuang decoction for 7 days after gavaged with 10g·kg-1 Gangjiang Fuzi Rougui decoction for 14 days, all volume was 10mL·kg-1 per day. The serum metabolites were identified by liquid chromatography-mass spectrometry(LC-MS). [Results] Dual luciferase reporter gene assay indicated that Zhibai Dihuang decoction stimulated the ARE element, significantly; and Zhibai Dihuang decoction treatment stimulated the transcriptions of nuclear factor erythroid-2-related factor 2(Nrf2) and musculoaponeurotic fibrosarcoma(Maf) , and inhibited the transcription of Kelch-like ECH-associated protein 1(Keap1) in HepG2 cells(P＜0.001); and in vitro, Zhibai Dihuang decoction treatment could increase the ATP content(P＜0.05), OCR(P＜0.001) and SOD activity(P＜0.05). In vivo, Zhibai Dihuang decoction treatment could improve the serum metabolites disorder in model rat,such as choline, glycocholic acid and palmityl-L-carnitine(PALC), and the difference of choline and PALC content was statistically significant(P＜0.001). [Conclusion] Zhibai Dihuang decoction can inhibit the oxidative stress response by enhance the activity of ARE signaling pathway, and thereby improve the disorder of metabolites in hyperactivity of fire due to Yin deficiency syndrome rat model, which should be one of the possible mechanisms of Zhibai Dihuang decoction in the treatment of hyperactivity of fire due to Yin deficiency syndrome. |
|
查看全文
查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|