datasheet1_Formononetin Activates the Nrf2/ARE Signaling Pathway Via Sirt1 to Improve Diabetic Renal Fibrosis.docx

Oxidative stress is the main factor responsible for the induction of diabetic renal fibrosis. Thus, improving the state of oxidative stress can effectively prevent the further deterioration of diabetic nephropathy (DN). Previous research has shown that formononetin (FMN), a flavonoid with significant antioxidant activity and Sirt1 activation effect, can improve diabetic renal fibrosis. However, the exact mechanisms underlying the effect of FMN on diabetic renal fibrosis have yet to be elucidated. In this study, we carried out in vivo experiments in a db/db (diabetic) mouse model and demonstrated that FMN activated the nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway and improved oxidative stress by increasing levels of sirtuin-1 (Sirt1) protein level in renal tissue. We also found that this process reversed the up-regulation of fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1) and led to an improvement in renal insufficiency. In vitro results further showed that FMN significantly reversed the upregulation of FN and ICAM-1 in glomerular mesangial cells (GMCs) exposed to high glucose. FMN also promoted the expression of Nrf2 and widened its nuclear distribution. Thus, our data indicated that FMN inhibited hyperglycemia-induced superoxide overproduction by activating the Nrf2/ARE signaling pathway. We also found that FMN up-regulated the expression of Sirt1 and that Sirt1 deficiency could block the activation of the Nrf2/ARE signaling pathway in GMCs induced by high glucose. Finally, we found that Sirt1 deficiency could reverse the down-regulation of FN and ICAM-1 induced by FMN. Collectively, our data demonstrated that FMN up-regulated the expression of Sirt1 to activate the Nrf2/ARE signaling pathway, improved oxidative stress in DN to prevent the progression of renal fibrosis. Therefore, FMN probably represents an efficient therapeutic option of patients with DN.

氧化应激（oxidative stress）是诱发糖尿病肾纤维化的核心致病因素。故而，改善氧化应激状态可有效阻断糖尿病肾病（diabetic nephropathy, DN）的进一步进展。既往研究表明，芒柄花素（formononetin, FMN）作为一类具备显著抗氧化活性与沉默信息调节因子1（sirtuin-1, Sirt1）激活作用的黄酮类化合物，可改善糖尿病肾纤维化。然而，芒柄花素调控糖尿病肾纤维化的确切作用机制仍有待阐明。本研究采用db/db（糖尿病）小鼠模型开展体内实验，证实芒柄花素可通过提升肾组织中沉默信息调节因子1（sirtuin-1, Sirt1）的蛋白表达水平，激活核因子E2相关因子2（nuclear factor E2-related factor 2, Nrf2）/抗氧化反应元件（antioxidant response element, ARE）信号通路，从而改善氧化应激状态。本研究同时发现，该过程可逆转纤连蛋白（fibronectin, FN）与细胞间黏附分子1（intercellular adhesion molecule 1, ICAM-1）的表达上调，并改善肾功能不全症状。体外实验结果进一步证实，芒柄花素可显著逆转高糖诱导下肾小球系膜细胞（glomerular mesangial cells, GMCs）中FN与ICAM-1的表达上调。此外，芒柄花素可促进Nrf2的表达，并扩大其核内分布范围。综上，本研究数据表明，芒柄花素可通过激活Nrf2/ARE信号通路，抑制高血糖（hyperglycemia）诱导的超氧阴离子（superoxide）过量生成。本研究同时发现，芒柄花素可上调Sirt1的表达，而Sirt1缺失可阻断高糖诱导下GMCs中Nrf2/ARE信号通路的激活。最后，本研究证实，Sirt1缺失可逆转芒柄花素诱导的FN与ICAM-1表达下调。综上，本研究数据表明，芒柄花素通过上调Sirt1的表达以激活Nrf2/ARE信号通路，改善糖尿病肾病中的氧化应激状态，从而延缓肾纤维化的进展。因此，芒柄花素或可成为糖尿病肾病患者的高效治疗候选方案。