关键词: 地菍总黄酮; 糖尿病肾病; Nrf2/NQO1/GPX4信号通路; 铁死亡

Abstract: 【Objective】 The aim of this study was to investigate the inhibitory effect of total flavone from Melastoma dodecandrum Lour.(TFMD) on iron death in diabetic nephropathy (DN) mice and its related mechanism.【Method】 In this study,MPC5 cells were used to establish an in vitro DN model with high glucose-induced.Cells were divided into six treatment groups: Control group (5.5 mmol/L glucose),DN model group (60 mmol/L glucose),TFMD low (60 mmol/L glucose＋25 μg/mL TFMD),medium (60 mmol/L glucose＋50 μg/mL TFMD) and high (60 mmol/L glucose＋100 μg/mL TFMD) dose groups,and Fer-1 group (60 mmol/L glucose ＋2 μmol/L Fer-1).Cell viability was detected by cytotoxicity assay.The activity of superoxide dismutase (SOD) and the levels of glutathione (GSH) and malondialdehyde (MDA) were detected by the kits.The level of reactive oxygen species (ROS) was detected by DCFH-DA fluorescent probe.The mouse in DN model was constructed by high-fat diet combined with streptozotocin (STZ),and then the experiment was carried out in vivo.Sixty mice were divided into six treatment groups: Control group,DN model group,metformin positive group (0.5 g/kg) and TFMD high (1.2 g/kg),medium (0.8 g/kg) and low (0.6 g/kg) dose groups,10 mice in each group.The contents of urinary microalbumin (MAU),urinary total protein (TUP),serum urea nitrogen (BUN),creatinine (SCr),SOD activity,GSH and MDA contents in each group were detected.The content of iron ion in serum and kidney was also detected.The pathological changes of podocyte mitochondria in kidney were observed by transmission electron microscopy.Western blotting was used to detect Nrf2,NQO1 and GPX4 proteins expression in kidney of mice in each group.【Result】 In vitro test results showed that,compared with control group,the survival rate of MPC5 cells in DN model group was extremely significantly decreased (P＜0.01),MDA and ROS levels were extremely significantly increased (P＜0.01),GSH content and SOD activity were extremely significantly decreased (P＜0.01).Compared with DN model group,survival rate of MPC5 cell in TFMD dose groups and Fer-1 group was extremely significantly increased (P＜0.01),MDA and ROS levels were extremely significantly decreased (P<0.01),GSH content and SOD activity were extremely significantly increased (P＜0.01). In vivo tests showed that,compared with control group,the contents of MAU,TUP,SCr,BUN,MDA and iron ion in serum and kidney of mice in DN model group were extremely significantly increased (P<0.01),while the SOD activity and GSH content in serum were extremely significantly decreased (P<0.01).Compared with DN model group,the levels of MAU,TUP,SCr,BUN and MDA, and the content of iron ion in serum and kidney of mice in each dose group of TFMD were extremely significantly decreased (P＜0.01),and the SOD activity and GSH content were extremely significantly increased (P<0.01).The pathological observation results showed that TFMD could ameliorate mitochondrial related pathological injury of podocytes and improve mitochondrial morphology in different degrees.Western blotting results showed that compared with control group,the expressions of Nrf2,NQO1 and GPX4 proteins in kidney of mice in DN model group were extremely significantly decreased (P<0.01).Compared with DN model group,the expressions of Nrf2,NQO1 and GPX4 proteins in kidney of mice in TFMD dose groups were extremely significantly or significantly increased (P<0.01 or P<0.05). 【Conclusion】 The intervention effect of TFMD on mouse DN was related to the regulation of iron metabolism and the improvement of lipid peroxidation imbalance，through the activation of Nrf2/NQO1/GPX4 signaling pathway in kidney,thereby inhibiting the occurrence of iron death in podocyte.

Key words: total flavonoids from Melastoma dodecandrum Lour.; diabetic nephropathy; Nrf2/NQO1/GPX4 signaling pathway; ferroptosis