Study On The Mechanism Of Notch1/Nrf2/GPX4 Signaling Pathway In Emodin Induced Nephrotoxicity

Objective:1.Emodin has been demonstrated to possess multiple pharmacological activities.However,emodin has also been reported to induce nephrotoxicity at high doses and with long-term use,and the underlying mechanism has not been fully disclosed.2.The current study aimed to investigate the roles of oxidative stress and ferroptosis in emodin-induced kidney toxicity.Finding the target Notch1,use agonists to activate Notch1 signaling pathway to enhance antioxidant capacity,combat the phenomenon of emodin induced oxidative stress injury and iron death,alleviate its nephrotoxicity,provide safety evaluation for reducing toxicity in clinical application,and also provide reference for early warning of nephrotoxicity of similar traditional Chinese medicine.Methods:1.Animals:BALB/c mice were randomly divided into blank control group（CON group）,group with low dose of emodin（0.4 g/kg）,medium dose group（0.6 g/kg）,high dose group（0.8 g/kg）,6 mice in each group,intraperitoneal injection（equal volume saline in control group）,injected once a day for 14 days,during which the general situation of mice were observed.2.Detection of renal function and pathological changes in mice:serum levels of BUN and Scr.After killing the mice,the mouse kidneys were weighed,the relative kidney weight index was calculated,and the mouse kidney histopathological damage was observed by HE staining.3.Culture NRK-52E cells:MTT and LDH methods were used to detect the effects of emodin at different concentrations（0,40,80,160,320 mmol/L）for 24 hours on the activity of NRK-52E cells.4.Oxidative stress,lipid peroxidation,Fe²⁺level and PTGS2 gene expression in mouse kidney and NRK-52E cells:Special kits including SOD,GSH,ROS and JC-1 were used to evaluate kidney oxidative stress,and MDA,LPO were used for lipid peroxidation,and iron ion determination and other related indexes.5.Western blot and q RT-PCR were used to detect kidney and NRK-52E cells treated with emodin at different concentrations The activity levels and gene expression levels of Notch1,c-Notch1,Hes1,Akt,p-Akt,t-Nrf2,HO-1,NQO-1,GPX4,p53,n-Nrf2 and other proteins.6.After Jagged1 pretreatment activated Notch1,SC79 pretreatment activated Akt,or t-BHQ pretreatment activated Nrf2,the effects of emodin on NRK-52E cell viability,oxidative stress,lipid peroxidation,iron indices,related protein expression levels and gene expression levels were detected.Result:1.Emodin can significantly up-regulate the levels of BUN,SCr,MDA,LPO and Fe²⁺in kidney tissue,decrease the expressions of SOD and GSH,and induce pathological injury of kidney in mice.2.Emodin can decrease the viability of NRK-52E cells,promote the release of LDH,induce the intracellular production of reactive oxygen species and lipid peroxides,promote the accumulation of Fe,and cause the depolarization of mitochondrial membrane potential（ΔΨm）.3.Emodin reduces the antioxidant capacity of the kidney by decreasing the activity of Notch1/Nrf2/GPX4 pathway axis,promoting the occurrence of intracellular oxidative stress and iron death,and ultimately inducing renal toxicity.4.The toxic effects of emodin on NRK-52E cells,oxidative stress,lipid peroxidation,mitochondrial damage and iron death were weakened by Jagged1 pretreatment to activate Notch1,SC79 pretreatment to activate Akt,or t-BHQ pretreatment to activate Nrf2.Discussion:This current study indicated that emodin was capable of inhibiting Notch1/Nrf2/GPX4 antioxidant system and induced renal injury through an ROS-mediated ferroptosis.Activation of Notch1 or Nrf2 can reverse the emodin-induced nephrotoxicity,suggesting that Notch1 or Nrf2 is a promising therapeutic target for emodin-induced kidney injury in clinical practice.