Paeoniflorin Alleviates Ischemia/reperfusion Induced Acute Kidney Injury By Inhibiting Slc7a11-mediated Ferroptosis

Background and objective:Acute kidney injury（AKI）is a group of clinical syndromes characterized by a rapid decline in renal function in a short period of time,leading to the accumulation of metabolic wastes and toxins in the body,which in turn leads to complications and failure of other organs.Clinically,the causes of AKI mainly include ischemia-reperfusion（IR）injury,sepsis,and various endogenous and exogenous nephrotoxins.AKI is a common complication in hospitalized patients,which is closely related to poor prognosis such as prolonged hospital stay,the occurrence of chronic kidney disease and even end-stage renal disease,and increased mortality.At present,there is still a lack of effective methods for the prevention and treatment of AKI.The pathophysiological mechanism of AKI is very complex,and previous studies have mainly focused on cell necrosis or apoptosis.However,in recent years,ferroptosis has been shown to play an important role in the development of AKI.Ferroptosis is a newly discovered regulatory cell death mode,which is characterized by intracellular iron accumulation,decreased glutathione（GSH）and glutathione peroxidase,and increased lipid peroxidation and reactive oxygen species（ROS）.Paeononiflorin（PF）,a water-soluble monoterpene glycoside,is the main bioactive component of Paeoniflorin.Many studies have shown that paeoniflorin has a wide range of pharmacological effects in vivo and in vitro.It has been documented that paeoniflorin can reduce ischemia-reperfusion injury in various organs such as heart,liver and brain.Paeoniflorin has been reported to alleviate pancreatitis induced AKI by inhibiting cellular inflammatory response and apoptosis.Previous studies from our group have shown that paeoniflorin can protect the kidney in diabetic nephropathy by regulating autophagy and inhibiting inflammatory response.The aim of this study is to further explore the protective effect and mechanism of paeoniflorin on acute kidney injury induced by ischemia-reperfusion,and to provide new ideas for the prevention and treatment of AKI.Methods:In vitro study:Human renal tubular epithelial cells（HK2）between the 6th and 15th passages were selected for this study.First,the drug toxicity was detected by MTT assay,and the hypoxia/reoxygenation time and the optimal drug concentration were selected by Western blot.The experiment was divided into seven groups:normal control group,single administration group,hypoxia/reoxygenation（HR）group,low-dose administration group,medium-dose administration group,high-dose administration group,ferroptosis inhibitor group.The protein and m RNA were extracted,and the expression of kidney injury factor 1（KIM-1）was detected by immunofluorescence and Real-time PCR.Real-time PCR was used to detect the changes of renal inflammatory factors IL-1β,IL-6 and TNF-α.Western blot was used to detect the expression of ferroptosis-related proteins Slc7a11 and GPX4.Finally,the expressions of GSH,MDA and Fe²⁺in the cells were detected,and the oxidative stress level in the cells was detected by fluorescent probe DHE.To explore the specific mechanism of paeoniflorin in protecting renal tubular epithelial cells by RNA-seq analysis.The results of RNA-seq were analyzed by KEGG and Heatmap to screen out the specific pathways and target proteins.Molecular docking experiments were performed to verify the binding of the drug to the target protein Slc7a11.The expression of Slc7a11 was silenced by si RNA transfection,and the effect of paeoniflorin on ferroptosis of renal tubular epithelial cells after Slc7a11 silencing was detected by Western blot.In vivo study:Male C57BL/6 mice（6-8 weeks old）underwent bilateral renal ischemia-reperfusion surgery to establish acute kidney injury models,and were treated with paeoniflorin（25,50,and 100 mg/kg）and ferroptosis inhibitor Fer-1（5mg/kg）,respectively.The experiment was divided into seven groups（n=6-8 each）:Sham operation group（Sham）,single administration group,I/R group,low-dose administration group,medium-dose administration group,high-dose administration group,and ferroptosis inhibitor group.After the model was successfully established,the renal function of the mice in each group was detected by serum creatinine and urea nitrogen.PAS staining and HE staining were used to evaluate the degree of renal tubular injury.The expression levels of kidney injury factor 1（KIM-1）and renal inflammatory factors（TNF-α,IL-1β,IL-6）were detected by immunohistochemistry and Real-time PCR.The expression of ferroptosis key protein GPX4 was detected by immunohistochemistry.The morphological changes of mitochondria in mouse renal tubular epithelial cells were observed by transmission electron microscopy.Western blot was used to detect the expression of ferroptosis-related proteins Slc7a11 and GPX4.Finally,the expression changes of GSH,MDA and Fe²⁺in renal tissue of mice were detected,and the oxidative stress level in renal tissue of mice was detected by fluorescent probe ROS.Results:In vitro study:MTT showed that 6.25μΜ,12.5μΜ,25μΜ,50μΜ,100μΜpaeoniflorin treated human renal tubular epithelial cells for 24 hours,cell survival rate did not change significantly.The results of Western blot showed that the expression of KIM-1was significantly increased under the condition of 24h hypoxia and 3h reoxygenation.A protective effect of paeoniflorin was observed at a concentration of 25μΜand the expression of KIM-1 was attenuated in a dose-dependent manner.Real-time PCR showed that paeoniflorin could reduce the changes of inflammatory factors IL-1β,IL-6and TNF-α.Western blot showed that paeoniflorin restored the expression of ferroptosis related proteins Slc7a11 and GPX4.The results of GSH,MDA and Fe²⁺in cell homogenate showed that paeoniflorin could reduce the expression of MDA and Fe2⁺and increase the expression of GSH.DHE staining confirmed that paeoniflorin could reduce the occurrence of oxidative stress.Through RNA-seq analysis and molecular docking experiments,we found that paeoniflorin may act on the glutathione pathway through Slc7a11 to inhibit ferroptosis and alleviate HR-induced renal tubular epithelial cell injury.Western blot results showed that paeoniflorin could not further reverse HR-induced ferroptosis in Slc7a11silenced cells.In vivo study:The results of serum creatinine and urea nitrogen showed that paeoniflorin reversed IR-induced renal dysfunction in a dose-dependent manner,and Fer-1 also had protective effect on renal function.Renal PAS staining and HE staining showed that the drug alleviated renal tissue injury.Immunohistochemistry and Real-time PCR showed that KIM-1 was also reduced.We also found that paeoniflorin alleviated IR-induced renal cell ferroptosis,inflammation and oxidative stress.Conclusion:（1）Paeoniflorin inhibited IR-induced renal tubular epithelial cell injury,inflammation,ferroptosis and oxidative stress in a dose-dependent manner,and the high-dose group had better cytoprotection than the ferroptosis inhibitor Fer-1;（2）Analysis of RNA-seq results and molecular docking experiments showed that paeoniflorin may inhibit ferroptosis and alleviate HR-induced renal tubular epithelial cell injury by acting on the glutathione pathway through Slc7a11.（3）Paeoniflorin could not further reverse HR-induced ferroptosis in Slc7a11-silenced cells.These results suggest that paeoniflorin may protect HK2 cells from HR induced injury through Slc7a11-mediated ferroptosis.