Scutellarin Protects The Kidney From Ischemia/reperfusion Injury By Targeting Nrf2/HO-1 Pathway

Objective:Acute kidney injury（AKI）is a disease characterized by a rapid decline of renal function,which has high clinical morbidity and poor prognosis.Ischemia-reperfusion induced AKI（I/R-AKI）is an important cause of AKI.Therefore,this article aims to explore the effect of Scutellarin on the prevention of I/R-AKI and its possible mechanism.Methods:（1）In the first part of this study,in order to explore the renal protective effect of Scutellarin,the I/R-AKI rat model was constructed using the classic bilateral renal pedicle clamping method after Scutellarin pretreatment.HE,PAS and Masson staining were used to evaluate the degree of kidney injury,and the changes of serum creatinine（SCr）,blood urea nitrogen（BUN）and related gene expression levels were detected.In the pre-experiment in vitro,we explored the suitable hypoxia time and optimal Scutellarin concentration in the hypoxia-reoxygenation model of human renal tubular epithelial cells（RTECs）.Then,after 20μmol/L Scutellarin pretreatment,RTECs were treated with hypoxia for 24 h and reoxygenation for 1 h.CCK8,q RT-PCR and Western blot were used to explore the effect of Scutellarin on hypoxic renal cells.And the level of reactive oxygen species（ROS）was detected by flow cytometry.（2）In the second part,in order to explore the mechanism,we employed differentially expressed gene analysis,protein interaction network analysis and reverse molecular docking to screen out the possible target proteins of Scutellarin.The bioinformatic results were verified by experiments using small interfering RNA（si RNA）.Results:（1）In the first part,the SCr,BUN levels and renal tubular injury scores of the rats in the Scu+AKI group were significantly decreased（P<0.05）compared with the AKI group.Scutellarin decreased the expression level of kidney injury molecular-1（KIM-1）,and increased superoxide dismutase 1（SOD1）and heme oxygenase 1（HO-1）in rat kidneys（P<0.05）,indicating that Scutellarin alleviated ischemic kidney damage and enhanced its antioxidant capacity.In the pre-experiment in vitro,the results of q RT-PCR showed that the expression levels of related genes were significantly changed compared with the control group under the hypoxia for 24 h and reoxygenation for 1 h condition.RTECs reached the highest activity in the hypoxic condition when they were incubated with 20μmol/l Scutellarin（P<0.01）,so 20μmol/L was determined as the optimal concentration of Scutellarin.After the application of Scutellarin,the expression level of hypoxia inducible factor 1 subunitα（Hif1α）was downregulated,while the levels of antioxidant genes including SOD1 and HO-1 were upregulated（P<0.05）.Flow cytometry showed that Scutellarin reduced the generation of ROS in hypoxic cells（P<0.001）,indicating that it enhanced the antioxidant capacity of renal cells.（2）In the second part,we used bioinformatics to explore the mechanism of Scutellarin.Microarray analysis results showed that a total of 468 differentially expressed genes,including 349 upregulated genes and 119downregulated genes,were identified to be significantly related to the occurrence of I/R-AKI（adj.P<0.05）.Protein interaction network showed that nuclear factor,erythroid derived 2,like 2（Nrf2）（log₂FC=2.935157,adj.P=0.000129 in differentially expressed gene analysis）is one of the hub genes in I/R-AKI,which may play a role in AKI treatment.The reverse molecular docking results showed that the binding energy between Nrf2 and Scutellarin was-9.84,suggesting stable binding and that Nrf2 might be a target of Scutellarin.After inhibiting the expression of Nrf2 by si RNA,Scutellarin could not reduce the production of ROS nor increase the expression level of HO-1,which verified that Scutellarin prevents I/R-AKI through the Nrf2/HO-1 pathway.Conclusion:Scutellarin has a significant preventive effect on I/R-AKI,which is mediated by reducing renal oxidative stress through the Nrf2/HO-1 pathway.