P0533SCUTELLARIN PROTECTS ISCHEMIA/REPERFUSION INDUCED ACUTE KIDNEY INJURY FROM OXIDATIVE STRESS BY TARGETING NRF2
Authors: Jie Dai 
Published: June 2020

Abstract

Background and Aims
Ischemia/reperfusion (I/R) may cause acute kidney injury(AKI) by mediating the oxidative stress and related inflammation then inducing apoptosis. The present study is to explore the preventive effect and mechanism of Scutellarin(Scu),on AKI induced by I/R.

Method
①The renal ischemia-reperfusion model GSE98622 data set was selected from NCBI GEO DateSets, and the genes with significantly increased expression in acute kidney injury were selected. Further, dozens of hub-genes were identified as candidate proteins by protein-protein interaction network(PPI network). The molecular docking between the protein and the Scu was performed using the Autodock software. As the results of the molecular docking,the binding energy between the protein Nrf2 and Scu molecule is -9.84, suggesting that there is a high probability of interaction between the two molecules.
②Experiment in vivo. Wistar rats were randomly divided into 5 groups: normal group, sham group, AKI group (bilateral renal pedicle clip 45 min), Scu + AKI group (Intraperitoneal injection of 50 mg/kg.d Scu daily for 7 days before surgery,the same surgery with AKI group),Saline + AKI group (the same concentration of DMSO-normal saline solution daily for 7 days before surgery, the same surgery with AKI group), 6 rats in each group.The rats were sacrificed together 24h after surgery.
③Experiment in vitro.Human kidney tubular epithelial cells (HK-2) were firstly divided into 7 groups:hypoxia for 3 hours (h), 6h, 9h, 12h, 24h, 36h and 48h reoxygenation for 1h. RT-PCR detect Hihf1α, Nrf2, HO1,SOD-1,caspase3, Bcl2/BAX, NF-κB and TNFα levels to determine the best hypoxia time. To screen the safe concentration of the drug,after pretreatment with 200μmol/l, 150μmol/l,120μmol/l, 100μmol/l,80μmol/l,60μmol/l, 40μmol/l,20μmol/l, 10μmol/l,5μmol/l Scu for 12 h, use CCK8 to measure the absorbance. Then the optimal protective concentration in hypoxia was searched in maximum safe concentration to determine the final drug concentration.Finally,after the cells were randomly divided into normal group, hypoxia group, hypoxia + DMSO group, hypoxia + Scu group, RT-PCR ,Western Blot and ROS probe was used to detect related indicators.

Results
①Bioinformatics analysis suggests that Nrf2 protein is one of the key factors in acute kidney injury induced by ischemia-reperfusion, which may be a target for the prevention and treatment of diseases.
②The results of experiments in vivo showed that compared with the normal group and the sham group, the serum creatinine level increased after AKI, and the HE staining showed that the renal tubular injury score increased . The creatinine and renal tubular injury score of Scu group were significantly relieved. Scu also reduces the level of MDA increased by AKI.KIM-1 increased, Nrf2 and HO-1 increased,SOD-1 decreased,Caspase3 increased, Bcl2/BAX decreased, NF-κB and TNFα increased after AKI, all P&lt;0.05, suggesting obvious renal injury.However, the Scu+AKI group significantly up-regulated the Nrf2/HO-1 to combat oxidative stress damage.Moreover,Scu further down-regulated the inflammatory and apoptotic level.
③The pre-test choosed ischemia for 24h and re-oxygenation for 1 hour as the experiment time. And Compared with the normal group,the PCR results showed that in the hypoxia group and the hypoxia+DMSO group, the expression of hif1α increased, Nrf2 and HO-1 increased, caspase3 increased, Bcl2/BAX decreased, and NF-κB and TNFα increased, both P&lt;0.05. The H+Scu group significantly reduced the oxidative stress through the results of ROS probe and increasing Nrf2/HO-1 and SOD-1 level.Scu also ameliorates the indicators of inflammation and apoptosis (P&lt;0.05). The WB results are consistent with the PCR.

Conclusion
Scu has obvious protective effect on acute kidney injury induced by ischemia-reperfusion. The mechanism is probably alleviating oxidative stress by targeting Nrf2/HO-1 pathway.