P0538HYPERIN PREVENTS ISCHEMIC/REPERFUSION AKI VIA STK40
Authors: Xu Yan , Zhang Yue 
Published: June 2020

Abstract

Background and Aims
Ischemia-reperfusion injury (IRI) is the outcome of an inflammatory process and tubular cell death that is triggered by undergoing a transient reduction or cessation of blood flow and following by reperfusion. Unresolved IRI can contribute to chronic kidney disease even death. Our aims is to investigate the protective effect of hyperin on ischemia-reperfusion renal injury (IRI) and its possible mechanism.

Method
① The transcriptome chip data of multiple IRI models were selected from the NCBI GEO DateSets database and a number of key proteins that could participate in IRI were screened out (the fold increase was greater than 2 fold and was statistically significant). Network and transcript binding motif analysis was performed to determine the best binding protein. ② C57BL / 6J mice were selected and randomly divided into normal group, sham operation group, IRI group (bilateral renal pedicle clamping for 45min), hyperin + IRI group (50mg / kg.d per day, 7 days before surgery ), DMSO + IRI group (7 days before the operation, the same amount of DMSO was administered to the stomach every day, and the operation was the same as AKI), with 6 rats in each group. Renal tissue and blood were collected 24 hours after operation for testing. ③ In vitro experiments, human proximal tubule epithelial cells (HK-2) were selected and divided into hypoxia 3, 6, 9, 12, 24, 36, and 48h for reoxygenation of 1, 3, and 6h respectively. Relevant indicators for RT-PCR detection were determined Optimal hypoxia time. The drug safe concentration was selected according to 0, 5, 10, 25, 50, 100, 200, 400 μg / ml hyperin pre-treatment for 12 hours, and the CCK8 reagent was added for 2 hours to measure the absorbance at 450 nm. The cells were randomly divided into normal group, hypoxia group, hypoxia + DMSO group, hypoxia + hyperin group, and related indexes were detected by RT-PCR and Western Blot. ④ Obtain the tertiary structure of the protein and the three-dimensional structure of the hyperin molecule from the RCSB Protein Data Bank website and the PubChem compound database, and use molecular docking technology to determine the proteins that can bind to hyperin using autodock software and analyze their binding ability.

Results
Bioinformatics analysis suggested that STK40 protein is one of the key factors of IRI and may be a target for preventing and treating diseases. In vivo experiments showed that compared with the normal group and the sham operation group, the levels of serum creatinine, blood urea nitrogen, and kim-1 in rats were significantly increased after AKI, and HE staining of pathological sections showed an increase in renal tubular injury scores. Significantly decreased (P&lt;0.05); RT-PCR results showed that kim-1, caspase-3, NF-κB, IL-6, TNF-α increased significantly after AKI, STK40, Bcl2 / BAX decreased, and the above after hyperin The indicators changed in opposite directions (P &lt;0.05). In vitro experiments: The best time for hypoxia is 24h hypoxia + 1h reoxygenation; compared with the control group, the drug concentration is &lt;100 μg / mL and the cell proliferation activity rate is&gt; 90%, so the hyperin concentration was selected as 50 μg / mL (P &lt; 0.05); RT-PCR results showed that Hif1-α, caspase-3, NF-κB, IL-6, TNF-α significantly increased, and STK40, Bcl2 / BAX decreased compared with the normal group. After administration of hyperin, the above indexes changed in opposite directions (P &lt;0.05).

Conclusion
In this study, using molecular docking technology and constructing IRI mice model, it was confirmed that hyperin can reduce IRI and exert a protective effect on IRI by inhibiting STK40 expression.