Baicalin Protects Tubular Epithelial Cells From Hypoxia/reoxygenation-induced Apoptosis Via Inhibiting ROS-endoplasmic Reticulum Stress PERK Pathway

Background: Acute kidney injury(AKI)is a common disease with high morbidity and mortality.Thrombolysis,vascular surgery,shock,organ transplantation,burns,frostbite and so on can lead to renal ischemia-reperfusion injury.Ischemia reperfusion(IR)-induced renal tubular epithelial cells(TECs)apoptosis plays an important role in the pathogenesis of IR-induced AKI.Severe or prolonged endoplasmic reticulum(ER)stress will trigger cell apoptosis.Baicalin,a flavonoid extracted from traditional Chinese medicine,has various pharmacological activities,including anti-oxidation,anti-inflammation and anti-apoptosis,and has a protective effect on IR injury of various organs.The aim of this study was to investigate the effect of Baicalin on IR-induced HK-2 cells apoptosis using a hypoxia/reoxygenation(H/R)cell model to simulate IR in vitro and to clarify its mechanism.Methods: Human kidney proximal tubular epithelial cell line(HK-2)cells were exposed to either normoxia or hypoxia/reoxgenation afterpretreatment with or without Baicalin,N-acetylcysteine(NAC)or 4-phenyl butyric acid(4-PBA,ER stress inhibitor).ER stress-related protein expression was measured by western blot analysis and immunofluorescence,reactive oxygen species(ROS)production was measured by DCFH-DA fluorescence staining,cell death was measured by Cell Counting Kit 8(CCK-8)colorimetric assay,cell apoptosis was measured by flow cytometry assay,and TUNEL assay,and the expression of Cleaved-caspase 3 was measured by western blot analysis.Results: H/R induced HK-2 cells an obvious increase in cell death and apoptosis,and pretreatment with Baicalin significantly attenuated H/R-induced cell apoptosis in HK-2 cells.H/R significantly increased the expression of BIP,PERK,ATF4,CHOP and Caspase12,whereas the protein expression of ATF6 and IRE1 did not correspondingly increase in HK-2 cells.H/R increased the production of ROS in HK-2 cells and H/R-induced increased expression of BIP,PERK,ATF4,CHOP and Caspase12 was suppressed in the presence of antioxidant reagent NAC.Blocking ER stress with 4-PBA significantly ameliorated H/R-induced apoptosis in HK-2 cells.Baicalin could significantly attenuate H/R-induced increased ROS production and ER stress-related proteins expression in HK-2 cells.Conclusions: Our findings indicated that H/R induced ER stress in HK-2 cells via PERK signaling pathway.H/R-induced ER stress in HK-2cells were associated with excessive ROS production.In addition,Baicalin served as a protective role against H/R-induced apoptosis,the inhibition of ROS-ER stress pathway might be the mechanism.