Baicalin ameliorates H2O2 induced cytotoxicity in HK-2 cells through the inhibition of ER stress and the activation of Nrf2 signaling.

Miao Lin,Tongyu Zhu,Long Zheng,Long Li,Ruiming Rong,Ming Xu,Yi Zhang

doi:10.3390/ijms150712507

Abstract

Renal ischemia-reperfusion injury plays a key role in renal transplantation and greatly affects the outcome of allograft. Our previous study proved that Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia-reperfusion injury. This study aimed to study the underlying mechanism in vitro. Human renal proximal tubular epithelial cell line HK-2 cells were stimulated by H2O2 with and without Baicalin pretreatment. The cell viability, apoptosis and oxidative stress level were measured. The expression of endoplasmic reticulum (ER) stress hallmarks, such as binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), were analyzed by western blot and real-time PCR. NF-E2-related factor 2 (Nrf2) expression was also measured. In the H2O2 group, cell viability decreased and cell apoptosis increased. Reactive Oxygen Species (ROS) and Glutathione/Oxidized Glutathione (GSH/GSSG) analysis revealed increased oxidative stress. ER stress and Nrf2 signaling also increased. Baicalin pretreatment ameliorated H2O2-induced cytotoxicity, reduced oxidative stress and ER stress and further activated the anti-oxidative Nrf2 signaling pathway. The inducer of ER stress and the inhibitor of Nrf2 abrogated the protective effects, while the inhibitor of ER stress and the inducer of Nrf2 did not improve the outcome. This study revealed that Baicalin pretreatment serves a protective role against H2O2-induced cytotoxicity in HK-2 cells, where the inhibition of ER stress and the activation of downstream Nrf2 signaling are involved.

Highlights

Renal ischemia-reperfusion injury (IRI) is an inevitable acute injury during renal transplantation, which greatly affects the outcome of allograft [1,2]
In order to observe the change of cell viability along with H 2O2 stimulation time, we compared the Optical density (OD) value by Cell Counting Kit 8 (CCK-8) assay at different time points after H2O2 stimulation (Figure 1C)
The largest difference of cell viability between the H2O2 + Baicalin group and the H2O2 group was observed after 4 h of stimulation, which suggested that 4 h of H2O2 stimulation is most appropriate for our study

Summary

Introduction

Renal ischemia-reperfusion injury (IRI) is an inevitable acute injury during renal transplantation, which greatly affects the outcome of allograft [1,2]. Renal tubular epithelial cells (TECs) suffer from cell injury and undergo apoptosis in renal IRI, contributing to the loss of graft function. The pathophysiological change and underlying mechanism of TECs apoptosis are quite complex [3], involving the caspase cascade, mitochondria dysfunction, immune injury and endoplasmic reticulum (ER) stress [4]. The prevention of TECs apoptosis is considered to be an effective method to alleviate renal IRI. A serial of conservative and complementary adaptive signaling pathways were activated to cope with the perturbations, which are generally called the unfolded protein response (UPR). The homeostasis in the ER is maintained through the adaptive response of UPR [7]. UPR gradually develops into the apoptosis phase in renal IRI.

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