Effects Of Endoplasmic Reticulum Stress On HK - 2 Cell Apoptosis Induced By Olaquindox

Olaquindox (OLA) is one of an o-nitroaniline, which was widely used as antibacterial and growth-promoting feed addictive for the prevention of dysentery, bacterial enteritis of food-producing animals. With the development of intensive culture, veterinary drugs and their metabolite can be eventually intook by human body via their transition in the environment and transferred in food chain, bringing damage to human health. Toxicology studies show that olaquindox has genotoxic, reproductive toxicity, cumulative toxicity, and is recognized as mutagens and suspected carcinogen by the European Union. The toxic kinetic data show that olaquindox is mainly excreted via the kidneys, and the highest concentration can be detected in kidney, in vivo experiments we also found its substantive damage to the kidneys of mice.Human renal tubular epithelial cells (HK-2) derives from normal human proximal tubular epithelial cells, which retains the renal tubular epithelial cells characteristics in a variety of enzymology, electrophysiology and phenotypic. HK-2is the typical cell lines in the study of mechanism of renal toxicity.In this study, we supposed the generation of reactive oxygen as the start event.We constructed the injury model of HK-2cells by using the method of cell culture in vitro and also detected some biomarkers which are related to endoplasmic reticulum stress and apoptosis by western blot to explore the role mechanism of the toxicity effect of olaquindox in HK-2cells, which eventually investigated whether endoplasmic reticulum stress (ERS) pathway had effects on the olaquindox induced apoptosis in HK-2cell1. The olaquindox induced HK-2cells apoptosis in time, dose-effect manner1) Viabilities of HK-2cells treated with olaquindox significantly decreased with concentration-dependent manner compared to the control group. The IC50(24h) was6.8μmol/ml (R=0.9721);2) Apoptosis rates of group olaquindox exposure (2,3and4μmol/ml) were higher than that of the control group (P<0.05). Significant statistics difference was found between group2μmol/ml and group3μmol/ml olaquindox exposure, group 3μmol/ml and group4μmol/ml in multiple comparison (P<0.05). There is dose-effect relationship between the dose of olaquindox and the apoptosis rates;3) Apoptosis rates of12and24h group was higher than that of the control group (P<0.05). Significant statistics difference was found between group6h and group12h olaquindox exposure, group12h and group24h in multiple comparison(P<0.05). There is time-effect relationship between the exposure time of olaquindox and the apoptosis rates.2. The olaquindox induced the increase of ROS in HK-2cells in time-effect, dose-effect manner1) The levels of the intracellular ROS of group olaquindox exposure (2,3and4μmol/ml) were higher than that of the control group (P<0.05). Significant statistics difference was found between group lμmol/ml and group2μmol/ml olaquindox exposure, group2μmol/ml and group3Lmol/ml, group3μmol/ml and group4μmol/ml in multiple comparison(P<0.05). There is dose-effect relationship between the dose of olaquindox and the ROS level;2) The levels of the intracellular ROS of6,12and24h groups were higher than that of the control group (P<0.05). Significant statistics difference was found between group Oh olaquindox exposure and group6h, group6h and group12h,group12h and group24h in multiple comparison(P<0.05). There is time-effect relationship between the exposure time of olaquindox and the ROS level.3. The expression change of endoplasmic reticulum stress and its related apoptotic proteins in HK-2damaged by olaquindox.1) The expression of GRP78, GRP94, CHOP and Caspase-4of olaquindox exposure (2,3and4μmol/ml) groups were increased than that of the control group (P<0.05);2) The expression of GRP78, GRP94and Caspase-4of12and24h groups were increased than that of the control group (P<0.05). The expression of CHOP of (6,12and24h) groups were increased than that of the control group (P<0.05);3) Olaquindox may induce ERS in HK-2cell, especially increase the expression of GRP78and GRP94, which is related to the olaquindox exposure time and dose; Olaquindox may induce ERS related apoptosis in HK-2cell, especially increase the expression of CHOP and Caspase-4, which is related to the olaquindox exposure time and dose.4. The translation change of endoplasmic reticulum stress and its related apoptosis gene in HK-2impaired by olaquindox1) The mRNA’s translation level of GRP78and GRP94of olaquindox exposure (3and4μmol/ml) groups were increased than the control group (P<0.05), The translation level of Caspase-4of olaquindox exposure (2,3and4μmol/ml) groups were increased than that of the control group (P<0.05);2) The mRNA’s translation level of GRP78and Caspase-4of12and24h groups were increased than that of the control group (P<0.05) The translation level of GRP94of6,12and24h groups were increased than that of the control group (P<0.05);3) Olaquindox may induce ERS in HK-2cell, especially increase the translation level of GRP78and GRP94, which is related to the olaquindox exposure time and dose; Olaquindox may induce ERS related apoptosis in HK-2cell, especially increase the translation level of Caspase-4, which is related to the exposure time and dose of olaquindox;4) The mRNA’s translation level Chang of GRP78, GRP94and Caspase-4is similar to the expression change of GRP78, GRP94and Caspase-4in HK-2.5. ROS inhibitors effect in the apoptosis of olaquindox induced in HK-21) The apoptosis rate and ROS level of NAC pretreatment group was significantly reduced compared to the control group(p<0.05); the expression of GRP78, GRP94, caspase-4and CHOP was decreased in NAC pretreatment group (p<0.05);2) The olaquindox induced HK-2cell to apoptosis and increased the intracellular ROS level, endoplasmic reticulum stress and its related apoptotic protein changes associated with the increase of intracellular reactive oxygen species. ROS may be a critical factor of the endoplasmic reticulum stress and its related apoptosis.