| Background:Zinc oxide nanoparticles (nano-ZnO) are one of the most commonly utilized materials in diverse industrial fields such as electronics, food additives, sunscreens, cosmetics. Along with intensive application of nano-ZnO in our life, growing concerns have arisen about its biological safety and environment impact. In recent years, it was reported that nano-ZnO can cause the change of cellular response, including cell metabolism, stress response, cell signaling conduction and apoptosis. In vivo, nano-ZnO can accumulate in heart, liver, spleen, lung, kidney through circulatory system after it enter the body through several routs, including oral ingestion, intratracheal instillation, inhalation and intravenous injection. Studies have demonstrated that oral administration of nano-ZnO can accumulate in the liver and cause liver cell apoptosis mediated by oxidative stress. However, the molecular mechanism of nano-ZnO induced hepatotoxicity is still unclear.Although oxidative stress is widely recognized as the major mechanism of nanomaterials, how it triggers apoptosis signaling pathways is still unknown. Apoptosis is programmed cell death, there are three apoptotic pathways, including death receptor pathway, the mitochondrial pathway and endoplasmic reticulum stress pathway. Endoplasmic reticulum (ER) is the eukaryotic cells organelle. It is responsible for protein translocation, protein folding, and protein post-translational modifications and also is the storage site of intracellular Ca2+, which is closely related to transport of proteins, exchange of materials and detoxification. There is a large amount of endoplasmic reticulum in hepatocytes, oxidative stress, ischemia reperfusion injury and various toxicants can affect the structure and function of the endoplasmic reticulum and trigger endoplasmic reticulum stress response, which in turn induce apoptosis, liver damage and a series of cascades. Therefore, we assumed that:ROS can damage the structure and function of the endoplasmic reticulum, thereby activating the endoplasmic reticulum stress signaling pathways which can cause hepatocytes apoptosis.Objective:The aim of this study is to investigate the effect and mechanism of the endoplasmic reticulum stress signaling pathways on the nano-ZnO induced hepatotoxicity in mice. In addition to reveal the pathogenesis of toxic liver damage induced by nano-ZnO, it also provide a new way to prevent and remedy the toxic liver injury and new data for the future research of nano-ZnO toxicity.Methods:Fifty male C57mice aged6weeks, weighing from20g to22g were randomly divided into five groups with, ten mice per group. Mice were gavaged with nano-ZnO100,200,400,600mg/Kg and normal saline for14consecutive days, to establish subacute liver injury model. The serum levels of ALT, AST, and ALP were determined with complete automatic biochemical analyzer. Histological analysis of HE stained liver tissue were performed. The liver tissue homogenate level of SOD, MDA was detected. The content of caspase3, caspase9, caspase12in liver tissue homogenate was determined utilizing ELISA procedures. The mRNA expression of bcl-1, bcl-2, bax and GRP94of liver tissue were measured by RT-PCR.Fifty male C57mice (6weeks old, weighing20g-22g) were randomly divided into five groups:nano-ZnO group (200mg/Kg,400mg/Kg), ZnO group (200mg/Kg,400mg/Kg) and control group with ten mice in each group. Mice were lavaged with nano-ZnO, ZnO and normal saline for90consecutive days, to establish chronic liver injury model. The serum levels of ALT, AST, and ALP were determined with complete automatic biochemical analyzer. Histological analysis of HE stained liver tissue were performed. The liver tissue homogenate level of SOD, MDA, GSH were detected to test oxidative stress. The content of caspase3, caspase9, caspase12was determined utilizing ELIS A procedures. The mRNA expression of bcl-1, bcl-2, bax, GRP94, GRP78, CHOP, PDI, XBP-1of liver tissue were measured by RT-PCR. The protein expression of PERK, p-PERK, Eif2a, p-Eif2a, JNK, p-JNK and CHOP were detected by western blot. We observe the changes of endoplasmic reticulum morphology and structure in the liver with transmission electron microscope. The expression level of apoptotic protein CHOP in liver was determined by immunohistochemistry.Results:The serum level of ALT was significantly elevated in nano-ZnO(200mg/Kg,600mg/Kg), while the level of ALP was significantly increased in nano-ZnO(400mg/Kg,600mg/Kg) in the subacute hepatotoxicity model induced by different concentration of nano-ZnO. Pathological results showed the infiltration of inflammatory cells and a few spotty necroses in liver only in the nano-ZnO group (100mg/Kg,200mg/Kg). The level of SOD and MDA was significantly higher in nano-ZnO group than the control group. The content of apoptotic protein caspase12was significantly elevated in nanoZnO(200,400,600mg/Kg), besides the level of caspase9was significantly increased in nano-ZnO (600mg/Kg). The mRNA expression of bcl-1and bcl-2was down-regulation meanwhile the expression of bax and GRP94was up-regulation in liver tissue of nano-ZnO group compared with control group, the difference was statistically significant.In the chronic hepatotoxicity model induced by nano-ZnO, the serum level of ALT and AST significantly increased in nano-ZnO group compared with control group. The liver tissue pathological results displayed focal necrosis and congestion of dilated central vein in nano-ZnO group. The level of GSH in liver tissue in nano-ZnO group was significantly decreased compared with control group, while the level of SOD and MDA was increased in nano-ZnO group, but only in nano-ZnO (400mg/Kg) the difference of SOD is statistically significant compared with control group. The content of caspase3, caspase9, caspase12in liver tissue was significantly increased in nano-ZnO group compared with control group. The mRNA expression of bcl-1and bcl-2was down-regulation meanwhile the expression of bax, GRP94, GRP78, CHOP, PDI, XBP-1was up-regulation in liver tissue of nano-ZnO group compared with control group, the difference was statistically significant. In western blot, the protein expression level of p-Eif2a, p-JNK, p-PERK, CHOP was higher in nano-ZnO group than control group. In transmission electron microscopy, we can observe the swelling of endoplasmic reticulum, ribosomes degranulation in nano-ZnO group, while the structure of endoplasmic reticulum was normal in control group; the expression level of CHOP in immunohistochemistry was significantly higher in nano-ZnO group than control group.Conclusion:The sub-acute hepatotoxicity induced by different concentrations (100,200,400,600 mg/Kg) of nano-ZnO was mediated by oxidative stress; there was no obvious dose-effect between the degree of liver injury and intragastric concentrations. In chronic hepatotoxicity model induced by nano-ZnO, it induced oxidative stress. Excessive ROS destroyed the structure and function of the endoplasmic reticulum thus activated the endoplasmic reticulum stress signaling pathway that lead to hepatocyte apoptosis. |
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