Abstract

BackgroundFree radicals generated in the biological system bring about modifications in biological molecules causing damage to their structure and function. Identifying the damage caused by ROS and RNS is important to predict the pathway of apoptosis due to stress in PC12 cells. The first defense mechanisms against them are antioxidants which act in various pathways through important cellular organelles like the mitochondria and endoplasmic reticulum. Specific biomarkers like Gadd153 which is a marker for endoplasmic reticulum stress, Nrf2 which responds to the redox changes and translocates the antioxidant response elements, and Btg2 which is an antioxidant regulator have not been addressed in different stress conditions previously in PC12 cells. Therefore, the study was conducted to analyze the gene expression pattern (SOD, Catalase, Btg2, Gadd153, and Nrf2) and the protein expression pattern (iNOS and MnSOD) of the antioxidant stress markers in differential stress-induced PC12 cells. Peroxynitrite (1 μM), rotenone (1 μM), H2O2(100 mM), and high glucose (33 mM) were used to induce oxidative and nitrosative stress in PC12 cells.ResultsThe results obtained suggested that rotenone-induced PC12 cells showed a significant increase in the expression of catalase, Btg2, and Gadd153 compared to the control. Peroxynitrite-induced PC12 cells showed higher expression of Btg2 compared to the control. H2O2 and high glucose showed lesser expression compared to the control in all stress marker genes. In contrast, the Nrf2 gene expression is downregulated in all the stress-induced PC12 cells compared to the control. Further, MnSOD and iNOS protein expression studies suggest that PC12 cells exhibit a selective downregulation. Lower protein expression of MnSOD and iNOS may be resulted due to the mitochondrial dysfunction in peroxynitrite-, high glucose-, and H2O2-treated cells, whereas rotenone-induced cells showed lower expression, which could be the result of a dysfunction of the endoplasmic reticulum.ConclusionDifferent stress inducers like rotenone, peroxynitrite, H2O2, and high glucose increase the NO and ROS. Btg2 and Gadd153 genes were upregulated in the stress-induced cells, whereas the Nrf2 was significantly downregulated in differential stress-induced PC12 cells. Further, antioxidant marker genes were differentially expressed with different stress inducers.

Highlights

  • Free radicals generated in the biological system bring about modifications in biological molecules causing damage to their structure and function

  • As described in the “Methods” section, NO generation was observed in PC12 cells after the exposure of rotenone (1 μM), peroxynitrite (1 μM), H2O2 (100 μM), and high glucose (33 mM) for 24 h

  • reactive oxygen species (ROS) production is expressed in relative fluorescent intensity (RFUROS) produced in PC12 cells after the exposure of different stress inducers against the control

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Summary

Introduction

Free radicals generated in the biological system bring about modifications in biological molecules causing damage to their structure and function. Specific biomarkers like Gadd153 which is a marker for endoplasmic reticulum stress, Nrf which responds to the redox changes and translocates the antioxidant response elements, and Btg which is an antioxidant regulator have not been addressed in different stress conditions previously in PC12 cells. Superoxide radicals, hydrogen peroxide, and peroxynitrite are the intermediate products of the metabolic pathway in cellular signaling cascades [3]. ROS and RNS were considered harmful by-products of the aerobic metabolism process of the cell. Mitochondrial ROS were considered as harmful species implied in a range of diseases and pathologies [6], but their importance in the cellular signaling pathway is being addressed and has given new scope to it. Excessive production of RONS is the primary reason leading to several degenerative pathologies/diseases [8]

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