Caspase-9Mediated Mitochondrial Signaling Pathway Of Arsanilic Acid-induced Apoptosis In NRK Cells

Objective:Aim of this study was to investigate the apoptotic signaling pathways in rat renal cells (NRK) induced by Arsanilic acid, and the protective effects of Trolox.Methods:NRK cells were cultured and stained by haematoxylin. Normal cells counted from dead cells after trypan blue staining, cells proliferation was evaluated by MTT assays. Then, cells were treated with0.005mg/ml,0.05mg/ml and0.5mg/ml Arsanilic acid, proportion of living cells, apoptotic cells and dead cells were preliminary computed by fluorescence microscopy after AO/EB dyeing. MDA content, SOD and GSH-Px activities were estimated by benzodiazepines sodium (TBA) method, nitrite develo ping method and nitro benzoic acid anion mehod. cells ROS was tabbed fluorescence probe DCFH-DA. Cell apoptosis was discussed by TUNEL assay and Annexin-FITC/PI staining. Mitochondrial membrane potential was tested by Rhodaminel23assay. Caspase-3,9protein expression were assessed by western blot. Z-VAD-FMK, Ac-LEHD-FMK and Trolox were applied in flow cytometric detection, and Trolox was employed in mitochondria membrane potential measurement as well.Results:NRK cells were adherent growth, were polygonal with round or oval nuclei. After haematoxylin staining, cells nuclei were light purple, and living cells ratio was up to90%from recovery by Trypan blue staining. With increase of Arsanilic acid concentration, inhibition effect gradually increased in NRK cells, the drawn the survival-doses-curve was "S" type, IC50of Arsanilic acid is about0.2mg/ml. Fluorescent microscope showed thativing cells rate declined, apoptotic cells is significantly increased in every0.005mg/ml,0.05mg/ml and0.5mg/ml Arsanilic acid groups and cells density was low in0.5mg/ml Arsanilic acid group. MDA content and active oxygen release increased, SOD and GSH-Px activities declinedin a dose-dependent relations. The mitochondrial membrane potential lost, apoptosis rates augmented, Caspase-9and Caspase-3protein expression promoted after Arsanilic acid treatment compared to colntrol. After Ac-LEHD-FMD and Z-VAD-FMK treatment, cells apoptotic rate decreased. Similarly, apoptotic rates descended after Trolox treatment, cells viabilities increased impacted by100μM-600μM Trolox and achieved peak in500μM, fell down from700μM compare to control.Conclusion:①Arsanilic acid toxicity on NRK cells is to inhibit cell growth and proliferation.②Arsanilic acid promotes oxygen free radicals generation and inhibits oxygen free radicals and their oxidation products elimination.③Arsanilic acid induces apoptosis in NRK cells.④ Arsanilic acid-induced apoptosis of NRK cells dependent on the mitochondrial pathway of caspase-9, caspase-3activation.⑤The Trolox clearance of ROS, reduction interaction between ROS and mitochondrial apoptotic factors effectively protects NRK cells from the Arsanilic acid-induced apoptosis.Summary:Arsanilic acid induced apoptosis in NRK cells through mitochondrial pathway mediated by caspase-9, Trolox reduced apoptosis in NRK cells by scavenging reactive oxygen species, reducing the role of ROS in the apoptosiss.