During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-gamma in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism.
Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion-cytochrome c-caspase protease mechanism.
Cerebrovascular endothelial cells (CECs) are crucial components of the blood-brain barrier. Our previous study showed that oxidized low-density lipoprotein (oxLDL) induces apoptosis of CECs. This study was designed to further evaluate the effects of resveratrol on oxLDL-induced CEC insults and its possible molecular mechanisms. Resveratrol decreased the oxidation of LDL into oxLDL. Additionally, the oxLDL-caused oxidative stress and cell damage were attenuated by resveratrol. Exposure of CECs to oxLDL induced cell shrinkage, DNA fragmentation, and cell apoptosis, but resveratrol defended against such injuries. Application of Lox-1 small interference (si)RNA into CECs reduced the translation of this membrane receptor, and simultaneously increased resveratrol protection from oxLDL-induced cell apoptosis. By comparison, overexpression of Lox-1 attenuated resveratrol protection. Resveratrol inhibited oxLDL-induced Lox-1 mRNA and protein expressions. Both resveratrol and Lox-1 siRNA decreased oxLDL-enhanced translocation of proapoptotic Bcl-2-associated X protein (Bax) from the cytoplasm to mitochondria. Sequentially, oxLDL-induced alterations in the mitochondrial membrane potential, cytochrome c release, and activities of caspases-9, -3, and -6 were decreased by resveratrol. Pretreatment with Z-VEID-FMK (benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone) synergistically promoted resveratrol's protection against DNA fragmentation and cell apoptosis. Therefore, this study shows that resveratrol can protect CECs from oxLDL-induced apoptotic insults via downregulating Lox-1-mediated activation of the Bax-mitochondria-cytochrome c-caspase protease pathway.
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