ALBERT

Description: (1) Aims: The present study aimed to observe the effects of Ginsenoside Rb1 on high glucose-induced endothelial damage in rat retinal capillary endothelial cells (RCECs) and to investigate the underlying mechanism. (2) Methods: Cultured RCECs were treated with normal glucose (5.5 mM), high glucose (30 mM glucose), or high glucose plus Rb1 (20 μM). Cell viability, lactate dehydrogenase (LDH) levels, the mitochondrial DNA copy number, and the intracellular ROS content were measured to evaluate the cytotoxicity. Superoxide dismutase (SOD), catalase (CAT), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), poly(ADP-ribose) polymerase (PARP), and sirtuin (SIRT) activity was studied in cell extracts. Nicotinamide adenine dinucleotide (NAD+)/NADH, NADPH/NADP+, and glutathione (GSH)/GSSG levels were measured to evaluate the redox state. The expression of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), SIRT1, and SIRT3 was also evaluated after Rb1 treatment. (3) Results: Treatment with Rb1 significantly increased the cell viability and mtDNA copy number, and inhibited ROS generation. Rb1 treatment increased the activity of SOD and CAT and reduced the activity of NOX and PARP. Moreover, Rb1 enhanced both SIRT activity and SIRT1/SIRT3 expression. Additionally, Rb1 was able to re-establish the cellular redox balance in RCECs. However, Rb1 showed no effect on NMNAT1 expression in RCECs exposed to high glucose. (4) Conclusion: Under high glucose conditions, decreases in the reducing power may be linked to DNA oxidative damage and apoptosis via activation of the NMNAT-NAD-PARP-SIRT axis. Rb1 provides an advantage during high glucose-induced cell damage by targeting the NAD-PARP-SIRT signaling pathway and modulating the redox state in RCECs.

Description: Maintenance of intracellular pH, a critical cellular function, is required for generation of proton gradients and platelet response to agonist. Previously, we have demonstrated that freeze-dried rehydrated platelets are able to respond to agonists generate a rise in intracellular calcium (Auh et al. 2004 Calcium mobilization in freeze-dried platelets. Cell Preservation Technology in press) and maintain normal membrane and protein secondary structure (Wolkers et al. 2002 Towards a clinical application of freeze-dried platelets. Cell Preservation Technology 1:175–188). As part of our ongoing studies of trehalose loaded freeze-dried rehydrated human platelets we examined their ability to maintain their intracellular pH. Platelet proton regulation is achieved through the Na-H exchanger (NHE1) which is dependent on the concentration of extracellular sodium. Freeze-dried and fresh control human platelets were loaded with the pH ratio dye bis-carboxyfluorescein acetomethyl ester (BCECF-AM), washed over a Sepharose 2B column and examined by fluorescence spectroscopy. Fresh and freeze-dried rehydrated human platelets maintained virtually identical resting intracellular pH, 7.273 +/− 0.015, and 7.270 +/− 0.034 respectively. Both cell types responded to increased extracellular sodium by increasing their pH in a virtually identical manner. The addition of 0.5U/ml thrombin (in the presence of 135 mM NaCl) resulted in an initial acidification and subsequent alkalinzation of both fresh and freeze-dried rehydrated cells. Prior to the addition of thrombin both cell populations had an [pH]i of 6.9, while after thrombin stimulation the pH rose to 7.012 for fresh cells and 7.001 for freeze-dried rehydrated cells. Thrombin stimulation in the absence of extracellular sodium resulted in a significant acidification of both cell populations to a final pH of 6.6 for fresh cells and 6.7 for freeze-dried rehydrated cells. Specific inhibition of the NHE1 transporter by 5-(N-methyl-N-isobutyl) amiloride (MIA) completely abolished the response of all cells to increasing concentrations of sodium. In the parallel control experiment both freeze-dried and fresh cells acidified to pH 6.2 and incubated with 135mM NaCl responded by generating a rise in intracellular pH to 7.1. These results demonstrate that freeze-dried rehydrated platelets are able to maintain normal pH homeostasis and respond to agonist in a specific manner. Studies funded by DARPA.

Description: This study aimed to evaluate whether ginsenosides Rb1 (20-S-protopanaxadiol aglycon) and Rg1 (20-S-protopanaxatriol aglycon) have mitochondrial protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in primary mouse astrocytes and to explore the mechanisms involved. The OGD/R model was used to mimic the pathological process of cerebral ischemia-reperfusion in vitro. Astrocytes were treated with normal conditions, OGD/R, OGD/R plus Rb1, or OGD/R plus Rg1. Cell viability was measured to evaluate the cytotoxicity of Rb1 and Rg1. Intracellular reactive oxygen species (ROS) and catalase (CAT) were detected to evaluate oxidative stress. The mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP) were measured to evaluate mitochondrial function. The activities of the mitochondrial respiratory chain (MRC) complexes I–V and the level of cellular adenosine triphosphate (ATP) were measured to evaluate oxidative phosphorylation (OXPHOS) levels. Cell viability was significantly decreased in the OGD/R group compared to the control group. Rb1 or Rg1 administration significantly increased cell viability. Moreover, OGD/R caused a significant increase in ROS formation and, subsequently, it decreased the activity of CAT and the mtDNA copy number. At the same time, treatment with OGD/R depolarized the MMP in the astrocytes. Rb1 or Rg1 administration reduced ROS production, increased CAT activity, elevated the mtDNA content, and attenuated the MMP depolarization. In addition, Rb1 or Rg1 administration increased the activities of complexes I, II, III, and V and elevated the level of ATP, compared to those in the OGD/R groups. Rb1 and Rg1 have different chemical structures, but exert similar protective effects against astrocyte damage induced by OGD/R. The mechanism may be related to improved efficiency of mitochondrial oxidative phosphorylation and the reduction in ROS production in cultured astrocytes.