Inhibition of Suicidal Erythrocyte Death by Indirubin-3'-Monoxime.

BACKGROUND/AIMS: Qing Dai is a prized traditional Chinese medicine whose major component, indirubin, and its derivative, indirubin-3'-monoxime (IDM), have inhibitory effects on the growth of many human tumor cells and pronounced anti-leukemic activities. However, the effects of IDM on mature human erythrocytes are unclear. This study aimed to evaluate the potential impact of IDM on erythrocytes and the mechanisms underlying that impact. METHODS: Utilizing flow cytometry and confocal laser scanning microscopy, phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate (FITC). The relative cell size, expressed in arbitrary units, was evaluated by forward scatter in a flow cytometer. Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca2+ activity, reactive oxygen species (ROS) formation was assessed by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence, and ceramide abundance was evaluated by FITC-conjugated specific antibodies. RESULTS: The 24-h exposure of human erythrocytes to IDM (12 µM) significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration ([Ca2+]i). IDM (3-12 µM) did not significantly modify the ceramide level or DCFH-DA fluorescence. Energy depletion (removal of glucose for 24 hours) significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter, and these effects were significantly mitigated by IDM (12 µM). Moreover, the Ca2+ ionophore ionomycin (1 µM, 60 min) and oxidative stress (30 min exposure to 0.05 mM tert-butyl hydroperoxide, t-BHP) similarly triggered eryptosis, which was also significantly suppressed by IDM. CONCLUSIONS: IDM is a novel inhibitor of suicidal erythrocyte death following ionomycin treatment, t-BHP treatment and energy depletion. Thus, IDM may counteract anemia and impairment of microcirculation, at least in part, by inhibition of Ca2+ entry into erythrocytes.

[C, N, P stoichiometric characteristics of tree, shrub, herb leaves and litter in forest community of Shaanxi Province, China].

A total of 121 sampling sites were selected to study the C, N, P stoichiometric characteristics among the leaves of tree, shrub, herb and the litter of the majority forest community of Shaanxi Province, China. We also studied their relationships with geographical factors. The results showed that C, N concentrations were highest in tree leaf, P concentration was highest in herb leaf, and the C, N, P concentrations were lowest in litter. Leaf C: N: P ratios of tree, shrub, herb leaves and litter were 439.4:14.2:1, 599.2:13.5:1, 416.5:13.3:1, 504.8:15.5:1, respectively. The N:P ratios of tree, shrub and herb leaves were not significantly different, but they were all significantly higher than that of litter layer, indicating N:P ratio of different living life forms' leaves was stable. Compared with N, C and P had better relationships in each layer, and the relationships of leaf C, N, P stoichiometry between tree and herb leaves were better than those between tree and shrub leaves. The correlations of C, N, P stoichiometry between litter and the tree or herb leaves were highly significantly positive, and the correlations of P stoichiometry between litter and shrub leaves were highly significantly positive. Shrub leaf N, P concentration increased with the increasing latitude, and herb leaf N, P concentrations decreased with the increasing longitude. Compared with the longitude and latitude, the effect of altitude was smaller, and only tree leaf N:P decreased with the increasing latitude. Multivariate linear regression analysis results showed that the influence of geographical factors on herb leaf was longitude > latitude > altitude, while was latitude > longitude > altitude for tree, shrub leaves and litter.