Journal List > Korean J Physiol Pharmacol > v.15(3) > 1025729

Shin, Cuong, Lee, Min, Jeon, Lim, and Ryoo: Arginase Inhibition by Ethylacetate Extract of Caesalpinia sappan Lignum Contributes to Activation of Endothelial Nitric Oxide Synthase


Caesalpinia sappan (C. sappan) is a medicinal plant used for promoting blood circulation and removing stasis. During a screening procedure on medicinal plants, the ethylacetate extract of the lignum of C. sappan (CLE) showed inhibitory activity on arginase which has recently been reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. CLE inhibited arginase II activity prepared from kidney lysate in a dose-dependent manner. In HUVECs, inhibition of arginase activity by CLE reciprocally increased NOx production through enhancement of eNOS dimer stability without any significant changes in the protein levels of eNOS and arginase II expression. Furthermore, CLE-dependent arginase inhibition resulted in increase of NO generation and decrease of superoxide production on endothelium of isolated mice aorta. These results indicate that CLE augments NO production on endothelium through inhibition of arginase activity, and may imply their usefulness for the treatment of cardiovascular diseases associated with endothelial dysfunction.


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Fig. 1.
CLE inhibits arginase activity in a dose-dependent manner. Arginase II solution was prepared from kidney lysate. Arginase activities were measured in the presence of different concentrations of CLE as described in Methods. Incubation of CLE significantly decreased arginase II activity (n=12 from 4 different experiments; 1-way ANOVA, p<0.01). DMSO (10 μM) was used as a control.
Fig. 2.
CLE-dependent arginase inhibition results in increased NOx production. HUVECs were incubated with 20 μg/ml of CLE for 18 hours. CLE significantly inhibited arginase activity (A, vs. untreated, p<0.01, n=4) and reciprocally increased NOx production in a dose-dependent manner (B, vs. untreated, p<0.05; # vs. untreated, p<0.01, n=4).
Fig. 3.
CLE enhances the formation of eNOS dimer without altering expression levels of arginase II and eNOS. Protein levels of arginase II and eNOS were analyzed after incubation with CLE (18 hours, 20 μg/ml). Arginase II and eNOS protein levels were not significantly changed by CLE treatment (A, n=3). CLE incubation (20 μg/ml, 6 hours), however, induced eNOS dimerization, as detected by low-temperature SDS-PAGE and Western blot analysis (B). The dimer to monomer ratio of eNOS was shown in the bar graph from 4 independent experiments ( vs. untreated, p<0.01, n=4). Boiled samples were used as a control.
Fig. 4.
Arginase inhibition results in increased NO production and decreased superoxide generation in isolated mice aorta. Incubation of mice aortic rings with CLE (20 μg/ml, 16 hours) resulted in a significant decrease in arginase activity (A, vs. untreated, p<0.01, n=4). (B) Pretreated aorta were loaded with DAF (5 μM) followed by measurement of fluorescence (endothelial side up). The graph shows representative traces of DAF fluorescence in CLE- and CLE plus L-NAME (10 μM)-treated aorta. (C) The slope of DAF fluorescence was monitored and then determined ( vs. untreated, p<0.01; # vs. CLE, p<0.01; n=4 mice). (D) ROS production in the aortic endothelium was traced at different time points after preloading with DHE (5 μM). (E) The slope of DHE fluorescence was determined based on cumulative data ( vs. untreated, p<0.01; # vs. CLE, p<0.01; n=4 mice).
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