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Holothuria leucospilota Extract Induces Apoptosis in Leishmania major Promastigotes.
Foroutan-Rad M
,
Khademvatan S
,
Saki J
,
Hashemitabar M
.
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BACKGROUND: The present study aimed to survey antileishmanial activity of methanolic Holothuria leucospilota extract against Leishmania major promastigotes in vitro.
METHODS: Promastigotes were cultured in RPMI 1640 and after reaching the stationary phase, the study was conducted with different concentrations of the extract. Afterwards, MTT colorimetric assay for the obtaining of 50% inhibitory concentration (IC50) was utilized. Furthermore, in order to determine the possible induction of apoptosis in L. major promastigotes, flow cytometry and DNA fragmentation methods were employed using annexin-V FLUOS staining kit and DNA ladder kit, respectively.
RESULTS: The IC50 value of H. leucospilota extract at three time points of 24, 48, and 72 h was estimated 2000, 300 and 85 μg/ml, respectively. In addition, the extract revealed a dose and time-dependent antileishmanial activity. Furthermore, various characteristics of apoptosis appeared after L. major promastigotes treatment, which included cell shrinkage, formation of apoptotic bodies, blebbing of the cell membrane, and externalization of phosphatidylserine, although no laddering pattern was observed.
CONCLUSION: The methanolic extract of H. leucospilota possesses lethal effect on L. major promastigotes and induces the apoptosis in parasites. Further studies are required to address the apoptosis mechanism in vivo.
Fig. 2:. The viability of L. major promastigotes in the presence of various concentrations of methanolic exctract of H. leucospilota which was assessed by MTT
Fig. 3:. Analysis of morphology in light microscopy (magnification, x 100) of L .major promastigotes following treatment with H. leucospilota extracts A. L. major 0 hours after treatment and B. L. major 72 hours after treatment
Fig. 4:. Flow cytometry analysis: Apoptosis occurrence in L. major promastigotes after treatment with H. leucospilota extract (2000 μg/ml) for 72 h
Fig. 5:. DNA ladder assay: Lane M. DNA size marker; Lane 1. Unexposed L. major promastigotes (DNA fragmentation did not occur); Lane 2. Positive apoptotic cell (positive control); Lane 3. Exposed L. major promastigotes (DNA fragmentation did not occur)
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