Silibinin pretreatment protects against Ochratoxin A-mediated apoptosis in primary rat hepatocytes |
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Authors: | E Essid E Petzinger |
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Institution: | (1) Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Street 107, 35392 Giessen, Germany; |
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Abstract: | The inhibitory effect of silibinin on ochratoxin A (OTA)-mediated apoptosis on primary rat hepatocytes was investigated. Rat
hepatocytes were prepared by two different methods: the classical enzymatic digestion method by collagenase perfusion and
a new EDTA-perfusion method. The EDTA-perfusion method yielded hepatocytes, which were stably cultivated without DNA fragmentation
for up to 96 h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation
even in the absence of OTA. Treatment with 12.5 μmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed
DNA-laddering after 24–36 h. Lipopolysaccharide (LPS) of 0.1 up to 12.5 μg/ml showed no apoptotic DNA-effects under these
conditions. A low concentration of 26 μmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas
a five times higher concentration of silibinin (130 μmol/l) completely inhibited OTA-mediated apoptosis. Under the same conditions,
caspase-3 activity in hepatocytes increased in a time-dependent manner under OTA exposure within 12–24 h but was blocked by
130 μmol/l silibinin. In contrast, LPS incubation for 12 and 24 h did not alter caspase-3 activity. To measure viability of
OTA-/LPS-treated hepatocytes, the MTT-test and Live/Dead kit were applied. The results demonstrated that the used OTA concentration
of 12.5 μmol/l only moderately decreased viability for up to 24 h but showed cytotoxic effects depending on longer incubation
times (≥36 h). In contrast, LPS up to 12.5 μg/ml exhibited no cytotoxic effects up to 48 h. In summary, our results showed
contrasting effects on apoptosis in primary rat hepatocytes by OTA (produces apoptosis) versus LPS (produces no apoptosis),
also depending on the method of hepatocyte preparation. Silibinin at 130 μmol/l showed significant hepatoprotective and antiapoptotic
effects against OTA-mediated cell damage on cultured rat hepatocytes. |
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