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 96h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation even in the absence of OTA. Treatment with 12.5μmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed DNA-laddering after 24-36h. Lipopolysaccharide (LPS) of 0.1 up to 12.5μg/ml showed no apoptotic DNA-effects under these conditions. A low concentration of 26μmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas a five times higher concentration of silibinin (130μmol/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 12-24h but was blocked by 130μmol/l silibinin. In contrast, LPS incubation for 12 and 24h 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 12.5μmol/l only moderately decreased viability for up to 24h but showed cytotoxic effects depending on longer incubation times (≥36h). In contrast, LPS up to 12.5μg/ml exhibited no cytotoxic effects up to 48h. 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 130μmol/l showed significant hepatoprotective and antiapoptotic effects against OTA-mediated cell damage on cultured rat hepatocytes.
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