Effects of varied EGCG and (+)-catechin concentrations on proinflarnmatory cytokines mRNA expression in ConA-stimulated primary white blood cell cultures

摘要

EGCG [(-)-epigallocatechin gallate] and (+)-catechin hydrate are flavanoids, which are known as anticancer and healthy drugs. To test the immune modulatory effects of EGCG and catechin, various concentrations were tested on primary white blood cells (WBC) in cell cultures stimulated with the T-cell mitogen concanavalin A (ConA). WBC from dairy cows (1 x 10(6) cells/mL) were cultivated using RPMI medium with FCS and gentamycin. First, WBC were stimulated with ConA, and 6 h later the flavanoid treatment was started. Cultivated WBC were treated with various physiological flavanol concentrations (0-100 mu M) in cross-combination with various ConA concentrations (0-1 mu g/mL). After 24 h, cells were harvested, cell viability was verified, and total RNA was isolated. Relative mRNA expression levels of proinflammatory cytokines TNF alpha, IL1 beta, IL6, and transcription factor cFos and of nucleosome component histon H3 were quantified with real-time qRT-PCR. High EGCG and catechin concentrations had inhibitory effects on total RNA expression. Low EGCG concentration can induce total RNA expression in WBC. EGCG reduced cFos mRNA expression, which can be abolished by high ConA concentrations in a reverse dose-dependent manner. TNF alpha showed a flavanoid-specific expression pattern. EGCG acts in blood physiological concentrations (micromolar range), and catechin acts in higher gut-relevant concentrations (millimolar range) and has the potential to influence the proinflammatory TNF alpha. expression. Higher flavanoid concentration had more pronounced effects than lower, whereas EGCG showed a more potent suppression of gene expression than catechin (toward TNF alpha). EGCG and catechin had no significant effects in primary WBC on the expression pattern of the proinflammatory cytokines lL1 beta and IL6 and on the expression of the housekeeping genes GAPDH and histon H3. It is presumed that both flavanoids have the potential to regulate total RNA expression and gene-specific expression in WBC.