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We investigated the effect of two immunosuppressant drugs, FK506 and rapamycin, on reactive oxygen species (ROS) generation, nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression and nuclear factor kappa B (NF-κB) activation in lipopolysaccharide (LPS)-activated rat hepatocytes. Primary culture of rat hepatocytes was treated with LPS in the presence and absence of FK506 or rapamycin. LPS increased the release of lactate dehydrogenase (LDH) and nitrite into the culture medium. Western blot and reverse transcription–polymerase chain reaction analyses demonstrated increased levels of iNOS protein and mRNA. Both immunosuppressant agents inhibited the induction of iNOS mRNA and protein stimulated by LPS. ROS generation, assessed by flow cytometry using dichlorodihydrofluorescein diacetate, was significantly decreased by FK506 and rapamycin. Moreover, electrophoretic mobility shift assay experiments indicated that both drugs blocked the LPS-induced activation of NF-κB. Inhibitor kappa B protein levels were decreased by LPS and this effect was partly blocked by FK506 or rapamycin. In summary, both immunosuppressant agents decreased the intracellular generation of ROS and inhibited NO production and iNOS expression at mRNA level in association to NF-κB activation. In addition to its capacity to reduce acute allograft rejection, this study highlights the anti-inflammatory properties of FK506 and rapamycin. [Copyright &y& Elsevier]

Abstract: The effects of polyoxyethylenglycerol triricinoleate 35 (Cremophor EL, CrEL) on markers of oxidative stress, nuclear factor kappa B (NF-κB) activation and inducible nitric oxide synthase (iNOS) expression were studied in the liver of male Wistar rats. Animals were randomly divided into three groups. Group Cr1 received, i.p., CrEL at 0.046ml/kg daily for 7 days, group Cr2 received CrEL at 0.33ml/kg and the controls were injected with CrEL vehicle (saline solution with 25% ethanol). Both alanine transaminase (ALT) and aspartate transaminase (AST) serum activities were significantly increased in the Cr2 group (+16% and +25%, respectively). AST activity was also higher in the Cr1 group when compared to control animals (+20%). The cytosolic concentration of thiobarbituric acid reactive substances (TBARS) increased in both groups of rats receiving CrEL (Cr1: +24%; Cr2: +33%). Reduced glutathione (GSH) concentration was not significantly modified at any of the CrEL doses, but both the hepatic concentration of oxidised glutathione (GSSG) (Cr1: +37%; Cr2: +84%) and the GSH/GSSG ratio (Cr1: −21%; Cr2: −45%) were significantly modified. CrEL induced no significant NF-κB activation, changes in p50 and p65 NF-κB subunits or induction of iNOS protein. Data obtained indicate that although high doses of CrEL cause oxidative stress, this is not enough to induce changes in NF-κB activation or iNOS expression. [Copyright &y& Elsevier]