For statistical analy sis, the number of cells with morphological features of apop totic cell death was determined by scoring selleck chem Romidepsin 100 cells from 20 different microscopic fields. The cell pellet was www.selleckchem.com/iap.html resuspended in 400 l hypotonic lysis buffer containing pro tease inhibitors and was incubated on ice for 15 minutes. The extracts Carfilzomib Phase 2 were cen trifuged for 1. After 2 hours of incubation, the beads were washed with lysis buffer and resuspended in a kinase assay solution containing 50 mM HEPES, 20 mM MgCl2, 2 mM dithiothreitol, 10 M unlabelled ATP and 2 mg substrate GST IB, and were incubated at 30 C for 30 minutes.
This was followed by boiling in SDS PAGE sample buffer for 5 minutes. The proteins were transferred to a nitrocellulose membrane after SDS PAGE under reducing conditions as described above. Phosphorylation of GST IBwas assessed using a specific antibody against phospho specific IB. To dem onstrate the total amounts of IKK and IKK in each sample, whole cell lysates were transferred to a nitrocellulose mem brane after SDS PAGE under reducing conditions as described above. Detection of IKK and IKK was performed by immunoblotting with either anti IKK or anti IKK antibod ies. Statistical analysis The results are expressed as the means standard deviation of a representative experiment performed in triplicate. The means were compared using Students t test assuming equal variances. P 0. 05 was considered statistically significant. Results Effects of resveratrol and curcumin on human chondrocyte viability and proliferation In previous studies we have demonstrated that IL 1 induced NFB activation is cytotoxic to human chondrocytes. In the present study we evaluated the effects of resveratrol and curcumin on this IL 1 induced cytotoxicity. Proliferation and viability assays performed with the MTT test demonstrated that both resveratrol and curcumin significantly decreased the cytotoxic effects induced by IL 1. As these data indicated that both phytochemicals have positive and similar properties on human chondrocytes, we investigated the effects of combining resveratrol and curcumin on chondrocyte viability and proliferation. The results showed a positive effect of combining both phytochemicals with regard to cell viability and proliferation on inhibiting the IL 1 induced cytotoxicity on human chondrocytes. Resveratrol and curcumin inhibit IL 1 induced mitochondrial changes and apoptosis in chondrocytes Work from our group previously demonstrated that phyto chemical agents such as resveratrol and curcumin suppress IL 1 induced apoptosis in human chondrocytes through inhi bition of NFB mediated signalling pathways. The objective of the present study was to determine whether cur cumin and resveratrol can act synergistically to modulate the cytotoxic effects of IL 1 in human chondrocytes. Primary human chondrocytes were exposed to the indicated concen trations of resveratrol andor curcumin alone or with IL 1 as described in Materials and methods, and the effect of resvera trol andor curcumin on IL 1 induced apoptosis was exam ined at the ultrastructural level using transmission electron microscopy. Untreated primary human chondrocytes exhibited a typical rounded or flattened shape with small cytoplasmic processes, a large mostly euchromatic nucleus with nucleoli and a well structured cytoplasm. Treatment of chondrocytes with 10 ngml IL 1 for 1, 12, 24 and 48 hours led to degenerative morphological changes such as multiple vacuoles, swelling of rough endoplasmic reticulum, clustering of swollen mitochondria and degeneration of other cell organelles.