Studies On The Mechanisms Of Oridonin-and TNFα-induced L929 Cell Death

This dissertation reports the mechanisms of L929 cell death induced by tumor necrosis factor a (TNFα ) and oridonin, respectively. Also, we investigated the mechanisms of resistance to oridonin in L929 cells.First, recombinant human TNFα was purified, and then in order to screen the hypersensitive cell line to TNFα , sub-cloning of L929 cells was carried out. The studies demonstrated that TNFα apparently inhibited the proliferation of L929 cells in vitro. Photomicroscopic observation and Hoechst 33258 staining showed typical apoptotic changes in TNFα -treated cells. Lactate dehydrogenase (LDH)-based assay and DNA fragmentation analysis further confirmed that TNFα induced L929 cell death by means of apoptosis. Usually, caspase is a mediator of apoptosis triggered by extracellular stimuli. However, in our study, caspase did not mediate apoptosis, but protected L929 cells from TNFα -induced cell death. Pan-caspase inhibitor, caspae-8 inhibitor and caspse-3 inhibitor made the L929 cells more sensitive to TNFα , however, caspase-9 inhibitor had no effect on this sensitivity. In mitochondrial signal pathway, TNFα did not change the expressions of Bcl-2 and Bax, indicating that mitochondrial signal was not involved in this process. During the course of TNFα -induced cell death, the DNA repair enzyme, poly (ADP-ribose) polymerase (PARP) was not degraded. In addition, TNFα induced p38 and ERK activation, and the activated p38 and ERK mediated apoptosis.Basted on the above mentioned results, we further investigated the mechanism of pan-caspase inhibitor (z-VAD-fmk)'s augmentation of TNFα -induced L929 cell death. In the presence of TNFα , z-VAD-fmk augmented the cell death in a dose-dependent manner, and it promoted apoptosis and necrosis as measured by agarose gel electrophoresis and LDH activity-based assay. Mitochondrial permeability transition (MPT) inhibitor cyclosporine A effectively inhibited z-VAD-fmk-augmented cell death. In addition, z-VAD-fmk plus TNFα increased Bax expression without affecting Bcl-2 and cytochrome c expression. Western-blot analysis showed that z-VAD-fmk...