Abstract：It has been clear that silkworm (Bombyx mori) 30K protein in larval hemolymph has the highest apoptosis inhibition activities. As one of the member of 30K apolipoproteins family, Bm30Kc6 has been reported to have obvious anti-apoptotic activity, but the anti-apoptotic mechanism was still not very clear. In this study, the silkworm BmN cells were infected with recombinant virus Bacmid-Bm30Kc6 constructed by our lab and the expressed proteins were purified by a Ni-NTA agarose affinity filler. SDS-PAGE and Western blot results showed that the Bm30Kc6 proteins were highly purified. Then BmN cells were treated with different concentrations of hydrogen peroxide (0.1, 1, 5 and 10 mmol/L) to induce cell apoptosis, the results showed that a final concentration of 5 mmol/L H2O2 incubated with BmN cells for 4 h could be successfully induced cell apoptosis. Based on this cell damage model, the anti-apoptotic mechanism of Bm30Kc6 was studied. Firstly, cell death detection ELISA kit was used to evaluate the cell apoptosis, the results showed that 5 μg/mL Bm30Kc6 could remarkably slow down DNA fragmentation in BmN cells. Then cell proliferation ELISA kit was used to test the cell viability, the results showed that Bm30Kc6 could also enhance the viability of BmN cells obviously. Moreover, 8-isoprostane EIA kit was used to test the concentration of 8-isoprostane inside BmN cells, the results showed that H2O2 treatment could enhance the formation of 8-isoprostane obviously, however, Bm30Kc6 protein could decrease the concentration of 8-isoprostane obviously. Finally, Western blot results showed that Bm30Kc6 could inhibited the H2O2-induced cytochrome c released from mitochondria to cytoplasm inside BmN cells. In conclusion, Bm30Kc6 may inhibit BmN cell apoptosis by decreasing the level of oxidative stress inside cells and inhibit cell apoptosis by blocking the mitochondrial signaling pathway. This study will provide basic information for further study on the anti-apototic mechanism of Bm30Kc6 portein.
