Transports lipids and inhibits cell Title Loaded From File apoptosis in the insect and mammalian cells [1?]. However, effects of 30Kc6 on cell apoptosis of human vascular endothelial cell (HUVEC) and the underlying mechanism are largely unknown. Atherosclerosis (AS) is a vascular system disease with characteristics of non-inflammatory state, retrogression and hyperplastic pathologies. It often occurs in carotid arteries, aortas and peripheral arteries and seriously threatens human health [6?]. Vascular endothelial cell (VEC) is a blood-brain barrier and a common target of Ox-LDL, angiotensin II (Ang II), high glucose and other risk factors [6]. Furthermore, VEC apoptosis plays a critical role in the pathogenesis of AS. It has been confirmed thatapoptosis of VEC was an important initiating step for AS and was further involved in the whole process. Moreover, the VEC apoptosis played a key role in induction of atherosclerotic lesion formation and plaque shedding. Therefore, Title Loaded From File prevention of the oxidative stress-induced HUVEC damage might be one of the methods in the prevention and treatment of AS [7?]. Mitogen activated protein kinases (MAPK), serine/threonine kinases in most cells, are important molecules that accept and transmit the receptor-mediated extracellular signaling into cytoplasm and nucleus in order to participate in the gene expression and regulation as well as cell proliferation and cell death especially in eukaryotes. Extracellular receptor-activated kinases (ERK), cJun N-terminal kinases (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) are three major signaling kinases that are involved in cell apoptosis [10]. Specifically, JNK and p38 are oxidative stress-induced MAPK and are activated by intracellular oxidative stress that leads to cell apoptosis [11?2].Functional Analysis of Silkworm Protein 30KcTherefore, in this study, the silkworm protein 30Kc6 was expressed and purified using the Bac-to-Bac Baculovirus expression system. The effects of 30Kc6 on Ox-LDL-induced VEC apoptosis and apoptotic signaling pathways were then investigated in HUVEC cells. In addition, the protective effects of the silkworm oral feeding with pupa meal containing the 30Kc6 protein were further analyzed in atherosclerotic rabbit animal models.Expression and Purification of the Silkworm Protein 30KcThe BmN cells in logarithmic growth phase were infected with recombinant virus Bacmid-30Kc6 with a multiplicity of infection (MOI) of 10. The infected BmN cells with obvious infection symptoms were harvested in 72 hours (h) and were centrifuged at 1000 rpm for 10 min. The harvested cells were washed with phosphate buffered saline (PBS) and were centrifuged at 1,000 rpm for 10 min. The cells were suspended in 200 mL PBS and were lysed by ultrasound on ice. Cell lysates were centrifuged for 20 min with a speed of 12,000 rpm and the supernatants were harvested. The 16 native binding buffer (pH 8.0) was used to balance nickel column and the cell lysates were loaded into the nickel column and were incubated overnight on ice. The native washing buffer (pH 8.0) with a concentration gradient of imidazole was employed to wash the columns in batches. Finally, 23977191 the silkworm protein 30Kc6 was purified by eluting the binding proteins with the native elution buffer (pH 8.0).Materials and Methods MaterialsThe cultured silkworm BmN cells, the recombinant prokaryotic expression vector pET-28a-30Kc6, plasmid pFastBac-HTB, virus vector Bacmid, Escherichia coli BL21 (DE3) and DH10Bac stains w.Transports lipids and inhibits cell apoptosis in the insect and mammalian cells [1?]. However, effects of 30Kc6 on cell apoptosis of human vascular endothelial cell (HUVEC) and the underlying mechanism are largely unknown. Atherosclerosis (AS) is a vascular system disease with characteristics of non-inflammatory state, retrogression and hyperplastic pathologies. It often occurs in carotid arteries, aortas and peripheral arteries and seriously threatens human health [6?]. Vascular endothelial cell (VEC) is a blood-brain barrier and a common target of Ox-LDL, angiotensin II (Ang II), high glucose and other risk factors [6]. Furthermore, VEC apoptosis plays a critical role in the pathogenesis of AS. It has been confirmed thatapoptosis of VEC was an important initiating step for AS and was further involved in the whole process. Moreover, the VEC apoptosis played a key role in induction of atherosclerotic lesion formation and plaque shedding. Therefore, prevention of the oxidative stress-induced HUVEC damage might be one of the methods in the prevention and treatment of AS [7?]. Mitogen activated protein kinases (MAPK), serine/threonine kinases in most cells, are important molecules that accept and transmit the receptor-mediated extracellular signaling into cytoplasm and nucleus in order to participate in the gene expression and regulation as well as cell proliferation and cell death especially in eukaryotes. Extracellular receptor-activated kinases (ERK), cJun N-terminal kinases (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) are three major signaling kinases that are involved in cell apoptosis [10]. Specifically, JNK and p38 are oxidative stress-induced MAPK and are activated by intracellular oxidative stress that leads to cell apoptosis [11?2].Functional Analysis of Silkworm Protein 30KcTherefore, in this study, the silkworm protein 30Kc6 was expressed and purified using the Bac-to-Bac Baculovirus expression system. The effects of 30Kc6 on Ox-LDL-induced VEC apoptosis and apoptotic signaling pathways were then investigated in HUVEC cells. In addition, the protective effects of the silkworm oral feeding with pupa meal containing the 30Kc6 protein were further analyzed in atherosclerotic rabbit animal models.Expression and Purification of the Silkworm Protein 30KcThe BmN cells in logarithmic growth phase were infected with recombinant virus Bacmid-30Kc6 with a multiplicity of infection (MOI) of 10. The infected BmN cells with obvious infection symptoms were harvested in 72 hours (h) and were centrifuged at 1000 rpm for 10 min. The harvested cells were washed with phosphate buffered saline (PBS) and were centrifuged at 1,000 rpm for 10 min. The cells were suspended in 200 mL PBS and were lysed by ultrasound on ice. Cell lysates were centrifuged for 20 min with a speed of 12,000 rpm and the supernatants were harvested. The 16 native binding buffer (pH 8.0) was used to balance nickel column and the cell lysates were loaded into the nickel column and were incubated overnight on ice. The native washing buffer (pH 8.0) with a concentration gradient of imidazole was employed to wash the columns in batches. Finally, 23977191 the silkworm protein 30Kc6 was purified by eluting the binding proteins with the native elution buffer (pH 8.0).Materials and Methods MaterialsThe cultured silkworm BmN cells, the recombinant prokaryotic expression vector pET-28a-30Kc6, plasmid pFastBac-HTB, virus vector Bacmid, Escherichia coli BL21 (DE3) and DH10Bac stains w.
