Cells treated with Ox-LDL. The HUVEC cells were treated with different

Cells treated with Ox-LDL. The HUVEC cells were treated with different concentrations of Ox-LDL (20, 50, 100 and 150 mg/mL) for 24 h. The cell Title Loaded From File viability and apoptosis were analyzed. The HUVEC cells that were treated with vehicle (control) served as negative controls. *P,0.05 vs. control group. **P,0.01 vs. control group. Values are expressed as mean6SEM. Similar results were Ves were generated by a cycle of 95uC for 1 min, 65uC obtained in three independent experiments. doi:10.1371/journal.pone.0068746.gFunctional Analysis of Silkworm Protein 30KcFigure 5. The effects of 30Kc6 on Ox-LDL-induced HUVEC cell apoptosis (A) and cell viability (B). “Untreated” represents the HUVEC group without treatment. The 30Kc6 represents the HUVEC group treated with 30Kc6. The Ox-LDL represents the HUVEC group treated with Ox-LDL. The 30Kc6+ Ox-LDL represents the HUVEC group treated with 30Kc6 and Ox-LDL. *P,0.05 compared with the indicated groups. Values are expressed as mean6SEM. Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0068746.gThe Effects of 30Kc6 on Atherosclerotic RabbitThe effect of the silkworm protein 30Kc6 was further examined in vivo. Atherosclerotic rabbit models were set up, treated with30Kc6 protein and analyzed. The levels of serum TC, TG, HDLC and LDL-C increased in the rabbits, which were fed with high fat diets in eight weeks, indicating that the experimental animalsFigure 6. The effects of 30Kc6 on levels of 8-isoprostane in HUVEC cells incubated with Ox-LDL. The level of 8-isoprostane was analyzed in different groups. “Untreated” represents the HUVEC group without treatment. The 30Kc6 represents the HUVEC group treated with 30Kc6. The OxLDL represents the HUVEC group treated with Ox-LDL. The 30Kc6+ Ox-LDL represents the HUVEC group treated with 30Kc6 and Ox-LDL. *P,0.05 compared with the indicated groups. Values are expressed as mean6SEM. Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0068746.gFunctional Analysis of Silkworm Protein 30KcFigure 7. The effects of 30Kc6 on p38 (A) and JNK MAP kinase (B) activity after exposure to Ox-LDL. The HUVEC cells were treated and analyzed by Western blot. “Untreated” represents the HUVEC group without treatment. The 30Kc6 represents the HUVEC group treated with 30Kc6. The Ox-LDL represents the HUVEC group treated with Ox-LDL. The 30Kc6+ Ox-LDL represents the HUVEC group treated with 30Kc6 and Ox-LDL. The right panels show the results of densitometric analyses of immunoblotting (mean6SEM, n = 3). **P,0.01 compared with the indicated groups. doi:10.1371/journal.pone.0068746.gwere in hyperlipidemia state. The aortas and livers in the randomly sacrificed rabbits showed some pathological changes. After the following four weeks of different diet feeding, rabbit serum biochemical indicators of six diet groups with different treatments were assessed (Table 1). The levels of TC, TG, HDL-C and LDL-C were decreased in the groups treated with low and high dose of 30Kc6 as well as the positive controls. Specifically, thelevels of TC, TG and LDL-C were significant different between the groups treated with low dose of 30Kc6 and Bacmid (p,0.05). Moreover, the levels of TC, TG and LDL-C (p,0.01) and HDL-C (p,0.05) were significantly decreased in the group treated with high dose of 30Kc6 as compared to the group treated with the Bacmid. This observation indicated that 30Kc6 had protective effects on decreasing the blood fat levels. The blood 30KcTable 1. Serum biochemical i.Cells treated with Ox-LDL. The HUVEC cells were treated with different concentrations of Ox-LDL (20, 50, 100 and 150 mg/mL) for 24 h. The cell viability and apoptosis were analyzed. The HUVEC cells that were treated with vehicle (control) served as negative controls. *P,0.05 vs. control group. **P,0.01 vs. control group. Values are expressed as mean6SEM. Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0068746.gFunctional Analysis of Silkworm Protein 30KcFigure 5. The effects of 30Kc6 on Ox-LDL-induced HUVEC cell apoptosis (A) and cell viability (B). “Untreated” represents the HUVEC group without treatment. The 30Kc6 represents the HUVEC group treated with 30Kc6. The Ox-LDL represents the HUVEC group treated with Ox-LDL. The 30Kc6+ Ox-LDL represents the HUVEC group treated with 30Kc6 and Ox-LDL. *P,0.05 compared with the indicated groups. Values are expressed as mean6SEM. Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0068746.gThe Effects of 30Kc6 on Atherosclerotic RabbitThe effect of the silkworm protein 30Kc6 was further examined in vivo. Atherosclerotic rabbit models were set up, treated with30Kc6 protein and analyzed. The levels of serum TC, TG, HDLC and LDL-C increased in the rabbits, which were fed with high fat diets in eight weeks, indicating that the experimental animalsFigure 6. The effects of 30Kc6 on levels of 8-isoprostane in HUVEC cells incubated with Ox-LDL. The level of 8-isoprostane was analyzed in different groups. “Untreated” represents the HUVEC group without treatment. The 30Kc6 represents the HUVEC group treated with 30Kc6. The OxLDL represents the HUVEC group treated with Ox-LDL. The 30Kc6+ Ox-LDL represents the HUVEC group treated with 30Kc6 and Ox-LDL. *P,0.05 compared with the indicated groups. Values are expressed as mean6SEM. Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0068746.gFunctional Analysis of Silkworm Protein 30KcFigure 7. The effects of 30Kc6 on p38 (A) and JNK MAP kinase (B) activity after exposure to Ox-LDL. The HUVEC cells were treated and analyzed by Western blot. “Untreated” represents the HUVEC group without treatment. The 30Kc6 represents the HUVEC group treated with 30Kc6. The Ox-LDL represents the HUVEC group treated with Ox-LDL. The 30Kc6+ Ox-LDL represents the HUVEC group treated with 30Kc6 and Ox-LDL. The right panels show the results of densitometric analyses of immunoblotting (mean6SEM, n = 3). **P,0.01 compared with the indicated groups. doi:10.1371/journal.pone.0068746.gwere in hyperlipidemia state. The aortas and livers in the randomly sacrificed rabbits showed some pathological changes. After the following four weeks of different diet feeding, rabbit serum biochemical indicators of six diet groups with different treatments were assessed (Table 1). The levels of TC, TG, HDL-C and LDL-C were decreased in the groups treated with low and high dose of 30Kc6 as well as the positive controls. Specifically, thelevels of TC, TG and LDL-C were significant different between the groups treated with low dose of 30Kc6 and Bacmid (p,0.05). Moreover, the levels of TC, TG and LDL-C (p,0.01) and HDL-C (p,0.05) were significantly decreased in the group treated with high dose of 30Kc6 as compared to the group treated with the Bacmid. This observation indicated that 30Kc6 had protective effects on decreasing the blood fat levels. The blood 30KcTable 1. Serum biochemical i.