The Mechanism Of Anti-appptosis And Protective Effect Of Silkworm30Kc6Protein On Human Vascular Endothelial Cells

30K apolipoproteins are a major group of low-density lipoprotein in larvae hemolymph of Bombyx mori. Recent evidence suggests that30Kc6as one of the members of30K apolipoproteins, besides the biological function of carrying lipoids, they also have previously anti-apoptotic activities in insect cells and HeLa cells, however,30Kc6on vascular endothelial cells(Human vascular endothelial cell, HUVEC) apoptosis effect and its underlying molecular mechanism is not reported recently. Cerebrovascular diseases, with atherosclerosis (AS) being the main patho-change, do harm to human being badly. The apoptosis of endothelial cells plays an important role in the development of atherosclerosis. Endothelial cells, as a barrier of vascular system, is the target of Oxidized low density lipoprotein(Ox-LDL). It was investigated that Ox-LDL can lead to the oxidative stress damage of endothelial cells, this is also regarded as one of the early effects of atherosclerosis. Therefore, intervene the damage of oxidative stress has to be considered one of the main means to prevent atherosclerosis. Because30K proteins have the biological activities of carrying lipoid and anti-apoptosis, we propose that they could not only decrease the blood fat, but also protect vascular endothelial cells. For this reason,30Kc6is a potential drug in treating and preventing atherogenesis.In this study, we used Bac-to-Bac baculovirus expression system was used to express the target30Kc6in BmN cells and the expression products was purified. Firstly, based on the self-constructed apoptosis and injured model of BmN induced by hydrogen peroxide (H2O2) in vitro, the protective effect of30Kc6on BmN cells apoptosis was analyzed. The result displayed that5μg/mL30Kc6protein could remarkably slow down DNA fragmentation and decreased the concentration of8-isoprostane (a marker of oxidative stress) in BmN cells. Moreover,30Kc6could protected BmN cells though inhibiting the H2O2-induced cytochrome c released from mitochondria to cytoplasm inside BmN cells. Then based on the self-constructed apoptosis and injured model of HUVEC induced by Ox-LDL and the animal model of atherosclerosis, we studied the effects of30Kc6on Ox-LDL-induced HUVEC apoptosis and further explore its underlying molecular mechanism of anti-apoptosis and medicinal qualities. It also showed that1.25μg/mL30Kc6had obviously protection to oxidative-stress induced HUVEC apoptosis. In addition, the formation of8-isoprostane showed that30Kc6proteins restrained8-isoprostane formation induced by Ox-LDL. It showed that30Kc6inhibited Ox-LDL-induced HUVEC apoptosis by decreasing the intracellular ROS produce. Western blotting analysis showed that Ox-LDL could actived the MAP Kinase signaling pathway. Otherwise,30Kc6could inhibited Ox-LDL-induced HUVEC apoptosis by decreasing the the activity of p38and JNK. The animal experimental results displayed that30Kc6take the positive effect on atherosclerosis of New Zealand rabbits.Our previous research results have shown that30Kc6can protect vascular endothelial cells by inhibiting cell apoptosis induced by Ox-LDL, and it plays an important role in regulating JNK and p38MAP Kinase pathway. And the animal experimental results showed that30Kc6take the positive effect on atherosclerosis of New Zealand rabbits. This study will provide a basis to further explore the medicinal values of30Kc6, and it may be a new type biocompatible drug which can prevent and cure atherosclerosis in the future.