NADPH Oxidase On Peritoneal Macrophage Foam Cell Formation Of ApoE Deficient Mice

Atherosclerosis(AS) is a progressive disease, which is a complicated and chronic process. The earlier period pathogenetic research of AS plays a significant role in both the early preservation and treatment of it.After the partly injury of endarterium, plasmic lipids infiltrate endarterium and deposite in the intimal layer of the artery; Low density lipoproteins (LDL) are oxidized or acetylated, and the modified LDL are infinitely uptaken by the macrophages and then transformed into foam cells. With the accmulation of lipids in macrophages and the number of macrophage foam cells increasing step by step, the states of lipid metabolism and inflammation change in focal cells, and the fatty streak of artery wall, which is the early affection, emerges gradually via a series of complicated interaction among cells and molecules.The formation of macrophage foam cell is one of the main events in the early atherosclerotic stages, in which reactive oxygen species (ROS) play a very important part. NADPH oxidase is a significant resource in macrophage. And the role of NADPH oxidase in the development of AS is still arguable. Some researches indicate: atherosclerotic lesions in p47phox-/-/apoE-/- mice is evidently less than that in apoE-/- mice. Moreover, in contrast of WT mice, the level of O2- in artery from p47phox-/- mice degrades significantly. It shows that O2- derived from monocyte/macrophage NADPH oxidase contributes to the development of AS to some extent. However,other studies display that the leions between gp91phox-/-/apoE-/- mice and apoE-/- mice have no obvious difference, and that atherosclerotic lesions and blood pressure between p47phox-/-/apoE-/- mice and apoE-/- mice display no noticeable difference. These show that the knockout of gp91phox or p47phox gene does not affect the development of AS in apoE-/- mice.Studies in vitro discovered that: ox-LDL can activate Nox2 in macrophage via signaling pathway mediated by Toll-like receptor 4, then ROS is generated and it may promote the occurrance and development of AS. ROS generated from the process of macrophage foam cell formation can regulate the activation of matrix metalloproteinase in vitro. Furthermore, it could influence the expression of scavenger receptors in THP-1 cell line. Above all, oxidative stress can affect the formation of macrophage foam cell via regulating major transcription factor as well as other genes about cholesterol efflux and cholesterol influx, and finally may contribute to the occurance and development of AS. However, the role of ROS derived form NADPH oxidase in the formation of macrophage foam cell remains unkown.Accordingly, in this trial, diphenyleneiodonium chloride (DPI), which is the inhibitor of NADPH oxidase, is employed to interfere in the apoE-/- and WT peritoneal macrophages in vitro. Some indexes are detected at the cellular and molecular level in different groups and times during the formation of macrophage foam cell via the method of pathomorphology, real-time RT-PCR and immunocytochemistry and so on, to experiment on the role of NADPH oxidase in the expression of genes related to the formation of apoE-/- murine peritoneal macrophage foam cell, and in order to approach deeply the possible mechanisms of not only the formation of macrophage foam cell but also the development of early atherosclerotic lesions.The results show:1. The result of pathomorphology: the formation of apoE-/- and WT peritoneal macrophage foam cell can be induced by ox-LDL in 24h. Moreover, it aggravates as the time goes on. In apoE-/- mice, the activation of NADPH oxidase exerts little influence on peritoneal macrophage foam cell formation in 48 h. However, it can obviously accelerate or promote the peritoneal macrophage foam cell formation.2. The level of ROS: the activation of NADPH oxidase can be obviously inhibited by DPI.And DPI could evidently decrease the level of O2- and the total ROS induced by ox-LDL in apoE-/- and WT peritoneal macrophages (p < 0.05). The level of O2- and the total ROS have no significant difference between apoE-/- and WT peritoneal macrophages (p > 0.05).3. The effects of NADPH oxidase on the expression of the genes related to macrophage foam cell formation in apoE-/- and WT mice:In WT peritoneal macrophage, in contrast of the group treated with ox-LDL, strikingly, DPI can down-regulate the mRNA levels of Nox2, PPARα, LXRα, CD36 and SR-A (p < 0.05), up-regulate the mRNA levels of PPARγ, NF-κB and SR-BI (p < 0.05), and makes no differences in the mRNA levels of ABCA1 and p47phox in 6h (p > 0.05). However, DPI can notably up-regulate the mRNA levels of p47phox, PPARα, PPARγ, LXRα, NF-κB, CD36, SR-A, ABCA1 and SR-BI (p < 0.05), but, the mRNA levels of Nox2 in 24 h has no changes. (p > 0.05).In apoE-/- peritoneal macrophage, in contrast of the group treated with ox-LDL, obviously, DPI can up-regulate the mRNA levels of Nox2, p47phox, PPARα, PPARγ, LXRα, NF-κB, CD36, SR-A, ABCA1 and SR-BI in 6h (p < 0.05); up-regulate the mRNA levels of PPARα, ABCA1 and SR-BI and down-regulate the mRNA levels of PPARγand Nox2 in 24 h (p < 0.05). However, the mRNA levels of p47phox, LXRα, NF-κB, CD36 and SR-A have no changes (p > 0.05).In apoE-/- and WT peritoneal macrophage, ox-LDL can activate NF-κB via ROS, and its activation can be inhibited by DPI at 24 h (p < 0.05). Morever, the protein levels of ABCA1 and PPARγhave no changes at 24 h (p > 0.05).On the whole, NADPH oxidase can accelerate or promote the process of peritoneal macrophage foam cell formation to some extent, and then influence the development of AS. In the process, O2- and other ROS participate in and regulate the expression of the genes related to macrophage foam cell formation, and also affect the functions of macrophage via adjusting NF-κB, PPARs and so on.