Nogo-B Upregulated By OxLDL In Macrophage

Backgrounds:Atherosclerosis (Atherosclerosis, AS) is a one of the most serious disease in human health, which is the pathological basis of cardiovascular and cerebrovascular diseases. Studies have shown that AS is a chronic inflammatory response. There is a large number of macrophages derived foam cells in AS plaque. Large amounts of data suggest that foam cells in plaque are associated with inflammation, endoplasmic reticulum stress, apoptosis and autophagy. Promoting cholesterol efflux from foam cells and suppressing the inflammatory response may be possible avenues for the treatment of AS.Nogo-B, one of the the three Nogo subtypes, belongs to Reticulon protein family. RTN is a family of proteins located in the endoplasmic reticulum, which involving the formation of tubular endoplasmic reticulum and maintaining membrane curvature. Nogo-B is widely distributed in the body. Studies have found that Nogo-B is highly expressed in vascular endothelial cells, smooth muscle cells, and inflammatory cells. It is involved in the reconstruction in response to vascular injury, and promotes the migration of endothelial cells and the recruitment of inflammatory cells. With the process of plaque, the protein levels of Nogo-B has gradually reduced in the AS plaque, but seems to highly expressed in foam cells. Current studies suggest that the decline of Nogo-B protein levels in AS plaque may be a pro-AS factor. However, the relationship between Nogo-B and foam cells has not been found. To further clarify the correlation of Nogo-B and AS, we first used oxLDL to stimulate macrophages into foam cell, then studied the Nogo-B function in mononuclear macrophages and foam cells, and elaborated the anti-atherosclerotic effect of Nogo-B from the perspective of endoplasmic reticulum stress and autophagy.Methods and Results:To study the relationship between oxLDL and Nogo-B, we first use oxLDL to stimulate macrophages into form foam cells, and then assay Nogo-B protein and mRNA levels by western blot and the Realtime PCR.We found that Nogo-B protein in mouse peritoneal macrophages expression significantly increased by oxLDL. In addition, both of Nogo-B mRNA and protein levels were increased in RAW264.7cells.To study the mechanism of increased Nogo-B, we detect the activation of ATF-6by immune-fluorescence. Results showed that oxLDL induces endoplasmic reticulum stress in RAW264.7cells. Then we used ATF-6siRNA to interference the expression of ATF-6and tread cells with oxLDL. We found that oxLDL did not induce Nogo-B expression in Nogo knockdown cells. Suggesting that Nogo-B expression induced by oxLDL was mediated by ATF-6.In order to detect the relationship between Nogo-B and endoplasmic reticulum stress, we first used Nogo siRNA to interference the expression of Nogo-B, then stimulated cells with oxLDL. The increased phosphorylation was seen in PERK, eif2α and JNK but decreased in mTOR when compared with the control group.RAW264.7cells were stimulated with oxLDL for24h. A clear increase occurred in LC3-II, Atg5and Beclin-1protein levels; Immuno-fluorescence results showed that LC3dots rose after oxLDL stimulation. After interference Nogo-B expression by Nogo siRNA, we treated cells with oxLDL and evaluate the level of autophagy between Nogo knockdown group and the control group. Transmission electron microscopy (TEM) results showed that knockdown Nogo increased the amount of autophagie vacuole. Western blot showed that knockdown Nogo increased LC3-Ⅱ and p62protein level, but LC3-Ⅱ levels were not changed obviously after chloroquine treating, which suggests that the increased levels of LC3-Ⅱ were due to defective autophagic lysosome degradation.In order to further determine whether the increased level of p62protein is because of defective autophagy degradation or the increased expression, we tested the p62protein and mRNA level in cells after oxLDL stimulation. Realtime PCR and Western blot showed that p62protein levels but not mRNA levels in Nogo knockdown group obviously increased after oxLDL treated, which suggests that Nogo knockdown inhibited the degradation of p62. Furthermore, we investigate whether Nogo-B knockdown impair the basic autophagy level. P62and LC3-Ⅱ levels in Nogo knockdown cells were slightly higher than the control group, but had no significant difference after chloroquine treated. Suggesting that Nogo knockdown also influences the basis cell autophagy.Using flow cytometry technology to detect whether Nogo knockdown affected the phagocytosis of macrophages. Results showed that Nogo knockdown did not affect the phagocytosis of macrophages when compared with the control group. RAW264.7cells stimulated with Dil-oxLDL for12h, and then treated with3-MA and Rapamycin for12h. Fluorescent particles inside the cell were more than which of control group after3-MA treated, but intracellular fluorescence was significantly weaker than the control group after Rapamycin treatment. Showing that autophagy promotes the intracellular lipid degration. In addition, we found that intracellular fluorescence granular in Nogo knockdown group were more than that of the control group. Furthermore, Adipophilin, specific surface proteins of lipid droplet, was clearly increased when compaired with the control group, which suggests that Nogo knockdown inhibited lipids autophagic degration.Macrophages after over expressed Nogo-B, were given oxLDL stimulating for24h, Adipophilin protein levels were significantly lower Nogo-overexpression cells when compaired with the control group.p62protein was increased slightly, but LC3protein levels decreased clearly, which suggests that Nogo-B promoted the mature of autophagosome rather than activation of autophagy. Immuno-fluorescence showed LC3was co-located with intracellular lipid when lipid-loading in macrophage, showing that autophagy is involved in lipid degradation in macrophage. Interestingly, both endogenous and exogenous Nogo-B co-located with intracellular lipid, but the co-localization phenomenon with lipid drops was gradually disappear with the intracellular lipid reduction, which suggests that Nogo-B is involved in lipid drops autophagic degradation.Conclusion:1、Nogo-B was increased by oxLDL through endoplasmic reticulum stress activating factor ATF-6in macrophagy.2、Nogo-B was involved in the autophagosome degradation of the foam cells,which impair the formation of foam cell.