| 1 Background and objectiveAs a chronic inflammatory disease of blood vessel, Atherosclerosis has a "common ground" of the chronic inflammation. It includes the sustained release of inflammatory mediators and the lack of pro-resolving mediators. Leukotriene B4 (LTB4) is a pro-inflammatory lipid mediator, and lipoxin A4 (LXA4) is a pro-resolving lipid mediator. Although both of them are the metabolite of 5-lipoxygenase (5-LO) metabolic pathways, they have opposite biological activities in the atherosclerotic inflammation. As endogenous lipid mediators, the imbalances of leukotriene B4 and lipoxin A4 contribute to the development of atherosclerosis. Therefore, based on Atherosclerosis model in apolipoprotein E-deficient (ApoE-/-) mice in vivo, and inflammatory damage model in RAW264.7 mice macrophage in vitro, this study investigate the effects of SL extract (i.e., a combination of Radix Salviae miltiorrhizae and Andrographis paniculata extract) on the atherosclerosis from two aspects of anti-inflammation and pro-inflammatory resolution. This study are focused on promote the inflammatory resolution in the atherosclerosis, observed the effect of SL extract on the macrophage death, the formation of foam cells, efferocytosis and its receptor in the lack of inflammatory resolution in the atherosclerosis. In addition, based on the 5-lipoxygenase metabolic pathways, synthetical key enzymes, receptor expression, biological effect, signaling pathways of LTB4 and LXA4 are observed in this study. All of them are to deal with the effect way of SL extract on the regulating of the rebalance of endogenous pro-inflammatory mediator and pro-resolving mediator in the atherosclerosis.2 Methods2.1 Effect of SL extract on the atherosclerotic formation in ApoE-/- miceAtherosclerosis model was established by placing a perivascular collar on the right common carotid artery and a high fat diet in ApoE-/- mice. Mice were divided into five groups that included the model group, three SL extract groups (i.e.,95,190 and 380mg/kg) and an atorvastatin (ATO) plus pioglitazone (PIO) group (at 4.6mg/kg), C57BL/6J mice were the normal group. Wall thickness of the aortic arch, plaque progression of the cartoid artery and local blood flow were observed by using high resolution ultrasound biomicroscopy (UBM). Pathological changes were observed by histochemical staining, and lipid levels were measured by respective commercial kits. All the measurements were to investigate the Effect of SL extract on the atherosclerotic formation in ApoE-/- mice.2.2 Effect of SL extract on the imbalance of pro-inflammatory and pro-resolving in ApoE-/- miceAtherosclerosis model was established by placing a perivascular collar on the right common carotid artery and a high fat diet in ApoE-/- mice. Focused on the atherosclerotic inflammation, the serological levels of monocyte chemotactic protein 1 (MCP-1), interleukin-10 (IL-10) and transforming growth factor betal (TGF-β1) in serum were measured by ELISA. Focused on the 5-lipoxygenase metabolic pathways, the serological levels of LTB4 and LXA4 in serum were measured by ELISA, the histological levels of LTB4 receptor (BLT1) in the cartoid artery was detected by immunofluorescence. All the measurements were to observe the Effect of SL extract on the atherosclerotic inflammation from two aspects of anti-inflammatory mediators and pro-resolving mediators in ApoE-/- mice.2.3 Effect of SL extract on the inflammatory resolution by RAW264.7 macrophagesThe inflammatory damage model was established by oxidized low density lipoprotein (ox-LDL)-stimulated RAW264.7 mice macrophages. Focused on the atherosclerotic inflammation resolution, macrophages survival rate and the formation of foam cells were observed by MTT and oil red O staining, the role of macrophages in the clearance of apoptotic cells (namely efferocytosis) was measured by fluorescence activated cell sorting (FACS) and laser scanning confocal microscope (LSCM), the expression of phagocytosis receptor Mertk in the membranes of macrophages was detected by immunofluorescence. Focused on the 5-lipoxygenase metabolic pathways, LXA4 production by macrophages was determined in the culture supernatant using an ELISA assay kit, and the expression of its receptor FPRL1 in the membranes of macrophages was detected by immunofluorescence. In addition, the expression of Leukotriene A4 hydrolase (LTA4H),12-lipoxygenase (12-LO) which were the synthetical key enzymes of LXA4 and Protein kinase A (PKA) in the macrophages were measured by ELISA and immunofluorescence. All the measurements were to investigate the mechanism of SL extract on the atherosclerotic inflammation based on regulating pro-resolving lipid mediator LXA4 and its receptor FPRL1 in RAW264.7 macrophages.2.4 Effect of SL extract on the pro-inflammatory lipid mediator by RAW264.7 macrophagesThe inflammatory damage model was established by 4-hydroxynonenal (4-HNE) and Arachidonic acid (AA)-induced RAW264.7 mice macrophages. LTB4 production by macrophages was determined in the culture supernatant using ELISA. In addition, another model was established by LTB4-induced RAW264.7 mice macrophages. The expression of MCP-1 in the culture supernatant, BLT1 and CD36 in the membranes of macrophages was detected by ELISA and immunofluorescence. All the measurements were to investigate the mechanism of SL extract on the atherosclerotic inflammation based on regulating pro-inflammatory lipid mediator LTB4 and its receptor BLT1 in RAW264.7 macrophages.3 Results3.1 Effect of SL extract on the atherosclerotic formation in ApoE-/- miceAtherosclerosis model was successfully established by using high-fat diet and perivascular collar placement in ApoE-/- mice. High-fat diet increases blood viscosity and placement of a constrictive collar results in site-controlled atherogenesis, and did so based on changing the hemodynamic state of the carotid artery. Therefore, this model is very physiologically relevant to the human condition seen in atherosclerosis development, and the model was suitable for our research.In our current study, (1) the serological levels of TC, TG LDL and ox-LDL were increased significantly and HDL was reduced significantly in the model group. The low-dose, medium-dose and high-dose SL groups significantly reduced levels of TC, TG and LDL as compared with the model group, by contrast, increased HDL levels were detected in the high-dose SL group. (2) The results of HE staining showed that in the model group, the structure of the vascular intima and media were damaged, the vascular intima became thicker and fewer smooth cells appeared in the vascular media, moreover, the carotid artery lumen was filled with atherosclerotic plaques that contained a large number of foam cells, lipid droplets and inflammatory cells. As compared with the model group, the SL treatment groups showed obvious and dose-dependent suppression of those changes and fewer atherosclerotic plaques were observed. (3) The results of UBM showed that in the model group, the wall thickness of the GC, LC and BC were significantly increased; the carotid artery lumen was filled with atherosclerotic plaques, particularly evident in the proximal area, where in the lumen was almost completely blocked by plaque formation, and the vessel walls were unclear, the cross-sectional lumen of the carotid artery was narrowed with plaque at rate of 90.1 ±4.3%; the region and velocity of the local blood flow in the proximal and distal carotid artery were small and low. Compared with the model group, SL groups showed significantly reduced wall thicknesses of the GC, LC and BC; no plaque formation was detected in the distal zone of the carotid artery, and presence of atherosclerotic plaque in the proximal carotid artery were less than were found in the model group, in which the lumen became clearer, the plaque area in SL groups were significantly reduced (52.6±15.7%,34.1±5.7%,21.8±16.4%, respectively) in a dose-dependent manner; the region and velocity of local blood flow in the SL treatment groups were all increased, and the medium and high-dose SL group resulted in a significant increase in local blood flow velocity.3.2 Effect of SL extract on the imbalance of pro-inflammatory and pro-resolving in ApoE-/- miceIn our current study, (1) The results of ELISA showed that the serological levels of pro-inflammatory mediators MCP-1 and LTB4 were increased significantly in the model group. Compared with the model group, all the SL groups significantly reduced the levels of LTB4, and the medium-dose and high-dose SL groups significantly reduced the levels of MCP-1. (2) The results of immunofluorescence showed that, the expression of BLT1 in the plaque and tunica adventitia of vessel were increased significantly in the model group. Compared with the model group, the expression of BLT1 in the plaque and tunica adventitia of vessel was reduced significantly in all the SL groups. (3) The results of ELISA showed that the serological levels of pro-resolving mediators IL-10 and LXA4 were increased significantly and TGF-β1 was reduced significantly in the model group, the medium-dose and high-dose SL groups showed significantly increase levels of IL-10 and the high-dose SL groups showed significantly increase levels of LXA4 as compared with the model group, while there was no statistical differences in the levels of TGF-β1 between model and SL group.3.3 Effect of SL extract on the inflammatory resolution by RAW264.7 macrophagesThe inflammatory damage model was successfully established by ox-LDL-stimulated RAW264.7 mice macrophages.In our current study, (1) after 12h or 24h of 25μg/ml and 50μg/ml ox-LDL stimulation, the results of MTT showed that no cell toxicity was detected using those concentrations of ox-LDL, when the concentrations of ox-LDL up to 100μg/ml and 200μg/ml, the results of MTT showed that the cell inhibition rate reached 29.7% and 65.6% respectively after 12h, reached 39.1% and 91.8% respectively after 24h. However, the cell inhibition rate of 100μg/ml ox-LDL stimulation after 12h or 24h was significantly reduced in SL treatment groups. (2) After 50μg/ml ox-LDL stimulation, in the model group, the morphological changes of macrophages showed that cells became bigger, pseudopodia were increased and a larger "nuclear" was detected in the cells; the results of oil red O staining found that ox-LDL internalization into macrophages was markedly increased in a time-dependent manner. Compared with the model group, the morphological changes of macrophages had improved and ox-LDL internalization into macrophages was markedly reduced in a dose-dependent manner. (3) The results of FACS showed that the apoptotic cells were focused on Q2 area after ultraviolet (UV)-induced RAW264.7 macrophages, and the apoptosis rate was 97.9%; the results of LSCM showed that the processes in clearance of apoptotic cells included:finding apoptotic, extending pseudopodia, Pseudopodia captured apoptotic cell and apoptotic cell was phagocytized by macrophages, the time of whole phagocytosis was about 3h; the results of FACS showed that the phagocytosis index was increased significantly in the model group, compared with the model group, the phagocytosis index was also increased significantly in the SL groups. (4) The results of ELISA and immunofluorescence showed that in the model group, the levels of LXA4 in the culture supernatant had no statistical differences and the expression of FPRL1 in the membranes of macrophages were increased significantly, compared with the model group, the levels of LXA4 in the culture supernatant and the expression of FPRL1 in the membranes of macrophages were increased significantly in the SL groups. (5) The results of immunofluorescence found that in the model group, the expression of LTA4H and 12-LO in the macrophages were increased significantly, compared with the model group, the expression of LTA4H were reduced significantly in the SL groups while the expression of 12-LO were no statistical differences. (6) The results of ELISA showed that in the model group, the expression of PKA in the macrophages were increased significantly, compared with the model group, the expression of PKA in the macrophages were also increased significantly in the SL groups.3.4 Effect of SL extract on the pro-inflammatory lipid mediator of 5-LO metabolic pathways by RAW264.7 macrophagesThe inflammatory damage models were successfully established by 4-HNE with AA-stimulated and LTB4-induced RAW264.7 mice macrophages.In our current study, (1) after 4-HNE and A A stimulation, the level of LTB4 in the culture supernatant were markedly increased, the maximum of LTB4 was at 3h after 4-HNE and AA stimulation, so the best stimulating time of 4-HNE and AA was 3h. Moreover, after 4-HNE and AA stimulation for 3h, the levels of LTB4 in the culture supernatant were markedly reduced in the SL groups with a dose-dependent manner. (2) The results of ELISA showed the level of MCP-1 in the culture supernatant were markedly increased in the model group, and in the SL groups, the level of MCP-1 in were markedly reduced as compared with the model group. (3) The results of immunofluorescence found that the expression of BLT1 and CD36 in the membranes of macrophages were increased significantly in the model group, compared with the model group, the expression of BLT1 and CD36 in the membranes of macrophages were reduced significantly in the SL group.4 ConclusionIn summary, (1) SL extract could markedly inhibit formation of atherosclerotic plaques in the perivascular collar-induced atherosclerosis in ApoE-/- mice. (2) SL extract could significantly improve the imbalance of pro-inflammatory mediators and pro-resolving mediators in the atherosclerotic inflammation in ApoE-/- mice. (3) Based on the 5-lipoxygenase metabolic pathways, SL extract could markedly regulate the rebalance of endogenous pro-inflammatory lipid mediator LTB4 and pro-resolving lipid mediator LXA4 to inhibit the atherosclerotic inflammation, and the mechanism might dependent on the improving the key enzymes, the expression of receptors, biological effects and downstream signaling pathways. |
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