| Atherosclerosis and its complications of cardiovascular disease is a leading cause of the burden of human resources and medical resources. Therefore, there is an urgent need for research and development of drugs to improve the treatment of atherosclerosis. The disease can generally be viewed as a form of chronic inflammation that occurs in large and medium arteries, the underlying pathogenesis involves lipid accumulation and a maladaptive immune response of the arterial wall. Only lipid-lowering or anti-inflammatory can not solve the treatment of this disease, it is necessary to find drugs that have the dual function of anti-inflammatory and lipid regulation.In previous study, we have demonstrated that Ilexgenin A (IGA) extracted from Ilex hainanensis Merr, can regulate lipid abnormality, this study aimed to explore whether it also has anti-inflammatory effects, thus provided experimental basis for the treatment of atherosclerosis. In addition, to improve water solubility of IGA and enhance its biological activity, the subject used cyclodextrin polymer (β-cyclodextrin polymer, CDP) to encapsulate IGA before the studies of IGA treatment for atherosclerosis; Furthermore, we investigate the protective effects of IGA-CDP on atherosclerosis in ApoE-/-mice and explore its anti-inflammatory mechanisms. Methods:Part one:Firstly, we used epichlorohydrin as crosslinkers to prepare CDP, then prepared IGA-CDP by way of non-covalent destruction, and used infrared spectroscopy、UV、1H NMR to characterize IGA-CDP.Part two:The hyperlipidemia model of mice was established, and the animals were randomly divided into model group, IGA24mg-kg-1·d-1group, IGA-CDP15、30、60mg-kg-1·d-1group. The normal group was given the normal diet; also, the other groups were fed a high-fat diet. The normal group and model group were received the same volume of distilled water. After4weeks, serum levels of total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), apolipoprotein A1(Apo Al), apolipoprotein B (Apo B) were measured by ELISA and the atherosclerosis index (AI) and ApoA1/ApoB were calculated, paraffin sections were stained with hematoxylin and eosin (H&E).Part three:The atherosclerosis model was established by ApoE-/-mice, all animals were dividend into model group, IGA12mg-kg-1·-d-1group, IGA-CDP60mg-kg-1·d-1group, atorvastatin5mg-kg-1·d-1group (n=7). The C57BL6mice were used as the control group; all groups were given the normal diet. After12weeks, the mice were sacrificed, thymus, spleen, liver, fat were weighed and the corresponding organ coefficients were calculated; the levels of serum lipids (TC, TG, LDL-C, HDL-C), serum interleukin-6(IL-6) and tumor necrosis factor-a (TNF-a) were measured by ELISA, aortic tissue sections were prepared and stained with Oil Red O and H&E. In vitro, RAW264.7macrophages were stimulated by LPS. The levels of IL-6, TNF-a were measured by ELISA, and NF-κB p65expression of the inner and outer nuclear, IKKa, Phospho-IKKa/β expression were evaluated using the Western Blot assay. ResultsPart one:Compared with IGA, the solubility of IGA-CDP was greatly enhanced due to the water-soluble CDP host. The ratio of P-cyclodextrin (β-CD) units in CDP to IGA was determined as2:1by Benesi-Hildebrand method and1NMR integration. KD of the inclusion complex was evaluated as2.6×10-3mol·L-1using Benesi-Hildebrand method. In addition, mice were orally administered2000mg-kg-1of IGA-CDP, and no apparent toxicity was observed. Thus, it is preliminarily suggested that oral administration of IGA-CDP might be safe.Part two:(1) Compared with the normal group, weight of mice in model group were significantly increased (P<0.05), and compared with mice fed with a normal diet, weight gain in mice of model group.60mg-kg-1-d-1of IGA-CDP could effectively inhibit the increase in body weight of mice (P<0.05), with no effect on normal body weight.(2) Compared with the normal group, the serum levels of TC and LDL in the model group were significantly increased (P<0.01, P<0.01).24mg-kg-1-d-1of IGA and each dose of IGA-CDP significantly reduced the serum level of TC (P<0.05); the serum levels of LDL were significantly reduced in mg-kg-1-d-1IGA and30,60mg-kg1·d-1IGA-CDP group (P<0.05).(3) Compared with the normal group, the level of Apo B was significantly increased (P<0.01), and the ratio of Apo Al/Apo B was significantly reduced (P<0.01) in model group, IGA and each dose of IGA-CDP significantly reduced the levels of Apo B (P<0.01), increased the ratio of Apo A1/Apo B (P<0.01).(4) Compared with the normal group, AI was significantly increased in the model group (P<0.01), IGA and each dose of IGA-CDP could significantly reduce the AI (P<0.01), combined with the above index calculation, IGA-CDP potency is about IGA2-3times.Part three:Animal experiments:(1) Compared with mice in the normal group, the fat coefficient of mice in the model group was increased significantly, IGA-CDP at a dose of60mg-kg-1·d-1significantly reduced the fat coefficient (P<0.05).(2) Compared with the normal group, levels of TC and LDL-C in serum (P<0.01, P<0.01) was increased significantly in the model group, and a reduction of HDL-C level (P<0.01) was detected in the model groups. IGA-CDP at a dose of60mg-kg-1·-d-1and IGA at a dose of12mg-kg-1·d-1significantly reduced TC, LDL-C (P<0.01, P <0.01), increased HDL-C (P<0.05), the potency of IGA-CDP respectively was about1.16、1.67、1.17times higher than that of the IGA. The levels of TC, LDL-C were significantly reduced (P<0.01, P<0.01), the level of HDL-C was significantly increased (P<0.05) in atorvastatin group, and the effect of atorvastatin was better than the IGA-CDP.(3) Compared with the normal group, the levels of IL-6, TNF-a were increased significantly (P<0.01, P<0.01) in model group, the levels of IL-6, TNF-a were significantly reduced (P<0.01, P<0.01) in IGA-CDP60mg-kg-1·d-1group and IGA12mg-kg-1·d-1group, the potency of IGA-CDP was about1.17、1.02times higher than that of the IGA respectively. IGA-CDP inhibitory effect on TNF-a was superior to atorvastatin group.(4) Compared with the normal group, apparent lipid deposition, inflammatory cell infiltration were observed in aortic root of mice in model group; lipid deposition and inflammatory cell infiltration in aorta were significantly reduced in IGA-CDP60mg·kg-1·d-1group and IGA12mg-kg-1·d-1group, and the effect of IGA-CDP was superior to IGA.Cellular experiments:(1) MTT experiments have shown that in the absence of LPS stimulation and LPS-stimulated conditions IGA of RAW264.7cells with IC50values of66.57,52.45μmol·L-1, thus5,10,20, and40μmol-L-1of IGA were used in the following experiments.(2) ELISA experiments have shown that the levels of IL-6, TNF-a (P<0.01) was significantly increased in LPS-stimulated RAW264.7cells, and IGA were significantly inhibited increase of IL-6, TNF-a in a dose-dependent manner.(3) Western Blot experiments have shown that IGA significantly inhibited NF-κB p65expression by suppressing the IKKa/β phosphorylation.Conclusion:(1) CDP significantly improved the solubility of the IGA, and oral administration of IGA-CDP is safer.(2) IGA-CDP can significantly improve hyperlipidemic in mice and atherosclerosis in ApoE-/-mice; it not only has a good role in lipid metabolism, but also effectively suppresses secretion of inflammatory cytokines and the inflammatory cell infiltration in the process of atherosclerosis. In addition, IGA-CDP is better than IGA.(3) IGA can suppress atherosclerotic inflammation by suppressing the IKKa/β phosphorylation, and the expression of nuclear NF-κB p65. |
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