| Background:Reverse Cholesterol Transport(RCT)is a relatively novel strategy for the treatment of atherosclerosis.The process was performed by HDL in vivo.The major structural and functional protein of HDL,apolipoprotein(Apo)A-I has high lipid affinity and potent capacity of transport cholesterol,but the sequence of it was too long to make drug.So it is necessary to develop a series of short-sequence ApoA-I mimetic peptides to mimic the full-length ApoA-I.Objective:The 10th α-helix segment of the ApoA-I was used as the parent peptide.On the basis of retaining the original sequence as much as possible,the hydrophilic surface sequence was fixed after replacing the hydrophilic amino acid.The 17 new ApoA-I mimetic peptides were designed and synthesized via replacing hydrophobic amino acids to gradually enhance the hydrophobicity of the peptides.The relationship among the hydrophobicity with secondary structure,cholesterol efflux activity and toxicity of the new ApoA-1 mimetic peptides were discussed.The ApoE-/-mice were used to study the anti-atherosclerotic activity of the new ApoA-1 mimetic peptides and the action mechanisms were discussed.Methods:The hydrophobicity of 17 new ApoA-1 mimetic peptides was calculated using HeliQuest online software,and the retention time was determined by analytical high performance liquid chromatography.The secondary structures of them were determined by circular dichroism,and their lipid affinity was determined by DMPC vesicle solubilization assay.The cholesterol efflux activities in RAW 264.7 cells of them were determined with fluorescent labeled 22-NBD cholesterol.The cytotoxicities were measured by MTT assay and their hemolytic toxicity were determined.Finally,an optimal hydrophobicity range was selected.P12,the best peptides,was selected to continue the subsequent experiments.In order to study its action mechanism,ABCA1-dependent cholesterol efflux activity of P12 was measured.P12 labeled with C-TPP were used to localize the action site on the cells by laser scanning confocal microscope.The cholesterol efflux in RAW 264.7 and THP-1 cells of P12 was determined by flow cytometry.The action mechanism of P12 remodeling a-HDL to Pre-β HDL was studied by size exclusion chromatography and Western Blot.Finally,the anti-atherosclerosis effect of P12 in animals was evaluated preliminarily.Results:Firstly,the retention time of P1-P12 became longer as the theoretical value of hydrophobicity increased,and the retention time of P12-P17 became shorter as the theoretical value of hydrophobicity increased.The spatial secondary structures,the lipid affinities,cholesterol efflux activities and toxicities of P1-P17 change with hydrophobicity in a certain regularity manner.Parent peptide and P1-P5 had no obvious cholesterol efflux activity and cytotoxicity.P6-P12 had obvious cholesterol efflux activity in a concentration dependence way,and they had no obvious cytotoxicity.P13-P17 had obvious cholesterol efflux activities,but their efflux rates of cholesterol were suddenly increased at a certain concentration without a concentration dependent way.The experiment also found that P13-P17 had obvious cytotoxicity which related to destruction cytomembrane.All of peptides had no obvious hemolytic toxicity at low concentrations,but P14-P17 has obvious hemolytic toxicity at a certain concentration.The experiments showed that P12 had obvious ABCA1 dependence and the action site was on the cytomembrane and did not rupture cytomembrane.Flow cytometry further verified that P12 had potent cholesterol-promoting efflux activity in RAW 264.7 or THP-1.Lipoprotein binding separation experiments showed that P12 bind to mature HDL to produce β-HDL,and the conversion rate was as high as 38.2%at the 500 μg/mL concentration.Animal experiments showed that low doses of P12(10 mg/kg)can significantly reduce the serum TC,TG and LDL-C in ApoE-/-atherosclerotic mice(17.7%,45.4%,28.3%in male mice,females were 11.1%,20.8%,27.1%,respectively),and the serum HDL-C was also slightly elevated(5.2%for males and 8.9%for females).Serum inflammatory factors CRP,IL-6,MCP-1 and TNF-a were greatly reduced by P12 at 10 and 20 mg/kg.P12 had obvious effect on improvement the liver pathology of ApoE-/-atherosclerotic mice,and the thickening of the intima of the artery,the calcification of the artery,the proliferation of arterial smooth muscle,the elastic fiber disorder,the accumulation of foam cells,and the thickening of the plaque all been obviously improved.Conclusion:When the hydrophobicity of the ApoA-I mimetic peptides are within a reasonable range,the stable a-helical structure can be maintained and the spatial structure is closely related to their cholesterol efflux activities.It is not only facilitates the binding of ApoA-I mimetic peptides to cell membrane,but also facilitates the transport of excess cholesterol in macrophages of the arterial plaque through ABCA1 transporter,and transports cholesterol to the liver with HDL to achieve the purpose of anti-atherosclerosis.Objective:To study the united anti-atherosclerotic effect of the mixture of Hongqu and gypenosides(3.6:1 weight ratio).Methods:Sixty-four Wistar rats were randomly divided into eight groups:normal,model,positive control(simvastatin,1 mg/kg),Hongqu-treated(72 mg/kg),gypenoside(total saponin)-treated(20 mg/kg),and three doses HG-treated(50,100,and 200 mg/kg).All of the rats were fed with basal diet.Additionally,the model group rats were intragastrically administered a high-fat emulsion and intraperitoneally injected with vitamin D3.In compared with the model group,the serum lipid profiles,oxidative stress,inflammatory cytokine,and hepatic antioxidant levels of the other groups were then determined.Furthermore,the liver histopathology and arterial tissue were analyzed,and the expression of hyperlipidemia-and atherosclerosis(AS)-related genes was measured using reverse transcription-polymerase chain reaction.Results:The AS rat model was established after 80 days.In compared with the model group,the HG-treated groups showed an obvious improvement in the serum lipid profiles,oxidative stress,and inflammatory cytokine levels,and showed markedly increased hepatic total antioxidant capacity.Moreover,In comparing with the model group,after HG-treated,the expression of genes related to lipid synthesis and inflammation reduced and the genes related to lipid oxidation increased in the liver and arterial tissue,which also reflected an improved health condition.Conclusion:The anti-atherosclerotic effects of HG were superior to those of simvastatin group,Hongqu group,and the gypenosides group.Therefore,HG showed excellent anti-atherosclerotic effect. |
PDF Full Text Request