The Mechanism Of P3 Peptide To Improve Hypercholesterolemia In ApoE^-/- Mice By Promoting Cholesterol Efflux

Objective:Elevated plasma cholesterol levels are one of the major factors in the development of atherosclerosis.In this study,we observed the effect of P3 peptide(Ala-Phe-Tyr-Arg-Trp)on lipids in Apo E-/-mice on a high-fat diet and investigated its molecular mechanism.Methods:(1)In vivo animal experiment: 40 6-week-old SPF male ApoE-/-mice were randomly divided into control group(ND: normal diet),model group(HFD:high-fat diet),P3 peptide low-dose group(HFD+AFY-L:P3 peptide low-concentration group 40 mg/kg/d)and P3 peptide high-dose group(HFD+AFY-H:P3 peptide low-concentration group 80 mg/kg/d),10 animals in each group.Except for the blank group and the model group,mice in the P3 peptide low-dose group and the P3 peptide high-dose group were given the prescribed dose of P3 peptide intraperitoneally for two weeks.When the mice reached 8 weeks of age,the three groups were fed a high-fat diet for 8 weeks,except for the blank group,during which the P3 peptide low-dose and P3 peptide high-dose groups were injected intraperitoneally with the corresponding dose of P3 peptide every day.After the high-fat diet was continued for 8 weeks,blood was collected from the eyes of each group to determine serum triglyceride(TG),cholesterol(TC),high density lipoprotein(HDL)and low density lipoprotein(LDL)levels.The levels of serum bile acids and gallbladder bile acids were measured by colorimetric method,and liver tissues were stained with oil red O and HE,and the m RNA expression levels of LXRα(NR1H3),ABCA1,ABCG5,ABCG8 and CYP7A1 were measured by quantitative real-time fluorescence PCR(q RT-PCR).(2)RAW264.7 macrophages were divided into control group(Control),model group(ox-LDL 80 μg/m L),P3 peptide low concentration group(50 μg/m L)and P3 peptide high concentration group(100 μg/m L).In the model group,oxidized low-density lipoprotei(ox-LDL)at a concentration of 80 μg/m L was used to induce foam cell formation in RAW264.7 macrophages.In addition to the control and model groups,the P3 peptide low concentration group and the P3 peptide high concentration group were pretreated with the corresponding concentrations of P3 peptide for 24 h and then stimulated with 80 μg/m L ox-LDL for 24 h.The intracellular lipid deposition was detected by cytosolic oil red O staining,and the cholesterol content in the cells was detected by colorimetric method.The m RNA expression levels of ATP-binding cassette transporter A(ABCA1)and ATP-binding cassette transporter G(ABCG1)were measured by real-time fluorescence PCR(q RT-PCR),and the protein expression levels of ABCA1 and ABCG1 were measured by Western blot.Results:(1)Animal level: Compared with the model group,P3 peptide effectively reduced serum TC and LDL levels in Apo E-/-mice,but P3 peptide did not significantly affect serum TG and HDL levels in mice;compared with the model group,P3 peptide reduced serum bile acid levels and increased gallbladder bile acid levels in Apo E-/-mice;compared with the model group,P3 peptide effectively reduced liver lipid deposition and improved liver Compared with the model group,P3 peptide effectively reduced liver lipid deposition and improved liver steatosis,and significantly upregulated the m RNA expression levels of LXRα(NR1H3),ABCA1,ABCG5,ABCG8 and CYP7A1 in mouse liver tissues.(2)Cellular level: In RAW264.7 macrophages,P3 peptide effectively inhibited cellular lipid deposition and reduced intracellular cholesterol content compared with the model group;meanwhile,P3 peptide significantly upregulated ABCA1 and ABCG1 m RNA and protein expression levels;compared with the model group,P3 peptide and LXR agonist GSK2033,T0901317 significantly increased the expression levels of ABCA1 and ABCG1 m RNA and protein;when LXR gene expression was inhibited with LXR inhibitor GSK2033,P3 peptide had no effect on the expression of ABCA1 and ABCG1.Conclusions:P3 peptide reduced serum cholesterol levels in high-fat fed ApoE-/-mice,decreased lipid accumulation in RAW264.7 macrophages and inhibited foam cell formation.The mechanism of action may be related to activation of the LXR-ABCA1/ABCG1 pathway,thereby ameliorating hypercholesterolemia.