The Underlying Mechanism Of Laminaria Japonica Polysaccharide On The Suppression Of Atherosclerosis Via Modulating Autophagy

Atherosclerosis is a dyslipidemia-driven chronic vascular inflammatory disease,which seriously threatens human health.More and more evidences indicated that macrophage played an important role in the initiation and progression of atherosclerosis.Autophagy is a critical pathway required for lipid metabolism.It has been reported that the dysfunction of autophagy can trigger the lipid accumulation in macrophages and induce the conversion of macrophages to foam cells,leading to the initiation and progression of atherosclerosis.In our previous work,we obtained a homogeneous polysaccharide(LJP61A)with the activity of anti-atherosclerosis from Laminaria japonica.The present work aims to clarify the underlying mechanism of LJP61A on the suppression of atherosclerosis via modulating autophagy in vivo and in vitro.The main result was shown as the following.The dynamic response of vascular macrophage autophagy for LJP61A inhibiting atherosclerosis.To study the dynamic response of vascular macrophage autophagy for LJP61A inhibiting atherosclerosis,LDLr^-/-mice were fed with high-fat diet for 4,8,16 and 24 weeks to establish atherosclerosis models at different stages.Compared with the high-fat-diet groups,LJP61A not only apparently decreased the body weight,epididymal fat weight,liver index,serum LDL-C,TC,TG,apoB,NEFA,glucose and TC/HDL-C levels,but also significantly reduced liver lipid accumulation.Pathological observations showed that LJP61A could effectively inhibit the formation of atherosclerotic plaque and the infiltration of monocyte macrophages into blood vessels induced by high-fat diet.Meanwhile,compared with the model groups,LJP61A significantly inhibited the phosphorylation of ERK1/2,JNK,p38,Akt and p65,down-regulated the mRNA levels of TNF-?,IL-1?,IL-6,MCP-1,and increased the protein expression of I?B-?.These results revealed that LJP61A could effectively alleviate the atherosclerosis progression and inflammation induced by high-fat diet in mice.Moreover,it was found that LJP61A inhibited the progression of atherosclerosis,accompanied by the increase of ATG5 and LC3II protein levels,the decrease of p62protein level,and the enhanced immunofluorescence co-localization of MOMA-2 and LC3 in aorta.These results implied that LJP61A might inhibit the occurrence and development of atherosclerosis by regulating the autophagy activity of vascular macrophages.The modulatory role of LJP61A on macrophage autophagy.To explore the effects of LJP61A-regulated autophagy on the macrophage-derived foam cell formation,the Ox-LDL induced conversion of macrophage to foam cell was used as the experimental model.Compared with the model group,LJP61A significantly suppressed the transformation of Ox-LDL induced macrophages into foam cells,down-regulated the expression of lipid uptake/transformation genes(CD36 and ACAT1),up-regulated the lipid metabolism gene LXR-?,increased the cholesterol efflux to HDL or apoA-I,and reduced cholesterol ester concentration in cells,indicating that LJP61A could improve the imbalance between lipid absorption and clearance induced by Ox-LDL.In addition,compared with the model group,LJP61A significantly inhibited the phosphorylation of p65,ERK1/2,JNK,p38,Akt proteins,down-regulated the mRNA levels of TNF-?,IL-1?,IL-6,and up-regulated the protein expression of I?B-?and the mRNA level of IL-10.In addition,it was found that LJP61A inhibited macrophage-derived foam cell formation,accompanied by the increase in the the number of intracellular autophagosomes and the elevation of autophagy flux.Western blot results showed that,compared with the model group,LJP61A significantly increased the protein levels of ATG5,LC3II,LAMP1,Cathepsin D,ABCA1,ABCG1,and decreased the protein level of p62.The mRNA changes of ATG5,LC3II,LAMP1,Cathepsin D and p62 were consistent with the results of Western blot.These results hinted that LJP61A might suppress macrophage conversion into foam cells via regulating autophagy.Moreover,LJP61A promoted the lipid droplets selectively targeted by autophagosomes,the effective fusion of autophagosomes and lysosomes,the interaction between lipid droplets and lysosomes.ATG5siRNA,Chloroquine and Leupeptin were used to inhibit the initiation of autophagy,the fusion of autophagosome and lysosome,and the degradation of lysosome,respectively.All the three inhibitors significantly reduced the promotion of LJP61A on macrophage autophagy and the stimulation of LJP61A on intracellular cholesterol efflux,thus aggravating intracellular lipid deposition.These results demonstrated that LJP61A inhibited the transformation of macrophages into foam cells induced by Ox-LDL via regulating lipophagy.LDLr^-/-mice were fed with high-fat diet and orally administrated with Laminaria japonica polysaccharide LJP61A for 4 weeks.After that,macrophages with ³H-cholesterol was injected to abdominal cavity of experimental mice.Compared with the high-fat-diet group,LJP61A markedly increased[³H]-cholesterol in the plasma,liver,gallbladder,and feces of mice.Consistent with these,the protein levels of ABCA1 and ABCG1 responsible for cholesterol efflux in the aortas were enhanced by LJP61A.Similar variation of HDL and apoA-I were also observed in the serum.These results suggested that LJP61A improved the cholesterol reverse transport of macrophages in vivo.Based on the above results,we concluded that LJP61A could restrain the conversion of vascular macrophages into foam cells by restoring Ox-LDL induced lipophagy deficiency.The LJP61A-regulated lipophagy further mobilized the macrophage-specific reverse transport of LD-associated cholesterol,ultimately suppressing the initiation and development of atherosclerosis.The regulatory mechanism of LJP61A on macrophage autophagy.To further explore the regulatory mechanism of LJP61A on macrophage autophagy,Ox-LDL induced macrophage foam cell model was used.It was found that LJP61A inhibited macrophage-derived foam cell formation,accompanied by significantly increased AMPK phosphorylation and TFEB dephosphorylation,and decreased mTOR phosphorylation,indicating that LJP61A markedly regulated AMPK-mTOR-TFEB signaling pathway.Furthermore,AMPK siRNA and Rapamycin,the inhibitors of AMPK and mTOR were used to vertify the correlation between LJP61A-regulated autophagy and AMPK-mTOR-TFEB signaling pathway.It was confirmed that LJP61A significantly enhanced the translocation of TFEB to nucleus,increased the number of GFP-LC3-positive cells,the number of autophagosomes,the protein levels of ATG5,LC3-II,LAMP1,Cathepsin D,ABCA1 and ABCG1.Meanwhile,LJP61A markedly heightened lipid droplets selectively targeted by autophagosomes,the effective fusion of autophagosomes and lysosomes,the interaction between lipid droplets and lysosomes,the degradation of autophagy substrate protein p62,and autophagy flux.Consistent with these,LJP61A promoted the cholesterol efflux to HDL or apoA-I,thereby inhibiting macrophage-derived foam cell formation.In vivo experiments also showed that LJP61A might inhibit the occurrence and development of atherosclerosis by regulating AMPK-mTOR-TFEB mediated macrophage autophagy.LJP61A modulated autophagy-mediated macrophage polarization.To explore the effects of LJP61A-regulated autophagy on macrophage polarization,Ox-LDL induced macrophage polarization was used as the experimental model.Compared with the model group,LJP61A remarkably decreased the expression of M1 macrophage markers(CD86,iNOs,IL-6,IFN-?,TNF-?,IL-1?)and increased the expression of M2macrophage markers(CD206,Arg1,IL-10),thus reducing the M1/M2 macrophage phenotype ratio.In the meantime,the autophagic flux of macrophages was also enhanced by the treatment of LJP61A in vitro.The 3-methyladenine is an autophagic inhibitor.We found that the effects of LJP61A on the polarization of macrophages were blocked by this inhibitor.The SIRT1 and FoxO1 are two key upstream genes which control the autophagy behavior.Results showed that LJP61A significantly up-regulated the expression of SIRT1 and FoxO1.However,these effects of LJP6A were abolished by SIRT1 siRNA and FoxO1 inhibitor AS1842856.In vivo,it has been proved that LJP61A targeted SIRT1 and FoxO1,promoted their protein expressions,which were associated with increased autophagy and M2 phenotype polarization of aortas,thus alleviating the progression of atherosclerosis in high-fat-diet induced LDLr^-/-mice.In summary,LJP61A resisted high-fat-diet induced atherosclerosis in LDLr^-/-mice via regulating autophagy,inducing the polarization of macrophages to M2 phenotype,and inhibiting macrophage foam cell formation.