Mechanism Of Chitosan Oligosaccharides Inhibiting Foam Cells Formation Via Autophagy

Objective::Atherosclerosis is a chronic and progressive multifocal arterial inflammatory disease closely related to hyperlipidemia,hypertension,diabetes and obesity,it has an important effect on vital organs of the body.With the progression of atherosclerosis,the accumulation of lipid deposition,inflammatory response and abnormal remodeling of the arterial wall in the vascular endothelium gradually increase the formation of plaque.In advanced atherosclerosis,this plaques are very unstable,prone to rupture and falling off to form arterial thrombus,which is often the main cause of acute cardiovascular events.The formation of atherosclerotic plaques is a complex process with numerous influencing factors.Among these factors,lipid and cholesterol metabolism abnormality plays an important role.Macrophages phagocytosis of large amounts of lipid-forming foam cells are the important components of atherosclerotic plaques and are the key factor that lead to plaques instability.Enhance autophagy to promote cholesterol efflux in RAW234.7 macrophages and inhibition foam cells formation,slow atherosclerosis plaques formation and reduce the instability of atherosclerotic plaques have very important significance.Chitosan oligosaccharide is a kind of natural alkaline polysaccharide,which has been found to have many biological activities,including antiinflammatory,anti-tumor,anti-virus and anti-Alzheimer’s disease.This study mainly examine the effect of oligosaccharide on macrophage derived foam cell and the regulation of autophagy and its possible mechanism.Methods: This study model is oxidized low density lipoprotein induced macrophage derived foam cells,The effects of chitosan oligosaccharide on lipid accumulation in macrophages were evaluated by oil red O staining and total cholesterol content.Using Western blot technique to measure cholesterol inflows and outflows related protein(ABCA1,ABCG1,SR-A,CD36)and autophagy related protein(LC3II/I,P62)expression level is order to detect oligosaccharide effect on cell autophagy and bubblization.In the end,specific interference was performed by western blot to detect the protein expression of p-mTOR,mTOR,LC3,p62,and quantitative analysis was conducted by image j.And oil red O staining techniques were used to explore the role of oligosaccharide on formation of foam cells,and to study the role of chit oligosaccharide by regulating macrophage autophagy to reduce lipid accumulation and foaming in macrophages and the possible molecular mechanisms.On the basis of this,we preliminarily discuss the possible molecular pathways of macrophage autophagy and the potential of inhibiting bubble.Results :The experimental results showed that the chit oligosaccharide of200mg/ml could significantly inhibit the lipid accumulation in the macrophages induced by ox-LDL and reduce the intracellular cholesterol level.And the results of Western blot showed that the chit oligosaccharide significantly regulated the expression of key protein which were close to the excretion and absorption of cholesterol in the RAW264.7 macrophage,and the chit oligosaccharide inhibited the lipid accumulation and formation of foam cells.Western blot proved that chit oligosaccharide regulated autophagy with the relations of time-effect and dosage-effect,the results show that 200μg/ml oligosaccharide significantly unregulated the level of LC3II/LC3 I and down-regulated thelevel of p62 which proved that oligosaccharide dramatically promoted autophagy in the macrophage.In the study of the molecular mechanisms of chit oligosaccharide promoting the autophagy in the RAW264.7macrophages,the corresponding compounds were used to interfere with the expression of mTOR and the autophagy.The result of the experiment showed that chloroquine could significantly reduce the autophagy of macrophage from the oligochitosan,which was due to the specificity of chloroquine as an autophagy inhibitor to inhibit autophagy in the cells.Leucine as an autophagy inhibitor can promote the expression of m-TOR,after the treatment of RAW264.7 macrophage using chit oligosaccharide and leucine,the level of p-mTOR was increased,and oligochitosan could not increase the autophagy marker protein.It is indicate that chit oligosaccharide may increase the autophagy level of RAW264.7macrophage by inhibiting the expression of p-mTOR.Treatment RAW264.7 macrophage with oligochitosan and autophagy inhibitor not only reduce macrophage autophagy but also lower the expression of key protein associated with cholesterol efflux in cells,and lead to lipid accumulation.Conclusions : The above results showed that chitosan could promote autophagy in RAW264.7 macrophage by regulating the expression of pmTOR/mTOR,thus further inhibiting foam cells formation.In conclusion,the study confirmed that chit oligosaccharide promotes autophagy in RAW264.7 macrophages by regulating the expression of m-TOR,and further exerts the inhibition in the progression of foam cells formation.This suggests that chitosan may be important in reducing atherosclerosis progression,and the results of this study provide an important reference for the reduction of atherosclerosis process.