Study On The Inhibition Of L-theanine On The Formation Of Macrophage-derived Foam Cells And Its Mechanism

Atherosclerosis(AS)is a common cardiovascular disease and an important cause of death.The formation of foam cell plays an important role in the early stages of atherosclerosis.The dysregulation of cellular lipid metabolism leads to the formation of macrophages containing many lipid droplets(foam cells)in the cytoplasm.Foam cells are characteristic pathological cells in atherosclerotic plaques.The production of foam cells is a key step in the early stage of atherosclerotic plaque formation.L-theanine is the most abundant free amino acid in tea,accounting for 1% to 2% of the dry weight of tea.LTheanine is recognized by the US Food and Drug Administration(FDA)as a generally recognized as safe(GRAS),and there is no restriction on the amount during use.L-Theanine was first found to be soothing and relaxing,enhancing learning and memory functions;later it was found to have anti-tumor,blood pressure and blood lipid-lowering activities.Given that L-theanine shows cardiovascular protection and hypolipidemic effects,we speculate that L-theanine may have an inhibitory effect on the formation of macrophage-derived foam cells in the early stage of atherosclerosis.Therefore,we conducted the following research.We first treated RAW264.7 cells and mouse peritoneal primary macrophages with oxidized low-density lipoprotein(ox-LDL)to induce the formation of foam cells.The oil red O staining results showed that ox-LDL increased lipid content in the cells,and the maximal effects were observed at the concentration of about 80 mg/L of ox-LDL for 24 h.Indicating that the macrophages were transformed into foam cells.The above phenomenon was significantly reduced by pretreated with 800μM L-theanine,indicating that L-theanine significantly hindered the accumulation of cholesterol in macrophages and inhibited the formation of foam cells.Immunofluorescence results showed that L-theanine significantly reduced the uptake of Dil-ox-LDL by RAW264.7 cells and mouse peritoneal primary macrophages.Studies have shown that macrophage scavenger receptors control the uptake of ox-LDL.So,we examined the effect of L-theanine on the major macrophage scavenger receptor proteins scavenger receptor A(SR-A)and macrophage scavenger receptor B(CD36).Western blot results showed that L-theanine could significantly reduce the level of SR-A protein,but had no obvious inhibitory effect on the protein level of CD36.Further q PCR results showed that L-theanine had no significant effect on the m RNA level of SR-A.These results suggest that the effect of L-theanine on SR-A is at its protein level.Cycloheximide(CHX)is a broad-spectrum protein synthesis inhibitor that can inhibit protein synthesis.Therefore,we used CHX to inhibit the synthesis of SR-A protein to test the effect of L-theanine on the stability of SR-A protein.The results of western blot showed that L-theanine can significantly reduce the stability of SR-A protein,and the maximal effects were observed at the 24 h.Studies have reported that ubiquitin-proteasome degradation pathway is the main degradation pathway of SR-A protein.Therefore,we used the proteasome inhibitor MG132 to inhibit the degradation of the proteasome pathway.The results of western blot showed that MG132 can reverse the decrease of SR-A protein level caused by L-theanine.Further immunoprecipitation results showed that L-theanine significantly promoted the ubiquitination and degradation of SR-A protein in vivo and in vitro.These results indicate that L-theanine can promote the degradation of SR-A depends on the ubiquitin-proteasome pathway.Finally,we transfected SR-A plasmid into RAW264.7cells.The results showed that overexpression of SR-A significantly reversed the inhibitory effect of L-theanine on the formation of macrophage-derived foam cells and the inhibitory effect on the uptake of Dil-ox-LDL by macrophages.In summary,this study proves that L-theanine can significantly reduce macrophage cholesterol uptake and thereby inhibit the formation of foam cells.Further studies on the molecular mechanism of the action of L-theanine found that L-theanine regulates the degradation of SR-A protein through the ubiquitin-proteasome pathway,and overexpression of SR-A reverses the formation of macrophage-derived foam cells induced by L-theanine.These results suggest that L-theanine can be used as a potential drug for the prevention and treatment of atherosclerosis.