Study On The Mechanism Of Fucoidan Decomposing Lipids In Foam Cells By Promoting Autophagy

Atherosclerosis(AS)is the common basis for the onset of cardiovascular events.As an important part of its complex etiology,lipid metabolism theory suggests that foam cells formed by macrophages through phagocytosing a large number of oxidized low-density lipoproteins are important starting signs of AS.Moreover,previous studies have shown that macrophages of AS plaques have an autophagy-lysosomal system disorder.Therefore,if the autophagy-lysosomal system of foam cells can be activated,the phagocytosed lipids may be broken down,thereby Treat AS.Fucoidan is a kind of sulfated polysaccharide with anti-inflammatory,hypolipidemic and anti-atherosclerotic activity.Previous laboratory studies have shown that fucoidan can inhibit the growth of human cervical cancer Hela cells and colon cancer HT29 cells by apoptosis and autophagic-programmed cell death.Therefore,this project intends to study the pharmacological effects of fucoidan on oxLDL-induced foam cells,and explore its mechanism.The main contents are as follows:1.Pharmacological activity of fucoidan on foam cellsIn this paper,ox-LDL and macrophagy co-incubation were used to make foam cell models.After oil red O staining and cholesterol ester content determination,the results showed that the optimal method for foam cell models was that macrophages RAW264.7 were treated with 80 μg/mL ox-LDL for 24 h.Compared with the model control group,the cholesterol ester content of foam cells decreased to 43.48%,34.38%,and 23.17%after treatment with fucoidan at 200,400,and 800μg/mL,and oil red O staining also showed a significant reduction in the number of lipid droplets,indicating that fucoidan can clear ox-LDL in foam cells.2.Study on pharmacological mechanism of fucoidan on foam cellsAfter foam cells were treated with fucoidan,autophagy-related proteins were detected by Western-Blot,autophagy-related genes by qRT-PCR,and autophagy fluxes by Mcherry-GFP-LC3B.The results showed that the mRNA content of autophagy related genes such as LC3 Ⅱ and the expression of ATGs protein,LC3 II/LC3 Ⅰ and autophagy flux all increased with concentration,revealing that fucoidan can promote autophagy in foam cells.In order to explore the pharmacological mechanism of fucodian,fucoidan was used in combination with two autophagy inhibitors 3-MA and Bafilomycin A1.Compared with the above-mentioned normal administration group,the cholesterol content increased to 57.79%,51.90%,and 45.75%after 3-MA treatment,and the cholesterol ester content increased to 53.25%,50.57%,and 45.97%after Bafilomycin A1 treatment,and oil red O staining also showed a significant increase in the number of lipid droplets,demonstrating that the lipid-scavenging activity of fucoidan sulfate was largely inhibited.At the same time,the cell autophagy-lysosomal system was detected by Western-Blot,qRT-PCR and Mcherry-GFP-LC3,the decrease in expression of LC3 Ⅱ mRNA and ATGs,LC3 Ⅱ/LC3 Ⅰ,and autophagy flux suggested that autophagy of foam cells was inhibited after inhibitor treatment,but the expression of TFEB still increased and was concentration-dependent.Taken together,the results disclosed that fucoidan mainly breaks down lipids by promoting cell autophagy,and may be partially achieved by promoting the expression of TFEB.3.Study on the Relationship between TFEB and Fucoidan Pharmacological ActivityTFEB siRNA was used to silence TFEB in foam cells.Compared with the above-mentioned normal administration group,the cholesterol ester content increased to 57.78%,50.13%,and 42.68%after silencing,and the red lipid droplets increased significantly.Meanwhile,Western-Blot was used to detect autophagy-related genes.The results showed that the expression of LC3 Ⅱ and LC3 Ⅱ/LC3 Ⅰ were significantly reduced,indicating that autophagy of foam cells was inhibited,while ATGs showed a weaker concentration dependence.The above results illustrated that TFEB is very important in the fucoidan-promoting cell autophagy pathway.In summary,this subject provides the first evidence that that fucoidan can clear the lipids of ox-LDL-induced foam cells by promoting autophagy of foam cells,and TFEB plays a crucial role in this.The findings provide new ideas and strategies for the recovery of foam cells into macrophages,and have important theoretical and practical significance for the further development and utilization of fucoidan.