Fucoidan Alleviates Atherosclerosis By Regulating NLRP3 Inflammasome And Autophagy

Objective:Atherosclerosis(AS)is a chronic inflammatory disease.Rupture of vulnerable plaques and thrombosis of AS are identified as the main pathogenesis of stroke.Recent studies have found inflammation is always the key factor in the initiation of AS,the progression of lesions and the formation of thrombotic complications.Macrophages play an important role in this process.Macrophage autophagy has been regarded as a highly conserved cellular degradation process that widely existing in eukaryotic cells,which dynamically removes invading microorganisms,denatured long-lived proteins and damaged organelles.A large number of studies have shown that autophagy can inhibit the activation of inflammasomes or even encapsulate the main components of inflammasomes and then degrade them with lysosomes,thus playing an anti-inflammatory and therapeutic role in a variety of diseases.Fucoidan is a sulfated polysaccharide based on L-fucose,which is purified from seaweed.Fucoidan has a variety of pharmacological properties,including anti-virus,anti-tumor,anti-lipid,anti-angiogenesis,and fucoidan can regulate the release of inflammatory cytokines from macrophages.Based on the above research,we established a vulnerable carotid atherosclerotic plaques model in ApoE-/-mice to analyse whether fucoidan can exert anti-AS effect.At the same time,a foam cell model was established in vitro to investigate the underlying mechanism.Methods:1.Animal experiments: The AS model was established by using ApoE-/-mice with ligation of right common carotid artery and fed with high-fat diet.The serum lipid level,the carotid artery plaque formation,the expression of NLRP3 inflammasome and autophagy-related proteins in the mice were deteced.(1)Thirty-six six-week-old male ApoE-/-mice were randomly divided into three groups,12 mice in each group,namely the control group,the model group and the fucoidan group.The control group did not receive any treatment,and the mice in the other two groups gradually switch to a high-fat diet(containing 0.25% cholesterol and 15% fat)after 2weeks of adaptive feeding.At 8-week-old,the right common carotid artery was inserted with silicone ring to induce the formation of carotid atherosclerotic plaques in all ApoE-/-mice.Mice in the control group were fed a normal diet,and mice in the other two groups were fed a high-fat diet for 8 weeks.Mice in the fucoidan group were intraperitoneal injected with fucoidan 60mg/kg/day.(2)After isoflurane anesthesia,1-2ml of inner epicanthus artery blood was taken to detectthe levels of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-c)and high-density lipoprotein(HDL-c)in the serum of the mice.(3)Morphological observation: Paraffin and frozen sections were prepared after fixation of the common carotid artery.HE staining and oil red O staining were performed to observe the the changes of vascular wall structure,carotid plaque formation and lipid accumulation.(4)After the cannula side common carotid artery was fixed,ultrathin sections were made and stained.The ultrastructure of macrophages in the common carotid artery tissues of each group of mice was observed by transmission electron microscopy.(5)Detection of expression levels of NLRP3 inflammasome and autophagy-related proteins: Arterial blood was taken from mice after anesthesia,and serum IL-1βlevels were detected by ELISA.Paraffin sections were prepared and the expression of NLRP3 and p62 in plaques was observed with immunohistochemical staining.Total proteins were extracted from the common carotid artery tissue,and the expressions of NLRP3,ASC,caspase-1,IL-1β,LC3 and P62 were detected by Western blot.2.Cell experiment: The cell model was established by oxLDL-induced differentiated THP-1 cells.After the treatment of fucoidan,autophagy activator and inhibitor,GFP-RFP-LC3 lentivirus and p62 siRNA,the autophagy flow was observed and the expression levels of NLRP3 inflamemosome and autophagy-related proteins were detected.(1)Establishment of foam cell model: THP-1 cells were differentiated into macrophages by 100ng/ml PMA for 48 h,then treated with 80 μg/ml oxLDL for 36 h.The formation of foam cell model was determined by oil red O staining.(2)Macrophage autophagy was observed by transmission electron microscopy(TEM)and GFP-RFP-LC3 lentivirus transfection.(3)Detaction of expression levels of NLRP3 inflammasome and autophagy-related proteins: IL-1β in supernatant was detected by ELISA.Western blot was used to detect the effects of different concentrations of fucoidan(0,50,100,200,300 μg/ml),autophagy activator rapamycin,autophagy inhibitor 3-ma and p62 small interfering RNA on the expression of NLRP3 infalmmasome and autophagy related protein.The co-localization of p62 and NLRP3 was observed by immunofluorescence method.Results:1.Animal experiments: Fucoidan significantly reduced the levels of TC,TG and LDL-c in ApoE-/-mice;HE and oil red O showed that fucoidan reduced the area of plaques in the carotid artery and reduced lipid accumulation;ELISA results showed that fucoidan significantly reduced the expression of serum IL-1β;Immunohistochemistry showed that the protein level of NLRP3 and p62 in plaques of fucoidan group was significantly reduced,and Western blot showed that fucoidan decreased the expression of NLRP3,ASC,caspase-1,IL-1β,p62 and increased LC3II/LC3I;The results of electron microscopy showed that fucoidan significantly increased the number of autophagosomes in plaques.2.Cell experiment: MTT detection indicated that fucoidan had no significant effect on cell viability;Oil red O staining showed that fucoidan could reduce the accumulation of lipid in macrophages;ELISA results showed that fucoidan significantly reduced the expression of IL-1βin the supernatant;Western blot results showed that fucoidan significantly decreased the expression of NLRP3,ASC,caspase-1,IL-1β,p62 and increased LC3II/LC3 I in a dose-dependent manner;The results of electron microscopy showed that fucoidan significantly increased the number of autophagosomes in foam cells;The transfection results of GFP-RFP-LC3 lentivirus showed that fucoidan could enhance autophagic flow;Rapamycin could enhance the anti-inflammatory effect of fucoidan,and3-MA could reduce the anti-inflammatory effect of fucoidan;The co-localization of NLRP3 and p62 was observed by fluorescence microscope;Knockdown of p62 by small interfering RNA significantly eliminated the inhibitory effects of fucoidan treatment on NLRP3 inflammasome.Conclusion:1.Fucoidan can regulate the lipid levels of ApoE-/-mice and alleviate the progression of AS vulnerable plaques.2.Fucoidan can exert anti-inflammatory effects by inhibiting the expression of NLRP3 inflammasome.3.Fucoidan can enhance macrophage autophagy and improve the level of autophagy flow.4.Fucoidan may inhibit NLRP3 inflammasome activation by enhancing p62/SQSTM1-dependent selective autophagy to alleviate AS.