FGF21 Induces Autophagy-mediated Cholesterol Efflux To Inhibit Atherogenesis Via RACK1 Upregulation

Lipid metabolism disorder leads to the deposition of a large number of lipids in the large and medium arteries,which is the primary pathophysiological basis of atherosclerosis?AS?.Macrophages uptake of excessive modified lipoprotein leads to the accumulation of total cholesterol?TC?and triglyceride?TG?in the form of lipid droplets and formation of foam cells,which is an essential process for the occurrence of AS.Fibroblast growth factor 21?FGF21?is an endocrine factor that lowers TC,TG and low-density lipoprotein cholesterol?LDL-c?level and increase the level of high-density lipoprotein cholesterol?HDL-c?,thus having anti-AS potential.Our previous study found that FGF21 can reduce lipid accumulation in foam cells.However,the underlying mechanism remains unclear.Lipid accumulation is the crucial process of foam cell formation,and lipid droplet metabolism and cholesterol efflux of foam cells will reduce the formation of foam cells.Therefore,an in-depth study of the mechanism by which FGF21 regulates lipid metabolism in foam cells is vital for the prevention of AS.Autophagy can degrade lipid droplets by acid lipase,promote cholesterol efflux,and maintain lipid metabolism balance;and autophagy damage will inhibit cholesterol reverse transport and promote the occurrence and development of atherosclerosis.Therefore,this study aimed to investigate whether FGF21 can influence themacrophage-derived foam cell cholesterol metabolism anti-AS by regulating autophagy and explore its underlying mechanisms.Part I: Effect of FGF21 on High Fat Diet Apo E^-/-Mice Atherosclerosis Lesions[Objective]To observe the effect of FGF21 on AS lesions in high-fat diet apo E^-/-mice.[Methods] 60 male adult apo E^-/-mice were randomly divided into four groups:?1?Control group: normal diet.?2?AS1: HFD,fed a high-fat diet;?3?AS2: AS1 mice received intraperitoneal injection of the same amount of saline as AS3;?4?AS3: AS1 mice received intraperitoneal injection of FGF21?10 mg/kg.d?.Body weight and food intake are recorded weekly.Twelve weeks after the injection,the mice were sacrificed under pentobarbital-induced general anesthesia,and blood were collected,and plasma concentrations of TC,TG,HDL-c,and LDL-c were measured.Aortic AS lesions were observed by Sudan IV staining.The aortic sinus of the mice was taken for frozen section,HE and oil red O staining were used to detect the AS lesion of aortic sinus.The above animal experiments determined the effects of FGF21 on the lipid profile and AS lesions of HFD-fed apo E^-/-mice.[Results] Compared with the control group,serum TG,TC and LDL-c levels were significantly increased in the AS1 and AS2 groups,and HDL-c was significantly decreased?P <0.05?.HE and oil red O staining showed manifest AS plaques in mice aortic.Sudan IV staining showed significant lipid deposition in the aorta,suggesting successful modeling,and the intraperitoneal route?normal saline?had no significant effect on AS lesions.Compared with the HFD group,the FGF21+HFD group had lower TG,TC,and LDL-C,higher HDL-C,decreased AS lesion area,and lower body weight than the HFD group.[Conclusion] FGF21 can improve lipid profile and inhibit AS lesions in HFD apo E^-/-mice.Part ?: The Potential Mechanism of FGF21 Against AS[Objective] To investigate the potential molecular mechanism of FGF21 against AS lesions.[Methods] First,to confirm whether the role of FGF21 anti-AS depends on the role of regulating autophagy: 90 apo E^-/-male mice are divided into six groups:?1?control group: ND;?2?AS1: HFD;?3?AS2: HFD + NS ip;?4?AS3: HFD+ FGF21 ip.?5?AS4: HFD + 3-MA ip;?6?AS5: HFD + FGF21 ip + 3-MA ip.Autophagy-associated proteins were detected by Western Blot,and AS lesions were detected by HE and oil red O staining.Furthermore,the effects of FGF21 on autophagy and autophagic flow were observed at the cellular level.The experiment was divided into four groups:?1?control group;?2?Baf A1 group;?3?FGF21 group;?4?FGF21+Baf A1 group.The effects of FGF21 on autophagy and autophagic flow were analyzed by Western Blot,confocal microscopy detect GFP-LC3,MDC staining and transmission electron microscope?TEM?.Further analysis of the effect of FGF21 on cholesterol efflux from THP-1 macrophage-derived foam cells,THP-1macrophage-derived foam cells were treated by FGF21 at different concentrations?0-400 ng/ml?and at different times?0-48 h?,cholesterol efflux was analyzed by HPLC,oil red O staining and Liquid scintillation counting.To clarify whether FGF21 promotes cholesterol efflux depending on its role in regulating autophagy,liquid scintillation counting,HPLC and oil red O staining were further used to analyze the cholesterol efflux after 3-MA and ATG5 interference inhibited autophagy.Further validation of our hypothesis that FGF21 upregulates autophagy to promote cholesterol efflux and anti-AS is achieved by activating RACK1.First,we detected the expression of RACK1 in the different group in apo E^-/-mice?control group,HFD group,HFD+saline group and HFD+FGF21 group?by immunohistochemistry,PCR and Western Blot.Then,we constructed RACK1 sh RNA lentivirus to give tail vein injection to HFD+sh RNA group.After the experiment,the autophagy level of each group was analyzed by Western Blot.The lesions of AS were analyzed by HE staining and oil red O staining.Then,RACK1 si RNA was transfected into THP-1 cells to analyze the effects of RACK1 knockdown on autophagy?flow?and cholesterol efflux,autophagy?flow?was detected by Western Blot,confocal microscopy detects GFP-LC3,MDC staining and Transmission electron microscope.HPLC detection,oil red O staining analysis of the degree of lipid accumulation in foam cells.[Results] The LC3 ?/? ratio and Beclin1 increased and P62 decreased in the intraperitoneal injection of FGF21?AS3?compared with AS1 group.The results of HE and oil red O staining showed that the AS lesion was significantly reduced in the AS3 group compared with the AS1 group,while the AS lesion was aggravated,and autophagy was impaired in the AS5 group compared with the FGF21 intraperitoneal injection group?AS3?.In summary,FGF21 can up-regulate HFD apo E^-/-autophagy to inhibit AS,and autophagy damage aggravates AS lesions and weakens the effect of FGF21 in inhibiting AS.MDC staining and TEM results showed that FGF21 could induce autophagosome formation in foam cells.When foam cells were co-incubated with bafilomycin A1,the conversion rates of LC3-I to LC3-? were significantly increased compared with the control group.The conversion rates of LC3-I to LC3-? were further increased in the bafilomycin A1 + FGF21 group compared with the FGF21 group.Therefore,these results suggest that FGF21 induces autophagic flow in foam cells.The results of HPLC showed that,compared with the control group,the levels of TC,FC and CE were significantly decreased after treatment with 200ng/ml and 400ng/ml FGF21 for24 h,and after treatment with 200ng/ml FGF21 for 24 h and 48 h.At the same time,the results of oil red O staining also showed that the cholesterol accumulation of foam cells was inhibited.These results suggest that FGF21 reduces lipid accumulation in foam cells.Pretreatment with 3-MA or ATG5 si RNA significantly reduced FGF21's role in promoting LC3-I to lc3-? transformation and correspondingly increased P62 expression.Autophagosome formation was also inhibited by 3-MA or ATG5 si RNA.HPLC detection showed that autophagy inhibition significantly weakened the role of FGF21 in reducing TC,FC and CE.The results of oil red O staining showed that inhibition of autophagy weakened the function of FGF21 in promoting cholesterol outflow and reducing lipid accumulation in foam cells.Compared with the control group and the HFD group,the level of RACK1 protein was significantly increased by intraperitoneal injection of FGF21 in apo E^-/-mice.Compared with HFD+FGF21 ip apo E^-/-mice,LC ?/? ratio and Beclin1 expression were decreased in the HFD+FGF21 ip+RACK1 sh RNA group,while P62 expression was increased.In addition,AS lesions were increased in the RACK1 knockdown group compared with the HFD group.RACK1 si RNA inhibited the transformation of LC3-I to LC3-? and beclin-1 expression while up-regulating P62 and inhibiting autophagosome formation.In addition,RACK1 knockdown significantly restrain the role of FGF21 in reducing TC,FC and CE levels and promoting cholesterol outflow from foam cells.