"Gualou-Xiebai" Reduces Vascular Smooth Muscle Cell-derived Foam Cell Formation By Down-regulating Rab18 Down-inhibiting Ferroptosis

Atherosclerosis(AS)is a chronic inflammatory disease characterized by abnormal deposition of lipids,inflammatory cells,calcium ions and other atherosclerotic plaques in the intima of arteries,which is the pathological basis of many cardiovascular diseases.Foam cell is a kind of cell filled with lipids,which is the main component of atherosclerotic plaque in the intima of blood vessels,and its formation is an important sign of atherosclerosis.Vascular smooth muscle cells(VSMCs)are the main cells forming the vascular medium membrane and one of the main sources of foam cells.Ferroptosis is an important player in the course of AS disease,and is a novel cell death procedure that depends on excess iron lipid peroxidation.Excess iron resulted in decreased expressions of glutathione peroxidase 4(GPX4)and solute carrier family 7member 11(SLC7A11),decreased levels of antioxidant superoxide dismutase(SOD)and glutathione(GSH),and inhibited the cellular antioxidant system.At the same time,excess iron and inhibition of antioxidant system led to increased expression of ACSL4,the level of lipid peroxides malondialdehyde(MDA)and reactive oxygen species(ROS),and increased lipid peroxidation.Increased lipid peroxidation caused damage to mitochondria and other organelles and cell membranes,and was the direct cause of endothelial cell injury,VSMCs proliferation and foam cell formation caused by ferroptosis,which promoted the occurrence of AS.It has been shown that increased lipid peroxidation caused by ferroptosis promotes the formation of macrophage-derived foam cells.The lipid peroxides produced by the death of iron catalyze the oxidation of low-density lipoprotein(LDL)and increase the expression of ox-LDL receptor(ox-LDL)in cells to promote the phagocytosis of macrophages to ox-LDL and accelerate the formation of foam cells.At the same time,excess iron in cells reduced the expression of transcription factor liver X receptor αand ATP binding box transporter,reduced cholesterol efflux in macrophages,and further promoted the formation of foam cells.In addition,excess iron in cells can also increase the level of ferrostatin by activating inflammatory pathways,promote iron absorption in cells,further aggravate ferroptosis in cells,and promote foam cell formation.Ferroptosis inhibitors can reduce iron excess and lipid peroxidation in macrophages,reduce lipid levels in cells,and reduce lipid deposition,suggesting that inhibiting ferroptosis is an important means to reduce the formation of macrophage derived foam cells.However,the role of ferroptosis in the formation of VSMCs-derived foam cells is rarely reported.Lipid peroxidation is an important link in the ferroptosis and A direct factor in the formation of foam cells.The long chain family of Acyl-Co A synthetase(ACSLs)is an important factor in the regulation of lipid synthesis and lipid peroxidation in cells.Studies have shown that Rab18 can participate in lipid metabolism and regulate lipid peroxidation by regulating ACSLs.These results suggest that Rab18 may be involved in ferroptosis by regulating lipid peroxidation.Fructus Gualsanthostemon(GLXB)first appeared in the classic prescription for the treatment of "chest Bi" in the Synopsis of the Golden Chamber in the Han Dynasty.It has the effects of dispersing Yang and clearing up tangles,removing phlegm and reducing turbidity,and is mainly used in the treatment of metabolic diseases such AS AS in modern clinic.Previous studies have shown that GLXB can lower blood lipid levels,reduce oxidative stress,inhibit foam cell formation and treat AS.Previous experiments have also shown that GLXB can reduce the expression of Rab18 in vascular smooth muscle cells,but the effect of GLXB on vascular smooth muscle cell-derived foam cells and the specific mechanism of reducing the formation of foam cells are not completely clear.Starting from the formation of foam cells caused by this course,the effect and specific mechanism of GLXB on the formation of VSMCs-derived foam cells were investigated based on ferroptosis,so AS to provide a scientific basis for the treatment of AS by GLXB.OBJECTIVEThe effect and mechanism of GLXB on the formation of VSMCs derived foam cells were investigated based on ferroptosis.METHODSOx-LDL induced VSMCs,and VSm CS-derived foam cell model was established.The formation of VSm Cs-derived foam cells was defined as the cell esterification rate was greater than 50%.CCK8 assay was used to detect cell viability and screen drug stimulation concentration.Lipid accumulation was detected by oil red O staining,and total cholesterol(TC)and free cholesterol(FC)levels were detected by enzyme method.Mitochondrial morphology and oxidative damage were observed by transmission electron microscopy.Ferrozine microplate method was used to detect iron content in cells.MDA and GSH levels were detected by microplate method,SOD levels were detected by hydroxylamine method,ROS levels were detected by fluorescent probe,and the degree of lipid peroxidation was detected.The expressions of GPX4,SLC7A11,ACSL4 and Rab18 were detected by Western blotting.Rab18 overexpression plasmid and ferroptosis agonist Erastin were used to investigate the effect of Rab18 on the inhibition of ferroptosis by GLXB and the formation of VSm Cs-derived foam cells.RESULTS1.GLXB reduces the formation of VSMCS-derived foam cellsOx-LDL induced the formation of VSm CS-derived foam cells.GLXB significantly reduced lipid levels,esterification rate and lipid droplet accumulation,and inhibited the formation of VSm Cs-derived foam cells.2.GLXB reduces the formation of VSMCs-derived foam cells by inhibiting ferroptosisGLXB significantly inhibited mitochondrial shrinkage and the reduction of "ridge" structure in VSMCs.The levels of total iron,MDA and ROS were decreased,the levels of SOD and GSH were increased,the expressions of antioxidant regulatory proteins GPX4 and SLC7A11 were increased,and the expressions of lipid peroxidation regulatory protein ACSL4 were decreased.After the treatment with Erastin,the inhibitory effect of GLXB on the formation of VSm CS-derived foam cells was reversed,the lipid level and lipid droplet accumulation were increased,and the esterification rate was significantly increased.The levels of total iron,MDA and ROS increased,the levels of SOD and GSH decreased,the expressions of GPX4 and SLC7A11 decreased,and the expressions of ACSL4 increased.3.GLXB inhibits ferroptosis and reduces the formation of VSMCs-derived foam cells by down-regulating Rab18GLXB significantly decreased the expression of Rab18 in VSMCs.After Rab18 overexpression,the inhibitory effect of GLXB on ferroptosis and the formation of VSm Cs-derived foam cells was cancelled,and the lipid level and lipid droplet accumulation in cells were increased,and the esterification rate was increased.The levels of total iron,MDA and ROS increased,the levels of SOD and GSH decreased,the expressions of GPX4 and SLC7A11 decreased,and the expressions of ACSL4 increased.CONCLUSION1.GLXB reduces ox-LDL-induced VSMCs-derived foam cell formation.2.GLXB inhibits ferroptosis of VSMCs and reduces the formation of VSm Cs-derived foam cells by inhibiting ferroptosis.3.GLXB inhibits ferroptosis and reduces the formation of VSMCs-derived foam cells by down-regulating Rab18.