Effects Of Smoke Exposure On Lipid Metabolism And Lipid Deposition In Arterial Wall And Protection Of Melatonin

BackgroundCigarette smoke(CS)exposure is an important risk factor for lipid metabolism disorders and atherosclerotic vascular diseases,but the specific mechanism of the effects of smoking on lipid metabolism and lipid deposition in arterial wall is still unclear.ObjectivesTo study the effects of CS exposure on lipid metabolism and lipid deposition of arterial wall in mice and its possible molecular mechanism,as well as the protective effect of melatonin.MethodsPart 11.C57BL/6J mice were randomly divided into three groups:CS exposure group,melatonin group and control group.The expression of CD68 and ICAM-1 in aorta was evaluated by immunohistochemistry.The expression of low density lipoprotein receptor(LDLR)and proprotein convertase subtilisin/kexin type 9(PCSK9)in liver were analyzed by Western blot(WB),to investigate the effects of CS exposure on lipid metabolism.2.HepG2 cells were cultured and treated with CS extract(CSE)in vitro.The cell viability was detected by CCK-8 kit.The total reactive oxygen species(ROS)in cells was measured by 2,7-dichlorofluorescein diacetate fluorescent probe.The expression of LDLR and PCSK9 genes and proteins was detected by RT-qPCR and WB.Part 21.ApoE-/-mice were used to construct animal models and were also divided into three groups.The contents of scavenger receptor class B type 1(SR-B1)and CD31 in the aorta of mice were evaluated by immunohistochemical staining,the lipid deposition in the arterial wall was evaluated by oil red O staining,and the level of ROS in the aorta was detected by dihydroethidine.2.Human umbilical vein endothelial cells(HUVECs)were cultured and treated with CSE.CCK-8 kit was used to detect cell viability,and WB was used to detect the expression of SR-B1.Transwell technique was used to evaluate the transcytosis of LDL-C of HUVECs.3.The carotid plaque samples of smoking and non-smoking patients with carotid endarterectomy were collected(5 smokers and 5 nonsmokers),and the expression of SR-B1 in plaque was detected by immunohistochemistry and WB.ResultsPart 1The levels of serum total cholesterol(TC),triglycerides(TGs)and low density lipoprotein cholesterol(LDL-C)in serum and the expressions of CD68 and ICAM-1 in arterial wall of CS exposed mice were significantly higher than those of control group(P<0.05),and the expression of LDLR was significantly lower than that of control group(P<0.05).The levels of TC,TGs and LDL-C in serum and the expressions of CD68 and ICAM-1 in arterial wall in melatonin group were significantly lower than those in CS exposure group(P<0.05).When HepG2 cells were treated with CSE,the expression of LDLR decreased and PCSK9 increased.Pretreatment with ROS scavenger NAC or NF-B inhibitor BAY11-7082 or melatonin significantly attenuated CSE induced changes in PCSK9 and LDLR expression.However,the therapeutic effect of melatonin can be blocked by SIRT1 inhibitor Inauhsin.Part 2Compared with the control group,the lipid deposition in the arterial wall of CS exposure group was significantly increased(P<0.05),and the expression of SR-B1 and CD31 and the level of ROS in the arterial wall were significantly increased(P<0.05).In melatonin group,the lipid deposition in arterial wall was significantly reduced(P<0.05),the expression of SR-B1 and CD31 and the level of ROS in arterial wall were significantly decreased(P<0.05).In vitro experiments showed that CSE could promote the expression of SR-B1 in HUVECs and enhance the transcytosis of LDL-C.Melatonin could inhibit the expression of SR-B1 in HUVECs and decreased the transcytosis of LDL-C.Clinical exploratory study found that SR-B1 was expressed in the carotid plaques of smoking and non-smokers,and the expression level in plaques of smoking patients was significantly higher than that of non-smokers(P<0.05).ConclusionsCS exposure increased the level of circulating LDL-C via reducing the expression of LDLR on the surface of hepatocytes,and increased the expression of SR-B1 in vascular endothelial cells,promoting lipid deposition in the arterial wall.Melatonin inhibited the expression of PCSK9 and increased the content of LDLR in hepatocytes,thereby regulating the dyslipidemia caused by CS exposure.It also inhibited the expression of SR-B1 to weaken the transcytosis of LDL-C by vascular endothelial cells,reducing arteries lipid deposition on the wall.This study reveals a new mechanism of CS exposure to aggravate atherosclerosis,and provides a new theoretical basis and therapeutic target for the treatment of vascular lesions caused by CS exposure.