| Melatonin（MT）is a neuroendocrine hormone,mainly synthesized in the pineal gland.Many studies have confirmed that the supplementation of melatonin plays a role in the prevention of coronary heart disease.In recent years,it has been discovered that melatonin has anti-atherosclerosis（AS）effects.Melatonin can inhibit the progression of AS inapoE-/-mice,and its mechanism is related to inhibiting the level of mitochondrial reactive oxygen species（mt ROS）of macrophages and the activation of NLRP3,and alleviating the dysfunction of endothelial cells and the release of inflammatory factors in hyperlipidemia rats.It has a protective effect on vascular endothelial cells（VECs）.Pyroptosis is a form of programmed cell death.Pyroptosis is an important form of newly discovered VECs damage.MT can antagonize VECs pyroptosis.Proprotein convertase subtilisin/kexin type 9（proprotein convertase subtilisin/kexin type 9,PCSK9）is closely related to the occurrence and development of AS.It mainly participates in the process of AS through lipid pathways and non-lipid pathways.PCSK9 has damage to VECs role,can PCSK9 cause pyroptosis damage to VECs? Can MT antagonize the damage of PCSK9 to VECs by regulating the expression of PCSK9,and antagonize the damage of PCSK9 to VECs by means of pyroptosis inhibition? These are not clear,so this study intends to conduct experiments on human umbilical vein endothelial cells（Human umbilical vein endothelial cells,HUVECs）andapoE-/-mouse AS animal models.First,analyze the effect of oxidized low density lipoprotein（oxLDL）on HUVECs pyroptosis and whether PCSK9 is involved in the process,and analyze the full transcriptome of HUVECs via oxLDL,including circRNA,miRNA and genes,and systematically observe their differential expression profiles,screen and search for the circRNA and miRNA corresponding to PCSK9,establish a circRNA-miRNA-PCSK9 regulatory roadmap,and further verify the existence of this pathway through silencing/overexpression,and its involvement in the pyroptosis of VECs caused by oxLDL,whether MT antagonizes the pyroptosis of VECs caused by oxLDL through the circRNA-miRNA-PCSK9 pathway.Based on the above work,the existence of this pathway and its participation in AS were verified on theapoE-/-mouse AS animal model.Part Ⅰ oxLDL promotes HUVECs pyroptosis by up-regulating PCSK9 expressionObjective: To observe the effects of oxLDL on the expression of pyroptosis and pyroptosis-related molecules,as well as the expression of PCSK9 at the cellular level,and to analyze the mechanism of action from the expression of the mitochondrial gene UQCRC1 and mitochondrial function,Methods: HUVECs were cultured,PCSK9 and UQCRC1 were silenced by small interfering RNA and lentivirus transfection,mRNA expression level was detected by fluorescence real-time quantitative PCR,protein level was detected by western blot,Mito SOX Red probe was detected for mitochondrial reactive oxygen species（mt ROS）,and mitochondria were detected by JC-1 method membrane potential,phosphomolybdic acid colorimetric method was used to detect mitochondrial ATP content,immunofluorescence method was used to detect PCSK9 protein level,and propidium iodide（PI）and Hoechst33342 were used to detect cell chromosome breakage.HUVECs were cultured,incubated with oxLDL（50 and 100μg/ml）and HUVECs for 24 hours,western blot was used to detect the expression of pyroptosis-related molecules NLRP3,caspase-1 and IL-18,and qRT-PCR and ELISA to detect the expression and expression of IL-1β Release,PI/Hoechst 33342 double staining to detect cell chromosome breaks,enzyme method to measure LDH content to analyze cell damage,western blot,qRT-PCR to detect the expression of PCSK9 at protein and mRNA levels;small interfering RNA technology was used to knock down the expression of PCSK9 in HUVECs,and the PCSK9 overexpression lentiviral vector constructed by transfection to make PCSK9 highly expressed,qRT-PCR and western blot detection of PCSK9 mRNA and protein levels,analysis of PCSK9 expression efficiency,western blot detection of pyroptosis-related protein and UQCRC1 expression,ELISA detection of cells IL-1β expression in the supernatant,qRT-PCR to detect cell IL-1β mRNA expression level,enzymatic method to determine the supernatant LDH level,propidium iodide（PI）/Hoechst33342 double staining to detect cell chromosome breakage,phosphomolybdic acid ratio color method was used to detect mitochondrial ATP content,Mito SOX Red probe to detect mt ROS,after JC-staining,the mitochondrial membrane potential level was measured with fluorescence microscope and flow cytometer respectively;to further analyze whether PCSK9 caused mitochondrial dysfunction by down-regulating UQCRC1,the UQCRC1 was further constructed Small interfering RNA knocks down the expression of UQCRC1.The experiment is divided into control group,oxLDL group,small interfering RNA control group,oxLDL+si PCSK9,oxLDL+si PCSK9+si UQCRC1 group,a total of 5 groups,qRT-PCR and western blot detection of UQCRC1 mRNA and protein level,analysis of silencing efficiency,detection of mt ROS,ATP,and mitochondrial membrane potential to analyze changes in mitochondrial function.Results: oxLDL can up-regulate the expression levels of NLRP3,caspase-1,and IL-18.The results of qRT-PCR and ELISA show that oxLDL can promote the expression and release of IL-1β.The damage level of HUVECs increases after oxLDL treatment,and is concentration-dependent.The best effect is when the concentration reaches 100μg/ml and the time to treat cells for 24 hours.After HUVECs are treated with different concentrations of oxLDL（50 and 100μg/ml）,PCSK9 expression increases,and the cell immunofluorescence results are the same,that is,after different concentrations of oxLDL treat HUVECs with the increase of fluorescence intensity,oxLDL can up-regulate the expression level of PCSK9 in HUVECs in a concentration-dependent manner.PCSK9 si RNA was transfected to knock down the expression of PCSK9 in HUVECs.The expression of PCSK9 in the interference-positive group was significantly decreased,indicating that the interference was successful;western blot results showed that compared with the oxLDL group,the expression level of NLRP3,caspase-1,IL-1β and IL-18 in the oxLDL+si PCSK9 group was significantly reduced,and the expression and release levels of IL-1β were also reduced,and the detection of LDH activity decreased.The results of PI/Hoechst 33342 double staining showed that the cell death rate was reduced after interference with PCSK9,and the results showed that knockdown of PCSK9 expression can inhibit the pyroptosis of HUVECs induced by oxLDL.The PCSK9 overexpression lentiviral vector constructed by transfection,qRT-PCR and western blot results showed that HUVECs were successfully transfected with overexpression lentivirus,and the expression levels of NLRP3,caspase-1,IL-1β and IL-18 were obvious after PCSK9 was overexpressed increased expression and release of IL-1β;after overexpression of PCSK9,LDH activity increased.The results of PI/Hoechst 33342 double staining showed that cell mortality was significantly increased.The above results indicate that up-regulating the expression of PCSK9 can promote the pyroptosis of HUVECs.The high expression of PCSK9 can promote the increase of ROS levels and the decrease of ATP production,indicating that the up-regulated PCSK9 may cause mitochondrial dysfunction,which in turn promotes the increase of ROS levels;after overexpression of PCSK9,the UQCRC1 level is found to decrease.The above results indicate that PCSK9 may mediate mitochondrial dysfunction by inhibiting the expression of UQCRC1,and promote the increase of mt ROS,which in turn leads to the occurrence of pyroptosis.To further verify that PCSK9 promotes mitochondrial dysfunction through UQCRC1,after transfection of PCSK9 si RNA to detect whether the expression of UQCRC1 can be restored,the results of western blot showed that the expression of UQCRC1 increased after interference with PCSK9 in the oxLDL treatment group.PCSK9 si RNA and UQCRC1 si RNA were transfected at the same time.It was verified that UQCRC1 si RNA was successfully transfected.After the cells transfected with PCSK9 si RNA and UQCRC1 si RNA were incubated with oxLDL,the mt ROS level was detected.The results showed that compared with the oxLDL+si PCSK9 group,it interfered with the increase of ROS levels in PCSK9 and UQCRC1 cells,and the degree of mitochondrial membrane potential disorder increased.The above results indicate that PCSK9 may promote mitochondrial dysfunction and increased ROS production by regulating UQCRC1,thereby promoting the occurrence of pyroptosis.Summaries: oxLDL can up-regulate the pyroptosis of HUVECs and promote the expression of PCSK9;PCSK9 mediates the pyroptosis of HUVECs induced by oxLDL through the UQCRC1/ROS pathway.Part Ⅱ Melatonin regulates endothelial cell pyroptosis through the circ＿0000033/miR-214-3p/PCSK9 pathwayObjective: Previous studies have found that melatonin（MT）has the effect of antagonizing oxLDL-induced pyroptosis of vascular endothelial cells and anti-AS,but its mechanism of action is not systematic.Therefore,this section intends to analyze the transcriptome analysis of HUVECs after oxLDL and MT are processed to screen.Differentially express circRNA,miRNA and differentially expressed genes,and systematically explore the molecular mechanism of oxLDL up-regulating PCSK9 to promote the pyroptosis of HUVECs.Take PCSK9 as the target gene,obtain the miRNA with PCSK9 as the target gene from the differential miRNA,and then trace the circRNA that binds to the miRNA to find the circ-miR-PCSK9 pathway of MT anti-vascular endothelial cell pyroptosis,and combine with the change of mitochondrial function.To explore its mechanism more systematically.Methods: For whole transcriptome analysis,HUVECs were divided into 3 groups: control group,oxLDL treatment group,and oxLDL+melatonin pretreatment group.After the cells were treated for 24 hours,RNA was extracted.Subsequently,after quality inspection and pre-processing on the machine,the BGI DNBseq platform was used to sequence the whole transcriptome,the Pearson correlation coefficient was used to analyze the correlation between samples,and the BGI Gene Dr.TOM system was used for differential gene and enrichment analysis,KEGG The pathway analyzes the cell biological processes involved in all differential genes,and screens differentially expressed circRNA,miRNA and differentially expressed genes.Find the mRNA sequence of human circ＿0000033（flank-hsa＿circ＿0000033）based on NCBI,design primers through Primer Premier 5.0,and add restriction enzymes Hind III and Bam HI to the 5’end.RNA was extracted from 293 T cells and then reverse transcribed into c DNA,PCR amplified and electrophoresed to obtain the target fragment（identification sequence is as follows）.After digesting pc DNA3.1 vector,the target fragment was recovered.After ligating the vector and the target gene fragment,the positive plasmid was screened and extracted and verified by sequencing.After transfection of 293 T cells,the multiplicity of infection（MOI）was analyzed,and the transfection effect was detected by qRT-PCR.Select JASPAR software to predict the targeted binding effect of circ＿0000033 with miR-214-3p,select the species and gene name,enter the predicted gene type to screen the combined transcription factor and the corresponding binding site.Use this database to analyze the targeted binding of circ＿0000033 to miR-214-3p and the binding site.The combination of miR-214-3p and PCSK9 uses Target Scan（http://www.targetscan.org/mamm＿31/）to predict targeted gene binding,and check the binding advantage score and binding site.qRT-PCR and western blot were used to detect the mRNA and protein levels of PCSK9,the level of LDH in the supernatant was determined by enzymatic method,the chromosome breaks were detected by double staining with propidium iodide（PI）/Hoechst33342,and the mitochondrial ATP content was detected by phosphomolybdic acid colorimetry,Mito SOX Red probe detects mt ROS,after JC-staining,the mitochondrial membrane potential level is detected by fluorescence microscope and flow cytometer respectively,and the dual luciferase gene reporter system analyzes the targeted binding of miR-214-3p and PCSK9,the fluorogen Position hybridization to detect the localization and binding of circ＿0000033 and miR-214-3p.OUP was significantly reduced,the expression of PCSK9 protein was decreased,and the expression of UQCRC1 protein was significantly increased.The above results are in the overall level of animals verified that MT can inhibit the occurrence of endothelial cell pyroptosis,and it is related to the expression levels of PCSK9 and UQCRC1.qRT-PCR was used to detect the effects of circ＿0000033 and miR-214-3p in the aortic tissues of the two groups of mice.The results showed that the level of circ＿0000033 in the melatonin group decreased significantly compared with the control group,while the level of miR-214-3p increased significantly.This shows that at the overall level of animals,MT can also inhibit the expression of circ＿0000033 and reduce its binding to miR-214-3p,thereby increasing the binding of miR-214-3p to target genes,thereby alleviating pyroptosis and ultimately inhibitingapoE-/-The progression of AS in mice.Results:A total of 1920 small RNAs were identified by sequencing on the whole transcriptome platform,and the correlations were all higher than 95.6%,which can be used for subsequent analysis.Compared with the control group,the oxLDL group had 14,356 genes down-regulated and 14,782 genes up-regulated（including PCSK9）;the oxLDL+melatonin group had 267 genes down-regulated and 479 genes up-regulated compared to the oxLDL group.The KEGG pathway analyzes the cellular biological processes involved in all differential genes,including transmission and metabolism,signal transduction,and transcription.The above results indicate that after the treatment of oxLDL and melatonin,the related genes in the cell changed significantly in response to external stimuli.oxLDL treatment significantly increased the expression of NLRP3,Caspase1 and IL-1β,but the expression of these molecules decreased after MT treatment.Compared with the oxLDL group,MT reduced the expression of IL-1β at the mRNA level.After pretreatment of HUVECs with 20μM MT for 2h,the expression of PCSK9 induced by oxLDL was significantly inhibited.These results indicate that MT can significantly alleviate the pyroptosis of vascular endothelial cells caused by oxLDL.To start with circRNA,explore the mechanism of ce RNA regulated by MT.First,after taking the average of the differential gene expression,take the logarithm of the expression of the oxLDL group/control group,and then take the logarithm of the expression of the oxLDL+MT group/oxLDL group for analysis.After the analysis,a total of 26 circRNAs were up-regulated by oxLDL,Can be down-regulated by MT processing.Among them,circ＿0000033 has the largest difference after taking the logarithmic value.The LV-circ＿0000033 lentiviral vector was constructed,and it was found that as the concentration of the viral vector decreased,the fluorescence intensity in the cells decreased,indicating that the transfection efficiency was significantly concentration-dependent.Compared with the blank control group,TUNEL positive cells increased after overexpression of circ＿0000033 alone,while the positive cells of the MT treatment group decreased.After overexpression of circ＿0000033 alone,the expression of pyroptosis-related molecules（NLRP3,Pro-caspase1,Caspase1,GSDMD,IL-18 and IL-1β）was significantly up-regulated,indicating that overexpression of circ＿0000033 can activate the pyroptosis pathway caused by NLRP3;while the expression of these pyroptosis-related molecules in the MT treatment group was reduced,indicating that MT can inhibit the pyroptosis pathway.In order to further detect pyroptosis cells,LDH and PI/Hoechst33342 double staining were used to measure dead cells and DNA fragmentation respectively.After the expression of circ＿0000033 increased,the content of LDH in HUVECs increased,indicating that the level of dead cells increased.The results of PI/Hoechst33342 double staining showed that compared with the blank control group,the positive area of the transfected LV-circ＿0000033 group increased,and the fluorescence intensity of the MT treatment group decreased.These results indicate that increased expression of circ＿0000033 can cause cell pyroptosis and activation of the pyroptosis pathway,and MT can inhibit this process.JC-1 staining flow cytometry results showed that compared with the control group,overexpression of circ＿0000033 can cause disturbance of mitochondrial membrane potential,increase of mt ROS level,and decrease of intracellular ATP content,while MT treatment can alleviate this trend.The above results indicate that MT can alleviate mitochondrial dysfunction caused by increased expression of circ＿0000033.Bioinformatics analysis of target miRNA that circ＿0000033 may bind.After transfection of circ＿0000033 wildtype（WT）,miR-214-3p mimic or miR-214-3p inhibitor was given.The results showed that the fluorescence intensity of the transfected miR-214-3p inhibitor group was significantly higher than that of the miR-214-3p mimic group,indicating that circ＿0000033 has a strong binding with miR-214-3p.In situ hybridization after fluorescent probes showed that both circ＿0000033 and miR-214-3p were distributed in the cytoplasm and their fluorescence was superimposed.These results indicate that circ＿0000033 can target miR-214-3p.After miR-214-3p inhibitor transfection,the TUNEL area increased.Compared with the miR-214-3p inhibitor group,the TUNEL area and pyroptosis-related molecules decreased after MT treatment,and the expression level of pyroptosis molecules increased in the miR-214-3p inhibitor group;Compared with the miR-214-3p inhibitor group,the miR-214-3p inhibitor+MT group had a significant decrease in pyroptosis molecules.MT also reduces the intracellular expression and extracellular release of IL-1β.Subsequently,LDH levels and PI/Hoechst33342 positive cells were detected,and it was found that inhibiting miR-214-3p could increase intracellular LDH levels and PI/Hoechst33342 double staining positive cells,and MT treatment could inhibit this phenomenon.This indicates that MT can inhibit the pyroptosis of HUVECs caused by the decreased expression of miR-214-3p.To explore whether MT regulates mitochondrial function through the miR-214-3p pathway,the miR-214-3p inhibitor was transfected.The results showed that compared with the control group,the mitochondrial membrane potential was disturbed,the ATP production decreased,the release of mt ROS increased,these can be alleviated by MT.The above results indicate that inhibiting the expression of miR-214-3p can cause mitochondrial dysfunction and the generation of ROS,and MT can inhibit this process.Bioinformatics analysis miR-214-3p and PCSK9 have targeted binding sites,dual luciferase reporter system analysis,using fluorescent probe to label miR-214-3p,immunofluorescence detection of PCSK9.The results showed that miR-214-3p and PCSK9 are mainly distributed in the cytoplasm,a small amount in the nucleus,and a large amount of binding in HUVECs.This also shows that MT can inhibit its expression through miR-214-3p targeting binding to PCSK9.Summaries: MT inhibits oxLDL-induced VEC pyroptosis and pyroptosis-related molecule expression;MT inhibits oxLDL-induced VEC pyroptosis through the circ-0033/miR-214-3p /PCSK pathway.Part Ⅲ The effect of melatonin on the circ＿0000033/miR-214-3p/PCSK9 pathway and the progression of AS inapoE-/-miceObjective: To verify the existence of the circ＿0000033/ miR-124-3p/PCSK9 pathway and the influence of MT on this pathway onapoE-/-mouse AS animal models,and to discover a new mechanism of MT’s anti-AS effect.Methods: 40 maleapoE-/-mice were fed on a high-fat diet for 8 weeks and randomly divided into 2 groups: 1.Control group（n=20）: high-fat diet,intraperitoneal injection of 200 μl of normal saline twice a week;2.MT intervention group（n=20）: high-fat diet,melatonin perfusion,10mg/kg/day each time,continuous treatment for 8 weeks,continued intraperitoneal injection of melatonin 10mg/kg,twice a week.They were put to death after completing various tests at 16 weeks.Eyeball blood is used to detect blood lipids and related inflammation indicators.Separate blood vessels and surrounding tissues,expose the aortic arch and main branches,take and record with a stereo microscope.The heart was removed,and the aortic sinus section was serially sectioned with a thickness of 7 μm.After the frozen section,HE,Oil Red O,and Masson staining were performed to measure the AS plaque area of the aorta.The full-length aorta was taken for mRNA and protein detection.Enzymatic oxidation method detects plasma TG,TC,LDL-C,HDL-C content.qRT-PCR detects the mRNA expression of aortic miR-214-3p,circ＿0000033,pyroptosis-related molecules（NLRP3,caspase1,IL-18,GSDMD）,and UQCRC1,and Western Blot detects aortic protein pyroptosis-related molecules（NLRP3,caspase1,IL-18,GSDMD）,PCSK9,UQCRC1 protein expression levels.Immunofluorescence was used to detect the protein expression levels of murine pyroptosis and pathway-related proteins（caspase1,GSDMD）,endothelial cell CD31 markers,PCSK9,and UQCRC1.ELISA was used to detect serum IL-1β and IL-18 levels in mice.Summaries: MT reduces apoE-/-mouse aortic AS lesions and lowers TG、TC and LDL-Clevels;MT reduces the expression ofapoE-/-mouse aortic pyroptosis-related molecules and PCSK9,and promotes the expression of UQCRC1;MT up-regulatesapoE-/-mouse aortic circ＿0000033,down-regulates the expression of miR-214-3p.Conclusions1.oxLDL up-regulates the expression of PCSK9,thereby activating the UQCRC1/ROS pathway,leading to pyroptosis of HUVECs;2.Melatonin inhibits the expression of pyroptosis-related molecules induced by oxLDL through the circ＿0000033/miR-214-3p/PCSK9 pathway,thereby inhibiting HUVECs pyroptosis;3.Melatonin down-regulates the aorta circ＿0000033 of high-fat diet fedapoE-/-mice,up-regulates the expression of miR-214-3p,thereby inhibiting the expression of PCSK9 and the expression of pyroptosis-related molecules,reducing vascular endothelial cell pyroptosis,and reducingapoE-/-AS lesions in mouse aorta. |
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