| BackgroundAtherosclerosis is the accumulation of fatty and/or fibrous material in the innermost layer(intima)of the artery.The term atherosclerosis is derived from the Greek and reflects the appearance of the lipid material at the core of a typical atherosclerotic plaque.Over time,atherosclerotic plaques can become fibrous and calcify after calcium accumulates.Severe atherosclerotic plaque will invade the lumen of an artery,impeding blood flow and leading to tissue ischemia.Atherosclerotic cardiovascular disease(CVD)has been a major cause of vascular disease worldwide.When it affects coronary arteries,it will lead to acute coronary syndromes such as myocardial infarction or stable angina,chest pain or discomfort caused by underperfusion of the heart muscle.As for the pathogenesis of atherosclerosis,in the past 20 to 30 years,many studies have proved that atherosclerosis is closely related to a variety of risk factors,such as smoking,obesity,aging,inflammation,diabetes and hemodynamic disorders.Although more and more key regulatory factors are being identified to treat and mitigate atherosclerosis,the incidence of atherosclerosis remains high worldwide.Therefore,it is urgent to explore as many pathological mechanisms as possible and find out more effective therapeutic targets in the field of atherosclerosis.Therefore,in order to identify a novel gene with significant susceptibility to coronary artery disease(CAD)in the Han Chinese population,we conducted a genome-wide association study of 200 "diseased individuals" from the Shandong Research Center as northern populations and included them from the Shanghai Center(293 CAD/293 controls)as southern populations.Six SNPS of NPRC(RS700926,RS1833529,RS2270915,RS17541471,RS3792758 and RS696831)were found to be associated with CAD in northern and southern China,and the other two SNPS were associated with CAD in central China.However,the molecular mechanism of the association between NPRC gene polymorphism and CAD remains unclear.Natriuretic peptide(NPs)family consists of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and C-type natriuretic peptide(CNP)and their three receptors(NPRA,NPRB and NPRC).NPs have common structural and functional characteristics.Protein sequences of ANP,BNP,and CNP have been reported following de Bold's discovery of the natriuretic effects of atrial extracts in 1981.Subsequently,numerous studies have demonstrated complex physiological effects of NPs on multiple systems.For example,NPs secreted by the heart regulated the diastolic function of arteries and thus lowered arterial blood pressure.NPs in blood were viewed to regulate adipose tissue metabolism and fibrosis.CNP affected bone growth and development through its receptor and so on.Among the receptors of NPs,NPRA and NPRB have been studied most extensively.Previously,it was widely believed that NPs functions mainly through the binding of NPRA or NPRB,while NPRC is only the scavenging receptor of NPs,neutralizing their physiological functions through endocytosis and lysosomal degradation to enable the system to maintain homeostasis.Moreover,the affinity of NPs for the three receptors is also quite different.For NPRA,the sequence of natriuretic peptide family affinity was ANP>BNP>>CNP.For NPRB,the order is CNP>>ANP>BNP.Interestingly,NPRC has similar affinity with ANP,BNP and CNP.In other words,ANP and BNP are most likely to bind to NPRA,while NPRB is CNP's preferred ligand.However,in recent years,a growing number of studies have questioned this claim and found that NPRC,unlike NPRA and NPRB,which acted as guanine cyclase receptors,had no guanine cyclase activity but inhibited the activity of guanine nucleotide regulatory protein(Gi).One study showed that CNP maintained vascular homeostasis,including atherosclerosis,by binding to NPRC.In contrast,it has been suggested that the binding of CNP and NPRC may induce angiogenesis by activating Gi,ERK1/2,and phosphoinositol 3-kinase ?/Akt at the molecular level.However,these findings suggest that in addition to be a clear receptor of NPs,NPRC has its own potential signaling pathway and is closely associated with cardiovascular disease.However,few studies have reported the role of NPRC deficiency in atherosclerosis.Since atherosclerosis development involves a variety of pathological process and molecular mechanism,in this study,we propose the following hypothesis:In a high-fat diet ApoE-/-mice model of atherosclerosis,the deletion of NPRC can reduce inflammatory reaction and apoptosis in atherosclerosis plaques,thus improve the degree of atherosclerotic lesions.Objectives1.To explore the correlation between NPRC and the pathological progress of atherosclerosis.2.To explore whether NPRC is involved in regulating the degree of atherosclerosis.3.To explore whether NPRC is involved in the regulation of inflammatory infiltration of atherosclerotic plaque.4.To explore whether NPRC is involved in the regulation of endothelial apoptosis in atherosclerosis.Methods1.AnimalsNPRC gene knockout(NPRC-/-mice were constructed on C57BL/6 background mice using CRISPR-Cas9 technique.After crossed with ApoE-/-mice,homozygous ApoE-/-mice and homozygous ApoE-/-NPRC-/-mice were obtained.ApoE-/-mice were purchased from Beijing Viewsolid Biotechnology Co.LTD.(Beijing,China).2.Animal experiments grouping(1)20 eight-week-old male ApoE-/-mice were randomly divided into two groups,10 mice in each group:Chow diet group and Western diet group.(2)50 eight-week-old male ApoE-/-mice and male ApoE-/-NPRC-/-mice were randomly divided into two groups,25 mice in each group:ApoE-/-group and ApoE-/NPRC-/-group.3.Mouse model of ASMale ApoE-/-mice and male ApoE-/-NPRC-/-mice aged 8 weeks were fed with western diet(0.2%cholesterol,21%milk fat)throughout the whole period,and adequate water was guaranteed.The state of the mice was observed every day during the period.After 12 weeks,the mice were euthanized,and blood,heart,liver,kidney,spleen,adipose tissue and aortic tissue samples were collected for subsequent experiments.4.Mouse genotype identificationThe mouse tail DNA was extracted with the mouse tail DNA extraction kit,and the mouse genotypes were identified by sequencing after PCR amplification.In addition,aortic tissue proteins were extracted from mice,and the protein expression level of NPRC was detected by Western blot analysis to evaluate the efficiency of gene NPRC knockout.5.Body weight and body length measurement of miceThe weight of ApoE-/-group and ApoE-/-NPRC-/-group mice in second part of in vivo experiments was weighed by an electronic balance(Shimadzu Corp).Each mouse was measured 3 times and the average was taken as the final weight value.Body length of ApoE-/-mice and ApoE-/-NPRC-/-mice was measured 3 times per mouse using a 30 cm scale,and the average was taken as the final value of body length.6.Blood pressure and heart rate measurement of miceThe blood pressure and heart rate of ApoE-/-group mice and ApoE-/-NPRC-/group mice were measured by a non-invasive mouse tail sphygmometer at the location of the caudal artery of the mouse.The blood pressure and heart rate of each mouse were measured continuously for 3 times and the average value was taken as the final value.7.Tissue sampling of miceAt the end of the animal experiment,the whole blood,serum,heart,liver,spleen,kidney,adipose tissue and aorta of the mice were collected after euthanasia.Part of each tissue was put into liquid nitrogen for quick freezing and stored at-80?,while part was fixed in 4%paraformaldehyde for subsequent experimental use.8.Blood lipid measurement in serumThe serum from ApoE-/-group mice and ApoE-/-NPRC-/-group mice were collected to detect the serum levels of total cholesterol,triglycerides,low density lipoprotein cholesterol,high density lipoprotein cholesterol and very low density lipoprotein cholesterol.9.Meso Scale Discovery(MSD)was used to detect inflammation-related indexes in serumThe serum levels of TNF?,IL-6,MCP1,ICAM1 and VCAM1 of ApoE-/-group mice and ApoE-/-NPRC-/-group mice were detected by MSD multifactor detection plate and MSD detector.10.NPs test in serumThe serum from ApoE-/-group mice and ApoE-/-NPRC-/-group mouse were collected and the levels of serum atrial natriuretic peptide(ANP),brain Natriuretic peptide(BNP)and C-type Natriuretic peptide(CNP)were detected by ELISA method.11.RNA-seqThe aortic tissues of five ApoE-/-group mice and five ApoE-/-NPRC-/-group mice fed with a western diet for 12 weeks were removed,infiltrated in RNA later liquid,frozed in liquid nitrogen,and sent to Hangzhou Lianchuan Biotechnology Co.LTD(Hangzhou,China)for RNA sequencing.12.Pathological examinationOil red O staining was performed in en face aorta.The aorta root was prepared by continuous frozen section,mainly by H?E staining,oil red O staining and Picrosirius Red staining.Immunohistochemical staining was performed for NPRC,?-SMA,MOMA2,TNF?,IL-6,MCP1,ICAM1,VCAM1,PPAR? and PGC1?,and immunohistofluorescence staining was performed for NPRC.13.Western blot analysisProtein extraction kit was used to extract mouse aortic tissue protein,or cell lysis was used to extract total HUVEC protein.Protein concentration was measured first through BCA method,followed by SDS-PAGE gel electrophoresis.The expression level of Protein NPRC,TNF?,IL-6,MCP1,ICAM1,VCAM1,PPAR?,PGC1?,P-CREB,CREB,P-VASP,VASP,eNOS,P-PKA,P-AKT1,AKT1,CASpase3,Cleaved-caspase3,Caspase7,Cleaved-caspase7,P-I???/?,I???,I???,p-P65,and P65 were measured.14.Tissue RNA extraction,reverse transcription and quantitative real-time PCR(RT-PCR)The aortic tissue RNA of ApoE-/-group mice and ApoE-/-NPRC-/-group mice and total cells were extracted,and the mRNA was reversely transcribed using TaKaRa#RR047A reverse transcription kit.Then the Ct values of target genes TNF?,IL-6,MCP1,PPAR?,PGC1? and eNOS were obtained by real-time quantitative PCR.With ?-actin as the reference gene,the obtained Ct values were substituted into the formula(2-??Ct)to calculate the relative expression levels of target genes.15.Cell culture and treatmentHuman primary umbilical vein endothelial cells(HUVEC)were cultured in basal medium containing 5%fetal bovine serum(FBS)and 1%endothelial growth factor(ECGF).Raw264.7 macrophages were cultured in DMEM containing 10%fetal bovine serum(FBS).All cells were proliferated in incubators containing 5%CO2 at 37?.The main treatments are as follows:(1)According to the oxLDL concentration gradient in preliminary experiments,HUVEC stimulated by 100mg/mL oxLDL for 12 or 24 hours expressed higher levels of TNF?,IL-6 and MCP1;When HUVEC was stimulated by 100mg/mLoxLDL for 0.5 h or 1 h,the phosphorylation level of pathway protein was higher.Therefore,the following cell experiments selected 100mg/mL oxLDL stimulation for 12 and 24 hours as the experimental conditions to detect protein expression,and for 0.5 and 1 hour as the experimental conditions to detect protein phosphorylation.(2)Experimental group HUVECs were added with 100mg/mL oxLDL,control group HUVECs were added with equal volume of 1XPBS.After stimulation for 12 and 24 hours,cells were collected;(3)Experimental group HUVECs were added with 100mg/mL oxLDL,control group HUVECs were added with equal volume of 1XPBS.After stimulation for 0.5 and 1 h,cells were collected;(4)Experimental group HUVECs were added with 100mg/mL oxLDL,and control group HUVECs were added with equal volume of 1XPBS.After stimulation for 24 hours,cell supernatants were collected.Macrophages with pre-planted plates were added in and stimulated for 12 hours to collect cells.16.Cell transfectionHUVECs were cultured in vitro and transfected with small interfering RNA of NPRC using RNAiMAX to knock down the expression of NPRC gene.17.Extraction of peritoneal macrophagesMale wild-type(C57BL/6)mice at 8-10 weeks were selected,and each mouse was intraperitoneally injected with lmL sterile 3%broth 3 days in advance.The peritoneum of mice was fully exposed after sacrificed.30mL serum-free DMEM was absorbed with a sterile syringe and lavaged into the abdominal cavity for 3 times until the DMEM became clear and cell suspension was collected.After centrifugation at 1,000 rpm for 5 min,the cells were re-suspended in a fresh complete medium and cultured for subsequent experiments.18.Cell migration assayHUVECs were stimulated with 100mg/mL oxLDL for 24 h and their supernatant was collected.Transwell cell migration assay was used to detect the migration ability of Raw264.7 cells and peritoneal macrophages under the supernatant of HUVECs stimulated by oxLDL.19.Macrophage oxLDL engulf assayHUVECs were stimulated with 100mg/mL oxLDL for 24 h and their supernatant was collected.The supernatant was added to the macrophage pre-planted on slides and stimulated with 100mg/mL oxLDL for 36 hours.After fixation with 4%paraformaldehyde,the macrophage slides were stained with oil red O.20.Detection and quantitation of apoptosisApoptotic levels of HUVECs were detected by TUNEL kit after treatment with 100mg/mL oxLDL for 24h.21.Statistics AnalysisAll data were analyzed using GraphPad Prism 8 software and expressed as meanąstandard error(SEM).First,the shapiro-Wilk test was used to evaluate the normality hypothesis of the data distribution.The unpaired T-test was used to analyze the statistical differences between two groups in the univariate data of normal distribution,while the univariate analysis of variance was used for the statistical differences between multiple groups.For multiple sets of data with two variables,after verifying that the data are normally distributed,two-factor analysis of variance is used to analyze the statistical differences.The kruskal-Wallis test was used for nonparametric statistical analysis of data that did not belong to the normal distribution.In all statistical comparisons,p values less than 0.05 were considered statistically significant.Results1.The expression level of NPRC in aorta tissue in a mouse model of atherosclerosis induced by a western diet was significantly up-regulatedWe successfully constructed a mouse model of atherosclerosis by feeding ApoE-/mice with a western diet for 12 weeks.Western blot and immunofluorescence staining of aortic tissue showed that NPRC expression in aorta tissues of ApoE-/-mice fed with western diet was significantly up-regulated compared with that of ApoE-/-mice fed with chow diet for 12 weeks.2.ApoE-/-NPRC-/-mice were constructed to establish atherosclerosis model induced by western diet(1)In the background of C57BL/6J mice,NPRC-/-mice were successfully constructed using CRISPR-Cas9 technique.ApoE-/-NPRC-/-mice were crossed with ApoE-/-mice to obtain ApoE-/-NPRC-/-mice and their ApoE-/-littermates.The genotypes of ApoE-/-NPRC-/-mice were confirmed by sequencing of mouse tail genomic DNA.Meanwhile,Western blot and immunohistochemical staining were used to futher verify the protein knockout efficiency of NPRC in aortic tissue of ApoE-/-NPRC-/mice.(2)The body weight and body length of two groups of mice(ApoE-/-group and ApoE-/-NPRC-/-group)after modeling were measured.It was found that NPRC gene deletion did not affect the body weight of mice,but significantly increased the body length of mice.3.NPRC gene deletion down-regulated serum triglyceride level in mice fed with western dietThe results showed that serum triglyceride level of ApoE-/-NPRC-/-group was significantly lower than that of ApoE-/-group after modeling.4.Deletion of NPRC gene did not affect serum cholesterol in mice fed with western dietAfter feeding with western diet for 12 weeks,there were no significant differences in serum total cholesterol,high density lipoprotein cholesterol,low density lipoprotein cholesterol and very low density lipoprotein cholesterol levels in the ApoE-/-NPRC-/group compared with the ApoE-/-group.5.Deletion of the NPRC gene slightly reduced systolic blood pressure in mice fed with western dietAfter feeding with western diet for 12 weeks,ApoE-/-NPRC-/-mice manifested with a slight decrease in systolic blood pressure compared with the ApoE-/-group.6.Deletion of NPRC gene did not affect the levels of heart rate,diastolic blood pressure and mean arterial pressure in mice fed with western dietAfter feeding with western diet for 12 weeks,there were no significant differences in heart rate,diastolic blood pressure,and mean arterial pressure in the ApoE-/-NPRC-/-group compared with the ApoE-/-group.7.Deletion of NPRC gene reduced the degree of atherosclerotic lesions induced by a western dietOil red O staining of en face aorta showed that the aortic plaque load of ApoE-/-NPRC-/-group was significantly lower than that of ApoE-/-group after feeding with western diet for 12 weeks.H?E staining was used to observe the plaque morphology,and it was found that the cross-sectional area of aortic plaques in the ApoE-/-NPRC-/-group was significantly decreased after feeding with western diet for 12 weeks compared with the ApoE-/-group.These results indicated that the deletion of NPRC gene significantly alleviated the lesion degree in atherosclerotic mice.8.Deletion of the NPRC gene affected tissue composition in atherosclerotic plaquesHistopathological staining results showed that compared with ApoE-/-group,the percentages of lipids and macrophages in the plaques of ApoE-/-PRC-/-group were significantly decreased,while the percentages of smooth muscle cells and collagen in the plaques were significantly increased in the ApoE-/-group after feeding with western diet for 12 weeks.Therefore,compared with the ApoE-/-group,the plaque instability index of the ApoE-/-NPRC-/-group calculated by the formula was correspondingly decreased.These results suggested that NPRC gene deletion affected the tissue composition of atherosclerotic plaques and increased plaque stability.9.Gene sequencing analysis of NPRC gene deletion on aortic tissue mRNA expression profile in atherosclerotic miceTranscriptome sequencing showed that the differentially expressed genes were enriched in inflammatory response,leukocyte recruitment and infiltration.10.Deletion of the NPRC gene reduced the levels of inflammatory and adhesion molecules in serum and plaquesAfter feeding with western diet for 12 weeks,the concentrations of TNF?,IL-6,MCP1,ICAM1 and VCAM1 in serum of ApoE-/-NPRC-/-group were significantly decreased.At the same time,IHC,Western blot and RT-PCR methods were used to test the aortic tissues of the two groups of mice,and the results also suggested that NPRC gene deletion significantly reduced the expression of inflammation-related molecules TNF?,IL-6,MCP1 and adhesion molecules ICAM1 and VCAM1 in mouse aorta tissue,compared with that of ApoE-/-mice.11.NPRC mRNA expression in endothelial cells was the highest among the three types of aortic wall cellsRT-PCR results showed that the NPRC mRNA expression was the highest in vascular endothelial cells,lower in vascular smooth muscle cells and little in macrophages.These results suggested that endothelial cells may be the main target of NPRC gene deletion in regulating atherosclerotic lesions.12.NPRC gene knockdown up-regulated eNOS expression in plaques and HUVECsWestern blot and RT-PCR were used to detect the expression of eNOS in plaques in the ApoE-/-NPRC-/-group after feeding with western diet for 12 weeks,compared with the ApoE-/-group.In addition,cell experiments also demonstrated that NPRC knockdown in HUVEC rescued eNOS expression down-regulated by oxLDL stimulation in vitro.13.NPRC gene knockdown alleviated OXLDL-induced HUVECs apoptosisTUNEL cell apoptosis assay showed that NPRC knockdown in HUVEC reduced the number of HUVEC apoptotic cells induced by oxLDL stimulation.In addition,western blot results showed that cleaved caspase3 and cleaved caspase7 were significantly reduced in HUVEC in the NPRC interference group compared with the negative control group after oxLDL stimulation for 24h.14.NPRC gene knockdown mitigated the down-regulation of P-AKT1 in HUVECs induced by oxLDL stimulationThe results showed that oxLDL stimulation of HUVEC resulted in decreased phosphorylation level of AKT1,which was significantly alleviated after NPRC knockdown in HUVEC.15.NPRC gene knockdown reduced the expression of oxLDL-stimulated HUVEC inflammatory and adhesion moleculesThe results of RT-PCR,Western blot and MSD multifactor analysis showed that after oxLDL stimulation,the mRNA,protein synthesis and secretion levels of inflammation-related molecules TNF?,IL-6 and MCP1 of NPRC knockdown HUVEC were significantly lower than those of the negative control group.In addition,Western blot also confirmed that compared with the negative control group,the adhesion molecules ICAM1 and VCAM1 induced by oxLDL stimulation were significantly downregulated in HUVEC after NPRC gene knockout.16.Effect of NPRC gene knockdown in HUVECs on macrophage migration under oxLDL stimulationTranswell macrophage migration experiment results showed that after Raw 267.4 cells and peritoneal macrophages exhibited less migration stimulated by the medium of NPRC-knockdown HUVECs than that by the medium of control HUVECs.17.Effect of NPRC gene knockdown in HUVECs on macrophage phagocytosis of lipidsOil red O staining of macrophages showed that macrophages stimulated by the medium from NPRC-knockdown HUVECs showed less phagocytosis of lipids than those stimulated by the medium from control HUVECs,thus reducing the formation of foam cells.18.Effect of NPRC gene knockdown in HUVECs on expression of inflammation related molecules in macrophagesRT-PCR demonstrated increased expression of TNF?,IL-6 and MCP-1 in peritoneal macrophages stimulated by the medium from control HUVECs after oxLDL treatment.In contrast,expression of these cytokines was downregulated in peritoneal macrophages stimulated by the medium from NPRC-knockdown HUVECs after oxLDL treatment.19.Under oxLDL stimulation,NPRC gene knockdown in HUVECs inhibited the activation of NF-?B signaling pathwayWestern blot results showed that oxLDL stimulated HUVEC to activate NF-?B signaling pathway,and knockdown of NPRC significantly inhibited the phosphorylation of I???/? and P65 in HUVECs.These results suggested that NPRC knockdown may inhibit oxLDL-induced HUVEC inflammation by inhibiting NF-?B signaling pathway.20.Deletion or knockout of NPRC gene activated the cAMP/PKA signaling pathway in vivo and vitrocAMP levels in the aorta tissues of the two groups of mice showed that compared with the ApoE-/-group,cAMP levels in the aorta tissues of the ApoE-/-NPRC-/-group were significantly up-regulated,which subsequently enhanced the phosphorylation level of PKA in the aorta tissues of the ApoE-/-NPRC-/-group.In addition,the same enhanced levels of PKA phosphorylation were observed in NPRC knockdown HUVECs.These results suggested that deletion or knockdown of NPRC gene activated the cAMP/PKA signaling pathway in vivo and vitro.21.PKA pathway activation up-regulated the expression of eNOS and P-AKT1 in HUVEC and inhibited NF-?B signaling pathwaysThe results showed that PKA agonists significantly upregulated the expression of eNOS and the phosphorylation of AKT1 in HUVEC,while PKA inhibitors significantly inhibited this effect.PKA agonists inhibited the phosphorylation of P65 in HUVEC,whereas PKA inhibitors activated the phosphorylation of P65 in HUVECs.22.Mechanism of NPRC gene deletion to reduce the severity of AS lesionsThrough the above in vivo and in vitro experiments,we can basically understand the mechanism of NPRC gene deletion in mitigating AS:NPRC gene deletion activates the cAMP/PKA pathway of endothelial cells,enhances the expression of eNOS in endothelial cells,inhibits apoptosis and inflammation of endothelial cells,thus reduces the inflammatory infiltration and lipid phagocytosis of macrophages in plaques,and ultimately alleviates the pathological degree of AS.Conclusions1.The expression level of NPRC was increased in aortic pathological tissues of atherosclerosis.2.NPRC gene deletion significantly allevioated the degree of AS.3.NPRC gene deletion relieved endothelial apoptosis in plaques.4.NPRC gene deletion reduced the inflammation in atherosclerotic mice.BackgroundAtherosclerosis,as a chronic inflammation-related disease,characterized by endothelial dysfunction,inflammatory cell recruitment,lipid oxidation and form cell formation.Obesity is a major risk factor for atherosclerosis.Adipose tissue exerts its efifect on energy metabolism,storing and releasing lipids,which is showed to be closely associated with the process of sorts of cardiovascular disease such as atherosclerosis,hypertension and coronary artery disease.In diet-induced obesity,adipose tissue was characterized as inflamed and dysfimctional,which released more proinflammatory adipocytokines and free fatty acids resulted in vascular inflammation and oxidative stress to enhance atherosclerosis in a systemic manner.More insight into the relationship between adipose tissue and atherosclerosis may provide new knowledge and measures to protect patients witii high mass of fat from the risk of atherosclerosis.Adipose tissue is found throughout the body,which is mainly composed of adipocytes.Adipocytes can be approximately classified into three phenotypes(white,beige and brown)according to their different natures.Moreover,the phenotypes of adipocytes are not set in stone and they can transform to each other in distinct condition.White adipose tissue(WAT)is the most abundant adipose tissue type.In pathological condition,besides releasing fatty acids,white adipocytes can secret lots of pro-inflammatory factors,which recruit several types of immune cells such as macrophages into adipose tissue.As a consequence,white adipose tissue gradually leads to systemic inflammation,which may play a role in the progress of atherosclerosis.In contrast to WAT,brown adipose tissue(BAT)is reported to alleviate hypertriglyceridemia and protect from atherosclerosis development in mice.Natriuretic peptides were found to regulate multiple physiological process including diuresis,natriuresis,vasorelaxant effects and vascular remodeling,which include atrial and brain natriuretic peptides(ANP and BNP)produced by cardiac cells and C-type natriuretic peptide(CNP)released by endothelium.Besides,three main receptors for natriuretic peptides has been found,which classified into natriuretic peptide receptor A(NPRA),natriuretic peptide receptor B(NPRB),natriuretic peptide receptor C(NPRC).NPRC,regarded as just a clearance receptor for ANP,BNP and CNP a few years ago,has been received more and more attention from researchers because of its undetected function,such as activity of inhibitory guanine nucleotide regulatory protein(Gi).There was a study revealing adipose specific deletion of NPRC enhanced the browning of white adipose tissue and protected against diet-induced obesity and insulin resistance.Moreover,CNP was showed associated with maintaining vascular homeostasis.However,few researchers reported that the role of NPRC played in adipose inflammation accompanied with atherosclerosis.Therefore,to clarify the effect NPRC exerts on adipose inflammation accompanied with atherosclerosis,we generate NPRC'-/-ApoE "7' mice to construct atherosclerotic models to explore adipose phenotype,metabolism and inflammation compared with ApoE";* mice,which not only uncover the roles NPRC plays in adipose inflammation,but also may provide new views for preventing atherosclerosis.Objectives1.To investigate the correlation between NPRC and fat inflammation in hyperlipidemia mice;2.To investigate whether NPRC is involved in regulating the pathological degree of fat inflammation in hyperlipidemia mice;3.To investigate whether NPRC is involved in regulating macrophage infiltration in fat of hyperlipidemia mice;4.To investigate whether NPRC is involved in regulating the apoptosis of adipocytes in hyperlipidemia mice;5.To investigate whether NPRC is involved in regulating oxidative stress in fat of hyperlipidemia mice.Methods1.Groups of experimental animals(1)10 eight-week-old male ApoE-/- mice were randomly divided into two groups,10 mice per group: chow diet group and high fat group.(2)Male ApoE-/-mice and littermate male ApoE'7' NPRC-/- mice aged 8 weeks were randomly divided into two groups,5 mice each group: ApoE7" group and ApoE-/-NPRC-/-group.2.Establishment of hyperlipidemia mouse modelMale ApoE-/- mice and littermate male ApoE-/- NPRC" /mice aged 8 weeks were fed with western diet(0.2% cholesterol,21% milk fat)throughout the whole period,and adequate water was guaranteed.The state of the mice was observed every day during the period.After 12 weeks,the mice were euthanized,and blood,heart,liver,kidney,spleen,adipose tissue and aortic tissue samples were collected for subsequent experiments.3.Mouse tissue samples were collectedAt the end of modeling,the mice were euthanized,and the adipose tissues of the mice were stored in liquid nitrogen or fixed in special fixation solution for adipose tissue for 24-48 h for subsequent experimental use.4.Preparation of mouse adipose tissueThe mouse adipose tissue fixed in special fixative for adipose tissue was dehydrated and embedded into wax blocks to prepare paraffin sections(thickness:4|xm)5.Immunohistochemical staining and immunofluorescence stainingThe protein expression level of Mac2 in white adipose tissue of ApoE-/-mice in chow diet group and ApoE/mice in western diet group was detected by IHC staining.The protein expression levels of NPRC,Ucpl,Mac2,IL-ip and Nlrp3 in white adipose tissue of ApoE-/-group and ApoE-/-NPRC-/- group were detected.The expression of Adiponectin in white adipose tissue of ApoE'7' group and ApoE-/-NPRC-/-group was detected by iramunohistofluorescence staining.6.TUNEL cell apoptosis detectionTUNEL kit was used to detect apoptosis in white adipose tissue of ApoE-7' group and ApoE-/-NPRC-/-group.7.ROS detectionThe levels of reactive oxygen species(ROS)in white adipose tissue of ApoE*A group and ApoE-/-NPRC'7" group were detected by DHE fluorescence staining kit.8.Tissue RNA extraction,reverse transcription and real-time PCR(RT-PCR)Adipose tissue RNA was extracted from western diet fed ApoE-A mice and ApoE /NPRC-/- mice,and the mRNA was reversely transcribed by TAKARA reverse transcription kit.Ct values of MCP1?TNFa?IL-6 ? IL-ip were obtained by RT-PCR.Using P-actin as internal reference,the relative expression of the target molecule was calculated by 2ACt formula.9.Tissue Protein extraction and Western blotAdipose tissue protein was extracted from western diet fed ApoE-/-mice and ApoE-/-NPRC-/-mice using protein extraction kit.After the protein concentration was adjusted by BCA,SDS-PAGE gel electrophoresis was performed to detect the protein content of each target molecule.10.Data analysisAll data were analyzed using GmphPad Prism 8 software and expressed as mean ?standard error(SEM).The shapiro-Wilk test was used to evaluate the assumption of chowity of data distribution.Unpaired T test was used to analyze the statistical difference between two groups in the univariate data of chow distribution,and univariate ANOVA was used to analyze the statistical difference between multiple groups of chow distribution.For multiple sets of data with two variables,after verifying that the data is chowly distributed,two-factor ANOVA is used to analyze the statistical differences.Kruskal-wallis test was used for non-parametric statistical analysis of data that did not belong to chow distribution.In all statistical comparisons,p values less than 0.05 were considered statistically significant.Results1.The expression level of NPRC in adipose tissue was significantly up-regulated in mice fed with a western dietWe successfully constructed a hyperlipidemia mouse model by feeding ApoE".mice with a western diet for 12 weeks.Western blot analysis showed that compared with ApoE-/-mice fed with chow diet for 12 weeks,the expression level of NPRC in adipose tissue of ApoE-/- mice fed with high fat diet was significantly up-regulated.2.Macrophage infiltration in adipose tissue was significantly increased in mice fed with a western dietImmunohistochemical analysis showed that compared with ApoE -/- mice fed with a chow diet for 12 weeks,macrophage infiltration in adipose tissue of ApoE-/-mice fed with a western diet was significantly increased.3.The number of apoptotic cells in adipose tissue increased significantly in mice fed with a western dietCompared with ApoE-/-mice fed with a chow diet for 12 weeks,the number of apoptotic cells in adipose tissue of ApoE-/- mice fed with a western diet increased significantly.4.Oxidative stress was significantly increased in adipose tissue in mouse... |
PDF Full Text Request