【作者】 赵红；

【导师】 陈临溪；

【作者基本信息】 南华大学 ， 基础医学， 2020， 博士

【摘要】 血管紧张素受体AT1相关的受体蛋白(putative receptor protein related to the angiotensin receptor AT1,APJ)是1993年首次发现的7次跨膜的G蛋白偶联受体,Apelin(APJ endogenous ligand)为APJ的内源性配体。Apelin分为不同的亚型,其中生物活性最强的为Apelin-13。Apelin/APJ系统广泛表达于人体多种组织和细胞,尤其高表达于血管内皮细胞和平滑肌细胞,并具有多种生物学效应,比如,促进单核细胞与血管内皮细胞的粘附、舒张血管、促进血管新生、促进血管平滑肌增殖和增强心肌收缩力等。鉴此,Apelin/APJ系统可能是抗动脉粥样硬化(atherosclerosis,As)的一个新的药物筛选和治疗靶点。As是导致心血管功能稳态失衡的一种慢性炎症过程,已成为危害人类健康的主要疾病之一。众所周知,As早期病变的重要环节是单核细胞向血管内皮细胞粘附。研究发现Apelin/APJ系统可以促进人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)细胞间粘附分子1(intercellular cell adhesion molecule-1,ICAM-1)和血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)的表达。近期,课题组报道自噬途径介导Apelin-13促单核细胞-人脐静脉内皮细胞粘附。但是何种自噬途径介导了Apelin-13促单核细胞-人脐静脉内皮细胞粘附并不清楚。糖自噬是最近发现的一种新型的选择性自噬,是指细胞中的糖原被自噬体包裹并被溶酶体内的酸性α-葡萄糖苷酶(lysosomal acid a-glucosidase,GAA)降解,从而释放葡萄糖的过程。淀粉结合域融合蛋白1(starch-binding domain-containing protein 1,STBD1),为糖自噬的标志蛋白。它含有一个N端Atg8-interaction-motif,通过与自噬体形成的Atg8家族蛋白的相互作用介导膜锚定,以及一个C端CBM20结构域,用于结合糖原和糖原相关蛋白,包括糖原脱支酶(glycogen debranching enzyme,GDE)、糖原合成酶(glycogen synthase,GS)和痫蛋白(Laforin)。自噬相关蛋白γ-氨基丁酸受体结合蛋白1(GABA Type A Receptor Associated Protein Like 1,GABARAPL1)属于Atg8家族成员。最近研究发现,GABARAPL1与STBD1共定位于内质网膜并相互作用,从而将糖原运送至自噬小体,并将糖原降解成葡萄糖。糖自噬在血管新生、迁移和增殖中发挥重要的作用。在糖尿病小鼠模型中,心肌细胞STBD1的表达水平明显上调。而在糖尿病人或小鼠模型中,Apelin的表达水平也明显上升。糖代谢异常可以引起单核细胞与血管内皮细胞的粘附,诱导动脉粥样硬化的发生。基于文献,推测是否糖自噬介导了Apelin-13促单核细胞-人脐静脉内皮细胞粘附呢?利用生物信息学分析软件STRING,分析发现STBD1和ICAM-1确实存在蛋白质相互作用。据此,提出糖自噬可能介导Apelin-13促单核细胞-人脐静脉内皮细胞粘附的新构想。为了寻找Apelin-13促糖自噬的调控机制,利用生物信息分析软件发现核糖体蛋白S27a(ribosomal protein S27a,RPS27a)和STBD1存在蛋白质相互作用,提示RPS27a可能了参与STBD1的调控。文献报道RPS27a具有类泛素化修饰蛋白底物的功能,而STBD1的184位赖氨酸为类泛素化修饰位点。提示RPS27a可能类泛素化修饰STBD1,发挥相应生物学效应。据此提出“RPS27a类泛素化修饰STBD1诱导糖自噬介导Apelin-13促单核细胞-人脐静脉内皮细胞粘附”的新假说。本研究应用Western blot、免疫荧光等技术,探讨Apelin-13是否促进人脐静脉内皮细胞糖自噬的发生;明确Apelin-13是否促人脐静脉内皮细胞RPS27a表达,并分析RPS27a是否类泛素化修饰STBD1锚定内质网膜参与糖自噬;分析糖自噬是否介导Apelin-13促单核细胞-人脐静脉内皮细胞粘附。课题将揭示RPS27a类泛素化修饰STBD1诱导糖自噬介导Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附的新机制,其有望成为抗As治疗的新靶点。第一部分Apelin-13/APJ系统促人脐静脉内皮细胞糖自噬的发生目的:探讨Apelin-13/APJ系统是否促人脐静脉内皮细胞糖自噬的发生。方法:1培养人脐静脉内皮细胞,按照实验目的,给予不同浓度的Apelin-13(0、0.001、0.01、0.1、1μmol/L)处理细胞,确定量效关系;0、3、6、12、24小时确定时效关系。Western blot检测STBD1、GABARAPL1、GAA和GDE在HUVECs中的表达情况;2应用APJ拮抗剂F13A观察Apelin-13对人脐静脉内皮细胞STBD1、GABARAPL1、GAA和GDE表达的影响;3免疫荧光观察细胞内LC3B与STBD1的共定位情况;4糖原定量实验观察Apelin-13对人脐静脉内皮细胞糖原含量的影响;5采用透射电镜技术观察人脐静脉内皮细胞在Apelin-13作用下是否能够诱导内质网发生糖自噬。结果:Western blot检测发现Apelin-13呈浓度和时间依赖性的促人脐静脉内皮细胞STBD1、GABARAPL1、GAA和GDE的表达,并且Apelin-13浓度为1μmol/L和作用24h时,蛋白在细胞中的表达最强。APJ拮抗剂F13A减弱Apelin-13促人脐静脉内皮细胞STBD1、GABARAPL1、GAA和GDE的表达。细胞荧光技术证实Apelin-13促LC3B与STBD1的共定位。糖原定量实验发现Apelin-13促人脐静脉内皮细胞糖自噬的发生,降低细胞内糖原含量,而APJ拮抗剂F13A明显升高细胞内糖原含量,抑制Apelin-13促人脐静脉内皮细胞糖自噬的发生。透射电镜技术观察到人脐静脉内皮细胞在Apelin-13作用下内质网发生糖自噬。小结:(1)Apelin-13上调STBD1、GABARAPL1、GAA和GDE蛋白水平;APJ拮抗剂F13A抑制STBD1、GABARAPL1、GAA和GDE蛋白表达。(2)Apelin-13促进人脐静脉内皮细胞STBD1与LC3B荧光共定位,诱导内质网发生糖自噬,降低细胞内糖原含量。第二部分核糖体蛋白S27a类泛素化修饰STBD1锚定内质网膜参与糖自噬目的:研究核糖体蛋白S27a(ribosomal protein S27a,RPS27a)参与糖自噬的可能机制。方法:1培养人脐静脉内皮细胞,按照实验目的,给予不同浓度的Apelin-13(0、0.001、0.01、0.1、1μmol/L)处理细胞,确定量效关系;0、3、6、12、24小时确定时效关系。Western blot方法检测RPS27a在HUVECs中的表达情况;2应用APJ拮抗剂F13A,Western blot方法观察Apelin-13对人脐静脉内皮细胞RPS27a表达的影响;3沉默和过表达RPS27a,观察对其Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B的表达的影响;4糖原定量实验,观察沉默或高表达RPS27a和STBD1对人脐静脉内皮细胞糖原含量的影响;5免疫荧光、激光共聚焦和免疫共沉淀技术检测RPS27a与STBD1的相互作用;6透射电镜观察沉默RPS27a对Apelin-13诱导的人脐静脉内皮细胞内质网糖自噬的影响;7将STBD1 184位K(AAA)换成E(GAA),Western blot方法检测其对Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B表达的影响。结果:Western blot检测发现Apelin-13呈浓度和时间依赖性的促HUVECs RPS27a的表达,并且Apelin-13浓度为1μmol/L和作用24h时,蛋白在细胞中的表达最强。APJ拮抗剂F13A减弱Apelin-13促人脐静脉内皮细胞RPS27a的表达。沉默RPS27a能够抑制Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B的表达;相反,高表达RPS27a促进Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B的表达。沉默RPS27a或STBD1抑制Apelin-13诱导人脐静脉内皮细胞糖自噬,增加细胞糖原含量;而高表达RPS27a或STBD1促人脐静脉内皮细胞糖自噬,降低细胞糖原含量。细胞荧光和激光共聚焦技术证实Apelin-13促RPS27a与STBD1的共定位,免疫共沉淀实验证实RPS27a与STBD1存在蛋白相互作用。透射电镜技术观察到沉默RPS27a后,Apelin-13诱导人脐静脉内皮细胞内质网糖自噬下降。点突变STBD1 184位赖氨酸后,抑制Apelin-13诱导的人脐静脉内皮细胞STBD1、GABARAPL1、Beclin-1和LC3B的表达。小结:(1)Apelin-13诱导人脐静脉内皮细胞RPS27a的表达。(2)RPS27a类泛素化修饰STBD1锚定内质网膜参与糖自噬。(3)STBD1K184为RPS27a类泛素化修饰STBD1的重要修饰位点,是STBD1发挥生物学功能的结构基础。第三部分RPS27a类泛素化修饰STBD1诱导糖自噬介导Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附目的:探讨RPS27a类泛素化修饰STBD1诱导糖自噬是否介导Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附方法:1细胞免疫荧光检测Apelin-13/APJ系统是否促粘附分子ICAM-1和VCAM-1的表达;单核细胞-人脐静脉内皮细胞粘附实验检测Apelin-13/APJ系统是否促单核细胞-人脐静脉内皮细胞粘附;2沉默RPS27a后,采用Western blot方法观察其对Apelin-13促粘附分子ICAM-1、VCAM-1的表达的影响;单核细胞-人脐静脉内皮细胞粘附实验检测其对Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附的影响;3过表达RPS27a后,采用Western blot方法检测其对Apelin-13促粘附分子ICAM-1、VCAM-1的表达的影响;4沉默STBD1后,采用Western blot方法观察其对Apelin-13促粘附分子ICAM-1、VCAM-1的表达的影响;单核细胞-人脐静脉内皮细胞粘附实验检测其对Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附的影响;5过表达STBD1后,采用Western blot方法观察其对其Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1的表达的影响;6细胞免疫荧光技术观察siRNA-RPS27a和siRNA-STBD1对Apelin-13促人脐静脉内皮细胞ICAM-1和VCAM-1的表达的影响;7将STBD1 184K(AAA)换成E(GAA),Western blot方法检测其对Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1表达的影响;单核细胞-人脐静脉内皮细胞粘附实验检测其对Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附的影响。结果:Apelin-13/APJ系统促人脐静脉内皮细胞ICAM-1和VCAM-1的表达,促单核细胞-人脐静脉内皮细胞粘附。SiRNA-RPS27a和SiRNA-STBD1,均抑制Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1的表达,同时抑制Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附。而过表达RPS27a和STBD1均促进Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1的表达。点突变STBD1 184位赖氨酸后,抑制Apelin-13诱导的人脐静脉内皮细胞ICAM-1、VCAM-1的表达,同时抑制Apelin-13/APJ系统促单核细胞-人脐静脉内皮细胞粘附。小结:(1)Apelin-13/APJ系统促人脐静脉内皮细胞ICAM-1和VCAM-1的表达,促单核细胞-人脐静脉内皮细胞粘附。(2)糖自噬介导Apelin-13促单核细胞-人脐静脉内皮细胞粘附。(3)RPS27a类泛素化修饰STBD1介导Apelin-13促单核细胞-人脐静脉内皮细胞的粘附。(4)STBD1 184位赖氨酸是RPS27a类泛素化修饰STBD1的重要位点,是STBD1发挥生物学功能的结构基础。结论1.Apelin-13上调STBD1和GABARAPL1蛋白水平,促进人脐静脉内皮细胞发生糖自噬。2.Apelin-13诱导人脐静脉内皮细胞RPS27a的表达,RPS27a类泛素化修饰STBD1锚定内质网膜参与糖自噬。3.RPS27a类泛素化修饰STBD1诱导糖自噬介导Apelin-13促单核细胞-人脐静脉内皮细胞粘附。4.STBD1 184位赖氨酸是RPS27a类泛素化修饰STBD1的重要位点,是STBD1发挥生物学功能的结构基础。更多还原

【Abstract】 APJ(putative receptor protein related to the angiotensin receptor AT1),first identified in 1993,is a seven trans-membrane G protein coupled receptor.Apelin is endogenous ligand of APJ.Apelin is divided into different subtypes,among which Apelin-13 has the strongest biological activity.Apelin/APJ system is extensively expressed in a variety of human tissues and cells,particularly in smooth muscle cells and vascular endothelial cells.And it has a variety of biological effects,such as promoting the adhesion of monocytes to vascular endothelial cells,relaxing blood vessels,promoting angiogenesis,enhancing vascular smooth muscle cell proliferation and strengthening myocardial contractility and so on.Therefore,Apelin/APJ system may become a new drug screening and therapeutic target for anti-atherosclerosis.Atherosclerosis,a chronic inflammatory process,results in the imbalance of cardiovascular function.It has become one of the main diseases harmful to human health.It is well known that the important link of early pathological changes to atherosclerosis is the adhesion of monocytes to endothelial cells.Numerous researches have confirmed that Apelin/APJ system induces the high expression of intercellular adhesion molecule 1(ICAM-1)and vascular cell adhesion molecule 1(VCAM-1)in human umbilical vein endothelial cells(HUVECs).Recently,our research group has reported that autophagy pathway mediates monocyte-HUVECs adhesion induced by Apelin-13.However,it is not clear which autophagy pathway mediating the adhesion of monocytes to HUVECs induced by Apelin-13.Glycophagy,a glycogen-specific autophagy,functions as degrading cell glycogen within autophagic vacuoles via lysosomal acid a-glucosidase,thus releasing glucose.Starch binding domain fusion protein 1(STBD1)is a marker protein of glycophagy.STBD1 contains an N-terminus Atg8-interacting-motif for mediating membrane anchorage via interaction with the autophagosome-forming Atg8 family proteins,and a C-terminal CBM20 domain for binding glycogen and glycogen-associated proteins including GDE(glycogen debranching enzyme),GS(glycogen synthase)and Laforin.Autophagy associated protein γ-aminobutyric acid receptor binding protein 1(GABARAPL1)belongs to the Atg8 family.Recently,it has been found that GABARAPL1 and STBD1 are co-localization in the endoplasmic reticulum membrane.Moreover,GABARAPL1 interacts with STBD1 then transporting glycogen to autophagy bodies.Glycogen issubsequently degraded into glucose.Glycophagy plays an important role in angiogenesis,migration and proliferation.Studies have confirmed that the expression of STBD1 is significantly up-regulated in cardiomyocytes in the diabetic mouse model.More importantly,in diabetic mouse models,the expression level of Apelin is also significantly increased.Furthermore,abnormal glucose metabolism can cause monocytes to adhere to endothelial cells and induce As.Based on these literature,we wondered whether glycophagy mediates monocyte-HUVECs adhesion induced by Apelin-13.Using bioinformatics analysis software STRING,we found that there is a protein interaction between STBD1 and ICAM-1.Thus,we proposed a novel idea that glycophagy may mediate the adhesion of monocytes to HUVECs induced by Apelin-13.In order to explore the regulatory mechanism of Apelin-13 promoting glycophagy,we found that there is an interaction between ribosomal protein S27a(RPS27a)and STBD1 by using bioinformatics analysis software.It suggests that RPS27 a may be involved in the regulation of STBD1.In addition,RPS27 a has the function of ubiquitin-like modified(SUMO)protein substrate.And the lysine at position 184 of STBD1 is an ubiquitin-like modification site.Then we suggested that RPS27 a may modify STBD1 by means of SUMOylation to play the corresponding biological effects.Therefore,we put forward a new hypothesis that RPS27 a SUMOylation STBD1 promotes glycophagy mediating monocyte-HUVECs adhesion induced by Apelin-13.The purpose of this study is to explore whether Apelin-13 induces glycophagy in HUVECs by means of Western blot method.We will explore whether Apelin-13 promotes the expression of RPS27 a in HUVECs.Besides,we will analyze whether RPS27 a SUMOylation modifies STBD1 then anchoring endoplasmic reticulum to participate in glycophagy.Furthermore,we will clarify whether glycophagy mediates the adhesion of monocytes to HUVECs.The purpose of this study is to reveal the novel mechanism of RPS27 a SUMOylation STBD1 inducing glycophagy.And glycophagy mediates Apelin-13/APJ system promoting monocyte-HUVECs adhesion.It may provide a theoretical basis for Apelin/APJ system to become a new target for anti-As therapy.Part Ⅰ Apelin-13/APJ system promotes glycophagy in humanumbilical vein endothelial cellsAim: To investigate whether Apelin-13/APJ system inducing the occurrence of glycophagy in HUVECs.Methods: HUVECs were cultured and treated with different concentration of Apelin-13(0,0.001,0.01,0.1,1μmol/L)to determine the dose-effect relationship.The time effect was determined at 0,3,6,12 and 24 hours.The expression of STBD1,GABARAPL1,GAA and GDE in HUVECs was detected by Western blot method.The effects of Apelin-13 on the expression of STBD1,GABARAPL1,GAA and GDEin HUVECs were observed by using APJ antagonist F13 A.The co-localization of LC3 B and STBD1 in cells was observed by immunofluorescence.The effect of Apelin-13 on the content of glycogen in HUVECs was observed by the quantitative experiment of glycogen.The effect of Apelin-13 inducing glycophagy was observed in endoplasmic reticulum by transmission electron microscope.Results: Apelin-13 promoted the expression of STBD1,GABARAPL1,GAA and GDE in HUVECs with concentration-and time-dependent manner.And the expression of these proteins was the strongest when the concentration of Apelin-13 was 1 μmol/L and incubated for 24 hours.APJ antagonist F13 A attenuated the expression of STBD1,GABARAPL1,GAA and GDE induced by Apelin-13 in HUVECs.Apelin-13 promoted the co-localization of LC3 B and STBD1 by fluorescence technique.In addition,Apelin-13 promoted the occurrence of glycophagy and decreased intracellular glycogen concentration in HUVECs.Moreover,the glycophagy of endoplasmic reticulum(ER)was induced by Apelin-13 in HUVECs by transmission electron microscope.On the contrary,APJ antagonist F13 A significantly increased intracellular glycogen content and inhibited the occurrence of glycophagy induced by Apelin-13 in HUVECs.Summaries:(1)Apelin-13 up-regulates the levels of STBD1,GABARAPL1,GAA and GDE proteins,and APJ antagonist F13 A inhibits the expression of STBD1,GABARAPL1,GAA and GDE proteins.(2)Apelin-13 promotes the co-localization of STBD1 and LC3 B,and induces glycophagy in endoplasmic reticulumin HUVECs.(3)Apelin-13 decreases the content of glycogen of HUVECs.Part Ⅱ Ribosomal protein S27 a SUMOylation STBD1 anchoring endoplasmic reticulum induces glycophagyAim: To determine the potential mechanism of how RPS27 a inducing glycophagy.Methods: HUVECs were cultured and treated with different concentration of Apelin-13(0,0.001,0.01,0.1,1μmol/L).The time effect was determined at 0,3,6,12 and 24 hours.The expression of RPS27 a in HUVECs was detected by Western blot method.The expression of RPS27 a induced Apelin-13 in HUVECs was detected by Western blot method.The effect of APJ antagonist F13 A on the expression of RPS27 a induced Apelin-13 in HUVECs was also detected by Western blot method.The expression of STBD1,GABARAPL1,Beclin-1 and LC3 B induced by Apelin-13 were observed after silence of RPS27 a and overexpression of RPS27 a in HUVECs.The effect of silence or overexpression RPS27 a and STBD1 on the content of glycogen in HUVECs was observed by glycogen quantitative assay.The interaction between RPS27 a and STBD1 was detected by immunofluorescence,laser confocal focusing and immunoprecipitation techniques.The effect of silence of RPS27 a on theglycophagy induced by Apelin-13 was observed by transmission electron microscope.The effects of replacing STBD1 184K(AAA)with E(GAA)on the expression of STBD1,GABARAPL1,Beclin-1 and LC3 B induced by Apelin-13 in HUVECs were detected by Western blot method.Results: Apelin-13 promoted the expression of RPS27 a with a concentration and time dependent manner in HUVECs.Besides,the expression of protein was the strongest when the concentration of Apelin-13 was 1μmol/L and incubated for 24 hours.APJ antagonist F13 A attenuated the expression of RPS27 a induced by Apelin-13 in HUVECs.The expression of STBD1,GABARAPL1,Beclin-1 and LC3 B induced by Apelin-13 were inhibited after silence of RPS27 a in HUVECs.On the contrary,overexpression of RPS27 a promoted the expression of STBD1,GABARAPL1,Beclin-1 and LC3 B induced by Apelin-13 in HUVECs.Moreover,silence of RPS27 a or STBD1 inhibited glycophagy induced by Apelin-13 thus increase the content of glycogen in HUVECs.Conversely,overexpression RPS27 a or STBD1 increased glycophagy induced by Apelin-13 thus decrease the content of glycogen in HUVECs.Cellular fluorescence and laser confocal focusing techniques confirmed that Apelin-13 promoted the co-localization of RPS27 a and STBD1.Moreover,there was a protein interaction between RPS27 a and STBD1 through immunoprecipitation method.Glycophagy induced by Apelin-13 in endoplasmic reticulum was observed by transmission electron microscope in HUVECs.The point mutation of 184 lysine of STBD1 inhibited the expression of STBD1,GABARAPL1,Beclin-1 and LC3 B induced by Apelin-13 in HUVECs.Summaries:(1)Apelin-13 induces the expression of RPS27 a in HUVECs.(2)RPS27a SUMOylation STBD1 anchoring endoplasmic reticulum induces glycophagy.(3)The position of 184 lysine of STBD1 is the main modification site of RPS27 a SUMOylation STBD1.It is the structural basis for the biological function of STBD1.Part Ⅲ RPS27 a SUMOylation STBD1 inducing glycophagy mediates Apelin-13/APJ system promoting monocyte-human umbilical vein endothelial cell adhesionAim: To investigate whether RPS27 a SUMOylation STBD1 inducing glycophagy mediating the adhesion of monocytes to HUVECs induced by Apelin-13/APJ system.Methods: Cell immunofluorescence method was used for detecting the expression of adhesion molecules such as ICAM-1 and VCAM-1induced by Apelin-13/APJ system.Monocyte-HUVECs adhesion assay was used for exploring the effect of Apelin-13/APJ system on monocyte-HUVECs cell adhesion.The expression of ICAM-1 and VCAM-1 induced by Apelin-13 were observed after silence of RPS27 a by Western blot method.Monocyte-HUVECs adhesion assay was usedfor testing the effect of Apelin-13/APJ system on monocyte-HUVECs adhesion after silence of RPS27 a.On the contrary,the expression of ICAM-1 and VCAM-1 induced by Apelin-13 was detected after overexpression of RPS27 a by Western blot method.Moreover,after silence of STBD1,the effects of Apelin-13 on the expression of ICAM-1and VCAM-1 were observed by Western blot method.Monocyte-HUVECs adhesion assay was used for exploring the effect of Apelin-13/APJ system on Monocyte-HUVECs adhesion after silence of STBD1.On the contrary,the effect of Apelin-13 on the expression of ICAM-1 and VCAM-1 induced by Apelin-13 was observed by Western blot method in HUVECs after overexpression of STBD1.Furthermore,the effects of siRNA-RPS27 a and siRNA-STBD1 on the expression of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells induced by Apelin-13 were observed by cell immunofluorescence technique.Lastly,the effects of replacing STBD1 184K(AAA)with E(GAA)on the expression of ICAM-1 and VCAM-1 induced by Apelin-13 was detected by Western blot method in HUVECs.Similarly,monocyte-HUVECs adhesion assay was used for detecting the effect of Apelin-13/APJ system on monocyte-HUVECs adhesion after point mutation of lysine at position184 of STBD1.Results: Apelin-13/APJ system upregulated the expression of ICAM-1 and VCAM-1.It also promoted the adhesion of monocytes to HUVECs.The expression of ICAM-1 and VCAM-1 induced by Apelin-13 was decrease after silence of RPS27 a or STBD1 in HUVECs.Similarly,the adhesion of monocytes to HUVECs induced by Apelin-13/APJ system was also lower after silence of RPS27 a or STBD1 in HUVECs.Conversely,overexpression of RPS27 a or STBD1 promoted the expression of ICAM-1 and VCAM-1 induced by Apelin-13 in HUVECs.Point mutation of lysine at position 184 of STBD1 inhibited the expression of ICAM-1 and VCAM-1 induced by Apelin-13 in HUVECs.It also inhibited the adhesion of monocytes to HUVECs induced by Apelin-13/APJ system.Summaries:(1)Apelin-13/APJ system induces the expression of ICAM-1 and VCAM-1 in HUVECs.It also promotes the adhesion of monocytes-HUVECs.(2)Glycophagy mediates Apelin-13 promoting the adhesion of monocytes to HUVECs.(3)RPS27a SUMOylation STBD1 mediates the adhesion of monocytes to HUVECs induced by Apelin-13.(4)Lysine at position 184 of STBD1 is an important point of RPS27 a SUMOylation STBD1.It is the structural basis for the biological function of STBD1.Conclusions(1)Apelin-13 upregulated the levels of STBD1 and GABARAPL1proteins and promoted glycophagy in HUVECs.(2)Apelin-13 induced the expression of RPS27 a in HUVECs.RPS27aSUMOylation STBD1 anchored endoplasmic reticulum to participatein glycophagy.(3)RPS27a SUMOylation STBD1 induced glycophagy mediatingApelin-13 promoting monocyte-HUVECs adhesion.(4)Lysine at position 184 of STBD1 is an important point of RPS27aSUMOylation STBD1 and essential for its function.更多还原