核因子-κB在非对称性二甲基精氨酸诱导大鼠主动脉粥样硬化中的作用机制
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摘要
研究背景和目的
动脉粥样硬化(atherosclerosis, AS)是一类发生在动脉血管壁的慢性进行性炎症反应性疾病,是众多心脑血管疾病共同的病理基础,严重危害人类健康。血管内皮细胞损伤、平滑肌细胞增殖及巨噬细胞转化为泡沫细胞是粥样斑块形成的三个最为关键环节,其中巨噬细胞转化为泡沫细胞是AS发生的始动环节。国内外大量研究表明,高胆固醇血症、高血压、糖尿病、高同型半胱氨酸血症等是AS致病性的危险因素,血浆非对称性二甲基精氨酸(asymmetric dimethylarginine, ADMA)与这些危险因素密切相关,被认为是一个新的心血管疾病的危险因子。ADMA是内皮源性一氧化氮合酶(nitric oxide synthase, NOS)竞争性抑制剂。正常情况下人体血浆ADMA不足以抑制NOS活性,但在机体应激损伤时,血浆ADMA浓度则会明显升高。高浓度的ADMA可抑制NO合成、损伤血管内皮、促进VSMCs增生,但在巨噬细胞转化为泡沫细胞中的作用研究相对较少,其具体机制尚不明确。NOS是NO生物合成的关键限速酶,分为两类：持续表达型(constitutive nitric oxide synthase, cNOS)和诱导型(inducible nitric oxide synthase, iNOS)。cNOS广泛存在于正常的组织细胞中,iNOS在正常情况下低表达甚或不表达,但在受到各种刺激时可诱导表达。血凝素样氧化低密度脂蛋白受体1(lectin like oxidized low density lipoprotein receptor-1, LOX-1)是氧化低密度脂蛋白(oxidized low density lipoprotein, oxLDL)的特异受体,在结构上与巨噬细胞经典的清道夫受体不具有同源性,属于E型清道夫受体。巨噬细胞通过清道夫受体无负反馈性地吞噬大量氧化低密度脂蛋白,导致细胞内脂质堆积,形成泡沫细胞。研究表明,ADMA可促进巨噬细胞LOX-1表达,但具体机制尚不清楚。iNOS和LOX-1的基因启动子区都含有核因子-κB (nuclear factor kappa B, NF-κB)结合位点,能够与核蛋白特异结合调节下游基因转录。NF-κB是一种具有基因转录多项调控作用的转录因子,广泛存在于各种细胞中。本研究拟采用分子生物学实验方法,分别在细胞水平和组织水平上探讨在动脉粥样硬化形成过程中,ADMA影响iNOS、LOX-1表达的机制,是否是通过激活NF-κB实现的。
方法
1.体外细胞学实验
健康雄性Wistar大鼠50只。分离培养大鼠腹腔巨噬细胞。实验分组及处理：①正常对照组：以含10% FBS DMEM培养液与巨噬细胞共孵育；②oxLDL组：在巨噬细胞培养液中加入oxLDL(终浓度50mg/L)；③A+O组：在巨噬细胞培养液中先加入ADMA(终浓度15μmol/L)共孵育2h后,再加入oxLDL(终浓度50mg/L)；④P+A+O组：在巨噬细胞培养液中先加入PDTC(终浓度25μmol/L)共孵育30min后,再加入ADMA(终浓度15μmol/L)共孵育2h,最后加入oxLDL(终浓度50mg/L)。②③④组细胞在加入oxLDL后,再继续孵育48h。然后分别收集各组细胞和培养液,测定细胞内胆固醇含量及培养液NO水平和iNOS活性,分别提取细胞总RNA、总蛋白及核蛋白。以实时荧光定量PCR和Westen blot分别检测iNOS、LOX-1的mRNA和蛋白表达,以Actin为内参进行标化；以凝胶电泳迁移率变动分析(EMSA)和增强化学发光法(ECL)检测各组NF-κB活性。每组重复3次。
2.动物实验：健康雄性Wistar大鼠50只,随机分为四组：①正常对照组(n=10)：标准饲料喂养。②H组(n=12)：高脂饲料喂养。③A+H组(n=14)：高脂饲料喂养,给予ADMA [0.2mg/(kg·d)]灌胃,每日一次。④P+A+H组(n=14)：高脂饲料喂养,给予ADMA [0.2mg/(kg·d)]灌胃,每日一次；PDTC [40mg/(kg·d)]腹腔注射,每日一次。正常对照组、H组均给予等体积的生理盐水灌胃和腹腔注射,A+H组给予等体积的生理盐水腹腔注射。饲养满18周后麻醉大鼠,采血检测大鼠血清NO及iNOS活性；取胸主动脉做病理切片,观察其病理变化。分别以实时荧光定量PCR和Westen blot检测iNOS、LOX-1的mRNA和蛋白表达,以Actin为内参进行标化；以凝胶电泳迁移率变动分析(EMSA)和增强化学发光法(ECL)检测各组NF-κB活性。每组重复3次。
结果
1.体外细胞学实验：
①oxLDL组和A+O组巨噬细胞内胆固醇含量较正常对照组明显增加(均为P<0.05),且A+O较oxLDL组升高更明显(P<0.05),而P+A+O组胆固醇含量较A+O组降低(P<0.05)。②与正常对照组和oxLDL组相比,A+O组NO含量降低(P<0.05或P<0.01)：P+A+O组NO含量较A+O组显著升高(P<0.05)。③与正常对照组和oxLDL组相比,A+O组iNOS活力明显升高(均为P<0.05), P+A+O组iNOS活力较A+O组明显降低(P<0.05)。相关分析显示：NO含量和iNOS活力呈显著负相关(r=-0.773,P<0.05)④与正常对照组和oxLDL组相比,A+O组iNOS mRNA和蛋白表达量显著增加,其差异均有显著性(分别为P<0.01和P<0.05)；P+A+O组iNOS mRNA和蛋白表达较A+O组明显降低(P<0.05)。⑤A+O组LOX-1 mRNA和蛋白表达较正常对照组和oxLDL组增强,其差异均有显著性(均为P<0.05)；与A+O组相比,P+A+O组LOX-1 mRNA和蛋白表达明显降低,两组差异有显著性(P<0.05)。⑥A+O组NF-κB活性较正常对照组和oxLDL组明显增强,其差异均有显著性(均为P<0.05)；与A+O组相比,P+A+O组NF-κB活性明显降低,两组差异有显著性(P<0.05)。⑦相关分析：NF-κB活性与iNOS mRNA和蛋白表达量呈正相关(相关系数r分别为0.82、0.81)；NF-κB活性与LOX-1 mRNA和蛋白表达量呈正相关(相关系数r均为0.82)。
2.动物体内实验：
①主动脉病理切片HE染色显示：正常对照组血管内膜光滑、完整,未见空泡形成；H组血管内膜毛糙、但尚完整、有少量空泡形成；A+H组血管内膜断裂,血管中层SMCs增生明显,且排列紊乱,内膜层与中膜层有大量空泡形成；P+A+H组血管内膜完整,SMCs增生程度较A+H组明显减轻,空泡数量也较A+H组明显减少。②与对照组和H组比较,A+H组大鼠血清NO水平和iNOS活性明显升高,差异均有显著性(均为P<0.05),P+A+H组NO水平和iNOS活性较A+H组明显降低(均为P<0.05)。相关分析显示,NO水平和iNOS活性呈正相关(r=0.68,P<0.05)。③与对照组和H组相比,A+H组大鼠主动脉iNOS mRNA和蛋白表达量显著增加,(分别为P<0.01和P<0.05), P+A+H组iNOS mRNA和蛋白表达量较A+H组明显降低,两组相比差异有显著性(P<0.05)。④与对照组和H组相比,A+H组大鼠主动脉LOX-1 mRNA和蛋白表达量显著增加,(均为P＜O.05), P+A+H组LOX-1 mRNA和蛋白表达量较A+H组明显降低,两组相比差异有显著性(P<0.05)。⑤A+H组NF-κB活性较正常对照组和H组明显增强,其差异均有显著性(均为P<0.05)；与A+H组相比,P+A+H组NF-κB活性明显降低,两组差异有显著性(P<0.05)。⑥相关分析：NF-κB活性与iNOSmRNA和蛋白表达量呈正相关(相关系数r分别为0.85、0.87)；NF-κB活性与LOX-1mRNA和蛋白表达量呈正相关(相关系数r分别为0.79、0.81)。
结论
ADMA能够通过激活NF-κB上调iNOS和LOX-1的表达,从而促进AS的发生发展。
Background & Objective
Atherosclerosis (AS) is a chronic progressive inflammatory response within the artary wall and is the common pathological foundation of many cardiovascular diseases, which seriously threatens people's health. Three important aspects in formation of atherosclerosis plaque are the proliferation of vascular smooth muscle cells (VSMCs),injury of vascular endothelium cells, and macrophages thransforming into foam cells which is the first step in development of AS. A large number of researches at home and abroad indicate that hypercholesterolemia, hypertension, diabetes mellitus and hyperhomocysteinemia are pathogenic risk factors of AS. Asymmetric dimethylarginine (ADMA) is closely related with these risk factors, and is considered as a new risk factor of cardiovascular diseases. ADMA is a competitive inhibitor of endothelium drived nitric oxide synthase (NOS). In normal circumstances ADMA in human plasma has no effect on the activity of NOS, but under the stress status and the injury site, the concentration of ADMA in plasma is obviously increased. High concentration of ADMA plays an important role in inhibiting the synthasis of nitric oxide (NO), injuring the vascular endothelium and inducing VSMCs. However, the study is relatively few about the effect of ADMA on the process of macrophages thransforming into foam cells, and the mechainism is not clear. NOS is the key rate-limiting enzyme of NO biosynthesis, including constitutive nitric oxide synthase (cNOS) and inducibe nitric oxide synthase (iNOS). cNOS is known in a wide variety of cells and tissues. Normally, there is minimal or no expression of iNOS, but iNOS is induced to express by a variety of stimulus. Lectin like oxidized low density lipoprotein receptor-1 (LOX-1) is the specific receptor of oxidized low density lipoprotein (oxLDL). LOX-1 is different in structure from the classical scavenger receptors, belonging to scavenger receptor type E. Macrophages ingest a great quantity of oxLDL via scaverger receptors when there is no a negitive feedback, which leads to accumulation of lipid in the cytoplasm of macrophages and formation of foam cells. Researches show that ADMA enhances the expression of LOX-1 and its mechanism is not very clear yet. The promotor regions of iNOS gene containing an active nuclear factory kappa B (NF-κB) binding site, which can specially combine to nucleoprotein and regulate transcription of down-stream responsive genes. NF-κB is a ubiquitous intracellular transcription factor, taking part in regulateing gene transcription. In the present study, we adopted molecular biological techniques to explore the mechanism of ADMA inducing to express iNOS and LOX-1 during development of AS in cell level and tissue level, and whether activation of NF-κB has a relationship with the mechanism.
Methods
1. Study in vitro
Fifty-Healthy, male wistar rats were enrolled. Celiac macrophages of rats were gathered and cultured. Groups and interference:①Control group:macrophages were incubated with 10% FBS DMEM.②oxLDL group:macrophages were treated by oxLDL (final concentration 50mg/L) for 48 hours.③A+O group:macrophages were pretreated with ADMA (final concentration 15umol/L) for 2 hours, then co-incubated with oxLDL (final concentration 50mg/L) for 48 hours;④P+A+O group:macrophages were pretreated with PDTC (final concentration 25umol/L) for 30 minutes, then co-incubated with ADMA (final concentration 15umol/L) for 2 hours, finally co-incubated with oxLDL (final concentration 50mg/L) for 48 hours. Each group was repeated for 3 times. Macrophages and medium were respectively collected. Intracellular cholesterol level, NO level, iNOS activity were detected separately. The total RNA, total proteins and nucleoprotein were extracted from macrophages respectively. Real time fluorescent quantitative PCR (real-time PCR) was used to detected the expression of mRNA, including iNOS mRNA and LOX-1 mRNA. The expressions of iNOS and LOX-1 protein were measured by Westen blotting, using rat actin as an internal standard. NF-κB activity was detected with gelelectrophorestic mobility shift assay (EMSA) and enhanced chemiluminescent (ECL) technique.
2. Study in vivo
Fifty-Healthy, male wistar rats were enrolled and randomly divided into four groups.①Control group (n=10):Fed with standard diet.②H group (n=12):Fed with high fat diet.③A+H group (n=4):Fed with high fat diet, and administered intergastrically with ADMA [0.2mg/(kg-d)] once a day.④P+A+H group (n=14):Fed with high fat diet, administered intergastrically with ADMA [0.2mg/(kg-d)] and given interperitoneal injection with PDTC [40mg/(kg-d)], respectively once a day. Both control group and H group were administered intergastrically and injected interperitoneally with the same volume of normal saline. A+H group was injected interperitoneal with the same volume of normal saline.18 weeks later, the rats were anesthetized, rat blood and aortas were gathered. Serum NO level and iNOS activity were measured respectively. Collected thoracic aortas were made pathology slices and observed the histological structure under microscope. The total RNA, total protein and nucleoprotein were separately extracted from rat aortas. The expressions of iNOS mRNA and LOX-1 mRNA were detected with real time fluorescent quantitative PCR. Westen blotting was used to examinate the expression of iNOS and LOX-1 protein, using rat actin as an internal standard. EMSA and ECL technique was used to detect NF-κB activity.
Results
1. Study in vitro
①Intracellular cholesterol level in oxLDL group and A+O group were higher than that in control group (P<0.05,either), and compared with oxLDL group, intracellular cholesterol level in A+O group significantly increased (P<0.05), but intracellular cholesterol level of P+A+O group was lower than that of A+O group (P<0.05).②Compared with control group and oxLDL group, NO level in A+O group was significantly lower (P<0.05,either), and NO level of P+A+O group was much higher than that of A+O group (P<0.05).③The activity of iNOS in A+O group significantly higher than that of in control group and that of in oxLDL group (P＜0.05,either), but in P+A+O group, iNOS activity significantly decreased than that in A+O group (P<0.05). The correlation analysis indicated that NO level had a obvious negitive correlation with iNOS activity (r=-0.773,P<0.05).④Compared with control group and oxLDL group, the mRNA and protein of iNOS expression in A+O group significantly increased (P<0.05,either), and in P+A+O group,it obviously decreased than that in A+O group (P＜0.05).⑤The mRNA and protein of LOX-1 expression in A+O group significantly increased than that in control group and oxLDL group (P<0.05,either), but decreased in P+A+O group compared with A+O group (P<0.05).⑥In A+O group, the NF-κB activity markedly increased compared with that in control group and oxLDL group (P<0.05,either), and decreased in P+A+O group compared with A+O group.⑦Correlation analysis showed that the NF-κB activity took on a significant positive correlation with the expression of iNOS mRNA and protein (r=0.82,r=0.81,respectively;P<0.05), and the NF-κB activity took on a significant positive correlation with the expression of LOX-1 mRNA and protein (r=0.82,either;P<0.05).
2. Study in vivo
①Pathological examination:the paraffin-cut slices of thoracic slices were stained with HE and inspected under optic microscope. In control group the intima was smooth and integrated, without the formation of vacuoles. In H group, the intima contained a small amount vacuoles. In A+H group, the intima and elastic fibers ruptured, there were proliferation and irregular assay of VSMCs and a lot of vacuoles in intima and media. Compared with A+H group, the intima was integrated, the degree of VSMCs proliferation and the amount of vacuoles was decreased in P+A+H group.②Sesum NO level and iNOS activity in A+H group were obviously higher than those in control group and H group (P<0.05,either), and which in P+A+H group was lower than in A+H group (P＜O.05). Correlation analysis indicated that serum NO level was positively associated with iNOS activity (r=0.68,P<0.05).③The mRNA and protein of iNOS expressions in A+H group increased compared with those in control group and H group (P<0.05,either), and which in P+A+H group decreased than those in A+H group (P＜O.05).④The mRNA and protein of LOX-1 expressions in A+H group increased compared with those in control group and H group (P<0.05,either), and which in P+A+H group decreased than those in A+H group (P<0.05).⑤The NF-κB activity was significantly higher in A+H group than that in control group and H group (P<0.05,either), and was lower in P+A+H group than that in A+H group.⑥Correlation analysis showed that the NF-κB activity was significantly and positively correlated with the iNOS mRNA and protein expression (r=0.85, r=0.87, respectively; P＜O.05, either),and which was also significantly and positively correlated with the LOX-1 mRNA and protein expression (r=0.79, r=0.81, respectively; P＜O.05, either).
Conclusion
ADMA up-regulates the expression of iNOS and LOX-1 mainly through the NF-κB pathway, which promotes the occurrence and development of atherosclerosis.
动脉粥样硬化(atherosclerosis, AS)是一类发生在动脉血管壁的慢性进行性炎症反应性疾病,是众多心脑血管疾病共同的病理基础,严重危害人类健康。血管内皮细胞损伤、平滑肌细胞增殖及巨噬细胞转化为泡沫细胞是粥样斑块形成的三个最为关键环节,其中巨噬细胞转化为泡沫细胞是AS发生的始动环节。国内外大量研究表明,高胆固醇血症、高血压、糖尿病、高同型半胱氨酸血症等是AS致病性的危险因素,血浆非对称性二甲基精氨酸(asymmetric dimethylarginine, ADMA)与这些危险因素密切相关,被认为是一个新的心血管疾病的危险因子。ADMA是内皮源性一氧化氮合酶(nitric oxide synthase, NOS)竞争性抑制剂。正常情况下人体血浆ADMA不足以抑制NOS活性,但在机体应激损伤时,血浆ADMA浓度则会明显升高。高浓度的ADMA可抑制NO合成、损伤血管内皮、促进VSMCs增生,但在巨噬细胞转化为泡沫细胞中的作用研究相对较少,其具体机制尚不明确。NOS是NO生物合成的关键限速酶,分为两类：持续表达型(constitutive nitric oxide synthase, cNOS)和诱导型(inducible nitric oxide synthase, iNOS)。cNOS广泛存在于正常的组织细胞中,iNOS在正常情况下低表达甚或不表达,但在受到各种刺激时可诱导表达。血凝素样氧化低密度脂蛋白受体1(lectin like oxidized low density lipoprotein receptor-1, LOX-1)是氧化低密度脂蛋白(oxidized low density lipoprotein, oxLDL)的特异受体,在结构上与巨噬细胞经典的清道夫受体不具有同源性,属于E型清道夫受体。巨噬细胞通过清道夫受体无负反馈性地吞噬大量氧化低密度脂蛋白,导致细胞内脂质堆积,形成泡沫细胞。研究表明,ADMA可促进巨噬细胞LOX-1表达,但具体机制尚不清楚。iNOS和LOX-1的基因启动子区都含有核因子-κB (nuclear factor kappa B, NF-κB)结合位点,能够与核蛋白特异结合调节下游基因转录。NF-κB是一种具有基因转录多项调控作用的转录因子,广泛存在于各种细胞中。本研究拟采用分子生物学实验方法,分别在细胞水平和组织水平上探讨在动脉粥样硬化形成过程中,ADMA影响iNOS、LOX-1表达的机制,是否是通过激活NF-κB实现的。
方法
1.体外细胞学实验
健康雄性Wistar大鼠50只。分离培养大鼠腹腔巨噬细胞。实验分组及处理：①正常对照组：以含10% FBS DMEM培养液与巨噬细胞共孵育；②oxLDL组：在巨噬细胞培养液中加入oxLDL(终浓度50mg/L)；③A+O组：在巨噬细胞培养液中先加入ADMA(终浓度15μmol/L)共孵育2h后,再加入oxLDL(终浓度50mg/L)；④P+A+O组：在巨噬细胞培养液中先加入PDTC(终浓度25μmol/L)共孵育30min后,再加入ADMA(终浓度15μmol/L)共孵育2h,最后加入oxLDL(终浓度50mg/L)。②③④组细胞在加入oxLDL后,再继续孵育48h。然后分别收集各组细胞和培养液,测定细胞内胆固醇含量及培养液NO水平和iNOS活性,分别提取细胞总RNA、总蛋白及核蛋白。以实时荧光定量PCR和Westen blot分别检测iNOS、LOX-1的mRNA和蛋白表达,以Actin为内参进行标化；以凝胶电泳迁移率变动分析(EMSA)和增强化学发光法(ECL)检测各组NF-κB活性。每组重复3次。
2.动物实验：健康雄性Wistar大鼠50只,随机分为四组：①正常对照组(n=10)：标准饲料喂养。②H组(n=12)：高脂饲料喂养。③A+H组(n=14)：高脂饲料喂养,给予ADMA [0.2mg/(kg·d)]灌胃,每日一次。④P+A+H组(n=14)：高脂饲料喂养,给予ADMA [0.2mg/(kg·d)]灌胃,每日一次；PDTC [40mg/(kg·d)]腹腔注射,每日一次。正常对照组、H组均给予等体积的生理盐水灌胃和腹腔注射,A+H组给予等体积的生理盐水腹腔注射。饲养满18周后麻醉大鼠,采血检测大鼠血清NO及iNOS活性；取胸主动脉做病理切片,观察其病理变化。分别以实时荧光定量PCR和Westen blot检测iNOS、LOX-1的mRNA和蛋白表达,以Actin为内参进行标化；以凝胶电泳迁移率变动分析(EMSA)和增强化学发光法(ECL)检测各组NF-κB活性。每组重复3次。
结果
1.体外细胞学实验：
①oxLDL组和A+O组巨噬细胞内胆固醇含量较正常对照组明显增加(均为P<0.05),且A+O较oxLDL组升高更明显(P<0.05),而P+A+O组胆固醇含量较A+O组降低(P<0.05)。②与正常对照组和oxLDL组相比,A+O组NO含量降低(P<0.05或P<0.01)：P+A+O组NO含量较A+O组显著升高(P<0.05)。③与正常对照组和oxLDL组相比,A+O组iNOS活力明显升高(均为P<0.05), P+A+O组iNOS活力较A+O组明显降低(P<0.05)。相关分析显示：NO含量和iNOS活力呈显著负相关(r=-0.773,P<0.05)④与正常对照组和oxLDL组相比,A+O组iNOS mRNA和蛋白表达量显著增加,其差异均有显著性(分别为P<0.01和P<0.05)；P+A+O组iNOS mRNA和蛋白表达较A+O组明显降低(P<0.05)。⑤A+O组LOX-1 mRNA和蛋白表达较正常对照组和oxLDL组增强,其差异均有显著性(均为P<0.05)；与A+O组相比,P+A+O组LOX-1 mRNA和蛋白表达明显降低,两组差异有显著性(P<0.05)。⑥A+O组NF-κB活性较正常对照组和oxLDL组明显增强,其差异均有显著性(均为P<0.05)；与A+O组相比,P+A+O组NF-κB活性明显降低,两组差异有显著性(P<0.05)。⑦相关分析：NF-κB活性与iNOS mRNA和蛋白表达量呈正相关(相关系数r分别为0.82、0.81)；NF-κB活性与LOX-1 mRNA和蛋白表达量呈正相关(相关系数r均为0.82)。
2.动物体内实验：
①主动脉病理切片HE染色显示：正常对照组血管内膜光滑、完整,未见空泡形成；H组血管内膜毛糙、但尚完整、有少量空泡形成；A+H组血管内膜断裂,血管中层SMCs增生明显,且排列紊乱,内膜层与中膜层有大量空泡形成；P+A+H组血管内膜完整,SMCs增生程度较A+H组明显减轻,空泡数量也较A+H组明显减少。②与对照组和H组比较,A+H组大鼠血清NO水平和iNOS活性明显升高,差异均有显著性(均为P<0.05),P+A+H组NO水平和iNOS活性较A+H组明显降低(均为P<0.05)。相关分析显示,NO水平和iNOS活性呈正相关(r=0.68,P<0.05)。③与对照组和H组相比,A+H组大鼠主动脉iNOS mRNA和蛋白表达量显著增加,(分别为P<0.01和P<0.05), P+A+H组iNOS mRNA和蛋白表达量较A+H组明显降低,两组相比差异有显著性(P<0.05)。④与对照组和H组相比,A+H组大鼠主动脉LOX-1 mRNA和蛋白表达量显著增加,(均为P＜O.05), P+A+H组LOX-1 mRNA和蛋白表达量较A+H组明显降低,两组相比差异有显著性(P<0.05)。⑤A+H组NF-κB活性较正常对照组和H组明显增强,其差异均有显著性(均为P<0.05)；与A+H组相比,P+A+H组NF-κB活性明显降低,两组差异有显著性(P<0.05)。⑥相关分析：NF-κB活性与iNOSmRNA和蛋白表达量呈正相关(相关系数r分别为0.85、0.87)；NF-κB活性与LOX-1mRNA和蛋白表达量呈正相关(相关系数r分别为0.79、0.81)。
结论
ADMA能够通过激活NF-κB上调iNOS和LOX-1的表达,从而促进AS的发生发展。
Background & Objective
Atherosclerosis (AS) is a chronic progressive inflammatory response within the artary wall and is the common pathological foundation of many cardiovascular diseases, which seriously threatens people's health. Three important aspects in formation of atherosclerosis plaque are the proliferation of vascular smooth muscle cells (VSMCs),injury of vascular endothelium cells, and macrophages thransforming into foam cells which is the first step in development of AS. A large number of researches at home and abroad indicate that hypercholesterolemia, hypertension, diabetes mellitus and hyperhomocysteinemia are pathogenic risk factors of AS. Asymmetric dimethylarginine (ADMA) is closely related with these risk factors, and is considered as a new risk factor of cardiovascular diseases. ADMA is a competitive inhibitor of endothelium drived nitric oxide synthase (NOS). In normal circumstances ADMA in human plasma has no effect on the activity of NOS, but under the stress status and the injury site, the concentration of ADMA in plasma is obviously increased. High concentration of ADMA plays an important role in inhibiting the synthasis of nitric oxide (NO), injuring the vascular endothelium and inducing VSMCs. However, the study is relatively few about the effect of ADMA on the process of macrophages thransforming into foam cells, and the mechainism is not clear. NOS is the key rate-limiting enzyme of NO biosynthesis, including constitutive nitric oxide synthase (cNOS) and inducibe nitric oxide synthase (iNOS). cNOS is known in a wide variety of cells and tissues. Normally, there is minimal or no expression of iNOS, but iNOS is induced to express by a variety of stimulus. Lectin like oxidized low density lipoprotein receptor-1 (LOX-1) is the specific receptor of oxidized low density lipoprotein (oxLDL). LOX-1 is different in structure from the classical scavenger receptors, belonging to scavenger receptor type E. Macrophages ingest a great quantity of oxLDL via scaverger receptors when there is no a negitive feedback, which leads to accumulation of lipid in the cytoplasm of macrophages and formation of foam cells. Researches show that ADMA enhances the expression of LOX-1 and its mechanism is not very clear yet. The promotor regions of iNOS gene containing an active nuclear factory kappa B (NF-κB) binding site, which can specially combine to nucleoprotein and regulate transcription of down-stream responsive genes. NF-κB is a ubiquitous intracellular transcription factor, taking part in regulateing gene transcription. In the present study, we adopted molecular biological techniques to explore the mechanism of ADMA inducing to express iNOS and LOX-1 during development of AS in cell level and tissue level, and whether activation of NF-κB has a relationship with the mechanism.
Methods
1. Study in vitro
Fifty-Healthy, male wistar rats were enrolled. Celiac macrophages of rats were gathered and cultured. Groups and interference:①Control group:macrophages were incubated with 10% FBS DMEM.②oxLDL group:macrophages were treated by oxLDL (final concentration 50mg/L) for 48 hours.③A+O group:macrophages were pretreated with ADMA (final concentration 15umol/L) for 2 hours, then co-incubated with oxLDL (final concentration 50mg/L) for 48 hours;④P+A+O group:macrophages were pretreated with PDTC (final concentration 25umol/L) for 30 minutes, then co-incubated with ADMA (final concentration 15umol/L) for 2 hours, finally co-incubated with oxLDL (final concentration 50mg/L) for 48 hours. Each group was repeated for 3 times. Macrophages and medium were respectively collected. Intracellular cholesterol level, NO level, iNOS activity were detected separately. The total RNA, total proteins and nucleoprotein were extracted from macrophages respectively. Real time fluorescent quantitative PCR (real-time PCR) was used to detected the expression of mRNA, including iNOS mRNA and LOX-1 mRNA. The expressions of iNOS and LOX-1 protein were measured by Westen blotting, using rat actin as an internal standard. NF-κB activity was detected with gelelectrophorestic mobility shift assay (EMSA) and enhanced chemiluminescent (ECL) technique.
2. Study in vivo
Fifty-Healthy, male wistar rats were enrolled and randomly divided into four groups.①Control group (n=10):Fed with standard diet.②H group (n=12):Fed with high fat diet.③A+H group (n=4):Fed with high fat diet, and administered intergastrically with ADMA [0.2mg/(kg-d)] once a day.④P+A+H group (n=14):Fed with high fat diet, administered intergastrically with ADMA [0.2mg/(kg-d)] and given interperitoneal injection with PDTC [40mg/(kg-d)], respectively once a day. Both control group and H group were administered intergastrically and injected interperitoneally with the same volume of normal saline. A+H group was injected interperitoneal with the same volume of normal saline.18 weeks later, the rats were anesthetized, rat blood and aortas were gathered. Serum NO level and iNOS activity were measured respectively. Collected thoracic aortas were made pathology slices and observed the histological structure under microscope. The total RNA, total protein and nucleoprotein were separately extracted from rat aortas. The expressions of iNOS mRNA and LOX-1 mRNA were detected with real time fluorescent quantitative PCR. Westen blotting was used to examinate the expression of iNOS and LOX-1 protein, using rat actin as an internal standard. EMSA and ECL technique was used to detect NF-κB activity.
Results
1. Study in vitro
①Intracellular cholesterol level in oxLDL group and A+O group were higher than that in control group (P<0.05,either), and compared with oxLDL group, intracellular cholesterol level in A+O group significantly increased (P<0.05), but intracellular cholesterol level of P+A+O group was lower than that of A+O group (P<0.05).②Compared with control group and oxLDL group, NO level in A+O group was significantly lower (P<0.05,either), and NO level of P+A+O group was much higher than that of A+O group (P<0.05).③The activity of iNOS in A+O group significantly higher than that of in control group and that of in oxLDL group (P＜0.05,either), but in P+A+O group, iNOS activity significantly decreased than that in A+O group (P<0.05). The correlation analysis indicated that NO level had a obvious negitive correlation with iNOS activity (r=-0.773,P<0.05).④Compared with control group and oxLDL group, the mRNA and protein of iNOS expression in A+O group significantly increased (P<0.05,either), and in P+A+O group,it obviously decreased than that in A+O group (P＜0.05).⑤The mRNA and protein of LOX-1 expression in A+O group significantly increased than that in control group and oxLDL group (P<0.05,either), but decreased in P+A+O group compared with A+O group (P<0.05).⑥In A+O group, the NF-κB activity markedly increased compared with that in control group and oxLDL group (P<0.05,either), and decreased in P+A+O group compared with A+O group.⑦Correlation analysis showed that the NF-κB activity took on a significant positive correlation with the expression of iNOS mRNA and protein (r=0.82,r=0.81,respectively;P<0.05), and the NF-κB activity took on a significant positive correlation with the expression of LOX-1 mRNA and protein (r=0.82,either;P<0.05).
2. Study in vivo
①Pathological examination:the paraffin-cut slices of thoracic slices were stained with HE and inspected under optic microscope. In control group the intima was smooth and integrated, without the formation of vacuoles. In H group, the intima contained a small amount vacuoles. In A+H group, the intima and elastic fibers ruptured, there were proliferation and irregular assay of VSMCs and a lot of vacuoles in intima and media. Compared with A+H group, the intima was integrated, the degree of VSMCs proliferation and the amount of vacuoles was decreased in P+A+H group.②Sesum NO level and iNOS activity in A+H group were obviously higher than those in control group and H group (P<0.05,either), and which in P+A+H group was lower than in A+H group (P＜O.05). Correlation analysis indicated that serum NO level was positively associated with iNOS activity (r=0.68,P<0.05).③The mRNA and protein of iNOS expressions in A+H group increased compared with those in control group and H group (P<0.05,either), and which in P+A+H group decreased than those in A+H group (P＜O.05).④The mRNA and protein of LOX-1 expressions in A+H group increased compared with those in control group and H group (P<0.05,either), and which in P+A+H group decreased than those in A+H group (P<0.05).⑤The NF-κB activity was significantly higher in A+H group than that in control group and H group (P<0.05,either), and was lower in P+A+H group than that in A+H group.⑥Correlation analysis showed that the NF-κB activity was significantly and positively correlated with the iNOS mRNA and protein expression (r=0.85, r=0.87, respectively; P＜O.05, either),and which was also significantly and positively correlated with the LOX-1 mRNA and protein expression (r=0.79, r=0.81, respectively; P＜O.05, either).
Conclusion
ADMA up-regulates the expression of iNOS and LOX-1 mainly through the NF-κB pathway, which promotes the occurrence and development of atherosclerosis.
引文
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