血管紧张素Ⅱ及其受体信号在佐剂性关节炎大鼠血管损伤中的作用
发布时间：2018-09-08 10:35
【摘要】：类风湿关节炎(rheumatoid arthritis,RA)被认为是增加心血管疾病(cardiovascular disease,CVD)发生率与死亡率的非传统风险因素。临床研究表明,与健康受试者相比,RA患者内皮功能异常,颈动脉粥样硬化程度和平均颈动脉中膜厚度增加,提示RA患者血管结构与功能异常可能是引起CVD发生率与死亡率增加的重要原因,探索RA炎症与CVD相互影响及其可能机制具有重要意义。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)是血管紧张素Ⅰ(angiotensin I,Ang I)在血管紧张素转化酶(angiotensin converting enzyme,ACE)的作用下水解产生的八肽物质,主要作用于细胞膜上的两个受体(angiotensinⅡreceptors,ATR)即AT1R、AT2R发挥生物学效应。AngⅡ对收缩血管、调节血压、维持电解质平衡起到重要作用。有研究表明,佐剂性关节炎(adjuvant-induced arthritis,AA)大鼠体内灌注AngⅡ可加重高血压反应、内皮功能紊乱及血管肥厚,但其对AA炎症严重程度及血管结构与功能产生怎样的影响及作用机制仍有待深入研究。RA患者CVD死亡率是正常人群的1.5~2倍,因此RA疾病的正确治疗对减少CVD的发生有重要意义。RA是一种慢性、系统性的自身免疫病,以受累关节的滑膜炎症和血管翳为基本病变。成纤维样滑膜细胞(fibroblast-like synovial cell,FLS)增生是RA疾病的主要病理特征之一。研究表明,RA患者血清、滑膜液中ACE水平升高,ACE能将Ang I转化为AngⅡ,间接提示AngⅡ参与了RA疾病病程。AngⅡ作用于AT1R发挥促炎、促增殖作用,作用于AT2R发挥抗炎、抗增殖作用。课题组前期研究表明,AT1R阻断剂氯沙坦(Losartan)、AT2R激动剂(CGP42112)均能改善AA大鼠的炎症反应。AngⅡ作用于ATR如何影响AA炎症及血管损伤的相关机制尚不清楚。目的:建立大鼠AHD模型,观察2K1C-HT与AA疾病之间的相互影响,探索高水平AngⅡ对AA大鼠炎症及血管损伤的影响及部分机制,为临床认识RA患者罹患CVD风险的机制提供实验依据。方法:分别在大鼠左后足跖皮内注射0.1ml完全弗氏佐剂(complete freund's adjuvant,CFA)建立AA模型,实施外科手术用0.25mm银夹缩窄大鼠左肾动脉建立两肾一夹高血压(two-kidney-one-clip hypertension,2K1C-HT)大鼠模型,以及同时施加上述两种方法建立类风湿关节炎复合高血压疾病(adjuvant-induced arthritis combined with two-kidney-one-clip hypertension disease,AHD)模型。免疫11天后,每三天测量大鼠的体重和足爪肿胀度,并进行炎症评分;用尾动脉测压仪每周检测大鼠的尾动脉收缩压;第35天处死大鼠,取出左肾、右肾和心脏并称重,同时收集踝关节、脾脏、胸主动脉用于病理检查;Western blot、激光共聚焦法检测AA、2K1C-HT、AHD关节FLS上AT1R、AT2R的表达与定位;血管环实验检测胸主动脉内皮依赖的血管舒张功能;酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)检测血清中AngⅡ、TNF-α的变化;免疫组化(Immunohistochemistry,IHC)法检测AngⅡ及其受体的表达及定位;Western blot法检测胸主动脉组织AngⅡ及下游信号分子如p-ERK1/2、p-NF-κB的表达变化;分别以CCK-8和Western blot法检测Losartan、CGP42112、PD123319对FLS增殖及AT1R、AT2R表达的影响。结果:1.采用CFA足跖免疫法联合2K1C法可成功建立AHD大鼠模型与正常组相比,AA大鼠体重明显降低,关节明显肿胀、炎症指标评分明显升高,HT大鼠左肾指数明显降低,血压值明显升高;AHD大鼠体重明显降低、关节炎指标评分明显升高;病理学检查可见关节腔有大量炎性细胞浸润、滑膜增生和软骨破坏,脾脏生发中心数量增多、淋巴细胞聚集成团、红髓弥漫性浸润,血清AngⅡ、TNF-α水平升高,同时,AHD大鼠第2周开始血压值升高,第5周显著升高,左肾指数明显降低,心脏指数明显升高,胸主动脉血管横截面积(cross-sectional area,CSA)、管壁中膜厚度(media thickness,MT)增大、管腔直径(lumen diameter,LD)降低,心肌细胞排列紊乱、断裂、肿大,CSA与轴长增加。提示,AHD大鼠同时具有RA与2K1C-HT模型的特征,表明AHD模型成功建立。2.2K1C-HT对AA炎症的影响及部分机制与AA大鼠比较,AHD大鼠发病时程提前,发病率有升高趋势,足爪肿胀度、关节炎指数、关节及脾脏病理评分明显升高,血清中AngⅡ水平明显升高,提示2K1C-HT加重了AA炎症病理情况。进一步研究发现,AA大鼠关节滑膜细胞上AT1R表达明显增加而AT2R无明显变化,AHD大鼠滑膜细胞的AT1R、AT2R表达均明显升高且AT1R与AT2R的比值明显升高,表明在炎症条件下,两肾一夹诱导的高水平AngⅡ主要作用于AT1R发挥促炎作用。同时,我们进一步观察了AngⅡ受体靶点药物对AA关节局部效应细胞—滑膜细胞增殖的影响,结果显示AT1R受体阻断剂Losartan(10-6mol/L)、AT2R受体激动剂CGP42112(10-6mol/L)均能抑制AA-FLS的增殖能力,而AT2R受体拮抗剂PD123319(10-6mol/L)对AA-FLS增殖无明显影响。Losartan联合PD123319与单独使用药物相比无明显差异,Losartan联合CGP42112抑制FLS增殖能力比单独使用药物强但无显著性差异。Losartan、CGP42112、PD123319均能降低AT1R的表达,Losartan联合CGP42112比单独使用CGP42112明显降低AT1R的表达。CGP42112能显著提高AT2R的表达,PD123319明显抑制AT2R的表达,Losartan联合CGP42112比单独使用Losartan、CGP42112明显提高AT2R的表达。3.AA炎症加重了2K1C-HT大鼠血管及心肌损伤与2K1C-HT大鼠相比,AHD大鼠血压值、心脏指数明显升高,血清中AngⅡ水平显著升高,胸主动脉的MT和CSA明显增加、LD明显降低。心脏组织心肌细胞排列紊乱、出现断裂、心肌细胞CSA和轴长明显增加,胸主动脉内皮依赖的血管舒张功能明显降低。提示,AA炎症加重了2K1C-HT大鼠的血管及心肌损伤。4.AngⅡ可能通过AT1R/ERK1/2信号通路参与了AHD大鼠的血管损伤AHD大鼠血清中AngⅡ水平显著升高,大鼠胸主动脉组织AngⅡ、AT1R、AT2R、GRK2的表达均明显增加,且主要在血管壁内侧表达。与2K1C-HT大鼠相比,AHD大鼠胸主动脉组织AngⅡ及下游信号分子(p-ERK1/2,p-NF-κB)的表达水平也明显增加,且AT1R/AT2R比值明显较高,提示AngⅡ可能主要作用于AT1R激活ERK1/2/NF-κB信号通路而加重了血管损伤。结论:1.采用CFA足跖免疫联合2K1C法可成功建立大鼠AHD模型;2.2K1C-HT可使AA发病率有增高趋势,并且加重AA大鼠关节炎病理情况;3.AngⅡ及其受体在2K1C-HT血管损伤中发挥了重要作用,AA炎症加剧了AngⅡ引起的血管损伤;4.AngⅡ激活AT1R/ERK1/2信号通路可能是其加重AA大鼠血管损伤的重要机制。
[Abstract]:Rheumatoid arthritis (RA) is considered to be an unconventional risk factor for increased incidence and mortality of cardiovascular disease (CVD). Clinical studies have shown that RA patients have endothelial dysfunction, increased carotid atherosclerosis and increased mean carotid media thickness compared with healthy subjects, suggesting that RA patients have increased risk of cardiovascular disease (CVD) and mortality. Abnormal vascular structure and function may be an important cause of increased CVD incidence and mortality. It is important to explore the interaction between RA inflammation and CVD and its possible mechanism. The octapeptide produced by hydrolysis of CE mainly acts on two receptors (ATR) on the cell membrane, namely AT1R and AT2R. Ang II plays an important role in contracting blood vessels, regulating blood pressure and maintaining electrolyte balance. Endothelial infusion of Ang II can aggravate hypertensive response, endothelial dysfunction and vascular hypertrophy, but its effect on the severity of AA inflammation, vascular structure and function remains to be further studied. Fibroblast-like synovial cell (FLS) hyperplasia is one of the main pathological features of RA. Studies have shown that serum and synovial fluid ACE levels in patients with RA increase, ACE can convert Ang I into Ang I I, indirectly. It is suggested that Ang II plays an important role in the pathogenesis of RA. Ang II acts on AT1R to promote inflammation, proliferation and anti-inflammatory and anti-proliferative effects on AT2R. Previous studies showed that AT1R blockers Losartan and AT2R agonists (CGP42112) can improve the inflammatory response of AA rats. How Ang II acts on ATR affects AA inflammation and vascular injury AIM: To establish a rat model of AHD, observe the interaction between 2K1C-HT and AA disease, explore the effects of high-level Ang II on inflammation and vascular injury in AA rats, and provide experimental evidence for clinical understanding of the risk of CVD in RA patients. METHODS: Intradermal injection of 0.1ml of Ang II into the left hind plantar of rats respectively. Complete Freund's adjuvant (CFA) was used to establish AA model, left renal artery was constricted with 0.25 mm silver clip for surgical operation to establish two-kidney-one-clip hypertension (2K1C-HT) rat model, and rheumatoid arthritis combined with hypertension (adjuvant-indu) was established by both methods. After 11 days of immunization, body weight and paw swelling were measured every three days, and inflammation scores were made; tail artery systolic blood pressure was measured weekly with tail artery manometer; rats were sacrificed on the 35th day, left kidney, right kidney and heart were taken out and weighed. Ankle, spleen and thoracic aorta were used for pathological examination; Western blot and laser confocal focusing were used to detect the expression and localization of AT1R and AT2R on FLS of AA, 2K1C-HT and AHD joints; vascular ring test was used to detect endothelium-dependent vasodilation of thoracic aorta; enzyme-linked immunosorbent assay (ELISA) was used to detect Ang II and TNF-alpha in serum. Immunohistochemistry (IHC) was used to detect the expression and localization of Ang II and its receptors; Western blot was used to detect the expression of Ang II and downstream signal molecules such as p-ERK1/2, p-NF-kappa B in thoracic aorta; CCK-8 and Western blot were used to detect the effects of Losartan, CGP42112, PD123319 on the proliferation of FLS and the expression of AT1R and AT2R, respectively. Results: 1. Compared with the normal group, the weight of AA rats was significantly reduced, the joint swelled, the inflammatory index score was significantly increased, the left kidney index of HT rats was significantly decreased, the blood pressure value was significantly increased, the weight of AHD rats was significantly reduced, and the arthritis index score was significantly increased. There were a lot of inflammatory cells infiltration in the joint cavity, synovial hyperplasia and cartilage destruction, the number of splenic germinal center increased, lymphocyte aggregation, diffuse infiltration of red pulp, serum Ang II, TNF-a levels increased. At the same time, AHD rats began to increase blood pressure in the second week, increased significantly in the fifth week, left renal index decreased significantly, heart index increased significantly. High, cross-sectional area (CSA), media thickness (MT) increased, lumen diameter (LD) decreased, myocardial cells arranged disorderly, ruptured, enlarged, CSA and axial length increased. These results suggest that AHD rats have the characteristics of both RA and 2K1C-HT models, indicating that the AHD model was successfully established.2.2K1C-HT on A. Compared with AA rats, the effect of A-inflammation and some mechanisms in AHD rats showed that the course of disease was earlier, the incidence of AHD rats increased, the degree of paw swelling, arthritis index, pathological scores of joints and spleens were significantly increased, and the level of Ang II in serum was significantly increased, suggesting that 2K1C-HT aggravated the inflammatory pathology of AA rats. The expression of AT1R and AT2R in synovial cells of AHD rats were significantly increased and the ratio of AT1R to AT2R was significantly increased. It indicated that the high level of Ang II induced by two kidneys one clip mainly acted on AT1R to promote inflammation under inflammatory conditions. The effect of AT1R receptor antagonist Losartan (10-6mol/L) and AT2R receptor agonist CGP42112 (10-6mol/L) on the proliferation of AA-FLS was observed, while AT2R receptor antagonist PD123319 (10-6mol/L) had no significant effect on the proliferation of AA-FLS. Losartan, CGP42112, PD123319 all decreased the expression of AT1R, Losartan combined with CGP42112 significantly decreased the expression of AT1R compared with CGP42112 alone. CGP42112 significantly increased the expression of AT2R, PD123319 significantly inhibited the expression of AT2R. Losartan combined with CGP42112 significantly increased the expression of AT2R compared with Losartan alone. 3. AA inflammation aggravated vascular and myocardial injury in 2K1C-HT rats. Compared with 2K1C-HT rats, the blood pressure and cardiac index of AHD rats were significantly increased, the levels of Ang II in serum were significantly increased, the levels of CT and CSA in thoracic aorta were significantly increased, and LD was significantly decreased. These results suggest that AA inflammation aggravates vascular and myocardial injury in 2K1C-HT rats. 4. Ang II may participate in the serum Ang II water in AHD rats through AT1R/ERK1/2 signaling pathway. The expression of Ang II, AT1R, AT2R and GRK 2 in the thoracic aorta of AHD rats was significantly higher than that of 2K1C-HT rats. The expression of Ang II and downstream signal molecule (p-ERK1/2, p-NF-kappa B) in the thoracic aorta of AHD rats was also significantly higher than that of 2K1C-HT rats, and the AT1R/AT2R ratio was significantly higher, suggesting that Ang II might be the main factor. Conclusion: 1. The rat model of AHD can be successfully established by CFA plantar immunization combined with 2K1C method; 2.2K1C-HT can increase the incidence of AA and aggravate the pathological condition of AA rat arthritis; 3. Ang II and its receptor play an important role in the vascular injury of 2K1C-HT, AA. Ang II activates AT1R/ERK1/2 signaling pathway, which may play an important role in aggravating vascular injury in AA rats.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22;R54
[Abstract]:Rheumatoid arthritis (RA) is considered to be an unconventional risk factor for increased incidence and mortality of cardiovascular disease (CVD). Clinical studies have shown that RA patients have endothelial dysfunction, increased carotid atherosclerosis and increased mean carotid media thickness compared with healthy subjects, suggesting that RA patients have increased risk of cardiovascular disease (CVD) and mortality. Abnormal vascular structure and function may be an important cause of increased CVD incidence and mortality. It is important to explore the interaction between RA inflammation and CVD and its possible mechanism. The octapeptide produced by hydrolysis of CE mainly acts on two receptors (ATR) on the cell membrane, namely AT1R and AT2R. Ang II plays an important role in contracting blood vessels, regulating blood pressure and maintaining electrolyte balance. Endothelial infusion of Ang II can aggravate hypertensive response, endothelial dysfunction and vascular hypertrophy, but its effect on the severity of AA inflammation, vascular structure and function remains to be further studied. Fibroblast-like synovial cell (FLS) hyperplasia is one of the main pathological features of RA. Studies have shown that serum and synovial fluid ACE levels in patients with RA increase, ACE can convert Ang I into Ang I I, indirectly. It is suggested that Ang II plays an important role in the pathogenesis of RA. Ang II acts on AT1R to promote inflammation, proliferation and anti-inflammatory and anti-proliferative effects on AT2R. Previous studies showed that AT1R blockers Losartan and AT2R agonists (CGP42112) can improve the inflammatory response of AA rats. How Ang II acts on ATR affects AA inflammation and vascular injury AIM: To establish a rat model of AHD, observe the interaction between 2K1C-HT and AA disease, explore the effects of high-level Ang II on inflammation and vascular injury in AA rats, and provide experimental evidence for clinical understanding of the risk of CVD in RA patients. METHODS: Intradermal injection of 0.1ml of Ang II into the left hind plantar of rats respectively. Complete Freund's adjuvant (CFA) was used to establish AA model, left renal artery was constricted with 0.25 mm silver clip for surgical operation to establish two-kidney-one-clip hypertension (2K1C-HT) rat model, and rheumatoid arthritis combined with hypertension (adjuvant-indu) was established by both methods. After 11 days of immunization, body weight and paw swelling were measured every three days, and inflammation scores were made; tail artery systolic blood pressure was measured weekly with tail artery manometer; rats were sacrificed on the 35th day, left kidney, right kidney and heart were taken out and weighed. Ankle, spleen and thoracic aorta were used for pathological examination; Western blot and laser confocal focusing were used to detect the expression and localization of AT1R and AT2R on FLS of AA, 2K1C-HT and AHD joints; vascular ring test was used to detect endothelium-dependent vasodilation of thoracic aorta; enzyme-linked immunosorbent assay (ELISA) was used to detect Ang II and TNF-alpha in serum. Immunohistochemistry (IHC) was used to detect the expression and localization of Ang II and its receptors; Western blot was used to detect the expression of Ang II and downstream signal molecules such as p-ERK1/2, p-NF-kappa B in thoracic aorta; CCK-8 and Western blot were used to detect the effects of Losartan, CGP42112, PD123319 on the proliferation of FLS and the expression of AT1R and AT2R, respectively. Results: 1. Compared with the normal group, the weight of AA rats was significantly reduced, the joint swelled, the inflammatory index score was significantly increased, the left kidney index of HT rats was significantly decreased, the blood pressure value was significantly increased, the weight of AHD rats was significantly reduced, and the arthritis index score was significantly increased. There were a lot of inflammatory cells infiltration in the joint cavity, synovial hyperplasia and cartilage destruction, the number of splenic germinal center increased, lymphocyte aggregation, diffuse infiltration of red pulp, serum Ang II, TNF-a levels increased. At the same time, AHD rats began to increase blood pressure in the second week, increased significantly in the fifth week, left renal index decreased significantly, heart index increased significantly. High, cross-sectional area (CSA), media thickness (MT) increased, lumen diameter (LD) decreased, myocardial cells arranged disorderly, ruptured, enlarged, CSA and axial length increased. These results suggest that AHD rats have the characteristics of both RA and 2K1C-HT models, indicating that the AHD model was successfully established.2.2K1C-HT on A. Compared with AA rats, the effect of A-inflammation and some mechanisms in AHD rats showed that the course of disease was earlier, the incidence of AHD rats increased, the degree of paw swelling, arthritis index, pathological scores of joints and spleens were significantly increased, and the level of Ang II in serum was significantly increased, suggesting that 2K1C-HT aggravated the inflammatory pathology of AA rats. The expression of AT1R and AT2R in synovial cells of AHD rats were significantly increased and the ratio of AT1R to AT2R was significantly increased. It indicated that the high level of Ang II induced by two kidneys one clip mainly acted on AT1R to promote inflammation under inflammatory conditions. The effect of AT1R receptor antagonist Losartan (10-6mol/L) and AT2R receptor agonist CGP42112 (10-6mol/L) on the proliferation of AA-FLS was observed, while AT2R receptor antagonist PD123319 (10-6mol/L) had no significant effect on the proliferation of AA-FLS. Losartan, CGP42112, PD123319 all decreased the expression of AT1R, Losartan combined with CGP42112 significantly decreased the expression of AT1R compared with CGP42112 alone. CGP42112 significantly increased the expression of AT2R, PD123319 significantly inhibited the expression of AT2R. Losartan combined with CGP42112 significantly increased the expression of AT2R compared with Losartan alone. 3. AA inflammation aggravated vascular and myocardial injury in 2K1C-HT rats. Compared with 2K1C-HT rats, the blood pressure and cardiac index of AHD rats were significantly increased, the levels of Ang II in serum were significantly increased, the levels of CT and CSA in thoracic aorta were significantly increased, and LD was significantly decreased. These results suggest that AA inflammation aggravates vascular and myocardial injury in 2K1C-HT rats. 4. Ang II may participate in the serum Ang II water in AHD rats through AT1R/ERK1/2 signaling pathway. The expression of Ang II, AT1R, AT2R and GRK 2 in the thoracic aorta of AHD rats was significantly higher than that of 2K1C-HT rats. The expression of Ang II and downstream signal molecule (p-ERK1/2, p-NF-kappa B) in the thoracic aorta of AHD rats was also significantly higher than that of 2K1C-HT rats, and the AT1R/AT2R ratio was significantly higher, suggesting that Ang II might be the main factor. Conclusion: 1. The rat model of AHD can be successfully established by CFA plantar immunization combined with 2K1C method; 2.2K1C-HT can increase the incidence of AA and aggravate the pathological condition of AA rat arthritis; 3. Ang II and its receptor play an important role in the vascular injury of 2K1C-HT, AA. Ang II activates AT1R/ERK1/2 signaling pathway, which may play an important role in aggravating vascular injury in AA rats.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22;R54
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