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目的建立高血压大鼠主动脉夹层(AD)模型,探讨富含亮氨酸重复结构域蛋白(NLRP)3炎症小体在高血压促AD形成中的作用及相关分子机制。方法 40只雌性SD大鼠(3周龄,体质量60g左右)随机分为4组,每组10只:对照组:正常喂食饮水;β-氨基丙腈(BAPN)组(AD模型组):0.25%BAPN喂食;醋酸去氧皮质酮(DOCA)组:0.25%BAPN喂食,并皮下注射DOCA油剂;左旋硝基精氨酸甲酯(l-NAME)组:0.25%BAPN喂食,饮含l-NAME水。记录各组大鼠饮水、体质量及血压;动物死亡后(对照组大鼠为处死,模型组为自然死亡)分离主动脉行Masson三色染色及EVG染色,分别观察主动脉内胶原纤维、弹力纤维的变化情况,行免疫组化染色观察主动脉中NLRP3炎症小体相关蛋白[NLRP3、Caspase-1、白细胞介素1β(IL-1β)]的表达情况,采用实时聚合酶链反应及Western blot法分别检测主动脉内NLRP3炎症小体相关蛋白的基因及蛋白表达情况。结果与对照组比较,BAPN喂养2周后大鼠体质量明显减轻,DOCA组大鼠饮水量明显增加。DOCA组、l-NAME组大鼠血压较BAPN组明显升高[干预第2周平均动脉压:(95.4±10.1)、(110.0±9.9)比(70.4±2.9)mm Hg;干预第3周平均动脉压:(128.4±13.8)、(118.5±8.1)比(76.4±5.3)mm Hg,均P<0.01]。Masson三色染色显示:DOCA组、l-NAME组大鼠主动脉中胶原纤维较BAPN组明显增多(均P<0.05)。EVG染色显示:DOCA组、l-NAME组大鼠主动脉中弹力纤维较BAPN组明显增多(均P<0.05),且排列更加紊乱,断裂情况更加严重。免疫组化显示:与BAPN组比较,DOCA组、l-NAME组大鼠主动脉中NLRP3炎症小体相关蛋白表达增加(NLRP3:6.63±0.51、6.78±0.47比3.32±0.39;Caspase-1:5.22±0.45、6.19±0.31比3.36±0.30;IL-1β:5.51±0.53、5.80±0.40比3.18±0.35;均P<0.05)。与BAPN组比较,DOCA组及l-NAME组大鼠主动脉内NLRP3炎症小体相关蛋白在基因水平、蛋白水平的表达显著增加(均P<0.05)。结论 NLRP3炎症小体参与高血压促大鼠AD的形成。
Objective To establish a model of aortic dissection (AD) in hypertensive rats and to explore the role and related molecular mechanisms of leptin-rich repetitive domain protein (NLRP) 3 inflammasome in hypertension-induced AD formation. Methods 40 female SD rats (3 weeks old and body weight about 60 g) were randomly divided into 4 groups with 10 rats in each group: control group: normal drinking water; β-aminopropionitrile (BAPN) group (AD model group) 0.25% BAPN feeding; DOCA group: 0.25% BAPN feeding, and subcutaneous injection of DOCA oil; L-NAME group: 0.25% BAPN feeding, -NAME water. The water and body weight and blood pressure of rats in each group were recorded. Masson’s trichrome staining and EVG staining of isolated aorta were performed after the animals were sacrificed (the rats in the control group were sacrificed and the model group died of spontaneous death). Collagen fibers in the aorta, (NLRP3, Caspase-1 and IL-1β) in the aorta were detected by immunohistochemical staining. Real-time polymerase chain reaction and Western blot Method were used to detect the gene and protein expression of NLRP3 inflammasome-related proteins in the aorta. Results Compared with the control group, the body weight of rats in the BAPN group was significantly reduced after 2 weeks of feeding, and the water intake of rats in the DOCA group was significantly increased. Compared with BAPN group, the blood pressure of DOCA group and l-NAME group was significantly higher than that of BAPN group [mean arterial pressure (95.4 ± 10.1), (110.0 ± 9.9) vs (70.4 ± 2.9) mm Hg in the second week of intervention; Arterial pressure: (128.4 ± 13.8), (118.5 ± 8.1) vs (76.4 ± 5.3) mm Hg, all P <0.01]. Masson’s trichrome staining showed that collagen fibers in the aorta of DOCA group and l-NAME group were significantly increased compared with BAPN group (all P <0.05). EVG staining showed that the elastic fibers in the aorta of DOCA group and l-NAME group were significantly more than those of BAPN group (all P <0.05), and the arrangement was more disordered and the fracture was more serious. Immunohistochemistry showed that compared with BAPN group, expression of NLRP3 inflammasome related protein increased in DOCA group and l-NAME group (NLRP3: 6.63 ± 0.51,6.78 ± 0.47 vs 3.32 ± 0.39; Caspase-1: 5.22 ± 0.45,6.19 ± 0.31 vs 3.36 ± 0.30; IL-1β: 5.51 ± 0.53,5.80 ± 0.40 vs. 3.18 ± 0.35; all P <0.05). Compared with BAPN group, the levels of NLRP3 inflammasome related proteins in DOCA group and l-NAME group were significantly increased at gene level and protein level (all P <0.05). Conclusion NLRP3 inflammasome is involved in the formation of AD in hypertensive rats.