富含亮氨酸重复结构域蛋白3炎症小体在高血压大鼠主动脉夹层模型形成中的作用

The role of nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome in the development of hypertensive rat models of aortic dissection

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 the hypertensive rat models of aortic dissection（AD）,and to explore the role of nucleotide-binding domain and leucine-rich repeat protein 3（NLRP3）inflammasome in the development of AD induced by hypertension and its related molecular mechanisms. Methods Forty female Sprague-Dawley（SD）rats（3weeks old,weighing about 60g）were randomly assigned to 4groups,with 10 rats in each group.The rats in control group were fed with normal diet and water;the rats inβ-aminopropionitrile（BAPN）group（AD model group）were fed with the diet containing 0.25% BAPN;the rats in desoxycorticosterone acetate（DOCA）group were fed with the diet containing 0.25% BAPN and administered DOCA subcutaneously;and the rats in Nω-nitro-larginine-methyl-ester（l-NAME）group were fed with the diet containing 0.25% BAPN and water containing l-NAME. Water intake,body weight and blood pressure of all rats were recorded. After the rats died（rats in control group were killed,and model rats died of natural causes）,the aortas of rats were separated,the changes of collagenous fibers and elastic fibers were observed by Masson＇s trichrome staining and Elastica-van Gieson（EVG）staining,respectively. The expressions of NLRP3 inflammasome related proteins including NLRP3,Caspase-1and interleukin-1β（IL-1β）were assayed by immumohistochemical staining. Real-time polymerase chain reaction（PCR）and Western blot were used to detect the mRNA and protein expressions of NLRP3 inflammasome related proteins,respectively. Results Compared to the control group,the body weight of rats fed with BAPN for 2weeks was significantly reduced,and water intake was significantly increased in DOCA group. Blood pressure in DOCA group and l-NAME group was significantly higher than that in BAPN group[mean arterial pressure in the second week of intervention：（95.4±10.1）,（110.0±9.9）vs（70.4±2.9）mm Hg;mean arterial pressure in the third week of intervention：（128.4±13.8）,（118.5±8.1）vs（76.4±5.3）mm Hg,all P0.01]. Masson＇s trichrome staining showed that the collagenous fibers of aortas in DOCA group and l-NAME group were significantly more than those in the BAPN group（both P0.05）. EVG staining showed that the elastic fibers of aortas in DOCA group and l-NAME group were significantly more than those in the BAPN group（both P0.05）,with more irregular arrangement and more serious fragmentation in these two groups. Immumohistochemical staining showed that the expressions of NLRP3 inflammasome related proteins in DOCA group and l-NAME group were significantly higher than those in BAPN group（NLRP3：6.63±0.51,6.78±0.47 vs 3.32±0.39;Caspase-1：5.22±0.45,6.19±0.31vs3.36±0.30;IL-1β：5.51±0.53,5.80±0.40 vs 3.18±0.35;all P0.05）. Additionally,the mRNA and protein expressions of NLRP3 inflammasome related proteins in DOCA group and l-NAME group were markedly higher than those in BAPN group（all P0.05）. Conclusions NLRP3 inflammasome played a role in the development of AD induced by hypertension in rats.