膳食辣椒素对醋酸脱氧皮质酮－盐型高血压大鼠肾脏氧化应激损害的保护作用

Renoprotective effects and mechanisms of dietary capsaicin in deoxycorticosterone acetate-salt hypertensive rats

目的观察膳食辣椒素对醋酸脱氧皮质酮(DOCA)-盐型高血压大鼠的肾脏保护作用。方法 10周龄雄性Sprague Dawley(SD)大鼠16只,术后随机分成普食组和辣椒素组,每组8只;另取8只大鼠行假手术作为对照(假手术组)。辣椒素组于术后给予含0.2g/kg辣椒素饲料喂养,普食组与假手术组给予普通饲料喂养,膳食干预时间为8周。每周监测血压、体质量;干预8周后收集24h尿液及取血浆观察肌酐、尿素氮、尿蛋白等指标;解剖显微镜下分离肾内小动脉行冰冻切片,利用超氧阴离子和一氧化氮的荧光探针染色后分析一氧化氮和超氧阴离子水平,Western blot法观察肾内小动脉解耦联蛋白2(UCP2)、硝基酪氨酸、磷酸化内皮型一氧化氮合酶(p-eNOS)和瞬时受体电位香草醛亚家族1(TRPV1)蛋白表达的变化。结果干预8周后,DOCA-普食组收缩压高于假手术组[(186.5±9.4)比(103.8±8.9)mm Hg,P<0.01];肾内小动脉超氧阴离子水平升高,一氧化氮水平下降[DHE荧光强度(69.1±3.0)比(18.3±1.6),P<0.01;DAF-2DA荧光强度:(15.0±1.1)比(27.0±0.8),P<0.01];Western blot显示肾内小动脉组织TRPV1、p-eNOS下降(P<0.01),硝基酪氨酸蛋白表达水平增加(P<0.01);单肾质量/体质量、血尿素氮、24h尿蛋白、血肌酐增加[分别(9.64±1.31)比(4.13±0.64)mg/g;(11.13±3.01)比(4.25±1.66)mmol/L,(44.85±8.50)比(7.48±1.07)mg/24h,(71.47±9.32)比(36.97±2.32)μmol/L,均P<0.05],肌酐清除率下降[(2.25±1.13)比(5.88±0.14)mL/min,P<0.05]。与DOCA-普食组相比,DOCA-辣椒素组肾内小动脉TRPV1和UCP2表达、肾内小动脉组织中p-eNOS蛋白表达水平、一氧化氮水平增加(均P<0.01),硝基酪氨酸蛋白表达、超氧阴离子水平降低(均P<0.01),血压[(160.3±7.3)比(186.5±9.4)mm Hg,P<0.01]和肾功能指标[单肾质量/体质量:(7.94±1.08)比(9.64±1.31)mg/g;血尿素氮:(8.25±1.16)比(11.13±3.01)mmol/L;24h尿蛋白:(31.54±6.45)比(44.85±8.50)mg/24h;血肌酐:(60.32±5.13)比(71.47±9.32)μmol/L,均P<0.05]降低。结论辣椒素对DOCA-盐型高血压大鼠肾脏氧化应激损害有明显的保护作用,其机制涉及激活TRPV1,上调UCP2的表达,从而减轻氧化应激对肾脏的损害。

Objective To investigate the effects of capsaicin, the agonist of transient receptor potential vanilloid 1 （TRPV1）, on the progression of renal injury in deoxycorticosterone acetate （DOCA）-salt hypertensive rats. Methods Sixteen Sprague-Dawley （SD） male rats { 10 weeks old） were unilaterally nephrectomized and randomly di- vided into the normal diet group （n=8） and the capsaicin group （n= 8）. Additional 8 SD male rats were treated with sham operation （the Sham group）. In the coming 8 weeks, rats in the eapsaicin group were fed with dietary capsaicin at 0.2 g/kg, and rats in both normal diet and Sham groups were fed with normal diet. For all rats, tail blood pressure and body weight were recorded every week. After 8-week intervention, 24-h proteinuria, blood urea nitrogen （BUN）, serum creatinine （Scr）, and urine creatinine {Ucr） were measured. Renal arteries were separated,and surperoxide anion and nitric oxide （NO） were detected by fluorescent probe. The expressions of TRPV1, p-eNOS, nitrotyrosine, and uncoupling protein 2 （UCP2） in renal arteries were determined by Western blot. Results Rats in the normal diet group had significantly higher levels of systolic blood pressure [（ 186.5± 9.4） vs （103.8±8.9）mm Hg, P〈0.05] and superoxide anion （DHE fluorescence intensity： （69.1±3.0） vs （18.3±1.6）, P〈0.01,], but lower levels of nitric oxide [DAF-2DA fluorescence intensity： （15.0±1.1） vs （27.0±0.8）, P〈 0.01] than rats in the Sham group. As shown in Western blot, .the levels of TRPV1 in intrinsic artery of kidney tis- sue and p-eNOS were lower, but expression of nitrotyrosine was higher （P〈0.01）, in the normal diet group than the Sham group （all P〈0.01）. Moreover, there were higher levels of kidney weight/body weight [（9.64±1.31） vs （4.13±0.64）mg/g-], BUN [（11.13±3.01） vs {4.25±1. 66）mmol/L], 24-h proteinuria [（44.85±8.50） vs {7.48±1.07}mg/24 h,] and Scr [（71.47±9.32} vs （36.97±2.32）μmol/L,] and lower levels of creatinine clearance rate [（2.25±1.13） vs （5.88±0.14）mL/min] in the normal diet group than the Sham group （all P〈0.05}. Com- pared with the normal diet group, expression levels of TRPV1, UCP2, and p-eNOS and NO were significantly in- creased in the capsaicin group （all P〈0.01）, while decreased levels were observed for tyrosine nitration and super- oxide anion （P〈0.01}, systolic blood pressure [（160.3±7.3} vs （186.5±9.4}mm Hg, P〈0.05-1, kidney weight/body weight [（7.94±1.08} vs （9.64±1.31）mg/g, P〈0.051, BUN [（8.25± 1.16） vs （11.13±3.01} mmol/L, P〈0.05-], 24-h proteinuria [（31.54±6.45} vs （44.85±8.50}mg/24 h-1, Scr （60. 32±5.13） vs （71.47± 9.32）μmol/L, P〈0.05]. Conclusion Capsaicin was observed to have obvious protective effect on renal damage in DOCA-salt hypertensive rats by improving oxidative stress, and the mechanisms mainly involved the activation of TRPV1 and up-regulation of UCP2.