基质金属蛋白酶-9抑制剂治疗自身免疫性心肌炎大鼠模型的实验研究
기질금속단백매-9억제제치료자신면역성심기염대서모형적실험연구
Effects of matrix metalloproteinase-9 inhibitor in Lewis rats with experimental autoimmune myocarditis
  • 万方数据
    中华心血管病杂志 중화심혈관병잡지
    Chinese Journal of Cardiology
    2011年 2期 118-123 ,共6页

    韩丽娜%李铁岭%张亚晶%杨庭树%丁宇%赵晓宁%郭树理

    한려나%리철령%장아정%양정수%정우%조효저%곽수리

    心肌炎%MMP-9%米诺环素 心肌炎%MMP-9%米諾環素
    심기염%MMP-9%미낙배소
    Myocarditis%Matrix metalloproteinase-9%Minocycline
    目的 观察基质金属蛋白酶(MMP)-9抑制剂米诺环素对实验性自身免疫性心肌炎(EAM)Lewis大鼠模型的治疗效果,并探讨其机制.方法 Lewis大鼠60只,6～8周龄,体重150～170 g.双足底注射心肌C蛋白片段和完全弗氏辅佐剂的油状混合物,腹腔注射百日咳毒素建立自身免疫性心肌炎大鼠模型.根据药物干预的时期不同将大鼠分为早期干预组、中期干预组和晚期于预组3组,每组20只,不同的干预组又分为米诺环素治疗组(治疗组)和对照组两个亚组,每个亚组10只.早期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第1～21天.中期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第8～28天.晚期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第15～35天.各对照组均为在相同时间给予与治疗组相同体积的生理盐水腹腔注射.干预结束后,处死动物,心脏取材,进行系列检测.组织病理学石蜡切片苏木素-伊红(HE)染色检测心肌炎症分级,天狼猩红染色检测心肌胶原纤维含量,免疫组织化学染色检测心肌巨噬细胞和T细胞浸润,实时定量PCR检测MMP-2、MMP-9的mRNA表达水平,冰冻切片用于原位明胶酶谱法检测明胶酶活性.结果 早期干预组和中期干预组的治疗组大鼠心肌组织石蜡切片显示心肌组织炎症积分均显著低于其相应对照组[分别为1.51±0.36比3.03±1.35(P＜0.05)和2.11±0.82比3.75±0.29(P＜0.01)],免疫组织化学显示心肌巨噬细胞和T淋巴细胞浸润数目均显著低于其相应对照组,天狼猩红染色显示心肌间质纤维积分和纤维含量均明显低于其相应对照组[分别为1.51±0.35比2.75±0.29(P＜0.01)和1.61±0.42比2.50±0.41(P＜0.05)],实时定量PCR检测心肌MMP-2和MMP-9 mRNA表达低于其相应对照组,原位明胶酶法显示心肌明胶酶活性显著低于其相应对照组[分别为62 366±2131比162 367±5095(P＜0.01)和113 197±4809比184 256±5427(P＜0.01)].晚期干预组治疗组与对照组相比较心肌组织炎症积分、心肌巨噬细胞和T淋巴细胞浸润数目、心肌间质纤维化积分和纤维含量、心肌组织MMP-2和MMP-9 mRNA表达及心肌明胶酶活性差异均无统计学意义.结论 MMP-9抑制剂抑制EAM的早期病理发展,其机制可能与降低心肌炎症细胞浸润,延缓心肌间质纤维化,从转录水平降低明胶酶mRNA的表达,同时从蛋白水平降低明胶酶活性表达有关.
    목적 관찰기질금속단백매(MMP)-9억제제미낙배소대실험성자신면역성심기염(EAM)Lewis대서모형적치료효과,병탐토기궤제.방법 Lewis대서60지,6～8주령,체중150～170 g.쌍족저주사심기C단백편단화완전불씨보좌제적유상혼합물,복강주사백일해독소건립자신면역성심기염대서모형.근거약물간예적시기불동장대서분위조기간예조、중기간예조화만기우예조3조,매조20지,불동적간예조우분위미낙배소치료조(치료조)화대조조량개아조,매개아조10지.조기간예조치료조위복강주사50 mg/kg적미낙배소,1차/d,련속21 d,면역주사후제1～21천.중기간예조치료조위복강주사50 mg/kg적미낙배소,1차/d,련속21 d,면역주사후제8～28천.만기간예조치료조위복강주사50 mg/kg적미낙배소,1차/d,련속21 d,면역주사후제15～35천.각대조조균위재상동시간급여여치료조상동체적적생리염수복강주사.간예결속후,처사동물,심장취재,진행계렬검측.조직병이학석사절편소목소-이홍(HE)염색검측심기염증분급,천랑성홍염색검측심기효원섬유함량,면역조직화학염색검측심기거서세포화T세포침윤,실시정량PCR검측MMP-2、MMP-9적mRNA표체수평,빙동절편용우원위명효매보법검측명효매활성.결과 조기간예조화중기간예조적치료조대서심기조직석사절편현시심기조직염증적분균현저저우기상응대조조[분별위1.51±0.36비3.03±1.35(P＜0.05)화2.11±0.82비3.75±0.29(P＜0.01)],면역조직화학현시심기거서세포화T림파세포침윤수목균현저저우기상응대조조,천랑성홍염색현시심기간질섬유적분화섬유함량균명현저우기상응대조조[분별위1.51±0.35비2.75±0.29(P＜0.01)화1.61±0.42비2.50±0.41(P＜0.05)],실시정량PCR검측심기MMP-2화MMP-9 mRNA표체저우기상응대조조,원위명효매법현시심기명효매활성현저저우기상응대조조[분별위62 366±2131비162 367±5095(P＜0.01)화113 197±4809비184 256±5427(P＜0.01)].만기간예조치료조여대조조상비교심기조직염증적분、심기거서세포화T림파세포침윤수목、심기간질섬유화적분화섬유함량、심기조직MMP-2화MMP-9 mRNA표체급심기명효매활성차이균무통계학의의.결론 MMP-9억제제억제EAM적조기병리발전,기궤제가능여강저심기염증세포침윤,연완심기간질섬유화,종전록수평강저명효매mRNA적표체,동시종단백수평강저명효매활성표체유관.
    Objective To investigate the effects of matrix metalloproteinase-9 (MMP-9) inhibitor minocyclin hydrochloride in Lewis rats with experimental autoimmune myocarditis (EAM). Methods EAM was induced by injection of cardiac C protein emulsified in completed Freund adjuvant in double footpad and intra peritoneal injection of pertussis toxin on 6- to 8-week old Lewis rats. Sixty EAM Lewis rats were dividedinto 3 groups (early, middle and late intervention groups, n =20 each: 10 minocyclin treated and 10 control rats). In early intervention group, rats in treatment group received intraperitoneal injection of minocyclin hydrochloride from 1st to 21st day after immunization; in middle intervention group, rats were treated from 8th to 28th day after immunization and in late intervention group, rats were treated from 15th to 35th day after immunization (50 mg/kg body weight, once daily). Control rats received intraperitoneal injection of same volumetric physiological saline at corresponding time periods. At the end of intervention, rats were euthanatized and hearts were harvested. Paraffin sections were used for hematoxylin and eosin stain to determine the inflammatory score, for picrosirius stain to determine fibrosis score and collagen content, and for immunohistological stain to determine macrophages and T lymphocytes. Real time PCR was used to detect mRNA expression of myocardial MMP-2 and MMP-9. Cryostat sections were used for in situ zymography to detect protein activity of gelatinase. Results Inflammatory score in cardiac paraffin slides, number of cardiac macrophages and T lymphocytes, cardiac interstitial fibrosis score and content, expression of MMP-2, 9 mRNA and activity of gelatinase in treatment group were all significantly lower than in control group for early and middle intervention groups ( inflammatory score: early control group vs. treatment group: 3.03 ± 1.35 vs. 1.51 ±0. 36,P ＜0. 05, middle control group vs. treatment group: 3.75 ±0. 29 vs. 2. 11 ±0. 82,P ＜0. 01; cardiac interstitial fibrosis score, early control group vs. treatment group: 2. 75 ±0. 29 vs. 1.51 ± 0.35, P＜0.01, middle control group vs. treatment group: 2.50 ±0.41 vs. 1.61 ±0.42, P＜0.05;gelatinase, early control group vs. treatment group: 162 367 ±5095 vs. 62 366 ±2131, P ＜0. 01, middle control group vs. treatment group: 184 256 ±5427 vs. 113 197 ±4809, P ＜0. 01 ) while these parameters were similar between minocyclin-treated and control rats in late intervention group ( all P ＞ 0. 05 ).Conclusions MMP-9 plays an important role in the pathogenesis of autoimmune myocarditis. Inhibition of MMP-9 in early and middle stage could significantly attenuate inflammatory responses and myocardial fibrosis in this experimental EAM model.
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