Han Li-na, Li Tie-ling, Zhang Ya-jing, Yang Ting-shu, Ding Yu, Zhao Xiao-ning, Guo Shu-li
First Department of Cardiovascular Internal Medicine of South Building, Chinese People's Liberation Army General Hospital, Beijing 100853, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2011 Feb;39(2):118-23.
To investigate the effects of matrix metalloproteinase-9 (MMP-9) inhibitor minocyclin hydrochloride in Lewis rats with experimental autoimmune myocarditis (EAM).
EAM was induced by injection of cardiac C protein emulsified in completed Freund adjuvant in double footpad and intraperitoneal injection of pertussis toxin on 6- to 8-week old Lewis rats. Sixty EAM Lewis rats were divided into 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 1(st) to 21(st) day after immunization; in middle intervention group, rats were treated from 8(th) to 28(th) day after immunization and in late intervention group, rats were treated from 15(th) to 35(th) 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.
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).
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.
探讨基质金属蛋白酶-9（MMP-9）抑制剂盐酸米诺环素对实验性自身免疫性心肌炎（EAM）Lewis大鼠的影响。
采用在6至8周龄Lewis大鼠双侧足垫注射完全弗氏佐剂乳化的心肌C蛋白并腹腔注射百日咳毒素的方法诱导EAM。60只EAM Lewis大鼠分为3组（早期、中期和晚期干预组，每组n = 20只：10只米诺环素处理大鼠和10只对照大鼠）。早期干预组，治疗组大鼠在免疫后第1天至第21天腹腔注射盐酸米诺环素；中期干预组，大鼠在免疫后第8天至第28天接受治疗；晚期干预组，大鼠在免疫后第15天至第35天接受治疗（50 mg/kg体重，每日1次）。对照大鼠在相应时间段腹腔注射相同体积的生理盐水。干预结束时，处死大鼠并摘取心脏。石蜡切片用于苏木精-伊红染色以确定炎症评分，用于天狼星红染色以确定纤维化评分和胶原含量，用于免疫组织化学染色以确定巨噬细胞和T淋巴细胞。实时PCR用于检测心肌MMP-2和MMP-9的mRNA表达。冰冻切片用于原位酶谱分析以检测明胶酶的蛋白活性。
早期和中期干预组治疗组心脏石蜡切片的炎症评分、心脏巨噬细胞和T淋巴细胞数量、心脏间质纤维化评分和含量、MMP-2、9 mRNA表达及明胶酶活性均显著低于对照组（炎症评分：早期对照组与治疗组：3.03±1.35 vs.1.51±0.36，P < 0.05，中期对照组与治疗组：3.75±0.29 vs. 2.11±0.82，P < 0.01；心脏间质纤维化评分，早期对照组与治疗组：2.75±0.29 vs.1.51±0.35，P < 0.01，中期对照组与治疗组：2.50±0.41 vs. 1.61±0.42，P < 0.05；明胶酶，早期对照组与治疗组：162 367±5095 vs. 62 366±2131，P < 0.01，中期对照组与治疗组：184 256±5427 vs. 113 197±4809，P < 0.01），而晚期干预组米诺环素处理大鼠和对照大鼠之间这些参数相似（均P > 0.05）。
MMP-9在自身免疫性心肌炎的发病机制中起重要作用。在本实验性EAM模型的早期和中期抑制MMP-9可显著减轻炎症反应和心肌纤维化。
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