环孢霉素A诱导大鼠心肌纤维化和对基质金属蛋白酶及其抑制剂表达的影响

目的:研究环孢霉素A(CsA)诱导的大鼠心肌损伤及心肌纤维化的程度,检测基质金属蛋白酶(MMP) 2和MMP9及其组织抑制物(TIMP) 2和TIMP1表达的变化,为探寻CsA诱导心肌纤维化的发生机制提供理论依据。方法:64只健康雌性6～8周龄Wistar大鼠随机分为4组:对照组、低剂量CsA组、中剂量CsA组和高剂量CsA组,分别腹腔注射生理盐水、5 mg·kg~(-1)·d~(-1)CsA、12. 5 mg·kg~(-1)·d~(-1)CsA和25 mg·kg~(-1)·d~(-1)CsA,分别在第1、2、3周时处死大鼠。利用HE染色观察心肌组织形态结构,Masson染色观察心肌胶原沉积情况,免疫组织化学和Western blot法检测MMP2、MMP9、TIMP2和TIMP1蛋白的表达情况。结果:HE染色可见,随着CsA作用时间的延长和剂量的增加,心肌组织形态结构损伤严重,逐渐出现心肌灶状坏死及纤维化。Masson染色结果亦可见心肌纤维化程度随着时间和剂量的增加逐渐加重。免疫组织化学及Western blot检测结果显示,在CsA导致大鼠心肌纤维化的过程中,同一时点MMP2和TIMP2随CsA剂量增加表达显著增高(P 0. 05)。第1周MMP2高表达,随着时间的延长表达逐渐减低; TIMP2则随时间延长表达逐渐增高(P 0. 05)。与对照组相比MMP9和TIMP1表达则减低。各用药组MMP9第1周低表达,第2周表达增高,第3周表达减低(P 0. 05);同一时点,CsA对于MMP9表达变化的差异无统计学显著性。TIMP1随CsA剂量增加表达增高(P 0. 05)。结论:CsA可引起大鼠心肌损伤及间质纤维化,随着用药时间的延长和剂量的增加,纤维化程度逐渐加重。CsA诱导的大鼠心肌纤维化与MMPs/TIMPs表达的改变及MMPs/TIMPs的失衡有关。

Cyclosporin A induces myocardial fibrosis and affects expression of matrix metalloproteinases and their inhibitors in rats

AIM: To investigate the changes of matrix metalloproteinases (MMP2 and MMP9) and tissue inhibitor of metalloproteinases (TIMP2 and TIMP1) in the myocardial fibrosis induced by cyclosporin A (CsA), and to explore the underlying mechanism. METHODS: Female Wistar rats (n=64, 6~8 weeks old) weighing (200±25) g were randomly divided into 4 groups[control, low dose of CsA (5 mg·kg^-1·d^-1), medium dose of CsA (12.5 mg·kg^-1·d^-1) and high dose of CsA (25 mg·kg^-1·d^-1)]. The rats were intraperitoneally injected with saline and different doses of CsA, respectively. CsA was continuously administered for 1, 2 and 3 weeks, and then the animals were killed to collect samples. HE staining was used to observe the morphological structure of myocardium. Masson staining was used to observe the deposition of myocardial collagen and the degree of myocardial fibrosis. The protein expression levels of MMP2, MMP9, TIMP2 and TIMP1 were determined by immunohistochemistry and Western blot. RESULTS: HE staining showed that CsA induced cardiomyocyte edema, eosinophilic changes in the cytoplasm, fine granular and vacuolar changes in cardiomyocytes, disappearance of myocardial striae, nuclear condensation, and myocardial focal necrosis and fibrosis. Masson staining showed that the degree of myocardial interstitial fibrosis was getting worse with the increase in the duration and doses of CsA exposure. The results of immunohistochemistry and Western blot showed that the expression of MMP2 in CsA groups was significantly increased in the process of CsA-induced myocardial fibrosis. In each CsA dose group, MMP2 was highly expressed at the first week, and the expression was gradually decreased over time. In contrast, the expression of TIMP2 was increased gradually in a time-and dose-dependent manner (P<0.05). Compared with control group, MMP9 expression level was low at the first week, increased at the second week, and decreased at the third week (P<0.05). Compared with control group, the expression of TIMP1 was dose-dependently increased (P<0.05). CONCLUSION: CsA induces myocardial injury and interstitial fibrosis in rats in a dose-and time-dependent manner. CsA-induced myocardial fibrosis is associated with changes of MMPs and TIMPs, and is affected by the imbalance of MMPs/TIMPs.