鸢尾黄素在基础理论抑制心肌纤维化实验研究中的应用

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中文摘要

　　　背景：心肌纤维化（MF)是各种心脏疾病发生发展的共同结局，同时也能诱发某些心脏疾病的发生。随着人类医学理念的进步，对心肌纤维化的发生发展倍受关注。对心肌纤维化的发生和发展采用相应的预防和延缓措施对临床上许多心血管疾病的预防和治疗具有极其重要意义，因此需要我们确切了解心肌纤维化的病因和发病机制，以便应用药物改善心肌纤维化的发展进程。鸢尾黄素具有心肌保护作用，同时对肝纤维化和肺纤维化都有改善作用，本实验探讨其对心肌纤维化的作用和机制。

　　　目的：通过体内实验**大鼠心肌纤维化的动物模型和体外实验原代培养心肌成纤维细胞两种方法。研究鸢尾黄素对心肌纤维化发生发展的抑制作用。

　　　实验方法：

　　　体内实验：实验取大鼠60只，雌雄各半，随机分为正常对照组、ISO模型组（5mg/kg）、卡托普利阳性药物组（10mg/kg）、鸢尾黄素低中高剂量组(25mg/kg、50mg/kg、100mg/kg)。除正常对照组外，其余组应用皮下多点注射 Iso 的方法建立大鼠心肌纤维化模型，连续给ISO 7天。于造模第二天给予阳性药和鸢尾黄素，每天灌胃给药一次。28天后麻醉大鼠做心电图、取血、取心脏，分光光度法检测心肌组织中SOD、MDA的含量，心脏标本进行 HE染色，应用免疫组织化学法、RT-PCR等方法检测 ColⅠ、ColⅢ、LDH、CK 以及 NO的含量等在大鼠心肌组织中的表达和影响。同时分析上述实验结果。

　　　体外实验：实验取出生1W内的乳鼠10只，无菌开胸取心肌，通过胰酶反复消化、离心，接种含10%血清的培养液中，放入37℃、5%CO2孵箱中培养，通过差速贴壁法去掉心肌细胞和其他组织，制备CFb。把第3代CFb分为正常组、模型组和鸢尾黄素低中高剂量组。接种于96孔板，除正常组外，以AngII(10-7mol/ml)**CFb增殖，同时给予相应的药物处理。通过MTT法检测CFb增殖的增值情况，ELISA法检测细胞悬液中各组COLⅠ型和COLⅢ型以及NO的含量。

　　　实验结果：

　　　体内实验：

　　　①HE染色显示，ISO模型组大鼠心肌纤维化明显。而治疗组给予鸢尾黄素28天后，纤维化程度明显减轻。

　　　②分光光度法结果显示，与正常对照组比较模型组大鼠心肌组织中MDA含量明显增加而SOD活性明显下降（P＜0.01），鸢尾黄素低中高剂量组与模型组相比MDA含量下降、SOD活性明显增加（P＜0.01）。

　　　③ELISA法结果显示：模型组血清中ColⅠ、ColⅢ、LDH、CK的含量明显增加（P＜0.01），鸢尾黄素低中高剂量组血清中ColⅠ、ColⅢ、LDH、CK的含量明显降低（P＜0.01或P＜0.05）。

　　　④酶标仪检测NO的含量：模型组血清中NO的含量增加（P＜0.01），鸢尾黄素组血清中NO的含量明显下降（P＜0.01或P＜0.05）。

　　　体外实验：

　　　①MTT结果表明：与正常对照组比较，模型组心肌成纤维增殖加快（P<0.01）；与模型组比较，鸢尾黄素中、高剂量组细胞增殖明显减慢，有统计学意义（P<0.05）和（P<0.01）

　　　②ELISA法结果显示：细胞悬液中NO的含量与正常对照组比较，模型组心肌成纤维NO的含量降低（P<0.01）；与模型组比较，鸢尾黄素高剂量组NO的含量明显增多（P<0.01）；细胞悬液中胶原蛋白的含量与正常组比较，模型组ColⅠ、Col III含量显著增加（P<0.01）；与模型组比较，鸢尾黄素中剂量组胶原蛋白Ⅰ的含量降低明显（P<0.01），鸢尾黄素高剂量组胶原蛋白Ⅰ和III的含量均明显减少（P<0.01）。

　　　结论：通过体内和体外实验研究表明鸢尾黄素对心肌纤维化具有抑制作用。可能通过其抗氧化性、清除自由基以及上调eNOS的活性及扩张血管等作用机制来抑制心肌纤维化的发生。

　　　关键词：

　　　　　　心肌纤维化，鸢尾黄素，异丙肾上腺素，血管紧张素II

Abstract

　　　Background:Not only myocardial fibrosis is common result of many developing heart disease,but it also can induce certain diseases. With the advancement of the concept ofhuman medicine, myocardial fibrosis had been paid much attention. Prevented anddelayed the development of myocardial fibrosis have great significance for theprevention and treatment of many cardiovascular diseases, therefore it alsoneed to know the exact pathogenesis of myocardial fibrosis, then used drug toprevent myocardial fibrosis. Iris Flavin has cardioprotective activity, whileit can also have the activity to decreasing both liver and pulmonaryfibrosis.The aim of our experiment to investigate the effect of Iris Flavin onmyocardial fibrosis and further to invest the related mechanisms.

　　　Objective:Through observed the change of  leftventricular mass index, Ⅰ collagen and Ⅲ type collagen, while also detected the concentration of SOD, MDA,LDH, CK, NO both in normal tissue and Myocardial fibrosis to invest theinhibition of Iris yellow on myocardial fibers.

　　　Methods: 60rats were randomly divided into six groups: normal control group, ISO modelgroup (5mg / kg), captopril positive drug group (10mg / kg), flavin Iris LowMedium High dose group (25mg / kg, 50mg / kg, 100mg / kg). To established ratmodel of myocardial fibrosis through subcutaneous multi-point injection Iso,and give positive drug and Iris flavin in following day, and administeredorally once a day. After 28 days the rats were anesthetized and put to death,and then rat’s heart,blood has been taken out. The spectrophotometric was used to detect theconcentration of SOD, MDA in myocardial tissue; HE staining l was used todetect heart specimens .Immunohistochemistry, RT-PCR and Western Blotting wereused to detect the expression of ColⅠ, ColⅢ in ratmyocardium; The kit were also used to detect the concentration of LDH ,CK andNO in rat myocardium. Correlations of above results were also **yzed.

Results:

　　　①HEstaining results showed that myocardial fibrosis was significantly in ISO modelgroup. While when given Iris Flavin, after 28 days the myocardial fibrosis wassignificantly reduced

　　　②Thespectrophotometry results showed that compared with the control group, theconcentration of MDA was significantly, while SOD activity was significantlydecreased (P <0.01) in the myocardial tissue of model group Otherwise whencompared to the model group, the concentration MDA was decreased and SODactivity was significantly increased in three different dose groups. (P <0.01).

　　　③ELISAassay results showed that: The serum Col, ColⅢ,LDH, CK levels were increased significantly in the model group (P<0.01),and Vice versa (P <0.01 or P <0.05).

　　　④ Theresults about microplate detection showed that the concentration of NO in serumof model group were increased (P <0.01), and Vice versa (P <0.01 or P<0.05).

Conclusion: Iris flavin could increase eNOSactivity and dilation of blood vessels through antioxidant activity andremoving free radical

Key words:

Myocardial fibrosis, Iris Flavin,isoproterenol