| Myocardial fibrosis(MF) is enhanced collagen fibers accumulation of normal cardiac tissue, increased collagen concentration or changed ingredients. it can be classified by elevated collagen concentration or collagen volume fraction(CVF) than normal tissues, its main representation is cardiac hypertrophy, cardiac fibroblast(CFb) proliferation and large amouts of collagen protein secretion. many researches in latest years indicate that myocardial fibrosis may results from hypertension, myocardial infarction, cardiac failure and many cardio-vascular diseases. MF is the common pathological changes for many cardiac diseases in certain periods of time, it is also one of the main manifestation of myocardiac remodeling,and becomes the independent dangerous factor for cardio-vascular diseases. MF may lead to increased myocardiac stiffness, decreased ventricular diastolic function, myocardial electrical physiological disorder and cardiac arrhythemia, decreased cardiac coronary artery reserve, even sudden cardiac arrest or death. many scientists lay emphysis on MF and give more and more deeper study on mechanism and reversal way. researches prove that rennin-angiotensin- aldosterone system(RAAS)takes an important part in MF during many leading factors. Angiotensin II joins with its type one receptor, activates transcription factor through activating MAPK signal pathway in oxidized stress responses, enhances cardiac cell and cardiac fibroblast to secret fibrolitic factor such as ET-1 and TGF-β1.Enalapril lays first place in treating myocardial fibrosis for its powerful inhibiting of cardiac hypertrophy and CFb proliferation, but its mechanism, especially molecular biology and signal transduction pathways is still not very clear. therefore this thesis targets on MF, observes the effect of Ena on MF and its mechanism from in vivo and vitro experiments, and aims at finding mechanism of MF, looking for the main interferring target of Ena, laying a solid foundation for preparing ACEI drugs for certain therapeutic effect and minor side effect. its principal research contents are as follows: inducing model of MF in rat by adopting Iso and surveying the the effect of Ena on MF befor and after drug administration detecting hydroproline content and SOD, MDA level in cardiac tissues by spectrophotometer observing the effect of Ena on morphological change, p-p38MAPK and TGF-β1 expression by HE, Masson special staining, transmission electroscope, immunochemistry staining through pathogogical staining technology and Western blot method, the results manifest that: 1. Ena can inhibit cardiac hypertrophy induced by Iso obviously decrease collagen fiber expression and hydroproline content increase SOD and drop MDA level diminish p-p38MAPK and TGF-β1 expression. 2. Culturing cardiac fibroblast in vitro, inducing CFb proliferation with AngII, tetromethyl thiazole blue colorimetric assay,fl ow cytometer (FCM) are used to observe the effect of Ena on CFb proliferation and cell cycle immunocytochemistry staining(IC), FCM, WB are applied to observe the effect of Ena on cyclinD1 and p27 kip1 protein expression. The results manifest that Ena can inhibit CFb proliferation dramatically and block cellcycle at phase G0/G1 and dropping cyclinD1 closely related to cell proliferation,e nhancing CDKI p27k ip1 expression,a nd take on dosage dependence. 3. Culturing CFb in vitro, IC, FCM, RT-PCR methods are applied to measure TGF-β1 protein expression, collagenα1(I) level and TGF-β1 mRNA level hydroproline method is deployed to detect myocardial collagen fiber content correlative analysis is taken between TGF-β1 mRNA, protein expression and collagenα1(I) level. Ena can lower TGF-β1 mRNA level and protein expression lowering hydroproline content and collagen(I) level there is a obvious positive relation between TGF-β1 mRNA, protein expression and collagen fiber level. 4. Molecular mechanism:fluorescenic microscope, FCM are adopted to observe the effect of Ena on ROS intensity the effect of Ena on phosphorylated P38MAPK nuclear translocation the effect of Ena on p-p38MAPK protein expression in IC the effect of Ena on p-p38MAPK protein expression in Western blot and the effect of Ena on TGF-β1 protein expression in IF and WB. The results demonstrate that Ena can decrease ROS level dramatically inhibit P-P38MAPK nuclear translocation diminish p-p38MAPK and TGF-β1 protein expression indicating that Ena can inhibit p-p38MAPK nuclear translocation through dropping ROS level, consequently reducing TGF-β1 protein expression, through which supressing MF occurance. |
