| BackgroundLeft ventricular hypertrophy is one of the most common changes in primary hypertension. The two aspects of pathological changes was brought forward: (1) myocardial cell's hypertrophy necrosis and apoptosis;(2) extracellular matrix remodeling of myocardium. Myocardial remodeling induced not only stiff cardiac ventricle and decreased cardiac function but also myocardial ischemia and cardiogenic sudden death. How to treat hypertension is very important. At present, one of the most significant gist is to prevent and reverse myocardial remodeling of hypertension patients. For recent decades, a number of basical and clinical studies on myocardial remodeling of hypertension have been founded. The phasic achievements were obtained, but the mechanism, early diagnosis and therapeutic measures of myocardial remodeling were still explored. It is important for treatment of myocardial fibrosis to explore deeply the mechanism and the methods to block the pathway of myocardial remodeling development.Myocardial remodeling means that excess collagen fibers accumulated and the contents of the collagen increased notably or the compositions of the collagen altered. The extracellular matrix (ECM) in most tissues is composed of a complex arrangement of fibrillar collagen, elastin, microfibrillar proteins, proteoglycans and adhesive proteins such as laminin and fibronectin. Among the ECM proteins, collagens constitute up to 85%, among which type I and type III constitute two-thirds. The degradation of the ECM are mainly modulated by matrix metalloproteinases(MMPs), and the activities of MMPs can be specifically inhibited by a family of protease inhibitors, termed tissue inhibitors of matrix metalloproteinase (TIMPs), which are endogenous physiological inhibitors of MMPs. That is, the delicate balance between MMPs and TIMPs is very important to maintain the normal metabolism of the collagen. In briefly, the development of myocardial remodeling in hypertension is a process that numerous active factors participated and co-regulated, and a result that collagenation exceeds its degradation. Accordingly, inhibiting proliferation of cardiac fibroblasts and deposition of collagen are the key of the precaution and reversion of myocardial remolding and of improving heart function . Peroxisome proliferator-activated receptors (PPARs) are a family of at least 3 nuclear receptors (α, β, and γ). PPARα express mainly in liver and kidney, and play an important role in catabolism of fatty acid. The hypolipidemic fibrate drugs (such as fenofibrate and bezafibrate) is the ligand of the PPARα. Some researches indicated fenofibrate can reverse myocardial hypertrophy and fibrosis that caused by pressure overload. But many problems is unclear, such as: (1)The way of PPARα agonist fenofibrate regulating myocardial remodeling. (2)The main intracellular signal transduce pathway of PPARα regulating ECM remodeling.The problems mentioned above formed the objectives. The deep study of such problems not only conduce to interpret the molecular-biological mechanism of ECM remodeling, but also can bring new application area for PPARα agonist. It is anticipated to establish new target point and method for early diagnosis and treatment to prevent and reverse myocardial remodeling of hypertension. Objectives1. To explore the effect of fenofibrate on myocardial remodeling in spontaneous hypertensive rats (SHR).2. To observe the effect of fenofibrate on expression of PPARαN C-Fos C-Jun TIMP-1 Collagen I and heterodimer of C-Fos/C-Jun, and to explore molecular biological mechanism of PPARα regulating ECM remodeling.Methods 24 male 8-week-old SHR were randomly divided into twogroup: SHR-controlled group(SHR group) (n=10) and Fenofibrate group(n=14). The same week-old Wistar-kyoto (WKY) rats group (WKY group) (n=10) was also randomly selected. The Fenofibrate group animals were dosed orally with Fenofibrate (60mg.kg-1.d-1) and the other group animals were given with 0.9% saline every day for 8 weeks. The detection contents were listed below. (1) The animals were weighed every week and the blood pressure through arteria caudilis were measured every two weeks.(2) The common echocardiographic indexes were detected before and at the end of the experiment. (3) Hemodynamics indexes were measured by cardiac catheterization at the end of the experiment. (4)After the rats were killed, we get left ventricle mass (LVM) and calculate the left ventricular relative mass (LVM/BW). (5) Myocardial ultrastructral and histopathological changes were observed. (6)The content of collagen were quantified by Masson-staining. (7) The protein levels of C-Fos, C-Jun were detected by immunohistochemistry. (8) The mRNA expression of Collagen I, Collagen III, MMP-9, TIMP-1, PPARα, C-Jun and C-Fos were detected by quantification real-time RT-PCR. (9) The protein levels of C-Jun/C-Fos heterodimerswere detected by Co-immunoprecipitation-Western-blot. Results1. Blood pressure measurementDuring the whole experiment, compared with the WKY group, the blood pressure in SHR group and fenofibrate group were significantly increased(P<0.01); The blood pressure in fenofibrate group were insignificantly different from SHR group (P>0.05).2. Body weight and left ventricular relative mass (LVM/BW) measurementDuring the whole experiment, compared with the WKY group, the body weight in SHR group and fenofibrate group were significantly decreased (P< 0.01). The body weight in fenofibrate group were insignificantly different from SHR-controlled group (P>0.05).At the end of the experiment, compared with WKY group, the left ventricular relative mass (LVM/BW) in SHR group were significantly increased(P< 0.01); Compared with SHR group, the LVM/BW fenofibrate group were significantly decreased (P< 0.01).3. Echocardiographic detectionThe common echocardiographic indices: Before the experiment, compared with WKY group, the IVSTd LVPWTd and RWT in SHR group and fenofibrate group were significantly increased(P<0.01); There were no significant difference (P>0.05)between SHR group and fenofibrate group. At the end of the experiment, compared with WKY group, the IVSTd and LVPWTd in SHR group were significantly increased (P<0.01), and RWT was also significantly increased(P< 0.05),E/A ratio was obviously decreased (P< 0.01)and DT of E wave was extended (P< 0.05); Compared with SHR group, the IVSTd and LVPWTd in fenofibrate group were significantly decreased (P< 0.01), and RWT was also significantly decreased(P< 0.05), E/A ratio was obviously increased (P< 0.01)and DT of E wave was shorten (P< 0.05).Integrated backscatter detection: Before the experiment, there was no significant difference of the integrated backscatter percent(IBS%) and cyclic variation of IBS(CVIB) in left ventricular posterior wall(LVPW) and interventricular septa(IVS) among the three groups (P>0.05) .At the end of the experiment, compared with WKY group, IBS% of SHR group was obviously increased (P< 0.01) and CVIB of the SHR group was obviously decreased (P< 0.01)in LVPW and IVS. Compared with SHR group, IBS% of the fenofibrate group was obviously decreased (P< 0.05) and CVIB of fenofibrate group was obviously increased (P<0.05) in FVS, and IBS% of the fenofibrate group was obviously decreased(P< 0.01) and CVIB of fenofibrate group was obviously increased(P< 0.05) in LVPW.4. Hemodynamic indices determinationAt the end of the experiment, compared with WKY group, left ventricular end-diastolic pressure(LVEDP)in SHR group increased significantly (P< 0.01), and the peak left ventricular pressure descending rate(-dp/dtmax)and the peak left ventricular pressure ascending rate(+dp/dtmax) in SHR group decreased significantly (P<0.01) . Compared with SHR group, LVEDP in fenofibrate group decreased significantly (P< 0.01), and ±dp/dtmax increased significantly(P< 0.01).5. Pathological detectionThe myocytes from WKY group arranged regularly in HE-staining slice. The size of the nuclear was uniform. The staining cytoplasm was homogeneous. The myocytes from SHR group arranged irregularly. The unclear was irregular and the interrupted myofibril arranged irregularly. The fenofibrate group were obviously improved. The myocytes from fenofibrate group arranged more regularly than SHR group. The interrupted myofibril was not common. The nuclear shape was more regular than SHR group.6. Ultrastructural change observation by transmission electron microscopyIn WKY group, the cardiac muscle fibers were clear. The uniformly sized mitochondria arranged regularly. A little fibroblast and collagenous fibers distributed regularly in extracellular matrix. In SHR group, the swelling mitochondria increased. A lot of fibroblast and collagenous fibers distributed irregularly in extracellular matrix. Compared with SHR group, the ultrastructural change of fenofibrate group were obviously improved. The mitochondria were more regular than SHR group. The fibroblast and collagenous fibers in extracellular matrix decreased obviously.7. The content of collagen detection by Masson-stainingThe myocytes was red and collagen was green or blue. The collagen tissue was appropriately arranged among cardiomyocytes in WKY group. However, collagen tissue increased markedly, and disrupted in some area in the SHR group. Compared with SHR group, the collagen tissue decreased and arranged regularly in fenofibrate group.Quantitative analysis results: The collagen volume fraction (CVF)in SHR group was higher significantly than that of WKY group (P<0.01) .The CVF in fenofibrate group was lower significantly than that of SHR group (P<0.05 ) .8. Immunohistochemistry detectionThe positive reaction of C-Fos and C-Jun protein were stained brown and mainly absent in myocardial cellular nucleus. There was uniform distribution of weak brown staining in WKY group, but C-Fos and C-Jun staining in SHR group was strong and located at fargoing cardiac muscle tissues, and the expression of C-Fos and C-Jun protein were higher significantly than that of WKY group (P<0.01); The staining was weak and located within limits in fenofibrate group, and the expression of C-Fos and C-Jun protein, and he expression of C-Fos and C-Jun protein were lower significantly than that of SHR group (P<0.01) .9. The results of RT-PCRThe expression of PPARα mRNA in SHR group was lower significantly than that of WKY group (P<0.05) . The expression of PPARα mRNA in fenofibrate group was higher significantly than that of SHR group and WKY group (P<0.01) .The expression of C-Jun and C-Fos mRNA in SHR group was higher significantly than that of WKY group (P<0.01) . The expression of C-Jun and C-Fos mRNA in fenofibrate group was lower than that of SHR group (P<0.01) .The ratio of MMP-9 / TIMP-1 in SHR group was lower significantly than that of WKY group (P<0.01). The ratio of MMP-9 / TIMP-1 in fenofibrate group was higher than that of SHR group (P<0.01) .The expression of collagen I and collagenlll mRNA in SHR group was higher significantly than that of WKY group (P<0.01) . The expression of collagen I and collagenIII mRNA in fenofibrate group was lower than that of SHR group (P<0.01) . The expression of collagen I and collagenIII mRNA in fenofibrate group was higher than that of WKY group (P<0.01) .10. The results of Co-immunoprecipitation-Western-blotCompared with WKY group, the formation of C-Fos/C-Jun heterodimers was increased in the SHR group (P<0.01) . Compared with SHR group, the formation of C-Fos/C-Jun heterodimers was decreased in fenofibrate group (P<0.01) . Conclusions1. Sixteen week-old SHR have obvious myocardial hypertrophy and myocardial fibrosis; The main histopathologic changes were collagen deposition and myocardial interstitial remolding.2. Fenofibrate has a protective effect on myocardial remodeling in the SHR. Inhibiting the synthesis of collagen was an important mechanism. 3. The activator of PPARα can decrease expression of C-Fos C-Jun and inhibit theformation of C-Jun/C-Fos heterodimers resulting inhibiting the transcription of itsdownstream gene ,which may be the mechanism of above 2.
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