| Myocardial fibrosis(MF) refers to cardiac fibroblast proliferation and the excessive deposition of extracellular matrix in myocardial interstitial. These pathological changes generally occur with various cardiovascular diseases. MF will lead to structural damage and dysfunction, even result in cardiogenic sudden death. Although the severity of myocardial fibrosis is intimately associated with development and prognosis of cardiovascular disease, the ideal therapeutic drugs have not been developed yet. Therefore, it is a hot research focusing on pathogenesis and therapeutic targets of myocardial fibrosis at home and abroad. An ethnopharmacological survey is carried out in Inner Mongolia, the result indicats that Gentianella acuta(Gentianella acuta(Michx.) Hulten) has been used as a herbal tea, which is considered beneficial to heart. But little literature was regarding this action of Gentianella acuta.In the present study, we first observed the inhibition effect of Gentianella acuta on myocardial fibrotic formation in animal model induced by isoproterenol(ISO). We further investigated underlying mechanisms involved in molecular and gene level. Importantly, we attempted to provid experimental and theoretical basis for Gentianella acuta as a potent and efficacious intervention against myocardial fibrosis.Objective: In the present study, a myocardial fibrotic rat model induced by ISO was used to assess the effect of Gentianella acuta. Cardiac function, antioxidative indices and histopathological changes were observed. Moreover, expression of nuclear factor κB-p65(NF-κB-p65), transforming growth factor-β1(TGF-β1) and connective tissue growth factor(CTGF) were detected using immunohistochemical and molecular biological techniques. Based on these experimental methods, we evaluated the underlying mechanisms of Gentianella acuta against myocardial fibrosis, especially about its mechanisms of anti-inflammatory and antioxidation.Method: SPF male Sprague-Dawley rats were randomly divided into five groups(n = 8), namely, normal group, ISO induced myocardial fibrotic group, ISO + 1.2 g/kg Gentianella acuta, ISO + 0.6 g/kg Gentianella acuta, ISO+ 0.3 g/kg Gentianella acuta. The rat myocardial fibrotic model was induced by hypodermic injection of ISO once daily(5 mg/kg) for 7 days. Rats in medicine-treated groups were given once a day of Gentianella acuta by gavage based on different doses(1.2-, 0.6-, 0.3- g/Kg). The treatment started 24 hours after the first injection of Isoproterenol(on the 2th day) and was maintained for a period of 21 days. During test, body weight(BW) and electrocardiogram(ECG) of each rat were measured pre-test and weekly thereafter. On the 22 th Day, hemodynamic signals were recorded in anesthetic rats after fasting for 12 hours. Finally, animals were sacrificed and the hearts were taken rapidly and weighed. Left ventricular samples were isolated for biochemical assay and histopathological examination. Fourthermore, Immunohistochemistry, western blotting and fluorescence quantitative polymerase chain reaction(PCR) analysis were performed to determine expression of NF-κB-p65, TGF-β1 and CTGF in myocardial fibrotic group and medicine-treated groups.Results:1 The effects of Gentianella acuta on body weight and cardiac mass indexeCompared with normal rats(325.71 ± 9.21 g, 2.74 ± 0.04 mg/g, 1.81 ± 0.02 mg/g), body weight of the ISO-induced myocardial fibrotic rats was significantly decreased(282.36 ± 8.14 g), and cardiac weight index(CWI) and Left ventricular weight index(LVWI) was markedly increased(2.97 ± 0.03 mg/g, 2.15 ± 0.03 mg/g). Compared with model rats, different doses of Gentianella acuta could significantly increase the body weight, as well decrease CWI and LVWI(P < 0.05).2 The effect of G.entianella acuta on eletrocardiograph patternCompared with normal rats, ISO induced myocardial fibrotic rats represent by positive T wave, ST segment elevation, and a decrease in R wave amplitude. These changes were restored to near normal in Gentianella acuta treated rats.3 The effect of Gentianella acuta on cardiac functionCompared with normal group, ISO-induced rats showed significant rise in left ventricular end-diastolic pressure(LVEDP) and Heart rate, meanwhile, values of left-ventricular systolic pressure(LVSP), maximum and minimum rate of developed left ventricular pressure(± d P/dtmax) were markedly increased. After addition of different doses of Gentianella acuta, a noticeable attenuation in LVEDP and heart rate, as well as an increment of LVSP and ± d P/dt max were observed.4 The effect of Gentianella acuta on antioxidant defenceCompared to normal rats(6.79 ± 0.36 nmol/mgprot, 12.21 ± 0.42 U/mgprot, 4.76 ± 0.25 mg/L), ISO-induced rats resulted in elevation of malondialdehyde(MDA) concentrations(11.28 ± 0.31 nmol/mgprot) as well as depletion of superoxide dismutase(SOD) and reduced glutathione(GSH) levels(6.84 ± 0.29 U/mgprot, 2.43 ± 0.14 mg/L). In marked contrast, a notable suppression of MDA, and augmentation of SOD and GSH were observed in different doses of Gentianella acuta-treated groups comparing with myocardial fibrotic group(P < 0.01).5 The effect of Gentianella acuta on collagen contentHydroxyproline level in myocardial tissue were significantly higher in model group(0.92 ± 0.03 ug/mg) than normal group(0.38 ± 0.02 ug/mg). Compared with ISO-induced myocardial fibrotic rats, myocardial hydroxyproline level was obviously lowered(P < 0.01) in the high, medium and low dose groups of Gentianella acuta(0.58 ± 0.02 ug/mg, 0.43 ± 0.01 ug/mg, 0.43 ± 0.02 ug/mg) and decreased by 36.96%, 53.26%, 53.26%, respectively.6 The Effect of Gentianella acuta on histopathologic changes in HE stainningHeart tissues from ISO-induced rats showed widespread myocardial structure disorder including extensive myofibrillar degeneration and infiltration with leukocyte as compared with normal group. Treatment with Gentianella acuta at different doses exhibit decreased degree of fibrosis and less in filtration of inflammatory cells compared to ISO administered group.7 The effect of Gentianella acuta on extent of myocardial fibrosis assessed in Masson’s trichrome stainingExcessive collagen accumulation(collagen staining in blue color) was evident in ISO-induced rats compared to normal rats. On the other hand, different doses of Gentianella acuta showed improvement of collagen accumulation as compared to the isoproterenol group.8 The effects of Gentianella acuta on the myocardial NF-κB-p65 and TGF-β1 protein expressionCompared with normal rats, ISO significantly increase the protein levels of nuclear factor κB subunit p65(NF-κB-p65), transforming growth factor-β1(TGF-β1) and connective tissue growth factor(CTGF). In the different doses groups of Gentianella acuta, the protein levels of NF-κB-p65, TGF-β1 and CTGF were markedly decreased compared to the ISO group.9 The effects of Gentianella acuta on the myocardial TGF-β1 and CTGF m RNA expressionCompared with rats(1.16 ± 0.17, 1.13 ± 0.17), ISO upregulate the gene expression of TGF-β1 and CTGF in the myocardium(4.25 ± 0.23,3.86 ± 0.31). whereas the TGF-β1 and CTGF m RNA expression was markedly diminished after addition different doses of Gentianella acuta(P < 0.01).Conclusions: Gentianella acuta could reverse ISO-induced myocardial fibrosis in rats, and its mechanisms might be related to the reduction of TGF-β1 and CTGF expression via inhibition of NF-κB-p65 in myocardial tissues. |
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