| Cardiovascular diseases have become the leading cause of death in the world. It is important to point out that myocardial infarction(MI) continues to be the major cause of this diseases, which occurred when the blood supply through a coronary artery to the heart is blocked, resulting in irreversible damage or death of heart tissue. Therefore, it is a hot research focusing on pathogenesis and therapeutic targets of acute myocardial injury at home and abroad. Isoproterenol(ISO), a synthetic catecholamine and a β-adrenergic receptor agonist, has been found to cause infarct-like necrosis in the myocardium when injected into rats, which resemble the signs of acute myocardial infarction produced by myocardial ischemia in human. The model of ISO-induced myocardial injury is considered as a suitable standardized model to investigate the beneficial effects of therapeutic strategies. Gentianella acuta(Michx.) Hulten belonging to the family of Gentianaceae is an annual plant mainly distributed in Hebei, Dongbei, Shanxi, Inner Mongolia and Shandong. The whole herb was used as folk medicine to treat hepatitis, jaundice, headache and fever in Mongolia native medicine. It was found to be protective effect of the heart when it is used for tea to drinking.In this study, we observed the protective effect of Gentianella acuta on acute myocardial damage model induced by ISO in rat. Then, we further investigated underlying mechanisms involved on molecular and gene level.Objective: We investigated the effects of Gentianella acuta on ISO-induced acute myocardial injury in rat by detecting Cardiac function, serum cardiac marker enzymes, interlukin-6(IL-6), tumor necrosis factor(TNF-α), and histopathological changes. Then we further measured expression of nuclear factor κB-p65(NF-κB-p65) and Toll-like receptors 4(TLR4) in rat myocardial tissue using immunohistochemical stains and molecular biological techniques. Based on these experimental methods, we evaluated the mechanisms underlying protective effect of Gentianella acuta on ISO-induced myocardial injury in rat.Method: Male Sprague-Dawley rats were randomly divided into the follow five groups(n= 9). Control group(Con), ISO induced acute myocardial injury group(ISO), ISO+0.3g/kg Gentianella acuta group(ISO+G0.3g/kg), ISO+0.6g/kg Gentianella acuta group(ISO+G.0.6g/kg), ISO+1.2g/kg Gentianella acuta group(ISO+G.1.2g/kg). Control groups and ISO group were treated with purified water, different dose of drug-treated groups were given Gentianella acuta by gavage for 28 days. The rats were intraperitoneal injected with ISO(85mg/kg) on days 29 and 30, and sacrificed at 24 hours after the last administration. The hearts were excised and washed with normal saline, dried with filter paper. Left ventricles were separated and weighed, cut into pieces and fixed in 4% paraformaldehyde, the rest myocardial tissue samples were immediately frozen in liquid nitrogen and stored at-80?C. Heart weight index(HWI), the left ventricular weight index(LVWI) and electrocardiogram(ECG) were measured in each group. The serum concentrations of LDH, CK and AST were detected by biochemistry assay and TNF-α and IL-6 in serum were measured by radioimmunoassay. The myocardial histological changes in rat were observed by hematoxylin eosin(HE) and Mason dyeing. The protein expression of TLR4 and NF-κB-p65 were detected by western blot and immunohistochemistry in rat myocardium. The m RNA expression of NF-κB-p65 and TLR4 were quantified by RT-PCR.Results: 1 Gentianella acuta inhibits ISO induced acute myocardial injury in rats Models of acute myocardial injury by ISO-induced in rats were successfully made. Comparing with the control group, the HWI and LVWI of the ISO group were respectively increased(P< 0.01). Comparing with ISO model group, there were no significant differences of HWI and LVWI in Gentianella acuta-treated group. 2 Effects of Gentianella acuta on electrocardiogram(ECG) in rats Compared with the control group, ST segment deviation was significantly higher in ISO model group. Gentianella acuta treated group can significantly reduce the ISO-induced abnormal ST segment elevation compared to model group. These changes were restored to near normal in high dose treatment group. 3 Effects of Gentianella acuta on serum myocardial marker enzymes activities in rat There are three kinds of heart marker enzymes(CK, LDH and AST) in the serum were determined and summarized. There was a significant(P< 0.01) rise in the levels of serum diagnostic marker enzymes(CK-MB, LDH, and AST) in ISO group. Treatment with Gentianella acuta showed a significant(P< 0.01) reduction in the levels of all serum diagnostic marker enzymes compared to ISO group. 4 Effects of Gentianella acuta on serum TNF-α and IL-6 levels in rat The serum levels of IL-6 and TNF-αwere determined and summarized. There was a significant increase in levels of IL-6 and TNF-α in the ISO group compared to the control group(P < 0.05). Treatment with different dose Gentianella acuta showed significant decrease of serum levels of TNF-α and IL-6 compared to ISO group(P < 0.05). 5 Effects of Gentianella acuta on histopathologic changes in rat Comparing with the control group, HE staining of control myocardium showed normal cardiac fibers and no inflammatory cell infiltration. ISO caused the formation of infarction areas, characterized by disordered arrangement of myocardial fibers, myocardial cell swelling, hyaline degeneration and infiltration of inflammatory cells. Treatment with low dose Gentianella acuta did not obviously attenuate the histological appearance. Treatment with middle or high dose Gentianella acuta could significantly decrease the infarct size and infiltration of inflammatory cells. Masson’s staining showed that there was excessive collagen accumulation(collagen staining in blue color) in ISO group, compared with the control group. Gentianella acuta treated group showed improvement of collagen accumulation as compared to the ISO group. The current work showed that the effect of Gentianella acuta(0.6 or 1.2g/kg) significantly reduced ISO-induced myocardial infarct size in rat with AMI, compared to model group. But administration of Gentianella acuta(0.3g/kg) could also reduce infarct size without statistical significance. 6 Effects of Gentianella acuta on the myocardial TLR4 and NF-κB-p65 protein expression and m RNA expression Immunohistochemistry shows that TLR4 and NF-κB-p65 were low expressed in the normal heart. The expression of TLR4 and NF-κB-p65 were elevated in the ISO group compared to the control group. The expression of TLR4 and NF-κB-p65 were decreased in different degree in Gentianella acuta-treated group. Western blot shows that the protein levels of TLR4 and nuclear factor κB subunit p65(NF-κB-p65) were increased significantly in the ISO group compared to the control group, In the Gentianella acuta-treated groups, the protein levels of TLR4 and NF-κB-p65 were markedly decreased as compared to model group. RT-PCR shows that the levels of TLR4 m RNA and NF-κB-p65 m RNA were up-regulated in the myocardium tissue of ISO group compared with the control group. Whereas the TLR4 m RNA and NF-κB-p65 m RNA expression was markedly diminished in Gentianella acuta-treated group(P< 0.01).Conclusions: The results of these studies indicate that Gentianella acuta has good protective effect on acute myocardial injury induced by isoproterenol, and the cardioprotection may be related with inhibiting the TLR4/NF-кB signaling pathway and attenuating inflammatory effect. |
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