| Objective:We aimed to explore the effect and mechanism of Ghrelin on myocardial fibrosis in myocardial infarction rat.Method:The rat model of myocardial infarction was established by ligation of the left anterior descending branch of the coronary artery(Left anterior descending artery,LAD)in SD rats.The sham operation group was only wearing suture without ligation.After 2 days of operation,serum was acquired through rat tail vein and troponin T levels was measured by methods of ELISA to determine whether the myocardial infarction was successful.The rats were randomly divided into the sham operation group,the myocardial infarction group,the myocardial infarction + Ghrelin group and the Ghrelin group,6 rats for each group.The myocardial infarction + Ghrelin group was treated with Ghrelin(100 ?g/kg,subcutaneous,twice daily for 4 weeks),and the myocardial infarction group was treated with the same volume of saline(subcutaneous,twice daily for 4 weeks).After 4 weeks,HR,LVEDP and dp/dtmax were measured through hemodynamic measurements,followed by the rats killed and the hearts acquired.The fibrosis degree was observed by Masson staining.BNP levels were measured using ELISA method,CTGF and collagen I/III mRNA expressions were measured using real-time PCR method,the protein expressions of NADPH oxidase,SGK1,CTGF,MMP-2,MMP-9,Nrf2 and HO-1 were measured by Western bloting.For in vitro experiments,Ang II was used to stimulate cardiac fibrosis,and the proliferation and migration as well as the protein expressions of SGK1,CTGF,MMP-2,MMP-9,Nrf2,HO-1,collagen I and collagen III of cardiac fibroblasts were assayed with or without Ghrelin pretreated.Result:1.Left ventricle(LV)sections were stained with Masson's trichrome to detect interstitial and perivascular fibrosis in the non-infarcted region 4w post-MI.Compared to the sham group,MI induced a robust fibrosis,and this fibrotic response was attenuated with Ghrelin treatment.In addition,hydroxyproline(an indicator of fibrosis)content,collagen I and III mRNA expression partial decreased by Ghrelin treatment.There is no significant difference between sham and Ghrelin group.2.The protein and mRNA expressions of NADPH oxidase-ROS signaling,including NADPH oxidase,SGK1,MMP-2,MMP-9 and CTGF,were all significantly increased in the LV of 4 weeks post-MI compared to sham group,with the effect partially attenuated by Ghrelin treatment.There were no significant difference between sham and Ghrelin groups.3.ROS production in the Ang II stimulated group was significantly higher compared to the control group.Pre-treatment with Ghrelin partial reduced ROS content in Ang II-stimulated cardiac fibroblasts.Concomitantly,NADPH oxidase activity was increased in cardiac fibroblasts stimulated by Ang II,an effect that was abolished by Ghrelin pre-treatment.4.Pre-treatment with Ghrelin,apocynin(a specific NADPH oxidase inhibitor),or tempol(a ROS scavenger)both markedly attenuated the induction of SGK1,CTGF,and MMP-2 and-9,collagen I and collagen III by Ang II.5.Transfection of SGK1 siRNA was shown to significantly reduce SGK1 protein expression compared to transfection of NC and non-transfected CFs.CTGF protein expression was markedly increased in the non-transfected and NC siRNA transfected cardiac fibroblasts stimulated with Ang II.This upregulation in CTGF protein expression was markedly reduced in the SGK1 siRNA transfected group.Conclusion:1.Fibrosis was significantly increased in both cardiac interstitial and perivascular regions of rat hearts at 4 weeks after MI,Ghrelin treatment reduced the degree of myocardial fibrosis in MI rats.Ghrelin has no effect on myocardial fibrosis of normal rats.2.Ghrelin showed anti-oxidation effects in the LV of MI rats and in the Ang II stimulated cardiac fibroblasts,through which prevented the progress of myocardial fibrosis.3.The expression of SGK1 protein was increased in the LV at 4 weeks post-MI compared to sham LV,an effect that was partially attenuated by Ghrelin treatment. |
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