| Objective: Diabetes prevalence rate increased year by year, according to the international diabetes federation in 2015, the latest figures show the whole world has 415 million diabetic patients between 20-79 years old, and by 2040 the number will rise to 642 million. Diabetes is a systemic disease, cardiovascular complication secondary to diabetes is of high incidence, and myocardial fibrosis is one of the important causes leading to ventricular remodeling in patients with diabetes. Currently there are no effective treatments for myocardial fibrosis, so it is particularly important to prevent and control its progression. Liraglutide belongs to GLP-1 agonists. In addition to the hypoglycemic effect, Liraglutide also has benefits on weight loss, blood lipids regulating, cardiovascular protecting and so on. Studies showed that Liraglutide has anti-fibrosis effect. Early research found that Liraglutide can relieve diabetic myocardial and pulmonary fibrosis in rats, Liraglutide can low expression of mi R-27 a in H9C2 rat myocardial cells. Micro RNAs(mi RNAs) represent a class of small noncoding RNAs that function as post-transcriptional regulators of gene expression. They play crucial regulatory roles in most cellular and developmental processes and have been implicatied in many diseases in humans and animals. In recent years, studies found that Micro RNA27a(mi R-27a) promote fibrosis of organs, such as liver and lung, and was associated with heart failure and myocardial hypertrophy. This study aims at observing the effects of mi R-27 a expression of fibrosis in H9C2 rat myocardial cells in vitro, the expression changes of mi R-27 a in high sugar condition and weather Liraglutide alleviate fibrosis of myocardial cells through down-regulating mi R-27 a expression, so that to provide new targets for myocardial fibrosis prevention and control.Methods: 1. H9C2 rat cardiomyocytes were divided into 6 groups: control group, lipofectamine 2000 group(lipo2000 group), transfection of mi R-27 a mimics low concentration group(mi R-27 a mimics low concentration group), transfection of mi R-27 a mimics high concentration group(mi R-27 a mimics high concentration group) and transfected into their respective negative control group(mi R-27 a N.C low concentration group and mi R-27 a N.C high concentration group). Low concentration groups 150 pmol mi R-27 a mimics or NC was transfected, High concentration group 300 pmol mi R-27 a mimics or NC was transfected.2. H9C2 rat cardiomyocytes were divided into 8 groups: normal sugar group(LG group), high glucose group(HG group), HG+mi R-27 a mimics group, HG+mi R-27 a inhibitor group, HG+mi R-27 a mimics N.C group, HG+mi R-27 a inhibitor N.C group, HG+lipo2000 group, High osmosis group(HO group), 300 pmol mi R-27 a mimics, inhibitor or its respective NC was transfected.3. H9C2 rat cardiomyocytes were divided into 6 groups: control group, Liraglutide group, Liraglutide+mi R-27 a mimics group, mi R-27 a mimics group, Liraglutide +mi R-27 a mimics NC group, Liraglutide + lipo2000 group, liraglutide intervention concentration was 1000 n M and was added 24 h before transfection.Cells were collected 48 hours after transfection, q RT-PCR method was used to detect the expression level of mi R-27 a and cell immunofluorescence staining was used to reflect the expression level of collagen I in three parts. Western Blotting method was used to detect the expression of peroxisome proliferator-activated receptor ?(PPAR?), transforming growth factor-?1(TGF-?1), connective tissue growth factor(CTGF) and Matrix metalloproteinase-9(MMP-9) in protein level related to myocardial fibrosis.Results: 1. In the groups of transfection of mi R-27 a mimics with low or high concentration, the mi R-27 a expression were significantly increased compared to control group, high concentration group was further increased compared to low concentration group(p < 0.05). There is no significant difference within control group, NC groups and lipo2000 group.2. PCR results showed that compared to normal sugar group, mi R-27 a expression was increased in high glucose group, and high glucose+mi R-27 a mimics group increased more significantly(p < 0.05). While in the high glucose+mi R-27 a inhibitor group, mi R-27 a expression was down regulatedly compared to normal sugar group(p < 0.05). The expression level of mi R-27 a was not changed in high glucose+mi R-27a NC groups and high glucose+lipo2000 group compared to high glucose group.3. Western Blotting results showed that a decreased PPAR? expression and increased TGF-?1, MMP-9 and CTGF expression at protein level in high glucose group and glucose+mi R-27 a mimics group compared to normal sugar group,more significant changes was observed in high glucose+mi R-27 a mimics group(p < 0.05). While increased PPAR? expression and decreased TGF-?1, MMP-9 and CTGF expression was observed in high glucose+mi R-27 a inhibitor group compared to normal sugar group(p < 0.05). There is no significant change within high glucose group, NC groups and lipo2000 group.4. With a intervention of 1000 nM Liraglutide, mi R-27 a expression level was decreased in, Liraglutide group, Liraglutide+mi R-27 a mimics NC group and Liraglutide+lipo2000 group compared to control group(p < 0.05). mi R-27 a expression level was increased in Liraglutide + mi R-27 a mimics group and mi R-27 a mimics group compared to the rest four groups(p < 0.05), but no difference was observed within these two groups.5. Cell immunofluorescence staining results showed that intracellular collagen I expression level was in consistent with mi R-27 a expression level. As intracellular mi R-27 a expression level increased, the fluorescence intensity in cells increased.Conclusions: 1. Mi R-27 a promotes myocardial fibrosis and shows a dependence on concentration.2. Mi R-27 a expression level is increased in high sugar condition. Moreover, mi R-27 a can regulate the expression of PPAR?, TGF-?1, MMP-9 and CTGF protein and promote cardiomyocyte fibrosis.3. GLP-1 can inhibit cardiomyocyte fibrosis by down reglating mi R-27 a expression. |
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