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The morbidity and mortality of cardiovascular diseases are the highest in the world, with approximately 17.3 million cases of death each year. Myocardial fibrosis is an important pathology characteristic for the chronic cardiovascular diseases, which is the cause of heart failure, malignant arrhythmia and sudden cardiac death. At present the precise mechanism of myocardial fibrosis is not clear. It is well known that aldosterone(Ald) antagonists either spironolactone or eplerenone have long been used to reduce myocardial fibrosis. Endothlin-1(ET-1)could promote myocardial fibrosis. We hypothesized that aldosterone promote myocardial fibrosis might be through Endothlin-1 pathway and siRNA might inhibit Endothlin-1 to reduce myocardial fibrosis.
To test our hypothesis, the following experiments were performed,
1. Endothelin-1 small interfering RNA transfection of cardiac fibroblasts, the transfection efficiency and time curve were evaluated by using green fluorescence intensity and fluorescence expression rate under the fluorescent inverted microscope.
2. The expression levels of endothelin-1 were determined with ELISA, MTT, and Hydroxyproline assay in transfected cells as well as in cell culture supernatant,
1. No statistical difference was found in the numbers of cardiac fibroblasts between control cells and cells with siRNA transfection by using LipofectamineTM RNAiMAX at any time points, no green fluorescent cells in the green fluorescent field, indicating that siRNA transfection was low toxicity to cells and specified.
2.Transfection efficiency and integrated optical density of green fluorescent protein were increased in a dose-dependent manner at 6h and 12h in cardiac fibroblasts. Transfection efficiency was significantly higher at 12 h, 24 h, 48 h and 72 h time points than at 6h point (P<0.05, respectively).
3.ELISA results showed that ET-1 content significantly increased in Ald+control-siRNA group compared with control group (P<0.01), whereas significantly decreased in Ald+ endothelin -1 siRNA 1/siRNA 2/siRNA 3 groups (P<0.01, respectively), and obviously lower than control group (P<0.01, respectively). Compared with Ald+control siRNA group, ET-1 content was significantly higher in supernatants of three Ald+ ET-1 siRNA groups than in respective cells.
4.MTT results showed that the average absorbance value in Ald+control siRNA group visibly increased compared with control group (P<0.01), decreased in Ald+ ET-1 siRNA 1/siRNA 2/siRNA 3 groups significantly (P<0.01, respectively).
5.Hydroxyproline content assay showed that collagen content significantly increased in Ald+control siRNA group than in control group (P<0.01), decreased in three Ald+ ET-1 siRNA groups (P<0.01, respectively).
The study showed that siRNA could be efficiently and specifically transfected into cultured cardiac fibroblasts with low toxicity. Endothlin-1 siRNA inhibited myocardial fibrosis induced by aldosterone, indicating that Endothlin-1 plays a vital role in development of myocardial fibrosis.