ET-1siRNA Inhibits Myocardial Fibrosis Induced By Aldosterone In The Cardiac Fibroblasts

Objective: The morbidity and mortality of cardiovascular diseases are thehighest in the world, with approximately17million cases of death each year.Myocardial fibrosis is an important pathology characteristic for the chroniccardiovascular diseases, which is the cause of heart failure, malignantarrhythmia and sudden cardiac death. The severity of myocardial fibrosis isclosely related to the development and prognosis of disease. Therefore, it isvery important to study the pathogenesis of myocardial fibrosis and ideal drugfor improving survival rate and reducing mortality rate.Myocardial fibrosis is characterized by proliferation of fibroblasts,deposition of extracellular matrix proteins in the myocardial interstitium.Cardiac fibroblasts play a pivotal role in the development of myocardialfibrosis. Myocardial fibrosis is the abnormal proliferation and excessivesynthesis and secretion of collagen of cardiac fibroblasts. Thus, it’s importantto research the changes of cardiac fibroblasts for exploring the mechanisms,prevention and treatment of myocardial fibrosis.The pathogenesis of myocardial fibrosis is complex. At present theprecise mechanism of myocardial fibrosis is not clear, but the impact ofcytokine in myocardial fibrosis has attracted increasing attention. There aresome cytokines in the process of fibrosis, such as transforming growthfactor-β1(TGF-β1) and connective tissue growth factor. Endothelin-1(ET-1)is an important cytokine and a critical autocrine and paracrine regulator oflocal blood vessel tone. ET-1is also a prolonged and powerful factor of cellproliferation. Thus, ET-1may play a role in process of myocardial fibrosis.Recent studies showed that aldosterone (Ald) also played an important role inthe process of myocardial fibrosis. Therefore, the present study was toinvestigate the effect of ET-1in myocardial fibrosis induced by Ald. Small interfering RNA (siRNA) can silence target genes, which is highefficiency, specificity and low toxicity. In recent years, siRNA technology hasbecome a powerful tool in the study of genetic function, disease mechanism,disease control and prevention. Shyu reported that Urotensin II was animportant medium in vitro myocardial fibrosis models under anoxic conditionby using siRNA. Another study showed that ET-1siRNA increased tubeformation and decreased Rho kinase activity in pulmonary artery endothelialcells of persistent pulmonary hypertension model. The above researchsprovided the experimental basis for ET-1siRNA application in the cardiacfibroblasts of myocardial fibrosis model.Therefore, the present study was undertaken to investigate the effect ofET-1on the cardiac fibrosis by transfection ET-1siRNA into cardiacfibroblasts. The results would provide new clues and more direct evidence forillustrating the pathogenesis and prevention of myocardial fibrosis.Experimental group and methods:1Transfection efficiency and duration time of siRNA transfection in ratcardiac fibroblastsTo investigate the best siRNA transfection concentration and time,transfection efficiency and duration time in cardiac fibroblasts wereobserved by using LipofectamineTMRNAiMAX transfection reagent anddifferent concentrations of green fluorescent labeled siRNA. This studyprovided the basis for the next step of ET-1siRNA transfection in cardiacfibroblasts. Cardiac fibroblasts were isolated from cardiac tissues of neonatalSD rats by the trypsin digestion and differential adhesion method.3rd and4th passages of the cells were used in experiments. Cells were randomlydivided into the following6groups.(1) Control group (n=6): Cells were only added Opti-MEM I medium.(2) LipofectamineTMRNAiMAX transfection reagent group (n=6):Cells were added Opti-MEM I medium and LipofectamineTMRNAiMAXtransfection reagent. (3)12.5nmol/L siRNA transfection group (n=6): Cells were addedOpti-MEM I medium, LipofectamineTMRNAiMAX transfection reagentand green fluorescent labeled siRNA (Block-iT)12.5nmol/L.(4)25nmol/L siRNA transfection group (n=6): Cells were addedOpti-MEM I medium, LipofectamineTMRNAiMAX transfection reagentand green fluorescent labeled siRNA25nmol/L.(5)50nmol/L siRNA transfection group (n=6): Cells were addedOpti-MEM I medium, LipofectamineTMRNAiMAX transfection reagentand green fluorescent labeled siRNA50nmol/L.(6)100nmol/L siRNA transfection group (n=6): Cells were addedOpti-MEM I medium, LipofectamineTMRNAiMAX transfection reagentand green fluorescent labeled siRNA100nmol/L.Transfection efficiency and duration time in cardiac fibroblasts wereevaluated by green fluorescence intensity and fluorescence expression rate atthe bright field and fluorescence under the fluorescent inverted microscope.The cells were transfected with siRNA at different concentrations (12.5nmol/L,25nmol/L,50nmol/L and100nmol/L) and different time points (6h,12h,24h,48h and72h), respectively. Integrated optical density value ofgreen fluorescent protein was calculated by JEDA-801morphology imageanalysis system.2ET-1siRNA inhibition of myocardial fibrosis induced by AldTo investigate the effect of ET-1on myocardial fibrosis, ET-1siRNAwas transfected in cardiac fibroblasts and myocardial fibrosis was inducedby Ald. Cardiac fibroblasts were isolated from cardiac tissues of neonatal SDrats by the trypsin digestion and differential adhesion method.3rd and4thpassages of the cells were used in experiments. Cells were randomly dividedinto the following5groups.(1) Control group: Cells were only added Opti-MEM I medium.(2) Ald+control siRNA group: Cells were added Opti-MEM I medium,LipofectamineTMRNAiMAX transfection reagent and control siRNA. Cellfibrosis was induced by Ald. (3) Ald+ET-1siRNA1group: Cells were added Opti-MEMI medium,LipofectamineTMRNAiMAX transfection reagent and ET-1siRNA1. Cellfibrosis was induced by Ald.(4) Ald+ET-1siRNA2group: Cells were added Opti-MEM I medium,LipofectamineTMRNAiMAX transfection reagent and ET-1siRNA2. Cellfibrosis was induced by Ald.(5) Ald+ET-1siRNA3group: Cells were added Opti-MEM I medium,LipofectamineTMRNAiMAX transfection reagent and ET-1siRNA3. Cellfibrosis was induced by Ald.ELISA method was to detect the change of ET-1in cardiac fibroblastsand cell culture supernatants (n=6/group). MTT method was to investigatethe cardiac fibroblasts proliferation (n=8/group). Hydroxyproline assay wasto detect the collagen content in cell culture supernatants (n=6/group).Results:1.1Transfection results of different siRNA concentrations at the same timepointCompared with control group and LipofectamineTMRNAiMAXtransfection reagent group, there was no statistical difference in the numbersof cardiac fibroblasts between the two groups at different time points. Thisresult indicated that the concentration of LipofectamineTMRNAiMAXtransfection reagent was low toxicity to cells in this experiment. There wereno green fluorescent cells in the green fluorescent field, which indicated thatsiRNA transfection was specified. After siRNA transfection at6h and12h,obvious green fluorescence could be observed in the majority cytoplasm andnucleus of cardiac fibroblasts. Fluorescence intensity obviously enhanced at24h, sustained to48h, and reduced at72h, but it was still obvious at72h.Transfection efficiency and integrated optical density of green fluorescentprotein were increased in a dose-dependent manner with the siRNAconcentration at6h and12h of cardiac fibroblasts. Transfectionefficiencies and integrated optical densities were all higher at24h and48h,and the100nmol/L group was significantly higher compared with the other groups (P<0.05). At72h time point, changes of transfection efficiency andintegrated optical density were significant difference between the lowconcentration groups (P<0.05), and no obvious difference between the highconcentration groups.1.2Transfection results of same siRNA concentration at different time pointsTransfection efficiency was low in12.5nmol/L siRNA concentration at6h group. Compared with6h group, transfection efficiencies weresignificantly increased at12h,24h,48h and72h groups (P<0.05，P<0.05，P<0.05，P<0.05). In12.5nmol/L siRNA concentration group, transfectionefficiency reached the peak at24h and48h, and gradually decreased at72h(P<0.05). The change trends of25nmol/L,50nmol/L and100nmol/L weresimilar to that of12.5nmol/L, but transfection efficiencies all decreasedslower than that of12.5nmol/L at72h group. In addition, there was nostatistical difference between72h and48h groups at50nmol/L.2.1ET-1expression in cardiac fibroblasts was influenced by Ald and ET-1siRNAELISA results showed that ET-1content significantly increased inAld+control siRNA group compared with control group (P<0.01), whereassignificantly decreased in Ald+ET-1siRNA1, Ald+ET-1siRNA2andAld+ET-1siRNA3groups (P<0.01, P<0.01, P<0.01), and significantlylower than control group (P<0.01, P<0.01, P<0.01). Ald+ET-1siRNA2group was reduced apparently than the other two groups, but there was nostatistical difference.2.2ET-1content in culture supernatant of cardiac fibroblasts was influencedby Ald and ET-1siRNAELISA results showed that ET-1content in culture supernatant ofcardiac fibroblasts in Ald+control siRNA group significantly increasedcompared with control group (P<0.01), whereas significantly decreased inAld+ET-1siRNA1, Ald+ET-1siRNA2and Ald+ET-1siRNA3groups(P<0.01, P<0.01, P<0.01). ET-1content in three Ald+ET-1siRNA groupswas significantly lower than that of control group (P<0.01, P<0.01, P<0.01). Ald+ET-1siRNA2group was reduced significantly than the other twogroups, but there was no statistical difference. Compared with Ald+controlsiRNA group, ET-1content changes in supernatants of three Ald+ET-1siRNA groups were more significant than those of cells.2.3Cardiac fibroblasts proliferation was influenced by Ald and ET-1siRNAMTT results showed that the average absorbance value in Ald+controlsiRNA group significantly increased compared with control group (P<0.01),whereas significantly decreased in Ald+ET-1siRNA1, Ald+ET-1siRNA2and Ald+ET-1siRNA3groups (P<0.01, P<0.01, P<0.01). The averageabsorbance value in Ald+ET-1siRNA1group was higher than that of controlgroup (P<0.05). Ald+ET-1siRNA2and Ald+ET-1siRNA3groups were nostatistical difference compared to control group.2.4Collagen content in culture supernatant of cardiac fibroblasts wasinfluenced by Ald and ET-1siRNAHydroxyproline content assay showed that collagen contentsignificantly increased in Ald+control siRNA group than that of controlgroup (P<0.01), whereas significantly decreased in three Ald+ET-1siRNAgroups (P<0.01, P<0.01, P<0.01). The collagen content in three Ald+ET-1siRNA groups was higher than that of control group (P<0.01, P<0.01,P<0.01).Conclusion:The present study suggested that siRNA could be efficiently transfected intocultured cardiac fibroblasts by application of LipofectamineTMRNAiMAXtransfection reagent. Transfection time was lasted for72h, and reagents werelow toxicity to cells. The ET-1siRNA application showed that ET-1mayplay an important role in myocardial fibrosis induced by aldosterone in thecardiac fibroblasts.