The Effects Of Epigallocatechin Gallate On Rat Cardiac Fibroblasts Induced By Angiotensin Ⅱ And The Potential Mechanism

BackgroundMyocardial remodeling is a continuum of changes in the structure and function of the myocardium that commonly occur as a result of a pathological process. During myocardial remodeling, cardiac fibroblasts (CFs) play a central role in the maintenance of extracellular matrix (ECM) and undergo hyperplasia in response to some humoral factors such as angiotensin Ⅱ (AngⅡ) and endothelin (ET). There are at least four receptors known to mediate AngⅡ function, the AngⅡ type-1receptor (AT1R), AT2R, AT3R and AT4R. The signal transduction pathways activated by AngⅡ and responsible for cardiac fibrosis have been deeply explored. The effects of Ang Ⅱ on CFs were found to be exclusively mediated via angiotensin type1a receptor (AT1aR), which is a G protein coupled receptor (GPCR).GPCRs are extremely important drug targets and the β-arrestins intracellular scaffolding and adaptor proteins regulate major aspects of their pharmacology. Exciting new data have revealed that β-arrestins play potential roles as key signaling molecules in the treatment of heart diseases. Experiments showed that β-arrestin-1and β-arrestin-2mRNA levels were induced in the hearts of rats with congestive heart failure (CHF). The β-arrestin-dependent and G-protein-dependent pathways differentially activate extracellular regulated kinase (ERK) signaling in heart, and the β-arrestin-mediated transactivation of the epidermal growth factor receptor (EGFR) is cardioprotective.Based on considerable evidence accumulated during the last few years, much attention is focused on the use of naturally occurring botanicals for the prevention of heart diseases. Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and is considered as a potent antioxidant that may have therapeutic applications in the treatment of cardiac disorders. The existing results mainly focused on the antioxidation effects of EGCG, less attention had been paid on its role in regulating AT1R mediated signaling. Accordingly, in the study, we isolate primary CFs from2-day-old neonatal Sprague-Dawley (SD) rat hearts, and investigate the effects of epigallocatechin gallate on rat CFs induced by AngⅡ and the potential mechanism by using small interfering RNA, and applying advanced techniques for instance real-time quantitative reverse transcription PCR (RT-qPCR), western blot, etc. The results would not only demonstrate the importance of β-arrestin-dependent regulation in AT1R-mediated responses of CFs, but also shed new light on the molecular pharmacological potential of EGCG in treating myocardial remodeling.Objectives1. To isolate CFs from neonatal SD rat hearts, and then investigate the effect of EGCG on the activation of normal CFs or AngⅡ-induced CFs.2. To elucidate the effect of EGCG on the expression of β-arrestin-land β-arrestin-2, and analyze the correlation between the level of β-arrestin and the viability of CFs.3. To evaluate the role of β-arrestin-1in AT1R distribution, the viability, proliferation and collagen production of AngⅡ-induced CFs and the effect of EGCG4. To reveal the effect of EGCG on ATIR-Gq-PKC signaling in AngⅡ-induced CFs.Methods1. Primary CFs isolation and identificationPrimary CFs were isolated from2-day-old neonatal SD rat hearts. Ventricles were removed under sterile conditions from neonatal rats, placed in cold sterile calcium-free phosphate buffered saline (PBS), minced into approximately2mm cubes, and treated with1mg/ml collagenase. Dissociated cells were preplated for 45min in Dulbecco’s modified Eagle’s medium (DMEM) containing10%heat-inactivated fetal bovine serum (FBS). This process was repeated twice, and cells were collected through0.25%trypsin treatment. The resulting CFs were resuspended in DMEM containing10%FBS,100U/ml penicillin and100μg/ml streptomycin and seeded in standard culture dishes. The expression of vimentin was examined immunohistochemically. If the cells we cultured showed intensely positive staining for vimentin confirming that the all cells appeared spindleshaped with a fibroblast-like morphology. All CFs used in our experiments were at passage2-4.2. Drug administration in vitroThe CFs were seeded into96-well plates and grown to confluence, synchronized and then were cultured with EGCG (1×10-9,1×10-8,1×10-7,1×10-6,1×10-5mol/L) or valsartan (1×10-6mol/L) in the presence of AngⅡ (1×10-7mol/L) for24hours. CFs were cultured with or without EGCG (1×10-5mol/L) to determine the effect of EGCG on normal CFs.3. CFs viability detectionThe viability of CFs was evaluated by MTT assay. MTT was added into each well and the plates were incubated for another6hours. The formazan crystals were dissolved by120μl dimethylsulfoxide. The absorbance (A) was measured at490nm using Bio-Rad EIA Analyzer.4. CFs proliferation and collagen synthesis measurementThe CFs proliferation and collagen synthesis were measured by [3H]-thymidine and [3H]-proline incorporation assay respectively. The CFs were cultured with different concentrations of EGCG or Val in the presence of AngⅡ for24hours in media containing1μCi/ml [3H]-thymidine or2μCi/ml [3H]-proline. Then culture supernatant was discarded, cell layer was washed with PBS, lysed in lysis buffer (0.1M NaOH containing0.1%SDS). Radioactivity was determined using a liquid scintillation counter.5. The determination of β-arrestin-1, AT1aR and AT1bR mRNA expressionsThe total mRNA was prepared using Trizol reagent. First strand cDNA was synthesized from total mRNA (1μg) by reverse transcription. The primers used for RT-qPCR were:AT1aR (forward:5’-GGATGGTTCTCAGAGAGAGTACAT-3’, reverse:5’-CCTGCCCTCTTGTACCTGTTG-3’), AT1bR (forward:5’-CTGCCCCAA GGCTGGCAGGC-3’, reverse:5’-GCCACCTACTGCCGGCCGTT-3’), β-arrestin-1(forward:5’-AAGGGACACGAGTGTTCAAG-3’,reverse:5’-GACTCGCCTTTCTT TGAGATAC-3’), GAPDH (forward:5’-TCAAGAAGGTGGTGAAGCAG-3’, reverse:5’-AGGTGGAAGAATGGGAGTTG-3’) served as housekeeping gene.6. Western blot analysisCFs were lysed in lysis buffer at4℃. The homogenate was centrifuged for4min at3000×g and the resulting supernatants were used to detect total protein expression. Part of the supernatant was subjected to centrifugation at37,000g for25min. The pellet was resuspended in lysis buffer for membrane protein detection. The supernatant was used to determine the expression of cytoplasmic protein. Pan cadherin was applied as a general membrane marker to identify the efficiency of protein extraction. Equal amount of the extracts were loaded to detect the expression of AT1R, Gq, β-arrestin-1, β-arrestin-2or phosphorylated PKC-delta.7. β-arrestin-1small interfering RNA (siRNA) transfectionCFs were seeded onto6-well plates at a density of2×104cells/well and allowed to grow until80-90%subconfluent, then transfected with20μg of siRNA using20μl of Lipofectamine2000. Six hours after transfection, cell layer was washed with PBS and cultured for another48hours. CFs were synchronized for at least48hours prior to stimulation. The transfection efficiency was determined by both RT-qPCR and western blot.8. Statistical AnalysisFor all experiments, at least triplicate determinations were made. All data are expressed as mean and standard deviation (SD). Comparisons between two experimental groups were performed using Student’s t test, for multiple group comparisons we used ANOVA test. Results were considered statistically significant for P values less than0.05. Results1. The culture of CFsFreshly isolated cells from2-day-old neonatal SD rat hearts grew fast to confluence after2or3days. CFs was flat, spindle-shaped cells with multiple processes emanating from the main cell body. CFs has a branched cytoplasm surrounding an elliptical, speckled nucleus having one or two nucleoli.2. The identification of CFsAccording to the immunohistochemical examination, the cells we cultured showed intensely positive staining for vimentin confirming that the all cells appeared spindleshaped with a fibroblast-like morphology.3. Effects of EGCG on the viability, proliferation and collagen synthesis of normal CFsAs expected, EGCG alone has no obvious effect on viability, proliferation and collagen production (P>0.05) of normal CFs.4. Effects of EGCG on viability, proliferation and collagen synthesis of AngⅡ-induced CFsEGCG of1×10-7-1×10-5mol/L concentration-dependently inhibited cell viability evoked by Angll, Val displayed a similar effect. In AngⅡ treated CFs,[3H]-thymidine and [3H]-proline incorporation obviously increased. EGCG and valsartan partially, but significantly attenuated the hyperplasia and collagen synthesis of CFs induced by Angll.5. Effects of EGCG on β-arrestin-1and β-arrestin-2expressions in AngⅡ-induced CFs and the correlation between β-arrestin and the inhibitory effect of EGCG on cell viability.The expression of β-arrestin-1mRNA was increased in CFs when were exposured to AngⅡ, but this abnormality was alleviated by the administration of EGCG (1×10-6,1×10-5mol/L), to varying degrees, however, we have not found any changes in β-arrestin-2protein expression in AngⅡ-induced or in EGCG treated cells. Subsequently, the correlation between the inhibitory effect of EGCG on CFs viability and β-arrestin-1mRNA levels was then analyzed. Interestingly, the scatter plot of β-arrestin-1mRNA levels and the viability of CFs revealed a significantly positive correlation (R2=0.9304, P=0.0019). This result indicates that the inhibitory effect of EGCG on CFs viability is related to β-arrestin-1gene expression.6. The role of β-arrestin-1in AT1R distribution under Ang II stimulation and CFs viability, proliferation and collagen synthesis and the effect of EGCGβ-arrestin-1gene expression was reduced about56.7%, while the protein expression in CFs were reduced by about55%in response to introducing β-arrestin-1siRNA as compared with the negative control cells. The β-arrestin-1gene disruption upregulated membrane AT1R distribution, and blocked the viability, proliferation and collagen production of CFs treated with Ang Ⅱ. In β-arrestin-1knocked down CFs, EGCG further increased the expression of AT1R on cell membrane, and reduced the viability and proliferation of Ang Ⅱ treated cells. These results indicate that β-arrestin-1is essential for AngⅡ-mediated activation of CFs via regulating AT1R internalization, and it substantially contributes to the function of EGCG.7. Effects of EGCG on ATIR, β-arrestin-1and Gq expressionAs detected, AT1aR mRNA was reduced by AngⅡ stimulation, but was restored to normal levels by the administration of EGCG (1×10-5mol/L), whereas expression of ATI b mRNA was unaltered. The purification of membrane protein by protocol described above is satisfied. In CFs cultured with AngⅡ, total and membrane β-arrestin-1levels were increased (P<0.01), on the contrary, the expressions of total and membrane AT1R and Gq protein were decreased.1×10-5mol/L of EGCG reduced total β-arrestin-1expression.1×10-6,1×10-5mol/L of EGCG decreased membrane β-arrestin-1levels, but promoted global and membrane AT1R expressions. EGCG had no obvious effects on Gq expression.8. Effects of EGCG on p-PKC-delta expressionThe results indicated that the p-PKC-delta expression in the cytosol was significantly inhibited by AngⅡ (1×10-7mol/L), as compared with control CFs. Administration of EGCG (1×10-6,1×10-5mol/L) significantly restored the reduced p-PKC-delta levels. Neither AngⅡ nor EGCG obviously affected the expression of membrane p-PKC-delta.Conclusions1. The cells we cultured were identified to be CFs according to cell morphology and the immunohistochemical examination.2. EGCG showed strong inhibitory effects on viability, proliferation and collagen synthesis of AngⅡ-induced CFs, however, it did not obviously influence the activation of normal CFs.3. The inhibitory effect of EGCG on CFs viability evoked by AngⅡ was related to β-arrestin-1gene expression but not P-arrestin-2.4. β-arrestin-1was essential for AngⅡ-mediated activation of CFs via regulating AT1R internalization and it substantially contributed to the function of EGCG.5. AngⅡ activated CFs mainly through AT1aR mediated signaling. EGCG exerted its inhibitory effects on activated CFs via reducing the total and membrane expressions of β-arrestin-1, and thus restoring AT1R signaling.