Lnterleukin17A Inhibits Mouse Cardiac Fibroblast Activation Via EphrinB2Signaling Pathway

ObjectiveMyocardial fibrosis is an important pathological process of ventricular remodeling, IL-17A plays a critical role in this process. However the exact mechanism has not been clarified. The studies show that EphrinB2signaling may participate in renal fibrosis, but its role in myocardial fibrosis is not clear. It was found in our preliminary experiment that IL-17A can reduce the expression of EphrinB2in fibroblast. Therefore, the aim of this study was to determine whether IL-17A regulates mouse cardiac fibroblast activation via EphrinB2signaling.MethodsIsolation and culture of mouse cardiac fibroblasts by digesting the ventricular tissue with collagenase II and the different attachment time of cells. The fibroblasts were identified by immune fluorescence with vimentin, CD31, troponin T and SM myosin antibodies. At the same time, EphrinB2was detected by immune fluorescence; The study includes the following groups:control, scramble/vector, lenti-efnb2, control+IL-17, scramble/vector+IL-17, lenti-efnb2+IL-17; CCK8and scratch assay were used to detect the capacity of proliferation and migration. The expression of a-SMA, collagen I and collagen III were measured by real-time PCR and western blot; the test was tripled, data were expressed as means±SEM. Student’s t-test or two-way ANOVA was used to assess the statistical differences. SPSS19.0was used for analysis, P<0.05was considered statistically significant.ResultsCultured cardiac fibroblast cells were identified with vimentin (+), CD31(-), troponin T(-), SM myosin(-) by immune fluorescence, which indicated more than95%purity of cardiac fibroblasts. Additionally, EphrinB2was massively expressed in cardiac fibroblasts; IL-17A (100ng/ml) incubation for24h obviously inhibited the capacity of proliferation and migration, and the expression of a-SMA, collagen I and collagen III of cardiac fibroblast. At the same time, the level of EphrinB2decreased compared with control group; EphrinB2-shRNA transfected with virus significantly decreased the proliferation and migration and the expression of a-SMA, collagen I and collagen III of cardiac fibroblast, while the opposite results were observed in EphrinB2over-expression situation; EphrinB2-shRNA further inhibited the IL-17A caused inhibition of proliferation, migration, and the expression of a-SMA, collagen Ⅰ/Ⅲ compared with scramble+IL-17and control+IL-17groups, while EphrinB2-overexpression counteracted the inhibitory role of IL-17A in the activation of cardiac fibroblasts compared with vector+IL-17and control+IL-17groups.ConclusionIL-17A can suppress the proliferation and migration of fibroblasts and inhibit the expression of aSMA and collagen Ⅰ/Ⅲ in vitro, which may be related to the inhibition of EphrinB2signaling.