EPI prevents cardiac fibrosis
To examine whether EPI protects heart against cardiac fibrosis, we firstly established the TAC mouse models. Then intragastric administration of EPI was conducted into mice for 28 days after established TAC models. Echocardiography was used to monitor the cardiac function (Figure 1). LVEF, LVFS, LVIDs, LVIDd, LVPWs, LVPWd, IVSs and IVSd were detected. Compared to control group, TAC decreased LVEF, LVFS and increased LVIDs, LVIDd, which were ameliorated by EPI treatment.
The protective effect of EPI on the TAC-induced alteration of cardiac structure was determined. As shown in Figure 2, the results showed that collagen content was increased in the heart tissues of TAC group, which was significantly reduced by EPI treatment (Figure 2A-C). In addition, administration of EPI substantially reduced HW/BW index in TAC group (Figure 2D).
EPI blocks AngII-induced myofibroblasts transformation
To examine whether EPI protects cardiac fibroblast (CFs) against AngII-induced myofibroblasts transformation, we used primary cultured neonatal rat cardiac fibroblast for vitro models of cardiac fibrosis. Cells were incubated using Ang II or/and EPI for 24 h. As indicated in Figure 3A-B, α-SMA-positive area was significantly increased in AngII-treated group, and EPI inhibited AngII-induced fibroblast transformation.
To further evaluate protective effect of EPI on cardiac fibrosis, protein of α-SMA and collagenI/III (COLI/III) were detected by western blot (Figure 3C-E). As we expected, Ang II increased the protein expression of α-SMA and COLI/III. EPI efficiently inhibited these changes, suggesting that the myofibroblasts transformation and collagen synthesis induced by AngII could be mitigated by EPI.
EPI activates SIRT1/AKT/GSK3β pathway
To determine the signaling pathway participated in EPI-blocked myofibroblasts transformation, we studied the effect of EPI on the protein expression of SIRT1, AKT, GSK3β, P-AKT and P-GSK3β (Figure 4). As shown in Figure 4A-C, western blot analysis indicated EPI significantly increased the protein expression of SIRT1, P-AKT and P-GSK3β in CFs.
Next, we explored whether SIRT1 participates in EPI-induced activation of AKT/GSK3β. As shown in Figure 4D, EPI increased the protein level of SIRT1. We used Si-RNA approach to knock down SIRT1, which blocked SIRT1 proteins increased by EPI. We tested whether knockdown of SIRT1 could affect activation of AKT/GSK3β induced by EPI. As we expect, knockdown of SIRT1 significantly decreased protein level of P-AKT and P-GSK3β (Figure 4E-F), which illustrated that EPI-induced activation of AKT and GSK3β was mediated by SIRT1. These data suggested that EPI might activate SIRT1/AKT/GSK3β pathway for inhibiting cardiac fibrosis. These observations also found that SIRT1/AKT/GSK3β pathway was not affected by AngII.
SIRT1 participates in EPI-inhibited myofibroblasts transformation
Furthermore, as SIRT1 has been shown to participate in EPI-induced activation of AKT and GSK3β, we tested whether SIRT1 was involved in EPI-inhibited myofibroblasts transformation. We used Si-RNA approach to knock down SIRT1, which removed SIRT1, P-AKT and P-GSK3β proteins increased by EPI, and abolished the protective role of EPI against AngII (Figure 5A-C).
Next, we used Si-RNA approach to knock down SIRT1 in CFs. Then cells were incubated using Ang II or/and EPI for 24 h. As indicated in Figure 6A-B, α-SMA-positive area was significantly decreased in EPI+AngII-treated group, and Si-SIRT1 blocked EPI-inhibited myofibroblasts transformation. These results even further suggested that SIRT1 was essential for EPI-induced activation of AKT and GSK3β, which was also required for EPI-inhibited myofibroblasts transformation.