Emodin alleviates cardiac fibrosis by suppressing activation of cardiac fibroblasts via upregulating metastasis associated protein 3

Figure 1. Emodin reduces the thickened left ventricle in pressure overload mice. (A)–(C) Effects of varying dosages of emodin (10, 20, and 40 mg/kg/day) and captopril (10 mg/kg) on diastolic and systolic left ventricular posterior wall (LVPWd, LVPWs) thickness, heart weight/body weight (HW/BW) in transaortic constriction (TAC) mice (n = 5 batches in each group). E0 represents the non-emodin treated group; E10, E20, and E40 represent mice treated with 10, 20, and 40 mg/kg emodin, respectively. CAPT represents the 10 mg/kg captopril group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with control group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with TAC+E0 group. (D) Statistic data of fibrotic area/total area and (E) representative images of Masson trichrome-stained sections showing the collagen deposition and relief of such adverse alteration by emodin (n = 3). ∗∗∗P < 0.001 compared with control group; ##P < 0.01, ###P < 0.001 compared with TAC+E0 group. Scale bar = 50 μm.

Figure 2. Emodin represses the activation of cardiac fibroblasts in pressure overload mice. (A) and (B) α-SMA and COL1A2 protein levels detected by Western blotting showing the inhibitory effects of emodin (40 mg/kg/day) on the expression of these biomarkers for fibroblast activation in heart tissues, normalized to GAPDH. The data are presented as relative levels (A, n = 14; B, n = 7). ∗∗∗P < 0.001 compared with control group; ##P < 0.01, ###P < 0.001 compared with TAC group. (C) Determination of α-SMA-positive cells by immunofluorescent staining in cardiac fibroblasts isolated from adult mice. (D) Statistical data on the number of α-SMA positive cells (n = 5 batches in each group). ∗∗∗P < 0.001 compared with control group; ##P < 0.01 compared with TAC group. Scale bar = 20 μm.

Figure 3. Emodin suppresses viability and migration of cardiac fibroblasts stimulated by angiotensin II (AngII). (A) and (B) Results of transwell analysis depicting the effects of emodin on the migration ability of cardiac fibroblasts stimulated by AngII. E0 represents non-emodin cardiac fibroblasts; E5, E10, and E20 represent 5, 10, and 20 μmol/L emodin, respectively. CAPT represents 10 μmol/L captopril treated group (n = 3 batches in each group). ∗∗∗P < 0.001 compared with control group; ###P < 0.001 compared with AngII+E0 group. (C) Effects of emodin on cell number measured by trypan blue staining analysis (n = 7). ∗∗∗P < 0.001 compared with control group; ##P < 0.01, ###P < 0.001 compared with AngII+E0 group. (D) Effects of emodin on the number of living fibroblasts detected by cell counting kit 8 (CCK8) assay (n = 5). ∗∗∗P < 0.001 compared with control group; ##P < 0.01, ###P < 0.001 compared with AngII+E0 group. Scale bar = 20 μm.

Figure 4. Emodin downregulates α-smooth muscle actin (α-SMA) and COL1A2 in AngII-stimulated cardiac fibroblasts. (A) and (B) α-SMA and COL1A2 protein levels detected by Western blot in cardiac fibroblasts, normalized to GAPDH. Data are presented as relative levels (n = 5). ∗P < 0.05, ∗∗∗P < 0.001 compared with control group; #P < 0.05, ###P < 0.001 compared with AngII group. (C) and (D) Immunofluorescent staining of collagen I and the statistical data (n = 5 in each group). ∗P < 0.05 compared with control group; ##P < 0.01 compared with AngII group.

Figure 5. Emodin upregulates metastasis associated protein 3 (MTA3) expression. (A) Comparison of MTA3 abundance in cardiac fibroblasts, cardiomyocytes, and endothelial cells, as determined by Western blot. Averaged data were normalized to GAPDH (n = 3). ∗∗∗P < 0.001. (B) and (C) Effects of emodin on MTA3 expression at both protein and mRNA levels in TAC mice, as determined by Western blot (B, n = 11) and real-time PCR (C, n = 5), respectively. ∗∗∗P < 0.001 compared with control group; ###P < 0.001 compared with TAC group. (D) and (E) Effects of emodin on MTA3 expression at both protein and mRNA levels in cardiac fibroblasts pretreated with AngII, as determined by Western blot (D, n = 5) and real-time PCR (E, n = 3), respectively. ∗∗∗P < 0.001 compared with control group; ###P < 0.001 compared with TAC group.

Figure 6. Silence of MTA3 blocks the inhibitory effect of emodin on cardiac fibroblasts overproliferation. Cardiac fibroblasts were transfected with Mta3 small interfering RNA (siMTA3) or negative control (NC) in the presence of AngII with or without emodin. (A)–(C) Effects of siMTA3 on the protein levels of MTA3, α-SMA and COL1A2 (n = 5). ∗P < 0.05, ∗∗∗P < 0.001. (D) Effects of siMTA3 on the number of living cells as detected by CCK8 assay (n = 5). ∗∗∗P < 0.001. (E) Effects of siMTA3 on cell number as measured by Trypan blue staining (n = 5). ∗∗∗P < 0.001.

Figure 7. Silence of MTA3 expression promotes cardiac fibrosis. Cardiac fibroblasts were transfected with siMTA3 or NC. (A) Verification of the efficacy of MTA3 silence by siMTA3 at both protein (upper panel) and mRNA (lower panel) levels (n = 5). ∗∗P < 0.01 compared with control group; #P < 0.05, ##P < 0.01 compared with NC group. (B) and (C) Effects of siMTA3 on the expression of α-SMA and COL1A2 at both protein (upper panels) and mRNA (lower panels) levels (n = 5; except for mRNA in C, n = 4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with control group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC group.

Figure 8. Overexpression of MTA3 in regulating cardiac fibrosis. (A) Verification of MTA3 overexpression at both protein (upper panel) and mRNA (lower panel) levels (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (B) and (C) Effects of MTA3 overexpression on protein (upper panels) and mRNA (lower panels) levels of α-SMA and COL1A2 in cardiac fibroblasts stimulated by AngII (n = 5 for protein; n = 5 for mRNA). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.