Astaxanthin promotes M2 macrophages and attenuates cardiac remodeling after myocardial infarction by suppression inflammation in rats
Authors: Pan Xia , Zhang Kai , Shen Cheng , Wang Xi , Wang Long , Huang Ya-Yi 
Published: July 2020

Background

Cardiac remodeling after acute myocardial infarction (AMI) is an important process. The present study aimed to assess the protective effects of astaxanthin (ASX) on cardiac remodeling after AMI.

Methods

The study was conducted between April and September 2018. To create a rat AMI model, rats were anesthetized, and the left anterior descending coronary artery was ligated. The rats in the ASX group received 10 mg·kg−1·day−1 ASX by gavage for 28 days. On the 1st day after AMI, but before ASX administration, six rats from each group were sacrificed to evaluate changes in the heart function and peripheral blood (PB) levels of inflammatory factors. On the 7th day after AMI, eight rats from each group were sacrificed to evaluate the PB levels of inflammatory factors and the M2 macrophage count using both immunofluorescence (IF) and flow cytometry (FC). The remaining rats were observed for 28 days. Cardiac function was examined using echocardiography. The inflammatory factors, namely, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-10, were assessed using enzyme-linked immunosorbent assay. The heart weight/body weight (BW), and lung weight (LW)/BW ratios were calculated, and myocardial fibrosis in the form of collagen volume fraction was measured using Masson trichrome staining. Hematoxylin and eosin (H&E) staining was used to determine the myocardial infarct size (MIS), and TdT-mediated dUTP nick-end labeling staining was used to analyze the myocardial apoptosis index. The levels of apoptosis-related protein, type I/III collagen, transforming growth factor β1 (TGF-β1), metalloproteinase 9 (MMP9), and caspase 3 were assessed by Western blotting. Unpaired t-test, one-way analysis of variance, and non-parametric Mann-Whitney test were used to analyze the data.

Results

On day 1, cardiac function was worse in the ASX group than in the sham group (left ventricular end-systolic diameter [LVIDs]: 0.72 ± 0.08 vs. 0.22 ± 0.06 cm, t = −11.38; left ventricular end-diastolic diameter [LVIDd]: 0.89 ± 0.09 vs. 0.48 ± 0.05 cm, t = −9.42; end-systolic volume [ESV]: 0.80 [0.62, 0.94] vs. 0.04 [0.03, 0.05] mL, Z 
= −2.89; end-diastolic volume [EDV]: 1.39 [1.03, 1.49] vs. 0.28 [0.22, 0.32] mL, Z 
= −2.88; ejection fraction [EF]: 0.40 ± 0.04 vs. 0.86 ± 0.05, t = 10.00; left ventricular fractional shortening [FS] rate: 0.19 [0.18, 0.20] %FS vs. 0.51 [0.44, 0.58] %FS, Z = −2.88, all P < 0.01; n 
= 6). The levels of inflammatory factors significantly increased (TNF-α: 197.60 [133.89, 237.94] vs. 50.48 [47.21 57.10] pg/mL, Z 
= −2.88; IL-1β: 175.23 [160.74, 215.09] vs. 17.78 [16.83, 19.56] pg/mL, Z 
= −2.88; IL-10: 67.64 [58.90, 71.46] vs. 12.33 [11.64, 13.98] pg/mL, Z 
= −2.88, all P < 0.01; n 
= 6). On day 7, the levels of TNF-α and IL-1β were markedly lower in the ASX group than in the AMI group (TNF-α: 71.70 [68.60, 76.00] vs. 118.07 [106.92, 169.08] pg/mL, F = 42.64; IL-1β: 59.90 [50.83, 73.78] vs. 151.60 [108.4, 198.36] pg/mL, F = 44.35, all P < 0.01, n 
= 8). Conversely, IL-10 levels significantly increased (141.84 [118.98, 158.36] vs. 52.96 [42.68, 74.52] pg/mL, F = 126.67, P < 0.01, n 
= 8). The M2 macrophage count significantly increased (2891.42 ± 211.29 vs. 1583.38 ± 162.22, F = 274.35, P < 0.01 by immunofluorescence test; 0.96 ± 0.18 vs. 0.36 ± 0.05, F = 46.24, P < 0.05 by flowcytometry test). On day 28, cardiac function was better in the ASX group than in the AMI group (LVIDs: 0.50 [0.41, 0.56] vs. 0.64 [0.56, 0.74] cm, Z 
= −3.60; LVIDd: 0.70 [0.60, 0.76] vs. 0.80 [0.74 0.88] cm, Z 
= −2.96; ESV: 0.24 [0.18, 0.45] vs. 0.58 [0.44, 0.89] mL, Z 
= −3.62; EDV: 0.76 [0.44, 1.04] vs. 1.25 [0.82, 1.46] mL, Z 
= −2.54; EF: 0.60 ± 0.08 vs. 0.50 ± 0.12, F = 160.48; %FS: 0.29 [0.24, 0.31] vs. 0.20 [0.17, 0.21], Z 
= −4.43, all P < 0.01; n 
= 16). The MIS and LW/BW ratio were markedly lower in the ASX group than in the AMI group (myocardial infarct size: 32.50 ± 1.37 vs. 50.90 ± 1.73, t = 23.63, P < 0.01, n = 8; LW/BW: 1.81 ± 0.15 vs. 2.17 ± 0.37, t = 3.66, P = 0.01, n = 16). The CVF was significantly lower in the ASX group than in the AMI group: 12.88 ± 2.53 vs. 28.92 ± 3.31, t = 10.89, P < 0.01, n = 8. The expression of caspase 3, TGF-β1, MMP9, and type I/III collagen was lower in the ASX group than in the AMI group (caspase 3: 0.38 ± 0.06 vs. 0.66 ± 0.04, t = 8.28; TGF-β1: 0.37 ± 0.04 vs. 0.62 ± 0.07, t = 6.39; MMP9: 0.20 ± 0.06 vs. 0.40 ± 0.06, t = 4.62; type I collagen: 0.42 ± 0.09 vs. 0.74 ± 0.07, t = 5.73; type III collagen: 0.13 ± 0.02 vs. 0.74 ± 0.07, t = 4.32, all P < 0.01; n = 4).

Conclusions

ASX treatment after AMI may promote M2 macrophages and effectively attenuate cardiac remodeling by inhibiting inflammation and reducing myocardial fibrosis.