Pirfenidone controls the feedback loop of the AT1R/p38 MAPK/renin-angiotensin system axis by regulating liver X receptor-α in myocardial infarction-induced cardiac fibrosis

Abstract

Pirfenidone (PFD), an anti-fibrotic small molecule drug, is used to treat fibrotic diseases, but its effects on myocardial infarction (MI)-induced cardiac fibrosis are unknown. The aim of this study was to determine the effects of PFD on MI-induced cardiac fibrosis and the possible underlying mechanisms in rats. After establishment of the model, animals were administered PFD by gavage for 4 weeks. During the development of MI-induced cardiac fibrosis, we found activation of a positive feedback loop between the angiotensin II type 1 receptor (AT1R)/phospho-p38 mitogen-activated protein kinase (p38 MAPK) pathway and renin-angiotensin system (RAS), which was accompanied by down-regulation of liver X receptor-α (LXR-α) expression. PFD attenuated body weight, heart weight, left ventricular weight, left ventricular systolic pressure, and ±dp/dt_max changes induced by MI, which were associated with a reduction in cardiac fibrosis, infarct size, and hydroxyproline concentration. Moreover, PFD inhibited the AT1R/p38 MAPK pathway, corrected the RAS imbalance [decreased angiotensin-converting enzyme (ACE), angiotensin II, and angiotensin II type 1 receptor expression, but increased ACE2 and angiotensin (1-7) activity and Mas expression] and strongly enhanced heart LXR-α expression. These results indicate that the cardioprotective effects of PFD may be due, in large part, to controlling the feedback loop of the AT1R/p38 MAPK/RAS axis by activation of LXR-α.

Figure 1. Balance between ACE-Ang II-AT1R and ACE2-Ang(1-7)-Mas axes in the development of cardiac fibrosis.

Figure 2. LXR-α involved in the feedback loop of AT1R/p38 MAPK-RAS axis and the interventional effect of PFD.

Myocardial injury activated the AT1R/p38 MAPK pathway that disrupted the ACE/ACE2 ratio and further imbalanced ACE-Ang II-AT1R and ACE2-Ang(1-7)-Mas axes, including increases in ACE, Ang II, and AT1R and decreases in ACE2, Ang(1-7) and Mas. Moreover, increasing Ang II and decreasing Ang(1-7) synergistically inhibited LXR-α expression. Consequently, the decrease in LXR-α further activated the AT1R/p38 MAPK pathway. This signalling created a positive feedback loop that amplified AT1R/p38 MAPK signalling, thereby disrupting the RAS balance and inducing cardiac fibrosis. Interestingly, PFD activated LXR-α expression, inhibited the AT1R/p38 MAPK pathway, and balanced the RAS in this rat model of cardiac fibrosis.

Figure 3. Effects of PFD on MI-induced cardiac fibrosis (×200).

(A) Sham group, (B) model group, (C) PFD group, and (D) losartan group. Data are reported as means ± SEM (n = 13 for sham group, 12 for model group, 13 for PFD group, and 13 for losartan group). Differences between groups were examined by ANOVA followed by Dunnett’s test. ^#P < 0.05, ^##P < 0.01 vs. model group.

Figure 4. Effects of PFD on MI-induced infarct size (IS) (×10).

(A) Sham group, (B) model group, (C) PFD group, and (D) losartan group. Data are reported as means ± SEM (n = 13 for sham group, 12 for model group, 13 for PFD group, and 13 for losartan group). Differences between groups were examined by ANOVA followed by Dunnett’s test. **P < 0.01 vs. Sham group. ^#P < 0.05, ^##P < 0.01 vs. model group.

Figure 5. Effects of PFD on MI-induced CVF (n = 13 for sham group, 12 for model group, 13 for PFD group, and 13 for losartan group).

Data are reported as means ± SEM. Differences between groups were examined by ANOVA followed by Dunnett’s test. **P < 0.01 vs. sham group. ^#P < 0.05 vs. model group.

Figure 6. Effects of PFD on collagen I and III expression.

Data are reported as means ± SEM (n = 5). Differences between groups were examined by ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. model group. Cropped blots are shown. Full length gels are included in the Supplementary information.

Figure 7. Effects of PFD on α-SMA expression.

Data are reported as means ± SEM (n = 5). Differences between groups were examined by ANOVA followed by Dunnett’s test. **P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. model group. Cropped blots are shown. Full length gels are included in the Supplementary information.

Figure 8. Effects of PFD on hydroxyproline concentrations (n = 13 for sham group, 12 for model group, 13 for PFD group, and 13 for losartan group).

Data are reported as means ± SEM. Differences between groups were examined by ANOVA followed by Dunnett’s test. *P < 0.05 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. model group.

Figure 9. Effects of PFD on AT1R and phospho-p38 MAPK (p-p38 MAPK) expression.

Data are reported as means ± SEM (n = 5). Differences between groups were examined by ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. model group. Cropped blots are shown. Full length gels are included in the Supplementary information.

Figure 10. Effects of PFD on ACE, ACE2, Ang II, Ang(1-7), and Mas expression. Data are reported as means ± SEM (n = 5).

Differences between groups were examined by ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01 vs. sham group. ^#P < 0.05, ^##P < 0.01 vs. model group. Cropped blots are shown. Full length gels are included in the Supplementary information.

Figure 11. Effects of PFD on LXR-α expression (n = 5).

Data are reported as means ± SEM. Differences between groups were examined by ANOVA followed by Dunnett’s test. **P < 0.01 vs. sham group. ^##P < 0.01 vs. model group. Cropped blots are shown. Full length gels are included in the Supplementary information.