This study explored the influence of SA on macrophage polarization and ventricular remodeling in rat with MI model. We provide evidence for the following: (1) SA increased M2 macrophages infiltration after MI; (2) SA attenuated myocardial interstitial fibrosis and neural remodeling after MI; (3) In vitro study we further found that SA induced and promoted macrophage M2 polarization in BMDMs and IL-4 induced BMDMs; and (4) Activation of PPARγ is a potential mechanism by which SA regulates macrophage polarization.
The plasticity of macrophages makes macrophages an important regulatory target for the treatment of myocardial infarction. After myocardial infarction, pro-inflammatory M1 macrophages aggravate inflammatory response and myocardial injury by releasing pro-inflammatory cytokines, exosomes and miRNA . An excessive inflammatory response and prolonged M1 macrophage accelerates myocardial injury and adverse cardiac remodeling [20, 21]. Previous studies have shown that SA can inhibit inflammation [5–7]. Our study found that SA can inhibit macrophage infiltration in peri-infarct area. MCP-1, a ligand of G protein coupled receptor CCR2, recruits monocytes to the infarcted myocardium and increases macrophage infiltration to promote the inflammatory microenvironment . The expression of MCP-1 continuously markedly up-regulated from 1 day to 4 weeks after myocardial infarction, and MCP-1/CCR2 inhibition significantly ameliorates macrophage recruitment and interstitial fibrosis, and improves heart function after ischemia and reperfusion injury and MI [22, 23]. We found that SA not only downregulated the inflammatory factors TNF-α, IL-1α and IL-1β but also modulated macrophage polarization after MI. Our in vivo and in vitro experiments investigated that SA can promote the polarization of M2 macrophages. M2 macrophages infiltration begin to activate at 5 to 7 days after myocardial infarction [24, 25], and we observe M2 macrophages at 1 week after MI and in IL-4 induced BMDMs. IL-4 was applied to modulate macrophage polarization toward an M2 phenotype. IL-4 administration can significantly increase M2 macrophages infiltration and reduce the area of myocardial infarction and improve cardiac function in mice with myocardial infarction, and this effect depends on M2 macrophages rather than the direct effect of IL-4 . M2 macrophages can secrete various anti-inflammatory cytokines, such as IL-10 and IL-1RA. We speculate promoting M2 cell polarization is one of the important anti-inflammatory mechanisms of SA and SA did not affect the polarization of M1 macrophages.
A few weeks and months after MI, the imbalance of homeostasis and increase in extracellular matrix synthesis and deposition play an important role in structural remodeling. Previous studies have shown that SA prevents cardiac fibrosis in a hypertensive animal model . Persistent inflammatory response recruits and activates myofibroblasts that synthesize extracellular matrix proteins, which are involved in myocardial remodeling. Our study demonstrated that not only cardiac fibrosis but also MMP-2 and MMP-9 expression was inhibited after SA treatment. MMP-2 and MMP-9 are key regulators of LV remodeling and were upregulated both in MI and heart failure . In MMP2 and MMP9 knockout mice, LV enlargement and collagen accumulation were significantly attenuated after MI [28, 29]. Clinical studies have also shown that MMP-9 is an independent risk factor for heart failure after acute MI . Our study indicated that MMP2 and MMP9 is a regulatory target of anti-fibrosis effect of SA. Interstitial fibrosis gradually leads to impaired cardiac function and eventually progresses to heart failure. Our study also demonstrated that ventricle dilatation and systolic dysfunction following MI were evidently ameliorated after SA treatment.
Inflammatory response contributes to sympathetic neural remodeling that plays an important role in cardiac arrhythmias and SCD after MI. Macrophages that synthesize and express NGF around sympathetic nerves participate in sympathetic sprouting after MI . Inflammatory factor TNF-α and IL-1 β also directly regulate the expression of NGF . Furthermore, macrophage reduction followed by intravenous injection of clodronate inhibited sympathetic hyperinnervation after MI . NGF secretion decreased significantly after M2 polarization of microglia . Another study showed that atorvastatin induced M2 macrophages and attenuated sympathetic hyperinnervation in rat post myocardial infarction . SA may inhibit NGF expression and regulate sympathetic remodeling and nerve sprouting by alleviating inflammatory response.
PPARγ is closely associated with M2 polarization . Some drugs, such as rosuvastatin and pioglitazone can improve M2 macrophage polarization by PPARγ activation [36, 37]. IL-4 stimulation can induce BMDMs to M2 polarized activation and elevate PPARγ expression . In IL-4 stimulated PPARγ null BMDMs, the expression of M2 macrophage marker Arg-1 were reduced by nearly half . More importantly, there is PPAR response element (PPRE) within upstream of Arg-1, Ym-1, and Fizz-1 promoter to regulate the transcription of target genes [12, 13]. One recent study found that SA possesses a PPARγ activation role and the antioxidant stress effect of sinapic acid was abolished by the PPARγ inhibitor BADGE . In our study, we also found SA that activated PPARγ in BMDMs in a concentration-dependent manner. More importantly, PPARγ antagonist GW9662 attenuated the IL-4 induced M2 macrophage markers expressions after SA treatment, which indicated that PPARγ is the core signaling pathway of SA in regulating macrophage polarization. However, the specific mechanism of SA-mediated PPARγ activation remains to be further studied.
In conclusion, SA alleviated inflammation by promoting M2 macrophage polarization via activating PPARγ pathway, and SA attenuated structural and neural remodeling by inhibiting inflammation. SA could be a therapeutic candidate for anti-inflammation and ventricular remodeling after MI.