Discussion
Serelaxin has been documented to have broad clinical application prospects Because it has a wide range of pharmacological effects, such as anti-inflammatory, anti fibrosis, anti heart failure and so on. In this paper, our results showed that LPS induced inflammatory cytokine IL-1β, IL-6 and TNF-α were increased significan in CFs, while serelaxin inhibited the expression of these inflammatory cytokines. In addition, the expression of MMP-2, MMP-9 and IL-10 were increased, collagen Ⅰ and Ⅲ were decreased and fibrosis was alleviated. The results showed that the antifibrosis effect of serelaixn was to reduce the release of inflammatory factors by activation of the PPAR-γ which following abolished the activation of NF-κB signalling pathways.
Increasing evidences show that CFs may be proinflammatory and can produce a wide range of proinflammatory cytokines and chemokines in response to stimulation with hypoxia, mechanical stress, or endotoxin[15]. The role of these cytokines or chemokines in cardiovascular system involves inspiring cell proliferation, division and differentiation. There are many reports that infammation plays a crucial role in the progress of cardiac disease [2]. Hence, controlling infammation may be helpful for treatment and prevent cardiac fibrosis [8]. And inflammatory response is mainly regulated by cytokine network[16]. It is common knowledge that a variety of harmful stimuli induce the release of inflammatory factors IL-1β, IL-6 and TNF-α and participate in the occurrence and development of acute inflammation. LPS is a potent stimulant that increases IL-1β, IL-6 and TNF-α expression by activating host immune and inflammatory cells. Expression of IL-1β was increased significant in the heart tissue of acute myocardial infarction. The experimental mouse model showed that IL-1β is very important for the recruitment of leukocytes, especially neutrophils and monocytes after myocardial infarction.[18]. Furthermore, it has been reported that blocking IL-1 signal can attenuate heart failure in mice and men after myocardial infarction[19]. TNF-α is known as the main cytokine because it plays an important role in initiating acute inflammatory response[20]. Emerging evidence demonstrates that TNF-α positively correlate with the progression of immune activation and cardiac remodeling[21]. IL-6 is an inflammatory cytokine with pleiotropic effects in diverse cells and organs, has been implicated in cardiovascular pathologies [22]. Previous research have shown that serelaxin could suppress IL-1β, IL-6 and TNF-α in renal ischaemia/reperfusion (I/R) injury[23]. In this paper, indeed, the results showed that IL-1β, IL-6 and TNF-α expression were significantly increased in LPS treated cells, indicating that CFs may be the source of proinflammatory cytokines in the development of cardiac disease. Besides, our results showed that serelaxin markedly inhibited LPS-induced productions of IL-1β, TNF-α and IL-6 in CFs, confirming our results in vitro are able to exert an anti-inflammatory effect on CFs stimulated by LPS. These results suggest that serelaxin can inhibit myocardial inflammation may help to avoid inflammation induced excessive proliferation of CFs and subsequent cardiac fibrosis and cardiac remodeling.
Studies have shown that the production of IL-1β, IL-6 and TNF-α is regulated by NF-κB[24]. CFs are the key cell mediators of injury and inflammation that cause cardiac fibrosis[25]. It is generally believed that MI leads to cytokine/chemokine gene expression in resident cardic cells by NF-κB-mediated signaling pathway [17]. NF- κB pathway has been considered as a typical pro-inflammatory signaling pathway, because of its role in increasing the expression of inflammatory cytokines including IL-1β, IL-6 and TNF-α [24]. In this study, expression of IL-1β, IL-6 and TNF-α were signifcantly elevated by LPS-induced but down-regulated by serelaxin, suggesting the anti-infammatory capability of serelaxin. In addition, the results also showed that serelaxin treatment can significantly promote the expression of anti-inflammatory cytokines IL-10, IL-10 is an anti-inflammatory cytokine that can suppress the expression of pro-inflammatory cytokines[26]. After treatment with serelaxin, it significantly inhibited the expression of collagen I/III, and increased the expression of MMP-2/9. These results indicate that serelaxin has the ability to reduce the inflammation and anti-fibrosis of CFs induced by LPS.
It is crucial that serelaxin inhibited the expression of NF-κB and its signal target gene TNF-α. It is very likely that the anti-inflammatory activity of serelaxin is mainly through the regulation of key components of NF-κB.
Inflammatory mediators such as LPS triggers the activation of a heterodimer composed of RelA/p65 and p50 subunits, which is usually defined as the classical NF-κB pathway[27]. Due to the presence of NF-κB (IκBs), the NFκB p65/p50 heterodimer is usually isolated in the cytoplasm, and the most distinctive member of IκBs is IκBα. Under the stimulation of LPS, IκBα was phosphorylation and degradation lead to the release of p65/P50 heterodimer, which translocates to the nucleus and interacts with NF-κB common site. As a result, the the target gene transcriptional activity of NF-κB is activated. Our study further explores the potential anti-inflammatory mechanism of serelaxin, we tested the activity of NF-κB. The results showed that the expression of IκBα and p65/p50 were increased in LPS group and decreased with the treatment of serelaxin. Thus, serelaxin may reduce inflammation by blocking the activation of NF-κB signaling pathway.
PPARγ is a key transcription factor appertain to nuclear receptor family that plays an important role in regulating a great quantity of biological processes including inflammation[28]. The linkage between serelaxin and PPARγ has been investigated in a model of hepatic fibrosis[29] and it has been reported that PPARγ played a key impact in anti-inflammatory activities [28]. It has been proved that PPAR-γ agonists could down regulate the inflammatory response induced by LPS [30]. Therefore, we further examined the effects of serelaxin on PPAR-γ expression. In this paper, the result showed that serelaxin significantly increases the expression of PPAR-γ. We also discovered that the inhibitory effect of IL-1β, IL-6 and TNF-α by serelaxin can be reversed by the PPAR-γ specific antagonist GW9662. Our results indicated that anti-inflammatory ability through serelaxin in CFs was rely on the PPAR-γ pathway. In the present study, the results show that the expression of IκBα was managed by PPARγ, and yet use of GW9662 abated the expression of IκBα and enhanced the expression of p-IκBα. In addition, the expression of p-p65, a subunit of NF-κB, was analogue to the level of p-IκBα. Besides, the activation of PPAR-γ has been shown to attenuate LPS induced inflammation is related to the inhibition of NF-κB [31]. Consequently, the results show that serelaxin debilitates LPS-induced infammatory response in CFs through triggering PPAR-γ which afterwards abrogated the activation of NF-κB. Actually, more inhibitory experiments are required in our future research to demonstrate whether serelaxin treatment could suppress the nuclear translocation of NF-p65 by immunofuorescence staining and the activation of NF-κB using a luciferase reporter gene assay. Furthermore, a NF-κB agonist can apply to demonstrate whether it can inverse the anti-inflammatory effects of serelaxin.
In conclusion, the results showed that serelaixn could reduce the inflammatory factor IL-1β, IL-6 and TNF-α in CFs induced by LPS. Serelaxin play anti-infammatory role in CFs by stimulating PPAR-γ means and suppressed NF-κB pathway. The anti-inflammatory mechanism of serelaxin needs more in vivo experiments to confirm. The current research shows that serelaxin has great potential in the treatment of myocardial fibrosis.