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.