Discussion
DSS-induced colitis results in
dysfunction of colon, including severe Intestinal barrier dysfunction,
inflammatory infiltration, rectal bleeding, diarrhea, and gut microbiota
changes (Dai et al., 2015; Rohwer et al., 2019; Wirtz et al., 2017),
which is similar to clinical and histological manifestations of human
ulcerative colitis (Li et al., 2020). Several studies have demonstrated
the antioxidant agents can be against colitis, through effects on
reducing inflammation and rebalancing gut microbiota (Liu et al., 2019;
Peng et al., 2019). In this study, we adopted in vivo acute and
chronic colitis mouse models induced by DSS (the concentration of 3%
and 5%).
Diosmetin has already been
demonstrated that exert protective effects against colon cancer and LPS
induced acute lung injury (Koosha, Mohamed, Sinniah & Alshawsh, 2019;
Liu, Ci, Wen & Peng, 2018). In this study, we illustrated that
diosmetin can protect against LPS-induced injury to Caco2 and IEC-6
cells in vitro and against DSS-induced colitis in mice in
vivo shown by alleviating histopathological changes and restoring the
length of the colon. These data suggested that diosmetin can inhibit
DSS-induced
colitis.
The disruption of the intestinal epithelial barrier has been found to be
relevant to the colitis (Su et al., 2009) which can lead to pathogenic
antigen invasion. Thus, new ways of repairing mucosal barrier, improving
mucosal healing and reducing mucosal permeability are considered to be
potential methods of treating
colitis. Among many proteins in
mucosal barrier, ZO-1 is a major tightly linked scaffold protein, which
associates with the epithelial integrity (Furuse et al., 1994). Occludin
is essential for barrier function and tight junction stability (Saitou
et al., 2000). Claudin-1 is a transmembrane protein and a component of
tight junction strands (Furuse, Fujita, Hiiragi, Fujimoto & Tsukita,
1998). Transmembrane proteins
Claudin, Occludin and perimembrane protein ZO form a closed complex. The
complex formation and breakage of ZO-1, Claudin-1 and Occludin in
intestinal mucosal epithelial cells can affect intestinal epithelial
barrier function greatly (Morini, Babini, Barbieri, Baiocco &
Ottolenghi, 2017). In our study, the expression levels of Claudin-1 and
Occludin can be significantly increased by
diosmetin in vitro andin vivo compared to DSS group. The expression of ZO-1 in
immunofluorescence also significantly increased by diosmetin in vitro
and in vivo compared to DSS group. These datas showed that diosmetin
could inhibit DSS-induced intestinal epithelial barrier dysfunction.
It has been pointed out that the processes of oxidative stress and
inflammation be relevant to
DSS-induced colitis (Macias-Ceja et al., 2017; Zhao et al., 2019). The
MDA is natural products of lipid hydroperoxide of organisms. Oxidative
stress causes some fatty acids in cells to be oxidized and broken down
into a series of compounds, including MDA, which has been frequently
used as an indicator of oxidative stress (Qian et al., 2010). SOD has
been pointed out that it can catalyze the disproportionation of
superoxide anions to generate hydrogen peroxide
(H2O2) and oxygen
(O2), which is an important antioxidant enzyme in the
body (Cao et al., 2015). GSH and GSH-Px can catalyze hydrogen peroxide
and other peroxides to produce water and organic alcohol (Maejima,
Kuroda, Matsushima, Ago & Sadoshima, 2011). In our study, we found DSS
promoted oxidative stress shown by the low levels of GSH, GSH-Px and SOD
and high level of MDA in colon tissue of mice, which were reversed by
diosmetin. In addition, diosmetin
significantly reduced ROS levels and mitochondrial ROS in Caco2 and
IEC-6 cells caused by DSS. Therefore, inhibition of oxidative stress may
be one mechanism by which diosmetin protects against DSS -induced
colitis.
The inflammatory cytokines
of
IL-1β, IL-6, COX-2 and IFN-γ play leading roles in the formation of
colitis (Biasi, Astegiano, Maina, Leonarduzzi & Poli, 2011). In
addition, these pro-inflammatory cytokines can stimulate the activation
of NF-κB transcription (Cao et al., 2015). Meanwhile, activation of
NF-κB pathway further releases
proinflammatory cytokines including TNF-α, IL-1β and IL-6 in DSS-induced
colitis
(Kim, Shin, Chung, Lee, Baek & Lee, 2019). IL-6 can stimulate the
secretion of electrolytes from intestinal epithelial cells, result in
increased permeability of endothelial cells, prompt the aggregation of
neutrophils to the inflammation site and cause IBD (Zhang et al., 2011).
Yamamoto (Yamamoto-Furusho, Santiago-Hernández, Pérez-Hernández,
Ramírez-Fuentes, Fragoso & Vargas-Alarcón, 2011) have demonstrated that
the expression of IL-1β in the lesion site of ulcerative colitis was
significantly higher than that in the normal site, and also found that
the gene polymorphisms of IL-1β and
IL-1RN are relevant to genetic susceptibility and hormone dependence of
ulcerative colitis. COX-2 plays a
dominant role in the regulation of the inflammatory state by stimulating
the biosynthesis of prostaglandins (PG) (Cianciulli, Calvello, Cavallo,
Dragone, Carofiglio & Panaro, 2012). IFN-γ is a mediator of the
development of DSS-induced colitis
(An, Li, Bhang, Song & Youn, 2020). Thus, suppression of these
inflammatory cytokine is an important target for the treatment of IBD.
Our studies found,
diosmetin
significantly downregulated the mRNA levels of IL-1β, IL-6, COX-2 and
IFN-γ, which suppress the inflammation signalling pathways, it may be
another pathway of diosmetin against DSS-induced colitis.
Many studies have shown that gut microbiota is a key factor in the
pathogenesis of IBD (Ni, Wu, Albenberg & Tomov, 2017; Zuo, Kamm,
Colombel & Ng, 2018). Robert W. Li’s study of porcine colitis model
demonstrated that mechanistic insights into the attenuation of
intestinal inflammation and modulation of the gut microbiome by krill
oil (Liu et al., 2020b). In the present work,
we investigated the differences in
gut microbiota of mice among control groups, DSS group and DSS with
diosmetin group. Alpha diversity index dilution curve of each sample
tend to be flat, indicating that the sequencing result was credible.
Alpha diversity index has indicated that microbial diversity in DSS
group significantly decreased compared with control group, after
diosmetin treatment, the diversity was increased and the community
composition was changed. From the
level at phylum, DSS group increased the ratio of Firmicutes to
Bacteroidetes due to the relative abundance decreased in Bacteroidetes
and the relative abundance
increased in Firmicutes compared with the control group. The similar
phenomena has also been found in other studies (Jeffery et al., 2012;
Peng et al., 2019). However, DSS with
diosmetin group increased the
relative abundance of Bacteroidetes and decreased the abundance of
Firmicutes compared with the DSS group.
The LEfSe analysis at OTU level exhibited different dominant bacteria in
different groups. Herein, Eggerthella lenta is kind of bacterial
genera typical for inflammation (Soltys et al., 2020), and it was
repressed in the DSS with
diosmetin group. Previous studies have found that Pelomonas(25%) and Flavobacterium (13%) dominated the bacterial
composition of patients with a high Crohn’s disease activity index,
while Bacteroidetes had a relatively low abundance (4%). And in
this study, diosmetin treatment observably decreased the bacteria
abundance of Flavobacterium . Our research also found that
diosmetin significantly increase
bacteria abundance of Odoribacteraceae, prevotella, Rikenellaceae,
Ruminococcus, Coprococcus, Roseburia, Oscillospira, Anaeroplasma
and Synergistales compared with DSS group. These bacteria may become
potential biomarkers of colitis in the future. Thus, these data
demonstrated that diosmetin can
modulate the community of gut
microbiota, especially certain specific microbiome. These specific
bacteria can be benefit for treating colitis.
Recently, circular RNA has emerged as a regulator of many biological
processes through the regulation of the circular RNA-miRNA-mRNA network
(Chen & Yang, 2015). Circ-Sirt1 (hsa_circ_0093887), one member of the
Sirt1 circRNA, directly binded to miR-132/212 and interferes with Sirt1
mRNA (Kong et al., 2019). In this study, the levels of circ-Sirt1 were
observably increased by diosmetin in vitro and in vivo, accompanied by
up-regulated expression of Sirt1. Subsequently, the acetylation of NF-κB
was inhibited and the Nrf2 pathway was activated. Of course, the
mechanism of circ-Sirt1 in colitis still needs to be further
demonstrated, and our research provides potential research directions
for drug development
In summary, our studies have
demonstrated that diosmetin
exerted the therapeutic effects in
DSS-induced colitis through several pathways, including reducing the
expression levels of pro-inflammatory cytokines (IL-6, IL-1β, IFN-γ and
COX2) and oxidative stress, increase expression of tight junction
proteins of claudin-1, Occludin and ZO-1 and modulate gut microbiota.
These pathways are partially mediated by diosmetin through the
circ-Sirt1/Sirt1 axis. Diosmetin can potentially use to treat Colitis or
become a lead compound for further optimization.