• Discussion
UC is an inflammatory disease characterized by inflammation of the colon
mucosa, which affects the rectum and extends proximally[19]. The
incidence of UC has been increasing every year due to the
industrialization of various countries and the westernization of
people’s eating habits [20]. Although there have been therapeutic
advancements in UC, there is still a treatment gap from people’s ideal
state[21]. Consequently, an increasing number of studies have
explored the potential use of active monomers in functional food or
traditional Chinese medicine to treat colitis[22]. Forsythia
suspen sa, a deciduous shrub with bitter and cold characteristics that
align with anti-inflammatory Chinese medicine, has been used
historically for fever, inflammation, gonorrhea, and erysipelas. Through
exploring the immunosuppressive activity of Forsythia suspensa’scomponents in abnormal splenic lymphocyte proliferation induced by ConA
and LPS, we identified a beneficial component known as PHI with an IC50
of 4.359 μg/mL and 1.539 μg/mL, respectively. Additionally, PHI
inhibited cytokine secretion in LPS-stimulated THP-1 cells, which
motivated us to explore its therapeutic effect on clinical inflammatory
diseases using mouse models.
Next, significant therapeutic effects of PHI were found in mouse models
of UC, as reflected by its ability to maintain body weight, reduce
disease activity index and mortality, restore the intestinal mucosal
barrier, and inhibit cytokine secretion (Fig. 2 and Fig. 3). To
investigate the protective effects of PHI on the gastrointestinal tract,
H&E staining, PAS-Alcian blue staining, and FITC-dextran were used to
prove the therapeutic effects of PHI on maintain intestinal morphology,
reduce goblet cell loss, inflammatory cell infiltration, and decrease
intestinal permeability. In inflammatory bowel diseases, barrier
function is maintained by the mucus layer and epithelial cells connected
by tight junction proteins. In DSS-induced colitis, the expression of
tight junction-associated protein E-cadherin and Occludin was decreased,
and PHI was able to restore their expression as shown in Fig.2E, which
reduced intestinal damage and restored mucosal barrier function.
Furthermore, Macrophages play a critical role in the progression of
inflammation and the disease’s remission during intestinal repair. Flow
cytometry analysis of MLN and PBMC showed that PHI could reduce
macrophage infiltration in both colitis models. Immunofluorescence
performed on colon tissues also demonstrated that PHI significantly
inhibited macrophage infiltration in DSS-induced colitis (Fig.4E).
Cytokines are also important mediators in UC’s enhancement and
continuation, and their presence directly causes mucosal and tissue
damage, inducing disease-specific immune responses in UC. The levels of
pro-inflammatory cytokines including IL-1β, IL-6, and TNF-α in serum and
colon were determined, and we found that PHI could effectively inhibit
their secretion, especially the secretion of IL-1β.
The activation of IL-1β in response to infection, mucosal injury, and
stress triggers a local mucosal immune response, recruiting neutrophils
to the affected site and activating the NF-κB pathway, which leads to
the upregulation of pro-inflammatory cytokines and chemokines [5].
Furthermore, IL-1β is the most likely effector molecule downstream of
the NLRP3 inflammasome. Given the significant impact of PHI on
macrophages and IL-1β, the current study focused on the mechanism of
action of the NLRP3 inflammasome. We found that PHI inhibited
inflammasome protein levels at both the genetic and protein levels.
Starting with its upstream pathway, macrophages exposed to stimuli, such
as ligands for TLR4, activate the transcription factor NF-κB, which then
upregulates NLRP3 expression [23]. Specifically, TLR4 and MyD88
expression increased, and the phosphorylation of NF-κB increased
markedly, but PHI inhibited this pathway and restrained the activation
of the NLRP3 inflammasome. Finally, we conducted in vitroexplorations with BMDM and found that PHI suppressed inflammation by
inhibiting the activation of the NLRP3 inflammasome through the
TLR4/MyD88/NF-κB pathway.