• 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.