Nowadays, TCM is being more frequently chosen by patients to treat UC, and many TCM formulas have been successfully utilized for the treatment of UC[32, 33], including GGQLD. Vivo and Vitro studies have provided multiple lines of evidence indicating that some ingredients of GGQLD, such as berberine and Glabridin, have definite beneficial effects against UC. Given the particularity of multi-component, multi-target, and multichannel of TCM formulae, the underlying molecular mechanisms triggered by GGQLD in the treatment of UC is far from being clarified. New approaches are now being considered for drug-target exploration and identification of potential active ingredients in TCM research, including network pharmacology. Here, we used network pharmacology technology to predict the putative mechanism involved within the therapeutic effect of GGQLD in UC. In this study, we identified 139 bioactive compounds and 259 targets from the 4 herbs in GGQLD. Furthermore, 83 potential therapeutic targets and 118 correspond active ingredients were identified, and a G-U network was established with 205 nodes and 866 interactions. Also, GO and KEGG enrichment was further enriched based on the 83 potential target genes and a PPI network was constructed.
BP enrichment results indicated that a large number of genes were associated with oxidative stress, a key etiological factor of UC, including “reactive oxygen species metabolic process”, “response to reactive oxygen species”, “response to oxidative stress”, “regulation of reactive oxygen species metabolic process”, “cellular response to oxidative stress”, et al. The gastrointestinal tract is prone to reactive oxygen species (ROS) attack. Alterations of the balance between ROS production and the capacity to rapidly detoxify reactive intermediates lead to oxidative stress, which is an essential factor in the pathogenesis of gastrointestinal mucosal disease. During inflammatory episodes, neutrophils and macrophages infiltrate intestinal mucosa at the sites of IBD and release large amounts of ROS and cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Excessive levels of ROS released by the inflamed stroma elicit oxidative damage to DNA, proteins, and lipids, ultimately, promote the initiation and progression of UC. Response to lipopolysaccharide (LPS) was also enriched in BP, which also could play a pathogenic role in UC. LPS is a major component of the outer membrane of Gram-negative bacteria, plays a key role in host-pathogen interactions with the innate immune system and development of inflammatory diseases. LPS impairs intestinal barrier function by causing inhibition of intestinal restitution and stimulates the release of proinflammatory cytokines. Therefore, our result indicates that GGQLD may also have a role in regulating intestinal microbiota.
Additionally, the KEGG result indicates that most of the disease-related pathways are about immune and inflammatory, tumor-related signaling pathways, and virus infection-related signaling pathways, most of them have been reported to be closely related to UC. For example, IL-17, a key mediator in the pathogenesis of intestinal inflammation, up-regulated in inflamed mucosa from UC patients. The disease severity in UC patients is also correlated with the IL-17 level in peripheral blood mononuclear cells. Besides, IL-17 is produced mainly by T helper 17 (Th17) cells and other sources including natural killer cells, mast cells, and neutrophils. Th17 cells are considered to be key effector T cells of UC pathophysiology. New data from mouse models of IBD suggest that T cell plasticity, particularly along the Th1-Th17 and Th17-Treg axes, plays an important role in the regulation of intestinal immune responses. Our functional enrichment analysis results indicated that anti-inflammation, antioxidation, and immunomodulatory may be the mechanism of GGQLD against UC.
Therefore, we further constructed a PPI network to unraveling the complex molecular relationships underline the potential targets, and 10 hub genes were identified by “cytohubba”, including IL6, TNF, STAT3, IL1β, CXCL8, CCL2, ICAM1, IL10, IL4, and IL2. Most of them are cytokines, such as proinflammatory cytokines (IL6, TNF IL1β, CXCL8, CCL2), and anti-inflammatory cytokines (IL10, IL4, IL2). Cytokines are structurally diverse proteins with profound functional relevance to the maintenance of physiological homeostasis, including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are involved in intestinal homeostasis and pathological processes associated with IBD . For example, M1 macrophages promote colonic inflammation via the production of the pro-inflammatory cytokine IL-6, CCL2, and TNF-α. IL6, a key proinflammatory cytokine in the pathogenesis of multiple inflammatory diseases, acts on mesenchymal and epithelial cells to induce the recruitment of polymorphonuclear leukocytes (PMNs) and macrophages essential for wound healing. Recently, In a genome-wide meta-analysis of 20,550 patients with CD, 17,647 patients with UC, and more than 40,000 individuals without IBD (controls) result indicate that therapeutics designed to block IL6R signaling might be effective in the treatment of IBD. Chemokines are small secreted proteins that orchestrate migration and positioning of immune cells within the tissues. During innate and adaptive responses, chemokines are essential for the function of the immune system. CXCL8 is one of the first and most intensively studied chemokines acting as a pro-inflammatory chemokine. Patients with UC have elevated levels of CXCL8 and CCL2 in colonic mucosa compared to healthy volunteers. TNF is another key cytokine in IBD pathology, induces intestinal epithelial cell apoptosis in the context of IBD and murine disease models. Furthermore, treatment strategies targeting TNF signaling are administered systemically and efficacious in UC, such as infliximab, adalimumab, and golimumab.
Cytokines are crucial for the maintenance of immune system. For example, IL-10 is a key immunosuppressive cytokine expressed by many cell types, particularly important in maintaining the intestinal microbe-immune homeostasis. Polymorphisms in the IL-10 locus confer risk for UC, deficient in either IL-10 or IL-10 receptor exhibit severe intestinal inflammation and marked pro-inflammatory cytokines secretion in mice and human. IL-10 production by Th17 cells has been strongly related to the acquisition of regulatory properties by Th17 cells and the resolution of intestinal inflammation. IL-4, the core signature of Th2 responses, induces differentiation of naive helper T cells to Th2 cells. Enormous reports have also provided pieces of evidence that IL-4 participates in the pathogenesis of IBD[55, 56]. Human IL-4-treated regulatory macrophages promote epithelial wound repair, reduce cytokine-induced epithelial barrier defects, and are beneficial in a murine model of acute colitis. IL-2 is known as a T cell growth factor, negatively regulate immune-mediated inflammation and stimulate tissue repair processes. low-dose IL-2 has therapeutic effects on DSS-induced colitis and potential clinical value in treating UC.
Cytokine signaling pathways involving transcription factors of the signal transducers and activators of transcription (STAT) family play a key role in the pathogenesis of IBD. STAT3 activation occurs as a result of cytokine binding (for example, IL6), several studies have reported an increased expression of STAT3 or STAT3 phosphorylation in human IBD[61, 62]. STAT3 has also been shown to be critical in modulating the balance of Th17 and regulatory T (Treg) cells, as well as in promoting CD4(+) T cell proliferation. Intracellular adhesion molecule-1 (ICAM-1), is a transmembrane glycoprotein of the immunoglobulin family, constitutively expressed on vascular endothelial cells and upregulated in inflamed colonic tissue. It has been demonstrated that ICAM-1 expression increased in colonic lysates from UC patients. ICAM-1 can also be up-regulated in response to proinflammatory stimuli, such as TNF-α, IL-1β, and IFN-γ. Particularly, ICAM-1 signaling seems to produce the recruitment of inflammatory immune cells such as macrophages and granulocytes. Meanwhile, the GO and KEGG analysis of the 10 hub nodes showed that a considerable number of genes were involved in pathways including “Inflammatory bowel disease”, and “Cytokine-cytokine receptor interaction”.
Sankey diagram indicated that quercetin corresponds to most of the hub targets. Quercetin is a plant-derived polyphenolic compound belonging to the flavonols (a subclass of flavonoids) and has shown beneficial effects in the prevention and/or treatment of several pathological conditions, due to its antioxidant, anti-inflammatory, anti-fibrotic, antimicrobial, and antitumoral activities[68–70]. In gastrointestinal tract, quercitrin can reduct the early stage inflammation by reducing IL-1β, IL-6, TNF-α levels, and NF-κB expression. Various evidence has shown the anti-inflammatory activity of Quercitrin in experimental colitis[71, 72]. Moreover, modulation of intestinal microbiota is another way for quercetin to exert a therapeutic effect. Recent studies showed that quercetin reshapes intestinal microbiota in several diseases, such as obesity, Non-alcoholic fatty liver disease, and atherosclerosis, suggesting a prebiotic effect of the flavonol[73, 74]. Furthermore, molecular docking was conducted between quercetin and the 10 hub genes to verify the binding activities between active ingredients and potential targets. The results showed that quercetin had good binding activities to IL6, TNF, STAT3, IL1β, CXCL8, CCL2, ICAM1, IL10, IL4, and IL2.
Cytokines that either promote or suppress intestinal inflammation has led to some efficacious therapeutics for IBD. In general, these therapies can be grouped into two categories: blockade of pro-inflammatory or enhancement of anti-inflammatory cytokine pathways. The discovery and characterization of these therapies indicate that simultaneous regulation of anti-inflammatory factors and pro-inflammatory factors may become a more potential treatment strategy for IBD. Our research suggests that GGQLD may have such a therapeutic effect.