TCM has the advantages of reducing side effects, improving therapeutic efficacy, and have low treatment cost. Nowadays, network pharmacology provides a new approach to study TCM formulas and the advantages of multi-compound, multi-target and multi-pathway characteristics of TCM formula.
Based on the ADEM properties, we have screened out 181 active compounds of RSBDP. Then, the H-T network of RSBDP had 159 active compounds, including β-sitosterol, Sitosterol, quercetin, kaempferol, Imperatorin, isoimperatorin, nodakenin, isorhamnetin, Stigmasterol, Phellopterin, naringenin, (+)-Anomalin, and the compounds as mentioned above can act on 2-5 Chinese herbal medicines simultaneously, and were divided into three major categories, including Phytosterols, Flavonoids and Coumarins. Phytosterols, the plant analogs of cholesterol, including β-sitosterol, Sitosterol and Stigmasterol. Recently, many studies have shown that phytosterols have anti-inflammatory functions36-37. β-sitosterol has shown many biological functions, including lowing cholesterol level, anti-inflammatory and anticancer effects. Oral β-sitosterol (20 mg/kg) may significantly inhibit the expression of cytokines TNF-α, IL-1β, and IL-6 in the colon of male C57BL/6 mice with TNBS-induced colitis and simultaneously inhibits the expression of cyclooxygenase (COX)-2 and the phosphorylation of p6538. β-sitosterol can inhibit the activation of NF-κB by inhibiting the binding between lipopolysaccharides (LPS) and toll-like receptor (TLR)4 to express anti-inflammatory effects39. Furthermore, Feng et al. studies have shown that dietary phytosterols, β-sitosterol and stigmasterol inhibited NF-κB activation in mice with DSS-induced colitis40. Flavonoids are widely distributed in the plant kingdom, including quercetin, kaempferol, isorhamnetin and naringenin. Quercetin has multiple biological functions, like anti-inflammatory, anticancer, antioxidant and anti-hypertensive. Quercetin regulates epithelial inflammatory genes associated with LPS-induced models, which down-regulated C/EBP-β, and Lcn-2, and up-regulated Hmox1, and Fpn141. The most important properties of kaempferol are anti-inflammatory, can be used to treat numerous diseases caused by acute and chronic inflammation. Park et al. have been confirmed that kaempferol down-regulated the colonic mRNA expression levels of the inflammatory cytokines (i.e., TNF-α, IL-1β, and IL-6) in the colons of female C57BL/6J mice with DSS-induced colitic and inhibited the activation of myeloperoxidase (MPO)42. Coumarins are plant-derived products, including Imperatorin, isoimperatorin, nodakenin, Phellopterin and (+)-Anomalin. The antioxidant, anticancer and anti-inflammatory effects have been recognized in many in vivo and in vitro studies43-44. Imperatorin (IMP) can effectively reduce the increase of IL-1β and TNF-α levels in LPS-induced mice45. Luo et al. reported that IMP inhibited TNF-α and IL-6 in TNBS-induced colitis mice and alleviated the symptoms of UC by regulating the Nrf-2/ARE/HO-1 pathway46.
According to the active compounds obtained above and matched with related UC targets, we were obtained 92 RSBDP-UC targets, and constructed the C-T network and PPI network. Cytokines such as TNF, IL-1, IL-6, IFN-γ, IL-2, and IL-8 (CXCL8) were produced by Th1 cells, which mediate cellular immune responses to pathogens and participate in promoting the development of CRC and CAC. In contrast, IL-4 and IL-10 were produced by Th2 cells, which are related to anti-inflammatory response47. If the balance between Th1 and Th2 is lost, inflammation of the tissues and viscera will occur, leading to the occurrence and development of diseases. TNF is a well-known pro-inflammatory cytokine, which is mainly secreted by monocytes or macrophages. Signaling pathways and transcription factors (i.e., NF-κB) were triggered by the activation of TNF-α48-49. Like TNF-α, IL-1 plays a crucial signal in the initial alarm, causing a series of up-regulation and anti-inflammatory responses to be activated. Among the IL-1 family, IL-1α and IL-1β are cytokines that have been widely studied. And evidence suggested that IL-1β can indirectly activate endothelial cells and angiogenesis by up-regulating pro-inflammatory effects and pro-angiogenic molecules. Moreover, the findings have shown that the activation blockage of IL-1β reduces tumorigenesis in a murine CAC model by impairing macrophage-dependent IL-6 secretion. These findings suggested that IL-1 has a significant effect on the initiation of colonic inflammation50-51.
IL-6 is essential for long-standing inflammation. It is used to inhibits the effect of regulatory T cells, and involved in activating STAT3, a major oncogenic transcription factor. STAT1 and STAT3 as a downstream targets of IL-6 signaling, which can prolong the survival period of CRC patients. The inhibition of colitis activity is related to the inhibition of STAT3 activation, the induction of T cell apoptosis and the reduction of pro-inflammatory cytokines49-50,52-53. Gordziel et al. have shown that the expression of STAT1 and STAT3 are correlated respectively with IL-6, and the significantly expression of IL-6 in CRC biopsies tissues are both shown to a tendency for association with longer survival55.
Then, the T-P network were constructed and performed enrichment analysis, and the results reflected that 85 targets could be mapped to 179 pathways. Five pathways of the top 20 pathways, including “TNF signaling pathway,” “Toll-like receptors signaling pathway,” “NOD-like receptors signaling pathway,” and “HIF-1 signaling pathway,” were closely related to inflammatory and immune function. TNF signaling pathway acts as an important role in a regulator of inflammation. TNF has two primary receptors, such as TNF receptors 1 and 2(TNFR1 and TNFR2). In UC, high tissue levels of TNF and cytokines (i.e., IFN-γ, IL-17 and IL-12) can cause a series of symptoms such as diarrhea. Bradford et al. studies have shown that TNF promotes the recovery of colitis mucosa by acting on the colonic epithelial stem and progenitor cell populations55. Pattern recognition receptors (PRPs) are essential for regulating the homeostasis of the intestinal immune system and are mediators of inflammation in the gastrointestinal tract, including Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-like receptors (RLR)56. TLRs signaling pathway mainly control immune function and can act as a risk modifier for UC and CAC. It has been found that TLRs expressed in the intestinal epithelium are related to the long-term existence of mucosal inflammation, result in a high degree of morbidity. Activation of the TLR signal transduction pathway induces pro-inflammatory cytokines, and transcription factors like NF-κB to target invaders57. Once activated, TLRs trigger the subsequent activation of downstream signaling cascades and induce the activation of NF-κB. TLR mutations and dysregulation are the main factors in the predisposition and susceptibility to IBD58-59. NLRs signaling pathways are involved in the regulation of inflammatory and apoptotic responses. A pivotal role of NOD2 in the inhibition of intestinal inflammation and tumorigenesis. Kurzawski et al. reported the NOD2 might be a risk factor for CRC60. Udden et al. studies in vivo have shown that NOD2-mediated protection against colorectal tumorigenesis, and the inhibition of TLR-mediated activation of NF-κB pathways and MAPK pathway, and promotion of the inflammatory cytokines61.
However, recent studies have provided evidence that a comparison of chronic inflammation and normal tissues found that hypoxic mainly exists in the former. The adaptive responses to hypoxic are mediated via the Hypoxia Inducible transcription Factors (HIFs)62. HIF-1 signaling pathway has been shown to have a protective effect in colitis in vivo. The HIF pathway is regulated by a family of oxygen-sensitive HIF prolyl hydroxylases. The expression and transcriptional activity of HIF-1 was inhibited by hydroxylases62-63. Cummins et al. studies in vivo and in vitro have demonstrated that the pan-hydroxylase inhibitor, dimethyloxalylglycine (DMOG), can induce the activation of HIF-1 in cultured intestinal epithelial cells, and accelerate recovery in DSS-induced female C57BL/6 mice, and promote the pro-inflammation cytokines in DMOG-treated mice64.
These results reveal that RSBDP not only can regulate pro- and anti-inflammatory, immune and anti-hypoxic effects but also can prevent the occurrence and development of tumors. Moreover, the occurrence and development of UC were related to the imbalance of cytokines and downstream mediators. These networks in this study illustrate the features of RSBDP multi-compounds, multi-targets and multi-pathways therapy of UC with the complicated and varied action mechanisms.