*5.2. Anti-Inflammatory*

Endotoxin produced by dysbacteriosis is considered as the major trigger of inflammation in intestines [87,88]. When gram-negative bacteria such as *Escherichia coli* and *Salmonella* predominate in gut, bacterial lipopolysaccharide (LPS) can form a complex called LPS binding proteins (LBP) to be associated with pattern recognition receptors (CD14) which locate on the cell membrane, and then activate toll-like receptors (TLRs), such as the TLR4 pathway, to induce inflammatory reactions in di fferent types of cells, such as epithelial cells and immune cells [24]. TLR4 dimerizes itself and induces two major pathways, the myeloid di fferentiation factor 88 (MyD88)-dependent pathway and MyD88-independent pathway. In the dependent pathway, MyD88-induced phosphorylation of interleukin receptor-associated kinases 1 (IRAK1) and IRAK4 can activate the tumor necrosis-associated factor 6 (TRAF-6) adapter protein, which forms a complex with the enzymes that activate transforming growth factor beta-activated kinase-1 (TAK1) during ubiquitination. Then TAK1 induces the phosphorylation of the inhibitor kinase complex (IKKβ), which further induces the decoupling of NFκB in the dimer of NFκBp50 and NFκBp65 by degrading its inhibitory protein IκB. Finally, NFκB enters the nucleus and modulates the expression of a series of inflammatory cytokine genes [24].

Overexpression of inflammatory cytokines largely influences the expression of epithelial tight junctions (TJs) such as zonula occludens-1 (ZO-1), occludin, and claudin [89,90], which increase cellular permeability and give more access for LPS to enter cells [91,92]. The pro-inflammatory cytokines, like TNFα, IFN-γ, and IL-1β, can induce an increase in intestinal TJ permeability potentially through the activation of myosin light chain kinase (MLCK), which appeared to be an important pathogenic mechanism contributing to the development of intestinal inflammation [93]. Another factor that aggravates intestinal inflammation is that macrophages can be recruited to adhere and infiltrate into inflammatory sites through chemokines and intercellular adhesion molecule-1 (ICAM-1), which is largely increased by the activation of the NF-κB signaling pathway [94] among several cell types including leukocytes, endothelial cells, and macrophages [95].

In addition to the influence on gu<sup>t</sup> microbiota, the inhibitory e ffect of anthocyanins on epithelial inflammation is another important factor that acts against intestinal injury [64,76]. Ferrari et al. have demonstrated that the main protective e ffect of C3G in chronic gu<sup>t</sup> inflammatory diseases is derived from the selective inhibition of the NF-κB pathway in epithelial cells [76]. Our previous studies have also shown that the *Lonicera caerulea* L. berry rich in C3G can inhibit LPS-induced inflammation potentially through TAK1-mediated mitogen-activated protein kinase (MAPK) and NF-κB pathways in an LPS-induced mouse paw edema and macrophage cell model [80]. Although the metabolites of C3G are complicated, recent studies have revealed that phenolic metabolites identified in blood circulation, such as PCA, PGA, VA, and FA, may modulate inflammatory signaling pathways. PCA, VA, and FA can suppress the production of ICAM-1, and thus, alleviate inflammatory infiltration and damage in vascular endothelial cells [52,96]. In a mouse colitis model, PCA can decrease both mRNA levels and protein concentration of Sphingosine kinases (SphK), which induces the phosphorylation of sphingosine to form sphingosine-1-phosphate (S1P), but increase the expression of S1P lyase (S1PL) which irreversibly degrade S1P, and thus, inhibit SphK/S1P pathway-mediated activation of the NF-κB pathway through S1P receptors (S1PR) [33]. The main mechanism of VA on inflammation is that it can down-regulate the MAPK pathway by suppressing the phosphorylation of ERK, JNK, and p38 [47]. It is reported that FA may prevent macrophages from responding to LPS, potentially through target myeloid di fferentiation factor 88 (MyD88) mediated pro-inflammatory signaling pathways [50,97], while other studies suggested that FA may increase the expression of TJs, such as occludin and ZO-1 via regulating HO-1 expression to prevent LPS enter the cells [52]. In our previous studies, both C3G and its phenolic metabolites showed inhibitory e ffects on LPS-activated inflammatory pathways in macrophages, and C3G can directly bind to TAK1 and ERK1/2, while PGA, one of phase I metabolites, can also directly bind to ERK1/2 [41,80]. These studies sugges<sup>t</sup> that C3G and its phenolic metabolites may attenuate both a primary and secondary inflammatory response and by inactivating pro-inflammatory pathways and enhancing cellular barrier function (Figure 4).

**Figure 4.** Potential mechanisms of C3G&C3G-Ms in attenuating intestinal inflammation. C3G and its phenolic metabolites mainly modulate inflammation by three ways, first, to suppress the production of chemotactic factors such as ICAM-1 and thus alleviate inflammatory infiltration, second, to down-regulate inflammatory pathways such as TAK1-mediated MAPK pathway and SphK/S1P mediated NF-κB pathway, finally, the down-regulated inflammatory pathways, and up-regulated antioxidant pathway, as mentioned in Figure 3, will maintain sufficient expression of tight junction proteins such as ZO-1 to promote normal intestinal barrier function, and thus prevent LPS from entering mucosal cells. C3G, cyanidin 3-glucoside; FA, ferulic acid; ICAM-1, intercellular adhesion molecule-1; PCA, protocatechuic acid; PGA, phloroglucinaldehyde; SphK, Sphingosine kinases; S1P, sphingosine-1-phosphate; TAK1, transforming growth factor beta-activated kinase-1; VA, vanillic acid.
