*5.1. Antioxidant*

The protective e ffect of C3G&C3G-Ms against intestinal injury is largely based on their antioxidant ability. On the one hand, C3G, along with its bioactive phenolic metabolites, including PCA, VA, and FA, can up-regulate the antioxidant enzyme system, such as increasing the activities of manganese-dependent superoxide dismutase (MnSOD) [34] and GSH [34,77]. On the other hand, they can also down-regulate the pro-oxidant system, such as decrease the expression of cyclooxygenase-2 (COX-2) [77,78] and inducible nitric oxide synthase (iNOS) [77,78], and thus, decreasing the production of free radicals, including ROS [79] and reactive nitrogen species (RNS) [78]. Our previous study has shown that the *Lonicera caerulea* L. berry rich in C3G may enhance the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and MnSOD during the earlier response in LPS-induced macrophages [80].

Nrf2 is a transcription factor with a basic leucine zipper (bZIP) that regulates the expression of antioxidant enzymes. Under normal conditions, Nrf2 is kept in ubiquitination by Cullin 3 and Kelch like-ECH-associated protein 1 (KEAP1), which facilitates ubiquitination of Nrf2. In this regard, Nrf2 forms a virtuous cycle so that it does not come into the nucleus to bind with the antioxidant response element (ARE) to modulate the transcription of down-stream genes. Once upon oxidative stress, Nrf2 can be released from KEAP1 to enter the nucleus with the disruption of cysteine residues in KEAP1 [81], or the activation of protein kinase C (PKC) [82], extracellular signal-regulated kinase (ERK) or p38 MAPKs [83], GSK-3β [84], and phosphoinositide 3-kinase (PI3K) [85]. In the nucleus, Nrf2 binds with ARE and other bZIP proteins (like small Maf) to induce down-stream genes to transcribe.

The bioactive phenolic metabolites of C3G have also been reported to activate Nrf2. PCA may increase the activities of glutathione reductase (GR) and glutathione peroxidase (GPx) by the c-Jun NH2-terminal kinase (JNK)-mediated Nrf2 pathway in murine macrophages, as silencing of the JNK gene expression can attenuate the PCA-induced nuclear accumulation of Nrf2 [86]. FA potentially induces the expression of Nrf2 and HO-1 via the activation of the PI3K/Akt pathway, as the specific PI3K/Akt inhibitor can suppress FA-induced Nrf2 and HO-1 expression, and block the FA-induced increase in occludin and ZO-1 protein expression in rat intestinal epithelial cells [49]. The potential mechanisms underlying the C3G-Ms induced expression of Nrf2 is summarized in Figure 3.

**Figure 3.** Potential mechanisms underlying the C3G-Ms regulated Nrf2 system. PCA and FA may induce the nuclear translocation of Nrf2 via JNK and PI3K/Akt pathways, respectively. FA, ferulic acid; GPx, glutathione peroxidase; GR, glutathione reductase; JNK, c-Jun NH2-terminal kinase; KEAP1, Kelch like-ECH-associated protein 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; PCA, protocatechuic acid; PI3K, phosphatidylinositol 3-Kinase.
