*2.7. HO-1/Nrf2 Axis Played a Key Role in Suppression of Oxidative Stress in LPS-Induced DCs*

Heme oxygenase 1 (HO-1) and its products can also provide beneficial protection against oxidative injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a cytoprotective factor that regulates gene expression for antioxidant and anti-inflammatory properties [30]. Therefore, we detected the expression levels of HO-1 and Nrf2 in DCs by Western blot. Our results demonstrated that astaxanthin treatment significantly upregulated the expression of HO-1 and Nrf2 in LPS-induced DCs (Figure 8A–C). We further investigated whether HO-1 played a significant role in the antioxidant effects of astaxanthin in LPS-induced DCs. We detected the NO production (Figure 8D), intracellular GSH (Figure 8E), GSSG (Figure 8F), the GSH/GSSG ratio (Figure 8G), and the SOD activity (Figure 8H). Significantly, tin protoporphyrin IX (SnPP, an inhibitor of HO-1) reversed the antioxidant effects of astaxanthin in LPS-induced DCs (Figure 8D–H), whereas the suppressive effect of astaxanthin was further aggravated by cobalt protoporphyrin (CoPP, an inducer of HO-1) (Figure 8D–H). Taken together, this showed that the HO-1/Nrf2 axis played a key role in the suppression of oxidative stress in LPS-induced DCs.

**Figure 8.** Astaxanthin suppressed oxidative stress via HO-1/Nrf2 axis in LPS-induced DCs. (**A**–**C**) After stimulation for 24 h with astaxanthin and LPS (100 ng/mL), HO-1 and Nrf2 levels were assessed by Western blot. (**D**–**H**) DCs were incubated with astaxanthin (10 μM) and LPS (100 ng/mL) in the presence or absence of SnPP (25 μM) or CoPP (50 μM) for 24 h. (**D**) NO production in DC supernatants was measured using the Griess reagent. (**E**–**H**) The levels of GSH (**E**), GSSG (**F**), and SOD (**H**), and the ratio of GSH/GSSG (**G**), in DCs were measured as described in the Materials and Methods section. Results are from one representative experiment of three performed. Data are presented as means ± SD. The comparisons were performed with analysis of variance (ANOVA) (multiple groups). Different lowercase letters indicate significant differences between groups (*p* < 0.05).

#### **3. Discussion**

Previously, we found that astaxanthin strongly inhibited the immune dysfunction of DCs induced by LPS [31]. Here, our work further shows the antioxidative effects of astaxanthin in DCs and mice, which is a potential key aspect of inflammatory control in the sepsis model (Figure 9). These investigational results demonstrated that astaxanthin reduced NO production, ROS production, and lipid peroxidation activities in LPS-induced DCs and LPS-challenged mice. Meanwhile, the GSH level, the GSH/GSSG ratio, and antioxidant enzyme (GPx, CAT, and SOD) activities were upregulated during the above processes. Based on these antioxidant properties, astaxanthin strongly inhibited the cytokine production (IL-1β, IL-17, and TGF-β) in LPS-induced DCs and LPS-challenged mice. Furthermore, we found that the antioxidation mechanism of astaxanthin depended on the HO-1/Nrf2 axis.

NO, an intracellular messenger, regulates cellular functions, such as inflammation and pathogen elimination [32]. However, excess NO can combine with O2 − to form ONOO−, which results in oxidative stress and cellular injury [33]. ROS, generated through a variety of extracellular and intracellular actions, have gained attention as novel signal mediators which are involved in growth, differentiation, progression, and cell death [34]. However, the overproduction of ROS induces significant oxidative stress, resulting in the damage of cell structures, including lipids, membranes, proteins, and DNA [35]. Lipid peroxidation can directly affect the biophysical properties and alter other biophysical characteristics of cell membranes. In addition, cell membrane fluidity is decreased by lipid peroxidation [36]. Meanwhile, ROS can react with polyunsaturated fatty acids of lipid membranes and induce lipid peroxidation [37]. In this study, our results suggested that astaxanthin exerts powerful

suppressive effects on NO production, ROS levels, and lipid peroxidation in vitro and in vivo, which play a key role in reversing overloaded LPS-induced oxidative stress.

**Figure 9.** Schematic of proposed mechanism of antioxidant protection of astaxanthin for inflammatory control in LPSinduced DCs. The HO-1/Nrf2 axis was activated by astaxanthin, which inhibited the oxidative stress of LPS-induced DCs, including NO production, ROS production, the lipid peroxidation activities, the GSH level, the GSH/GSSG ratio, and antioxidant enzyme (GPx, CAT, and SOD) activities. These antioxidant properties are conducive to inflammatory controls in DCs, including decreases in levels of activation marker (CD69), the release of cytokines (IL-1β, IL-17, TGF-β, TNF-α, IL-6, and IL-10), phenotypic markers (MHCII, CD40, CD80, and CD86), and a migration marker (CCR7) by astaxanthin.

Previous studies have found that high concentrations of glutathione within cells provide protection against different ROS [32]. GSH, a ubiquitous tripeptide thiol, is known as a vital intracellular and extracellular protective antioxidant, which plays a series of key roles in the control of signaling processes, detoxifying certain xenobiotics and heavy metals [38]. Furthermore, GSH is considered to be one of the most important scavengers of ROS, and its ratio with GSSG may be used as a marker of oxidative stress [38]. The GSH/GSSG redox couple can readily interact with most of the physiologically relevant redox couples, undergoing reversible oxidation or reduction reactions, thereby maintaining the appropriate redox balance in the cells [39]. Under oxidative stress conditions, the GSH can convert itself to GSSG, and the reduction of H2O2 is catalyzed by the GPx enzyme [40]. Importantly, the addition of astaxanthin to DCs was shown to dramatically attenuate intracellular oxidative stress, indicative of an increase in GSH levels, the GSH/GSSG ratio, and GPx enzyme activity.

Apart from the GPx, other antioxidant enzymes, including CAT and SOD, also play a very important role in the defense of cells against oxygen-derived free radicals. CAT is a ubiquitous enzyme found in all known organisms, and can transform two H2O2 into two H2O and O2 [41]. SOD activity was discovered by McCord and Fridovich in 1969, which can dismutate two superoxide anions (O2 −) into H2O2 and O2 [42]. Our results indicated that astaxanthin significantly upregulates the activities of CAT and SOD, suggesting that the increase in antioxidative enzyme activity might be beneficial to the suppression of oxidative stress.

LPS, derived from Gram-negative bacteria, interacts with Toll-like receptor 4 (TLR4) to cause phagocytic cells to robustly generate a variety of proinflammatory cytokines [43]. Interleukin-1β (IL-1β) is a key proinflammatory cytokine involved in host responses to pathogens and tissue injury [44]. Monocytes, macrophages, and DCs are major IL-1β sources and release this cytokine in response to stimuli such as pathogen-associated or danger-associated molecular patterns (PAMPs or DAMPs) mediated by signaling via several TLR pathways [45]. IL-17 is not only a proinflammatory cytokine, but also a potent mediator of inflammatory responses in various tissues [46]. IL-17 induces multiple genes associated with inflammation, including interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor (GM-CSF) [47–49]. In addition, IL-17 enhances the proinflammatory responses induced by IL-1β [50,51], implying that astaxanthin might downregulate the production of IL-1β and IL-17 to protect LPS-induced sepsis. TGF-β is required for IL-17 to produce T helper cell (Th-17 cell) differentiation [52]. In our data, astaxanthin reduced the production of IL-1β, IL-17, and TGF-β in LPS-induced DCs and in LPS-challenged mice, which is in line with our previous findings that showed a decrease in TNF-α, IL-6, and IL-10 caused by astaxanthin in an LPS-induced DC model [31]. These data suggest that astaxanthin, as an antioxidant, can effectively mitigate overloaded cytokine production in vitro and in vivo. The TLR family of receptors can activate the innate immune system by DAMPs that are released during conditions of oxidative stress [53]. ROS from NADPH oxidase can signal the commencement of inflammatory pathways through TLRs. Therefore, we speculated that astaxanthin utilizes its antioxidant property to control inflammation, which might be a promising strategy for treating sepsis. However, the mechanism needs to be further investigated.

Previously, astaxanthin was shown to suppress an LPS-induced increase in inflammatory factors via mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor-κB (NF-κB) activation in vivo [54]. Here, we demonstrated that astaxanthin inhibited the oxidative stress in LPS-induced DCs and LPS-challenged mice via the activation of the HO-1/Nrf2 pathway. Nrf2 is a transcription factor responsible for the regulation of cellular redox balance and protective antioxidant and phase II detoxification responses [55,56]. Several studies have demonstrated that HO-1 genes are regulated through Nrf2 and play a crucial role in the development of oxidative stress [57]. HO-1, a stress-inducible enzyme, cooperates with NADPH cytochrome P450 to degrade heme in order to produce three bioactive products: iron ions, carbon monoxide (CO), and biliverdin, with the latter being rapidly converted to bilirubin. Biliverdin and bilirubin are potent antioxidants; meanwhile, the other products of HO-1 activity regulate inflammation, apoptosis, and angiogenesis [30]. In addition, CO, an end product of HO-1, can also inhibit NO production and inducible nitric oxide synthase (iNOS) expression via the inactivation of NF-κB [58]. It has been reported that the activation of Nrf2 may prevent an increase in ROS generation through NADPH oxidase [59]. Additionally, the overexpression of HO-1 was also able to inhibit NO production and iNOS expression [58]. Therefore, the activation of the Nrf2/HO-1 axis plays a significant role in protecting host cells against oxidative stress [60].

### **4. Materials and Methods**

#### *4.1. Ethics Statement*

Animal studies were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number: SYXK(SU)2017-0044) and complied with the guidelines for laboratory animal welfare and ethics of the Jiangsu Administrative Committee for Laboratory Animals.
