**1. Introduction**

Sepsis is an organic dysfunction caused by a disordered host response to infection by viruses, fungi, and bacteria [1–4], which remains a major cause of morbidity and mortality worldwide, with increased burden in low- and middle-resource settings [5]. In the United States, the treatment of sepsis accounted for more than USD 20 billion (5.2%) in total hospital expenses in 2011 [6]. An extrapolation from high-income country data suggests that on a yearly basis, there are an estimated 31.5 million sepsis and 19.4 million severe sepsis cases, with a potential 5.3 million deaths globally [7]. Although more than 100 clinical therapeutic trials have been conducted, no treatment options for sepsis are currently approved by the US Food and Drug Administration (FDA) [8].

After infection, the components of the pathogen, such as lipopolysaccharide (LPS), a key component of the bacterial cell wall, are recognized by macrophages, dendritic cells (DCs), and other immune cells, and then the overloaded inflammatory immune response is activated in early septic patients [9]. Historically, direct anti-hyperinflammatory strategies

**Citation:** Yin, Y.; Xu, N.; Qin, T.; Zhou, B.; Shi, Y.; Zhao, X.; Ma, B.; Xu, Z.; Li, C. Astaxanthin Provides Antioxidant Protection in LPS-Induced Dendritic Cells for Inflammatory Control. *Mar. Drugs* **2021**, *19*, 534. https://doi.org/ 10.3390/md19100534

Academic Editors: Donatella Degl'Innocenti and Marzia Vasarri

Received: 31 August 2021 Accepted: 21 September 2021 Published: 23 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

that attempt to block cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been the main therapeutic pathway against sepsis. However, the outcome of life-threatening infection is determined by the endogenous complicated inflammatory response. Therefore, using a conventional anti-inflammatory strategy in sepsis cases has to date failed to improve outcomes [8]. Notably, exposure to LPS can induce the rapid and robust production of reactive oxygen species (ROS), which is also a critical pathological feature in septic patients [10]. Oxidative stress is induced by an imbalanced redox state, involving either the excessive generation of ROS or dysfunction of the antioxidant system [11], resulting in the induction of cellular damage, impairment of the DNA repair system, and mitochondrial dysfunction [12]. A growing number of studies agree that interdependence and interconnection are not to be neglected between oxidative stress and inflammation, which co-exist in the inflamed microenvironment. Abundant ROS are released by inflammatory cells at the inflammatory site, which results in exaggerated oxidative injury. Meanwhile, a large amount of ROS and oxidative stress products strengthen proinflammatory responses [13]. Therefore, versatile antioxidants need to be developed to help control overwhelming oxidative stress and hyperinflammatory responses.

DCs are key regulators of innate and adaptive immunity [14]. The maturation of DCs is directed by signal transduction events downstream of Toll-like receptors (TLRs) and other pattern recognition receptors, following an increase in the production of cytokines, chemokines, and costimulatory molecules [15]. Just as importantly, DCs that possess strong antioxidant systems not only regulate the balance of oxidative stress but also influence the levels of inflammatory responses through the polarization of T cells. Therefore, DCs are an ideal target to manage both oxidative stress and inflammatory responses by some multifunctional antioxidants. Astaxanthin originates from seafood, such as microalgae, trout, yeasts, salmon, and krill [16,17]. Of note, a freshwater unicellular alga, named *Haematococcus pluvialis* (*H. pluvialis*), contains abundant natural astaxanthin [17,18]. Its structure is a xanthophyll carotenoid with hydroxyl and keto moieties on both ends (Figure 1) [19], which effectively scavenges free radicals, thereby protecting fatty acid and biological membranes from oxidative damage [20]. Astaxanthin also can attenuate inflammatory injury caused by diabetes-induced sickness and urate crystal-induced arthritis [21,22].

**Figure 1.** Chemical structure of astaxanthin.

Here, the antioxidant ability of astaxanthin was systematically evaluated on DCs for inflammatory control, which provides evidence that a DC-targeting strategy could be effectively applied in sepsis treatment.

#### **2. Results**

#### *2.1. Astaxanthin Suppressed NO Production in LPS-Induced DCs and LPS-Challenged Mice*

Nitric oxide (NO) plays a significant role in killing pathogens; however, excessive NO production has been identified as a key pathogenic factor in most immune-mediated diseases [23]. As shown in Figure 2A, LPS was shown to strongly stimulate NO production in DCs compared with an untreated group. Of note, astaxanthin was shown to remarkably suppress NO production in LPS-induced DCs. Many studies have documented an increase in NO production in response to severe sepsis or LPS administration [24]. Therefore, we further examined whether astaxanthin could affect NO levels in LPS-challenged mice. Mice were pre-treated with astaxanthin for 2 days and then injected with LPS. After LPS injection for 4 h, serum samples were collected for NO detection. We found that the administration of astaxanthin significantly decreased NO production in serum after LPS challenge (Figure 2B). Collectively, these findings suggested that astaxanthin strongly inhibited NO production in LPS-induced DCs and LPS-challenged mice.

**Figure 2.** Astaxanthin suppressed the NO production in LPS-induced DCs and LPS-challenged mice. (**A**) DCs were incubated with the indicated concentrations of astaxanthin and LPS (100 ng/mL) for 24 h. (**B**) C57BL/6 mice were orally given astaxanthin before LPS injection. NO production in DC supernatants and serum was detected using the Griess reagent. Results are from one representative experiment of three performed. All of the 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).

### *2.2. Astaxanthin Decreased ROS Levels in LPS-Induced DCs*

Oxidative stress refers to elevated intracellular levels of ROS, which result in damage to cellular lipids, proteins, and DNA. Next, intracellular ROS was measured as described previously, with some modifications [25]. As shown in Figure 3, ROS levels were remarkably increased after exposure to LPS for 24 h, whereas astaxanthin strongly attenuated the LPS-induced ROS production in a dose-dependent manner.

**Figure 3.** Astaxanthin suppressed the ROS production in LPS-induced DCs. (**A**) After stimulation for 24 h with astaxanthin and LPS (100 ng/mL), DCs were stained with 2 ,7 dichlorofluorescein diacetate (DCFH-DA) and analyzed by flow cytometry (FCM) for ROS detection. (**B**) Results are from one representative experiment of three performed. All of the 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).

#### *2.3. Astaxanthin Exhibited Anti-Lipid Peroxidation Activities in LPS-Induced DCs and LPS-Challenged Mice*

Maleic dialdehyde (MDA) is commonly known as a marker of oxidative stress and antioxidant status in cells [26]. To investigate whether astaxanthin modulated lipid peroxidation activities in LPS-induced DCs, the intracellular level of MDA was measured. Compared with the control group, the MDA level was significantly elevated in the LPSonly group, while it was remarkably inhibited by the treatment of astaxanthin in a dosedependent manner (Figure 4A). The serum MDA is a marker of lipid peroxidation in sepsis [27]. Our murine serum results showed a significant decrease in MDA levels after the administration of astaxanthin in LPS-challenged mice (Figure 4B).

**Figure 4.** Astaxanthin suppressed lipid peroxidation in LPS-induced DCs and LPS-challenged mice. (**A**) DCs were incubated with the indicated concentrations of astaxanthin and LPS (100 ng/mL) for 24 h. (**B**) C57BL/6 mice were orally given astaxanthin before LPS injection. Blood was sampled at 4 h after LPS injection. The MDA contents in DC lysate supernatants and murine serum were measured as described in the Materials and Methods section. Results are from one representative experiment of three performed. The comparisons were performed with analysis of variance (ANOVA) (multiple groups). Different lowercase letters indicate significant differences between groups (*p* < 0.05).

### *2.4. Astaxanthin Exhibited Modulating Effects on Intracellular GSH, GSSG, and the GSH/GSSG Ratio in LPS-Induced DCs*

We further investigated the effects of astaxanthin on the cellular levels of reduced glutathione (GSH), oxidized glutathione (GSSG), and their ratio (GSH/GSSG) in LPSinduced DCs. As shown in Figure 5, LPS significantly decreased the GSH level, increased the GSSG level, and reduced the GSH/GSSG ratio compared with the control. However, astaxanthin remarkably reversed this process in a dose-dependent manner.

**Figure 5.** Astaxanthin modulated the intracellular GSH, GSSG, and GSH/GSSG ratio in LPS-induced DCs. After stimulation for 24 h with astaxanthin and LPS (100 ng/mL), the levels of GSH (**A**) and GSSG (**B**), and the ratio of GSH/GSSG (**C**), in DCs were measured as described in the Materials and Methods section. Results are from one representative experiment of three performed. The comparisons were performed with analysis of variance (ANOVA) (multiple groups). Different lowercase letters indicate significant differences between groups (*p* < 0.05).
