*2.3. IL-10-Inducing Action of the CRGs and the Ech on the Human Blood Cells*

The action of the carragenans, Ech, and their complexes on the pro-inflammatory (IL-6 and TNFα) and anti-inflammatory (IL-10) cytokines induction was conducted. In this experiment, Ech was used at one concentration, 1 μg mL−<sup>1</sup> whereas, CRG concentrations individually and in complexes, varied in the following row 5.0, 10.0, and 20 μg mL<sup>−</sup>1. As seen in Figure 3, κ- and λ-types (10 and 20 μg mL<sup>−</sup>1) induced the expression of IL-10 in cells, by approximately 120 and 100 pg mL<sup>−</sup>1, in comparison to the negative control, respectively. Ech significantly inhibited the synthesis of IL-10, reducing the induction of this anti-inflammatory cytokine by 50%, compared to the control. At the same time, the inclusion of Ech into the CRG complex increased the induction of IL-10 synthesis, compared to Ech. The greatest effect was shown by the complex of Ech with ι/κ-CRG (Figure 3). Regarding the pro-inflammatory cytokines, Ech (1 μg mL−1) was a strong inductor, in comparison to the highest concentrations of the CRGs, but its action was decreased, especially in the complexes with the κ- or λ-types, by about 300 pg mL−<sup>1</sup> for IL-6 and 350 pg mL−<sup>1</sup> for TNFα. However, the combined action of the CRNs and the Ech complexes on the IL-6 still remained high, compared to the control. The ι/κ-CRG influenced the

effect of Ech with less degree than the others, as complexes had formed. Thus, the CRGs modified the activity of Ech by decreasing its pro-inflammatory effect.

**Figure 3.** The induction of the necrosis factor-α, IL-6, and IL-10, in the presence of λ-, κ-, ι/κ-CRGs, Ech, and their complexes. The concentration of the LPS was 0.01 μg mL−<sup>1</sup> and that of Ech was 1 μg mL<sup>−</sup>1, which were the final value. The concentrations of the CRGs, alone and in complexes with the Ech (1 μg mL<sup>−</sup>1), changed in the following rows: = control (saline/1 μg mL−<sup>1</sup> of Ech); = 5 μg mL<sup>−</sup>1, final value; = 10 μg mL<sup>−</sup>1, final value; = 20 μg mL<sup>−</sup>1, final value. \* *p* < 0.05.

#### *2.4. Influence of the CRGs, the Ech and Their Complex, on the HT-29 Tumor Cells*

The effect of the Ech, alone and in carrageenans complexes, on the HT-29 cells treated with ethanol was investigated. The exposure of cells to EtOH permits an assessment of the samples' ability to affect cell viability and, as a result, the permeability of the epithelial monolayer. All of the investigated samples were inert, in response to the intestinal epithelial HT-29 cells, under normal conditions. Under stress conditions, only the κ/β-CRG and the Ech, as well their complex, restored the cell viability after exposure to the EtOH. As to the CRG, the most prominent action was detected for the

lowest concentrations, where protective effect preserved. The complex of κ/β-CRG with the Ech, also possessed an ability to restore the HT-29 cells after an exposure to ethanol (Figure 4).

**Figure 4.** Time and dose-dependent cellular response profiling of HT-29 intestinal epithelial cells, in the presence of the κ/β-carrageenan, Ech, and their complex. Representative data are averaged from five wells. All experiments were repeated at least two times. Four stages of the experimental design are indicated with dashed lines: **1**—Growth of HT-29 cells to confluence; **2**—the stage of samples addition; **3**—incubation with ethanol; **4**—after the ethanol exposure. Concentration of the Ech (1 μg mL<sup>−</sup>1, final value) was fixed. After each stage, the culture medium (McCoy's 5A Modified) was refreshed.

#### **3. Discussion**

Ech, a water-insoluble compound, is the active substance (P N002362/01) of the drug Histochrome®, registered in the Russian Federation. Earlier we have shown that Ech is soluble in aqueous solutions of CRGs, up to the concentration of 0.1 mg mL<sup>−</sup>1. Moreover, the CRG environment protects the Ech from autooxidation [11]. In this work, we showed that carrageenans modified the biological activity of the Ech.

One of the manifestations of the biological effect of the drug is its ability to cause some disorders in the development and death of embryos (spermotoxic, embryotoxic, and cytostatic activities). The widespread use of the sea urchin embryos to test the toxicological and pharmacological effects of various drugs is due to the simplicity of the incubation of the synchronously developing embryos, under controlled conditions, and the ease of the intravital observation. The influence of the Ech and its complex with CRG, on sperm, was determined by the degree to which it inhibited the ability of spermatozoa to fertilize the sea urchin eggs and the further development of the early embryos of sea urchins, in comparison to the control. As the results showed, the CRGs significantly decreased the spermotoxicity of the Ech, towards the sea urchin *S. intermedius* sperm. Furthermore, neither the Ech, nor its complex with CRG, affected the division and development of the early embryos of the sea urchin.

Oxidative processes occurring during the neutrophils activation could be traced by the change in the ROS production [25]. In the case of the phagocytosis of the pathogens, the ROS were produced by the nicotinamide adenine dinucleotide phosphate oxidase (NOX) in a small volume of the phagosome [26]. During our study, we used an APF fluorescent probe (2-[6-(4-amino)phenoxy-3H-xanthen-3-on-9]benzoic acid), with a strong specificity towards the species of the reactive oxygen, localized predominantly in the phagosomes [27]. It should be noted that, in general, the activating effect of the CRGs was dependent on the polysaccharide concentration and the sulfation degree. In this study, a positive correlation between the impact on the ROS formation and the sulfation degree of the CRG (except for its highest concentration) was observed. Generally, the complex of the λ-CRG, with the Ech, was the most active out of all three types, their effect (complexes of the CRGs and the Ech with concentrations of 200:5 and 100:5 μg mL<sup>−</sup>1) was about 15%, compared to control (Figure 2). The importance of the CRG sulfation degree, with regards to monocyte behaviour, have also been observed, previously [28].

In the complexes of the Ech with polysaccharides, containing 3,6-anhydrogalactose and the lower sulfate group contents (κ, ι/κ), the resultant action was closer to the level of the Ech alone. This was supported by results from the literature, which reported that the Ech significantly prevented an increase in the ROS levels in rat cardiac myoblast H9c2 cells and cardiomyocytes induced by some cardiotoxic agents [3], as well as in intraocular inflammation caused by endotoxin-induced uveitis [29]. Overall, this experiment indicated the modulation of the inductions of the ROS, in complexes with substances containing pro- (CRGs) and anti- (Ech) activating properties.

Depending on the ROS location in cells, the function of these molecules changes enormously. For example, mitochondrial ROS have a particularly interesting role in the immune response, since these ROS are currently considered essential for pathways initiating the production of pro-inflammatory cytokines [30]. The influence of the investigated samples on the synthesis of the immune mediators enabled the study of another facet of their immune activity, both separately and as complexes. Pro-inflammatory cytokines were exemplified by the IL-6, the most important inducer of the acute-phase proteins, and the TNFα, another pro-inflammatory molecule with cytotoxic effects in antitumor immunity, whereas the IL-10 is an important immunoregulatory cytokine with multiple biologic effects and strong tendencies of anti-inflammatory action [31]. The CRGs stimulated the induction of anti-inflammatory IL-10, whereas, the Ech inhibited the synthesis of this cytokine, and the addition of the CRGs to the Ech increased the induction of the expression of the anti-inflammatory IL-10 (Figure 3). Ech, at a concentration 1 μg mL−1, increased the IL-6 and TNFα synthesis; however, the complexes with CRGs exhibited much less activity in the case of synthesizing the pro-inflammatory cytokine TNFα. The effect of the Ech on the cytokine balance towards the pro-inflammatory response corresponded to the literature data, which reported that spinochromes act as inductors of TNF-α production in LPS-stimulated macrophage cell cultures [5]. Another study underlined a pro-inflammatory action of the naphthoquinones, in mice [32].

Literature data suggest that the CRGs do not affect the epithelial cells of human gastrointestinal tract [33], but the influence of the Ech towards these cells, which is of special interest when one considers an oral administration of a drug, has not been investigated, to our knowledge.

HT-29 is a colorectal cancer cell line used as an in vitro model, for the intestinal epithelium, because it is a mucin secreting cell line which retains many features attributed to the lower small intestine [34]. Previously we have studied the influence of CRGs on these cells, under stress conditions and have found out that only the low-sulfated CRG had a protective action towards the HT-29 intestinal epithelial cells [35]. Our purpose in the study described in this report, was to determine the protective action of the Ech alone and in combination with the low-sulfated CRG on the survival of monolayers of these cells, treated with EtOH (Figure 4). The stress effect of ethanol on the state of the HT-29 cells provided an opportunity to assay the protective properties of polysaccharides from the red algae and the Ech. The Ech (1 μg mL−1) also preserved the HT-29 cells, under stress conditions, to an extent similar to the κ/β-CRG (25 μg mL−1). These results provide an opportunity to propose the CRGs as a possible matrix system, for oral delivery of Ech, which preserves the Ech-favorable qualities and mitigates its negative biological properties.

In general, the CRGs modified the Ech toxicity and the immunological properties. Our results showed that the CRGs significantly decreased the spermotoxicity of the Ech, against the sea urchin *S. intermedius* sperm. The Ech, as well as its complex with CRG, did not affect the division and development of the early embryos of the sea urchin. The influence of the investigated substances on the induction of the ROS, in the neutrophils, confirmed that Ech in a complex with a polysaccharide inhibited the induction of ROS induced by CRG.

The complexes obtained by us illustrated the modulation of the ROS induction, by these substances, with pro- (CRGs) and anti- (Ech) activating properties of the initial components. The CRG decreased the Ech's ability to induce the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. Whereas, the Ech's protective action towards the intestinal cells exposed to EtOH, remained invariable in the complex with the κ/β-CRG.
