**3. Discussion**

The emergence of multi-drug resistant Gram-negative bacteria "super bugs" are becoming a serious therapeutic problem. Animal and human studies indicate LPS as an antigen that activates the immune system, playing an important role in the pathogenesis of metabolic chronic diseases [11]. The application of bactericidal antibiotics, apart from killing the bacteria, may lead to the massive release of endotoxin. The search for non-toxic effective endotoxin binding and neutralizing compounds is an ongoing process.

One of the promising ways to inhibit the harmful inflammatory responses of endotoxins is to bind LPS with certain polycations, which can interact with lipid A as a result, blocking this toxic center of the endotoxin. In recent years, it has been demonstrated that antimicrobial peptides from the Pep 19-2.5 family, which were designed to bind to LPS, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. The peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the LPS [40]. It is known that LPS as amphiphilic molecules tend to form supramolecular aggregates in aqueous solutions at concentrations above the critical micellar concentration. LPS molecules may aggregate into different physical structures, including micelles, inverted micelles, or bilayers and undergo lamellar to inverted hexagonal or cubic phase transitions depending on the physiochemical environment [41]. The complex hydrodynamic geometry exhibited by LPS in dilute suspensions may have consequences for the interpretation of LPS biological activity in the host immune response [42].

As shown by our previously obtained data using dynamic light scattering, CRGs changed the surface charge and the particle size of LPS. This process depended on the structural type of carrageenan, the initial concentration of the components, and their proportion in the solution [43]. In the current work, AFM was used to study the probable modification of LPS morphology due to the effect of CRGs. Compared with other microscopic methods, AFM is characterized by minimal artifacts related to the fixation and staining of samples. Furthermore, the height and width of objects on AFM images may provide additional information about the degree of association and polymer heterogeneity. The macromolecular structures of LPS with κ-, <sup>κ</sup>/β-, and λ-CRGs were investigated. Comparative

AFM examination of LPS and its mixture with CRGs showed that LPS morphology is significantly changed under the action of the polysaccharide. The macromolecular structure of LPS, determined using AFM, shows a change in LPS aggregates of the micellar structure into worm-shaped formations in the presence of CRGs. In the case of κ- and κ/β-CRGs, the number of separate fibers of CRG is significantly decreased. The AFM images show that the LPS vermicular structure is integrated into a three-dimensional network of κ- and κ/β-CRGs (incorporated into the network or just lying over it). In a mixture with λ-CRG, a change in the LPS macromolecular structure is also observed (Figure 2c). However, in this case, the absence of the three-dimensional structure of λ-CRG does not limit the assembly of these vermiform LPS particles into larger associates. It is worth nothing that the worm-like structures form the densest agglomerates in λ-CRG. We suppose that the worm-like structures consist of LPS, which has changed its morphology under the influence of the CRG network consistent with the electron microscopy data we obtained earlier [43].

It is known that, when entering the bloodstream during local or systemic Gram-negative infections, endotoxin has an impact on almost all the systems of an organism, causing a number of pathophysiological changes [44]. Recently, in a LPS-induced endotoxemia mouse model, it has been shown that hirsutanol A (isolated from the red-algae-derived marine fungus *Chondrostereum* sp.) pretreatment improved endotoxemia-induced acute sickness behavior, including acute motor deficits and anxiety-like behavior [45].

A nonspecific resistance of the organism to *E. coli* LPS induced by CRG was studied in the present work. We investigated the e ffect of di fferent structural types of CRGs on the LPS-induced intoxication of animals by the degree of variability of biochemical and pathological parameters to those most adequately responding to any valid stressor, including bacterial endotoxin. In our experiments, LPS parenterally injected into mice (in a nonlethal dose) caused significant changes of these parameters. Preventive oral administration of CRGs significantly reduced the morphological, endocrine, and metabolic disorders caused by endotoxin in the liver, thymus, spleen, adrenal glands, and blood of animals (Table 2). The results of these studies have shown that biochemical and pathomorphological manifestations of endotoxemia induced by intraperitoneal injection of bacterial LPS were less pronounced in the mice that received CRGs. --, κ-, and κ/β-CRGs increase the resistance of the host organism to bacterial LPS. As seen in these experiments, these CRGs are more active than λ-CRG, which has a high degree of sulfation and does not form a three-dimensional structure. As shown by the AFM data (Figure 2), LPS is embedded in the three-dimensional structure of κ- and κ/β-CRGs, which probably contributes to a greater screening of its toxicity. Moreover, as we recently showed, κ- and κ/β-CRGs have the most powerful mucoadhesive properties, which may prevent LPS from landing on the epithelial layer [46].

Our data are consistent with results that demonstrate how fucoidan prevented endotoxin-induced damage in a mouse model of endotoxemia and increased the mice's resistance to LPS. The parenteral or per os administration of fucoidan resulted in decreasing the degree of microcirculatory disorders and secondary dystrophic-destructive changes in parenchymal organs of mice with endotoxemia [47].

Although the mechanisms responsible for the CRG e ffects require further study, the data obtained provide strong evidence for a normalizing e ffect of CRGs on the state of organs of mice with endotoxemia. The CRG-induced host resistance to endotoxin may result from a variety of reasons, one of which may be associated with a change in the macromolecular structure of LPS. The resistance to endotoxin induced by CRGs can be attributed to the immunomodulating e ffect of polysaccharide. As is known, CRGs and endotoxins also stimulate the biosynthesis of di fferent mediators of the immune system, such as IL-10, IL-8, IL-6, and TNFα [6,44]. Since cytokines play a critical role in regulating inflammatory and immunological processes of the host, in vivo administration of CRGs may influence antibacterial host-defense systems. It is known that IL-10 has grea<sup>t</sup> potential for use in the treatment of inflammatory and immune illnesses. It has been shown that IL-10 protects mice against lethal doses of endotoxin [48]. In addition, IL-10 treatment inhibits the activation of cytokine synthesis during experimental endotoxemia in primates [49] and humans [50] and in microglia cell cultures [51].

Previously we have shown [22] that CRGs of different structural types induced the synthesis of the anti-inflammatory cytokine IL-10 in human blood cells, which rose with an increase in polysaccharide concentration. κ/β-CRG showed fairly high activity independent of the concentration. κ/β-CRG under oral administration into mice possesses a protective effect and reduces an intensity of inflammatory response induced by LPS probably due to some modulating effect on the cellular activity of peritoneal leukocytes and to a greater degree on cytokine production [52].

In the present work, κ/β-CRG caused an increase in the level of the cytokine IL-10 in blood compared with the control (Figure 3). The effect of --CRG on the induction of IL-10 synthesis exceeded the LPS effect at the same concentration by almost more than 1.5 times. Along with this, --CRGs and κ/β-CRG at concentrations of 1–100 ng/mL showed weak activity to stimulate the synthesis of pro-inflammatory cytokine TNF-<sup>α</sup>. These polysaccharides also enhanced the effect of LPS, increasing the synthesis of the anti-inflammatory cytokine IL-10 in a test with the pre-treatment of blood cells with *E. coli* LPS. λ-CRG showed a different effect on the stimulation of cytokine synthesis in cells. This polysaccharide did not have the ability to stimulate the synthesis of anti-inflammatory cytokine IL-10, but it possessed an adequate ability to induce pro-inflammatory cytokine synthesis increasing the level of TNF-α in serum at 1 μg/mL and 100 ng/mL, which was comparable to the effect of LPS at the same concentrations.

Known disorders of coagulation or disseminated intravascular coagulation (DIC) induced by endotoxin lead to multiple-organ dysfunction [53]. An important role in the development of DIC has been shown to belong to platelets and LPS-induced platelet aggregation [54]. Earlier in the in vitro experiment, we showed that CRGs significantly inhibited LPS-induced upregulation of reactive oxygen species reduced or completely inhibited collagen-induced platelet aggregation and decreased their aggregation activity caused by the cooperative effect of LPS and collagen [43]. In the clinical study, we showed the effects of food supplement Carrageenan-FE on the immune system and lipid profile in patients with cardiovascular disease. Carrageenan-FE moderately modulated all of the immunity system markers and caused statistically significant decreases in the biomarkers of chronic inflammation [55].

It is known that the overreaction of the immune system into fragments of bacterial cells, such as LPS, underlies many inflammatory bowel diseases. In this regard, in the complex treatment of intestinal infections, the use of agents that restore normal microflora is indicated [56]. In the current study, we investigated the therapeutic effect of CRG in complex therapy of patients with enteric infections of Salmonella etiology. Our results show that the application of food supplement Carrageenan-FE in the standard therapy of toxicoinfections contributes to the correction of hemostasis. In this case, the action of CRG had a modulating regulatory pattern: in patients with hypercoagulation, a decrease in platelet aggregation activity was observed, and, in patients with hypocoagulation, the degree of aggregation increased. The application of Carrageenan-FE in the standard therapy scheme corrected some biochemical indicators and parameters of the immune system of the patient organism more actively in comparison with a control. A quick recovery of the immune system of patients taking CRG is likely due to its immunoregulatory properties.

These results allow us to hope for the practical application of CRGs for lowering the endotoxemia level in patients under the development of the infectious process caused by Gram-negative bacteria.

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