The effect of specific therapeutic agents on inflammation in sepsis-induced neonatal rats

Introduction

Affecting multiple bodily systems, neonatal sepsis is a serious condition with a high mortality rate and leads to hemodynamic changes as shock, organ dysfunction, and organ failure. The most discussed agents in the pathogenesis of sepsis are gram-negative bacteria [1], [2]. Lipopolysaccharide (LPS) is the main component of gram-negative bacteria’s cell outer wall, and is responsible for biological processes that can lead to fever, septic shock, and even death [3]. When LPS is injected to the body, defense cells trigger a series of inflammatory responses, including forming a LPS-LPB-CD14 complex, which is made up of LPS, LPS-binding proteins (LPB), alongside forming CD14 glycoprotein, which serves as a specific receptor task for LPS and LPB. This complex activates the inflammatory cascade, upon which monocytes and macrophages cause the release of proinflammatory cytokines and interleukins. This leads to the fragmentation of CD14 from cell membranes and the hepatocyte cells and fragments are known as soluble-CD14 (sCD14) [4]. A subtype of sCD14 (sCD14-ST) results from plasma protease activity during inflammation and is an N-terminal fragment of sCD14 composed of 64 amino acids and found in blood circulation. This subtype contributes to the transmission of intracellular signals, thus playing an important role in the occurrence of septic shock [5], [6].

Presepsin is a polypeptide in sCD14-ST group. It weighs 13 kDa and is located on the N-18 terminal of CD14 [7]. The synthesis of presepsin in the bloodstream increases in the early stages of sepsis and during systemic bacterial infections. The release of presepsin, synthesized from the liver into the bloodstream, in turn rises. Sepsis studies have revealed that presepsin directly modulates both humoral and cellular immune responses by interacting with T and B cells. Moreover, researchers have found that presepsin significantly reduces the mortality rate resulting from the severity of endotoxemia and gram-negative bacterial infections, and thus have suggested that presepsin may serve as a potential biomarker for routine use [6], [8], [9], [10], [11], [12].

The Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a dimeric transcription factor associated with regulating the genes responsible for many pathological events such as apoptosis and inflammation. It is believed that many pathogens and cytokines activate NF-κB signaling pathways. In the presence of sepsis, NF-κB is involved in two major components of the host response: (1) the stimulation of monocyte and macrophage activation resulting from rapid production of large amounts of inflammatory cytokines and (2) the intense activation of endothelial cells [13], [14].

Thalidomide is a glutamic acid derivative associated with bemegride (alpha-ethyl-alpha methyl glutarimide) and glutethimide (beta-ethyl beta-phenylglutarimide), albeit containing different pharmacological properties. For example, the use of Thalidomide inclinicalpractice as a therapeutic drug for multiple myeloma and erythema nodosum leprosum has been approved. It has a number of side effects, including teratogenicity, sedation, and redness [15]. Thalidomide inhibits NF-κB activation, even in the presence of potent NF-κB activators by inhibiting kappa-light-polypeptide gene enhancers during B-cell (IκB) kinase activity. Additionally, NF-κB prevents transcription events by inhibiting itself to DNA. Thus, the inhibited activation of NF-κB results in the down-regulation of the target transcriptional genes. Tumor necrosis factor alpha (TNF-α) is another physiological activator of NF-κB as well. The release of TNF-α during inflammatory responses up-regulates the expression of NF-κB. Thalidomide specifically inhibits monocytes to release TNF-α as well as NF-κB activation [16]. It is likely to consider Thalidomide as an alternative therapeutic agent for sepsis since it acts as anti-inflammatory activities by reducing TNF-α expression, inhibiting NF-κB, and downregulating inflammatory events.

Etanercept is a fusion protein that is produced using recombinant DNA technology, and is obtained by combining the human tumor necrosis factor receptor (TNFR2/p75) with the human IgG1-Fc (immunoglobulin) protein. It competitively inhibits the binding of TNF-α to cell surface receptors [17]. Etanercept being an anti-TNF-α monoclonal antibody has been widely discussed in the literature as a therapeutic agent that can be used to treat the immune modulation of numerous systemic inflammatory and autoimmune diseases [18].

Therefore, we believe that if we can indirectly downregulate NF-κB expression using Etanercept, we then can develop an alternative treatment to improve sepsis. By considering the potential role that both Thalidomide and Etanercept play in inflammation, this study was conducted to investigate the possible protective effects of these drugs in LPS-induced rat sepsis model.

Materials and methods

Results

Table 1 shows the levels of tissue Presepsin and NF-κB for all groups. It was found that presepsin levels increased statistically significantly in the Lipopolysaccharide (LPS)-induced sepsis group (109.42±27.33 pg/mg protein) compared to the sham group (41.08±19.79 pg/mg protein) (p<0.001). Administration of Thalidomide, Etanercept and Thalidomide+Etanercept along with LPS provided a significant reduction in presepsin levels (67.92±15.16, 69.59±19.68, and 59.67±23.09 pg/mg protein, respectively) in the rats compared to the rats in LPS group (p<0.01, p<0.01, p<0.005, respectively) (Table 1).

There was a statistically-significant increase in NF-κB levels in the sepsis group compared to sham group (p<0.001). NF-κB levels increasing due to LPS exposure significantly decreased by the use of treatment agents (p<0.005 for all treatment groups) (Table 1).

However, the expression of NF-κB protein was higher in the LPS group than the sham group (p<0.001). The administration of Thalidomide, Etanercept, and Thalidomide+Etanercept significantly decreased the expression levels of NF-κB protein (p=0.001 for all treatment groups) when compared to the LPS group (Figure 1).

Figure 1:

Western blot results for NF-κB cleavage according to groups.

The analysis showing the effect of LPS and co-administered Thal, Eta and Thal+Eta on NF-κB protein expression (A). Western blotting was normalized to β-actin expression and expressed relative to the control sample (B). The corresponding densitometric analysis is also shown (C). Data are means±SD of three independent experiments. ^*p<0.001, compared with the sham group; ^**p=0.001 compared with LPS alone (Tukey HSD).

Discussion

According to current sepsis hypotheses proposed by various experimental studies and theories, the immune system’s excessive proinflammatory response following infection leads to multi-organ failure or further damage. Despite recent advances in antimicrobial therapies, better-defined pathophysiology, and technological developments with regards to diagnostic methods in recent years, sepsis continues to be defined as a systemic inflammatory response with serious clinical conditions.

Lipopolysaccharide (LPS)-[E. coli, 0111: B4] is easy and reliable to use; therefore, we selected this agent to induce experimental neonatal rat sepsis model [21]. This study investigated the effects of Thalidomide and Etanercept, which are known as TNF-α blockers in sepsis treatment, on inflammatory parameters such as presepsin and NF-κB.

Various studies have examined the diagnostic value of presepsin, and have revealed meaningful results. First, Okamura and Yokoi [22] investigated presepsin levels and the value of diagnosis in neonatal sepsis patients, and provided prospective ancillary diagnostic information. Afterwards, the studies have also revealed remarkable differences in presepsin levels in healthy people, in patients with sepsis who have local infections, and in severe sepsis patients who systemic inflammatory response syndrome (SIRS). Endo et al. [23] studied presepsin, procalcitonin, and IL-6 levels in patients suffering from suspected sepsis, and found an increase in presepsin concentrations in predominantly bacteremic patients. Presepsin is used to investigate adult and neonatal cases with sepsis as well as deaths due to sepsis [6], [7], [23], [24].

The present study showed that presepsin levels were higher in the LPS-induced sepsis group than both sham and treatment groups, which supported that presepsin could serve as a biomarker candidate for neonatal sepsis. In the light of these results, it has been observed that both Etanercept and Thalidomide may have beneficial effects in reducing presepsin levels, however it was thought that Thalidomide+Etanercept combination was more effective than individually Etanercept and Thalidomide treatments.

Although animal experiments and in vitro studies have supported the hypothesis that different inhibitors stop the sequence of events that causes sepsis by inhibiting the NF-κB pathway, there is still no consensus on the mechanism involved. For example, in a study it was observed that the pyrrolidine dithiocarbamate (PDTC), being an antioxidant substrate, protected mice from septic shock without affecting lymphocyte infiltration, tissue interleukin-1 (IL-1), and IL-6 production against a lethal dose of LPS. Another in vivo study revealed that PDTC directly affected the translocation of NF-κB into the nucleus (breaking), and inhibited the gene expression of proinflammatory proteins such as intercellular adhesion molecule-1 (ICAM-1) and TNF-α, cyclooxygenase-2 (COX-2), which both use the NF-κB pathway during sepsis [25]. The NF-κB-dependent synthesis mechanism of adhesion molecules during endothelial activation has also been studied extensively. Some in vitro studies have used protein kinase inhibitors and NF-κB inhibitors, which block TNF-α-activated signal transduction. Moreover, there have been ongoing clinical trials that use neutralizing antibodies against adhesion molecules in order to reduce the damage that LPS causes to the body [26]. In their studies, both Von Moos et al. [27] and Teo [28], identified Thalidomide as an immunomodulatory agent that inhibits the release of cellular adhesion molecules, angiogenesis, and the production of various anti-inflammatory mediators. Other studies have also reported that Thalidomide and Etanercept prevent NF-κB and inhibit the production of various cytokines, thereby eliminating immune and inflammatory responses [18], [29]. In the present study, increased NF-κB levels in the LPS group showed a statistically significant decrease in all the treatment groups and this suggested that anti-inflammatory functions of treatment agents were controlled by the transcriptional factor NF-κB. This situation may be regarded to support that thalidomide and etanercept mentioned in our previous study are an efficient anti-inflammatory efficient drug [30].

NF-κB is a generic term for a family of transcription factors consisting of various homo- and/or hetero-dimeric complexes belonging to the Rel family such as NF-κB 1 (p50 and its precursor p105), NF-κB 2 (p52 and its precursor p100), RelA (p65), RelB, and c-Rel [31], [32]. This transcription factor and its complexes have been investigated in various inflammation studies. For instance, NF-κB p50 and its precursor p105 can be regarded as the master regulator of the inflammatory process [33]. Another study showed that p50-deficient mice heterozygous for RelA were extremely sensitive to LPS-induced shock and P50/105 subunits of NF-κB having no transactivation domains repressed the expression of NF-κB target genes and inhibited inflammation [34]. However, other gene knockout studies have revealed that the protein expression of NF-κB can have both pro- and anti-inflammatory roles. However, other gene knockout studies have revealed that the protein expression of NF-κB can have both pro- and anti-inflammatory roles.

In the present study, NF-κB p-105 (the precursor of P50) expression levels in liver tissue homogenates were determined by using Western blot method. Relative protein expression values were similar to the ELISA results. NF-κB expression was higher in the LPS group than the sham group. Similarly, a significant decrease was found in NF-κB p-105 levels in all of the treated groups compared to LPS group. Despite the fact that there are a limited number of studies on tissue expression of NF-κB levels, this present study supports the results of our previous study which also investigated neonatal sepsis [19]. Moreover, in their study, Yang et al., evaluated NF-κB expression in cecal ligation puncture (CLP)-induced sepsis model in lung tissue. According to their results, the Liriodendrin treatment inhibited the sepsis- dependent inflammation by decreasing NF-κB activation in the lung tissue [13]. These results were compatible with the results of the present study. Considered that the specific treatment agents applied in the present study prevented sepsis-induced inflammation by downregulating tissue NF-κB expression levels and by inhibiting TNF-α pathways. In other words, it can be asserted that blocking NF-κB nuclear translocation and inhibiting NF-κB expression may serve as another way to prevent neonatal sepsis development. However, we need to acquire more knowledge about these pathways in the presence of neonatal sepsis, which then can help us develop specific blocking drugs and use them in animal-based experiments in order to obtain better results.

It is thought that while increased NF-κB levels and NF-κB expression are both related to LPS-induced sepsis inflammation, evaluation of these parameters may be useful to estimate the inflammation in LPS-induced newborn sepsis. It is also suggested that agents like Etanercept and Thalidomide show anti-inflammatory activity by blocking this pathway directly and/or indirectly. Therefore, the results of the present study offer new perspectives on the effects of Thalidomide and Etanercept alone or in combination, which could be used to treat neonatal sepsis by suppressing the inflammatory response.

In conclusion, we can say that these results may lead us to an alternative treatment protocol for sepsis in the near future in the hope of significantly lowering both mortality and morbidity rates.