The Combined Effect of STAT3 and NF-Κb Inhibitors (The Last Links of the Cholinergic Anti-Inflammatory Pathway) on Mouse Mortality and Blood Concentration of Proinflammatory Cytokines in Sepsis

Introduction

Mortality from sepsis, depending on various factors, ranges from 12 to 60% of all deaths associated with diseases and their complications [1], and it is noted as an increase in the number of cases of sepsis, and mortality rate from it [2, 3, 4, 5]. For the first time in 1987, it was found that cholinergic stimulation significantly reduces the mortality of albino mice from sepsis [6], and later proved the feasibility of using cholinomimetics for emergency activation of the body’s antimicrobial resistance in sepsis [7]. The cholinergic anti-inflammatory mechanism was named after the study of its implementation on the organismic [6], cellular and subcellular levels in 2000 as the “cholinergic anti- inflammatory pathway” [6, 7, 8].

Currently, there is a great interest of researchers associated with the study of the possibility of reducing mortality in sepsis and various pathological processes by stimulating or inhibiting various elements of the cholinergic anti-inflammatory pathway [3, 4, 5, 9, 10], in particular, the possibility of achieving a therapeutic effect while inhibiting of NF-Kb and STAT3 transcription factor [9, 11, 12] (the last links of the cholinergic anti-inflammatory pathway) [8].

Aim of the Study

The aim of the study was to assess the effect of the inhibitor of the STAT3  transcription  factor (S3I-201 drug) and the NF-Kb inhibitor (BAY 11-7082), as well as their combined effect (the last links of the cholinergic anti- inflammatory pathway) on the mortality of mice in the early phase of sepsis caused by experimental peritonitis, and the content of Proinflammatory cytokines TNF -α, IL-1β, IL-6 in the blood.

Materials and Methods

The experiments were performed on random-bred albino mice of both sexes weighing 18-22g. The control group of mice (control group 1, n=8) received intraperitoneally 2.0ml of isotonic sodium chloride solution (saline) 10-15 minutes after the last intraperitoneal the introduction of 0.5ml of 0.05% aqueous solution of dimethyl sulfoxide-DMSO (Sigma-Aldrich), which was used daily for 4 days. The second group of mice (control group 2, n=47) was injected for 4 days with 0.5ml of a 0.05% aqueous solution of DMSO (i.p., once daily). 30-45 min after administration of this solution, mice in this group received (i.p.,) 2.5×109 CFUs diurnal culture of E. coli in 2.0ml of saline (sepsis modeling) [4, 5, 6, 7]. The third group (n=25) was administered (i.p., once daily) for 4days STAT3 inhibitor (S3I-201-2-Hydroxy-4-[[[[(4-methylphenyl) sulfonyl] oxy] acetyl] amino]-benzoic acid) (Sigma-Aldrich) at a dose of 5mg/kg in 0.5ml of a 0.05% aqueous solution of DMSO [11]. The fourth group (n=33) was administered (i.p., a single dose) the NF-Kb inhibitor BAY 11-7082-(E)-3- (4-Methylphenylsulfonyl)-2-propenenitrile-(Sigma-Aldrich) at a dose of 10 mg/kg in 0.5ml of a 1% aqueous solution of DMSO (Sigma-Aldrich) [5]. Prior to the administration of the NF-Kb inhibitor this group of mice received 0.5 ml of a 0.05% aqueous solution of DMSO for 3 days (i.p., once daily). In the 5th group (n=25) the STAT3 inhibitor (S3I-201) was administered in combination with the NF-Kb BAY inhibitor (11-7082). NF-Kb inhibitor was administered 10-15 minutes after the last injection of the STAT3 inhibitor. In groups 3 and 5, after 30-45 minutes after the administration of drugs, sepsis was modeled (in group 3, after 30-45 minutes after the last injection of the drug).

The mortality of mice (groups 2-3) was recorded after 4 and 24h after the sepsis modeling. The concentration of TNF-α, IL1β and IL-6 were measured in the blood plasma of all groups of mice (groups 1-5) using by ELISA (My Bio Source) according to manufacturer’s instructions. Determination the concentrations of Proinflammatory cytokines used monoclonal antibodies My Bio Source (cat.N- MBS494184, MBS494492, MBS335516 for TNF-α, IL-1β, and IL-6, respectively). Blood for research was taken from the retroorbital venous sinus. The obtained data were processed statistically using Student’s t-test (program STATISTICA 6.0).

Results

The STAT3 inhibitor (S3I-201) caused a decrease in the mortality of mice 4 hours after the administration of the daily culture of E. coli (sepsis modeling) compared to the control group 2 (sepsis) by 2.4 times (by 28.1%-p<0.05), and after 24hours-by 1.4 times (by 24.9%-p<0.05). The NF-Kb inhibitor reduced mortality from sepsis after 4 and 24hours after its modeling compared with group 2, respectively, 2.0 times (p<0.05) (by 24.7%) and 1.8 times (by 35,4%) (p<0.05). The administration of the STAT3 inhibitor in combination with the NF-Kb inhibitor caused an additive effect. Thus, the mortality of mice compared with the control after 4 and 24hours after the administration of E. coli compared with the control (group 2) decreased, respectively, by 3.1 times (by 32.9%) (p<0.05) and by 2, 9 times (by 52.9%) (p<0.05) (Table 1).

* -p<0.05 as compared to group 2); ** - p <0.05 in comparison with the control group 2 and group 3. Table 1: Effect of STAT3 inhibitor (S3I-201, 5mg/kg, once daily for 4 days) and NF-Kb inhibitor (BAY 11-7082, 10mg/kg, and a single dose) and their combined effect on mice mortality in sepsis, % (М±m).

The combined effect of the drugs compared to mortality with only STAT3 inhibitor or NF-Kb inhibitor caused a statistically insignificant reduction of the studied parameter after 4 hours, respectively, by 1.3 times (by 4.0%-p>0.05) and by 1.5 times (by 8.2%-p>0.05), After 24 hours, a significant effect of the combination of drugs was observed compared with the effect of only the STAT3 inhibitor (a decrease in mortality by 2.0 times (28.0%-p <0.05), respectively. The obtained data show that the combined effect of the STAT3 and the NF-Kb inhibitors exceeds the effect of the STAT3 inhibitor, and insignificantly (p>0.05) the effect of the NF-Kb inhibitor.

The concentration of cytokines TNF-α, IL-1β and IL-6 after the sepsis modeling (control group 2) in the blood of mice after 4h compared with the control group 1 (intact animals), increased respectively to 19.7; 19.8 and 59.3 times (p<0.05), and after 24hours, the content of these cytokines compared to their level after 4h decreased, respectively, to 23.0; 4.4 and 8.4 times (p<0.05). The content of IL-1β

and IL-6 after 24h remained higher than in group 1 by 1.6 times (p>0.05) and 3.2 times (p<0.05), respectively, and the concentration of TNF-α in groups 1 and 2 did not differ significantly (Table 2).

Note: 4 and 24-time after modeling of sepsis, h; a -p<0.05 compared to control (group 1); b -p<0.05 compared with corresponding parameter for sepsis (control group 2); с -p<0.05 compared with parameter after 4h; d -p<0.05-in comparison with group 3. Table 2: Effect of STAT3 inhibitor (S3I-201, 5mg/kg, once daily for 4 days) and NF-Kb inhibitor (BAY 11-7082, 10mg/kg, a single dose) and their combined effect on the concentration of Proinflammatory cytokines in blood of mice after modeling of sepsis, pg/ml (М±m; n=7-8).

The NF-Kb inhibitor (BAY 11-7082) 4 h after sepsis modeling reduced the blood levels of TNF-α, IL-1β and IL-6 (group 4) compared to the control group 2, respectively, by 7.0; 4.4 and 8.9 times (p<0.05). After 24 hours, the content of these cytokines compared with their level after 4 hours decreased, respectively, by 3.0; 2.4 and 3.3 times (p<0.05). The concentrations of IL-1β and IL-6 statistically significantly (p<0.05) exceeded those of the control group 1 by 1.8 and 7.8 times (p<0.05), respectively, and compared to the parameters of group 2, the content IL-1β and IL-6 were reduced by 2.4 and 3.5 times, respectively (p<0.05). The value of TNF-α was not significantly different from the levels in groups 1 and after 24h after the sepsis modeling.

The concentrations of TNF-α, IL-1β and IL-6 in group 5 (combined effect of STAT3 and NF-Kb inhibitors) compared with parameters of group 3 (effect of STAT3 inhibitor) 4h after sepsis modeling were lower respectively by 1.6 (p<0.05); 1.3 (p>0.05) and 1.5 times (p<0.05), and after 24h the levels of IL-1β and IL-6 were less than the corresponding values ​in the group of 3 by 1.5 (p>0, 05) and by 1.8 times (p<0.05).

The blood levels of TNF-α, IL-1β, and IL-6 in group 5 compared with those of group 4 (the effect of NF-Kb inhibitor) did not differ significantly (p>0.05) after 4 h after the sepsis modeling, and after 24 h IL-1β and IL-6 levels were less than the corresponding values ​in group 4 by 1.7 (p<0.05) and by 1.6 times (p>0.05). The blood concentrations of TNF-α, IL-1β and IL-6 in group 5 with a combined effect of STAT3 inhibitor (S3I-201) and of NF-Kb inhibitor (BAY 11-7082) 4h after modeling sepsis compared with the control group 2 (sepsis without the use of drugs) decreased, respectively, by 8.9; 5.0 and 5.5 times (p<0.05). The content of cytokines IL-1β and IL-6 significantly (p<0.05) exceeded the corresponding parameters of group

1. The concentration of pro-inflammatory cytokines after 24h after modeling sepsis significantly decreased (p<0.05) compared with these parameters after 4h. The content of TNF-α did not differ from those in groups 1-4, and the levels of IL-1β and IL-6 remained lower than the values ​in group 2 by 4.0 and by 5.5 times (p<0.05), respectively.

The data obtained suggest that the combined action of STAT3 inhibitor and NF-Kb inhibitor (the last links of the cholinergic anti-inflammatory pathway) causes an additive effect.

Discussion

The cholinergic anti-inflammatory mechanism (“cholinergic anti-inflammatory pathway”) [6, 7, 13, 14, 15], includes: acetylcholine m-acetyl cholinergic receptor type 1 (m1AChR) activation of the brain, modulating the immunoregulatory function of the vagus nerve [4, 5, 8, 13]; excitation of efferent fibers n. vagus; effect of acetylcholine on α7n-acetylcholine receptors (α7nAChR) of cells of the macrophage-monocytic system (MMS) [3, 5, 15]. The occurrence of anti-inflammatory effect in cells of MMS is provided by JAK2 kinase; STAT3  transcription  factor; NF- κB transcription factor [4, 5, 8, 13, 14]. These effects lead to a decrease in mortality from sepsis due to the reduction of the production of pro-inflammatory cytokines TNF-α, protein B1-HMGB1, macrophage-inflammatory protein-2-MIP-2, interleukins-IL-1β, IL-6 [5, 7, 8, 13, 14]. We have shown a decrease in the mortality of mice from sepsis after activation of α7nAChRs [3, 4], m1AChRs [4], α2-adrenoreceptors (β2ARs) [5], inhibition of NF-κB factor [5], and also their combined action was evaluated.

The transcription factor NF-Kb modulates the synthesis of pro-inflammatory cytokines involved in development of sepsis. Signal pathways initiated by Toll-like receptors (TLR2 and TLR4) to which bacterial products bind, in particular, E. coli lipopolysaccharide, lead to enhanced transcription of genes responsible for expression of cytokines, chemokines, adhesion molecules, apoptotic factors and other mediators of inflammatory response associated with sepsis [5].

Conclusion

The STAT3 inhibitor (S3I-201) and the NF-Kb inhibitor (BAY 11-7082) the last links of the cholinergic anti- inflammatory pathway-reduce the mortality of mice, the content of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in the blood in sepsis, and their combined action causes an additive effect.