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Background/aims: There are evidences that a decrease in the functional activity of pancreatic β-cells under type 2 diabetes conditions may be associated with their senescence, therefore, senotherapy may be a prospective strategy for the diabetes treatment., Methods: The senotherapeutic potential of peroxiredoxin 6 (PRDX6) was studied in RIN-m5F pancreatic β-cells with streptozotocin-induced senescence by measuring markers, associated with senescence., Results: Exposure to streptozotocin (STZ) resulted in the senescence of the β-cells. The addition of PRDX6 to the culture medium of RIN-m5F β-cells before treatment with STZ decreased the levels of the following senescence markers: the percentage of SA-β-Gal-positive cells, the phosphorylation of histone H2AX and p21 proteins, and the secretion of the proinflammatory cytokine IL-6 but not the anti-inflammatory cytokine IL-10. These effects were accompanied by a decrease in the production of reactive oxygen species (ROS) and the restoration of impaired NF-κB activation. In addition, PRDX6 altered the production of the heat shock protein HSP90: the production of the constitutive form of HSP90-beta decreased, while the level of inducible HSP90-alpha increased., Conclusion: PRDX6 prevented the senescence of RIN-m5F cells in response to the DNA damage-inducing agent streptozotocin, indicating a potential protective role of PRDX6 in type 2 diabetes mellitus., Competing Interests: The authors declare that they have no competing interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Diabetes mellitus type 1 (T1D) and type 2 (T2D) develop due to dysfunction of the Langerhans islet β-cells in the pancreas, and this dysfunction is mediated by oxidative, endoplasmic reticulum (ER), and mitochondrial stresses. Although the two types of diabetes are significantly different, β-cell failure and death play a key role in the pathogenesis of both diseases, resulting in hyperglycemia due to a reduced ability to produce insulin. In T1D, β-cell apoptosis is the main event leading to hyperglycemia, while in T2D, insulin resistance results in an inability to meet insulin requirements. It has been suggested that autophagy promotes β-cell survival by delaying apoptosis and providing adaptive responses to mitigate the detrimental effects of ER stress and DNA damage, which is directly related to oxidative stress. As people with diabetes are now living longer, they are more susceptible to a different set of complications. There has been a diversification in causes of death, whereby a larger proportion of deaths among individuals with diabetes is attributable to nonvascular conditions; on the other hand, the proportion of cancer-related deaths has remained stable or even increased in some countries. Due to the increasing cases of both T1D and T2D, these diseases become even more socially significant. Hence, we believe that search for any opportunities for control of this disease is an overwhelmingly important target for the modern science. We focus on two differences that are characteristic of the development of diabetes's last periods. One of them shows that all-cause death rates have declined in several diabetes populations, driven in part by large declines in vascular disease mortality but large increases in oncological diseases. Another hypothesis is that some T2D medications could be repurposed to control glycemia in patients with T1D., Competing Interests: The authors declare that the article was prepared in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

The aim of this work was to study the effects of thymosin-1 alpha (Tα1) on the anti-inflammatory response of RAW 264.7 macrophages cultured in the presence of lipopolysaccharide (LPS) from the walls of gram-negative bacteria. As well, we evaluated production of pro-inflammatory cytokines and the activity of the NF-κB and SAPK/JNK signaling pathways. In addition, the level of expression of a number of genes that regulate cell apoptosis, as well as the activity of receptors involved in the pro-inflammatory response, was determined. First, the addition of Tα1 normalized the level of cytokine production to varying degrees, with a particularly noticeable effect on IL-1β and IL-6. Second, the addition of Tα1 normalized the activity of the NF-κB and SAPK/JNK signaling cascades and the expression of the Tlr4 gene. Third, Tα1 significantly reduced p53 and the activity of the P53 gene, which is a marker of cell apoptosis. Fourth, it was shown that the increase in Ar-1 gene expression under the influence of LPS was significantly reduced using Tα1. Thus, it was found that the presence of Tα1 in the RAW 264.7 cell culture medium significantly reduced the level of the pro-inflammatory response of cells.

This study aimed to assess the effects of the immunomodulator thymulin, a thymic peptide with anti-inflammatory effects, and peroxiredoxin 6 (Prdx6), an antioxidant enzyme with dual peroxidase and phospholipase A2 activities, on the blood‒brain barrier (BBB) condition and general health status of animals with relapsing-remitting experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis in humans. Both thymulin and Prdx6 significantly improved the condition of the BBB, which was impaired by EAE induction, as measured by Evans blue dye accumulation, tight-junction protein loss in brain tissue, and lymphocyte infiltration through the BBB. The effect was associated with significant amelioration of EAE symptoms. Thymulin treatment was accompanied by a decrease in immune cell activation as judged by interleukin-6, -17, and interferon-gamma cytokine levels in serum and NF-kappaB cascade activation in splenocytes of mice with EAE. Prdx6 did not induce significant immunomodulatory effects but abruptly decreased EAE-induced NOX1 and NOX4 gene expression in brain tissue, which may be one of the possible mechanisms of its beneficial effects on BBB conditions and health status. The simultaneous administration of thymulin and Prdx6 resulted in complete symptomatic restoration of mice with EAE. The results demonstrate prospective strategies for multiple sclerosis treatment., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The aim of the work was to study effects of peroxiredoxin 6 (PRDX6), a recombinant antioxidant protein, on the level of pro-inflammatory responses of RAW 264.7 macrophages to endotoxin exposure. Addition of LPS to the RAW 264.7 cell culture medium expectedly increased production of TNF-α, and addition of PRDX6 led to a significant (15-20%) decrease in its production. The level of production of another pro-inflammatory cytokine, IL-1β, which was significantly activated by endotoxin, was completely normalized under the PRDX6 action. Moreover, addition of PRDX6 reduced production of reactive oxygen species (ROS) induced by endotoxin and also prevented overexpression of the iNos gene in the RAW 264.7 cells. The results showed that PRDX6 had a suppressive effect on the expression of Nrf-2 gene and production of the transcription factor NRF-2 during the first 6 h of cell cultivation. Addition of endotoxin caused activation of the NF-κB and SAPK/JNK signaling cascades, while in the presence of PRDX6, activity of these signaling cascades decreases. It is known that the pro-inflammatory response of cells caused by exposure to bacterial LPS leads to activation of apoptosis and elimination of the damaged cells. Our studies confirm this, since exposure to LPS led to activation of the expression of P53 gene, a marker of apoptosis. Peroxiredoxin 6 added within the first hours of the development of acute pro-inflammatory response suppressed the P53 gene expression, indicating protective effect of PRDX6 that reduced apoptosis in the RAW 264.7 macrophages.

This minireview discusses the very important biomedical problem of treating type 2 diabetes mellitus (T2D). T2D accounts for more than 90% of the total number of diagnosed cases of diabetes mellitus and can result from aging, inflammation, obesity and β-cell senescence. The main symptom of both T2D and type 1 diabetes (T1D) is an increase in blood glucose concentration. While T1D is insulin-dependent and is associated with the destruction of pancreatic β-cells, T2D does not require lifelong insulin administration. In this case, pancreatic β-cells are not destroyed, but their functional activity is deregulated. In T2D, metabolic stress increases the number of senescent β-cells while impairing glucose tolerance. The potential paracrine effects of senescent β-cells highlight the importance of the β-cell senescenceassociated secretory phenotype (SASP) in driving metabolic dysfunction. We believe that the main reason for the deregulation of the functional activity of pancreatic β-cells in T2D is associated with their "aging" or senescence, which may be induced by various stressors. We propose the use of peroxiredoxin 6 as a new senolytic drug, and the role of β-cell senescence in the development of T2D is discussed in this review., Competing Interests: No potential Disclosure Statement was reported by the authors., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Peroxiredoxin 6 (Prdx6) is a multifunctional eukaryotic antioxidant enzyme. Mammalian Prdx6 possesses peroxidase activity against a wide range of organic and inorganic hydroperoxides, as well as exhibits phospholipase A2 (aiPLA2) activity, which plays an important role in the reduction of oxidized phospholipids and cell membrane remodeling. Exogenous Prdx6 has recently been shown to be able to penetrate inside the cell. We hypothesized that this entry may be due to the phospholipase activity of Prdx6. Experiments using exogenous Prdx6 in three cell lines (3T3, A549, RAW 264.7) demonstrated that it is the phospholipase activity that promotes its penetration into the cell. Overoxidation of Prdx6 led to a suppression of the peroxidase activity and a 3-to-4-fold growth of aiPLA2, which enhanced the efficiency of its transmembrane transport into the cells by up to 15 times. A mutant form of Prdx6-S32A with an inactivated phospholipase center turned out to be unable to enter the cells in both the reduced and oxidized state of the peroxidase active center. Previously, we have shown that exogenous Prdx6 has a significant radioprotective action. However, the role of phospholipase activity in the radioprotective effects of Prdx6 remained unstudied. Trials with the mutant Prdx6-S32A form, with the use of a total irradiation model in mice, showed a nearly 50% reduction of the radioprotective effect upon aiPLA2 loss. Such a significant decrease in the radioprotective action may be due to the inability of Prdx6-S32A to penetrate animal cells, which prevents its reduction by the natural intracellular reducing agent glutathione S-transferase (πGST) and lowers the efficiency of elimination of peroxides formed from the effect of ionizing radiation. Thus, phospholipase activity may play an important role in the reduction of oxidized Prdx6 and manifestation of its antioxidant properties.

The aim of the study was to evaluate the possibility of increasing the radioprotective potential of peroxiredoxin 6 (Prdx6) and its mutant form S32A by their combined use with geldanamycin (GA) for 3T3 fibroblasts irradiated with X-rays at a dose of 6 Gy. The mutant enzyme S32A, which does not have phospholipase activity, exhibits a more pronounced radioprotective activity when combined with GA. The use of this combination of radioprotective drugs completely abolishes the peak of NF-κB activity in irradiated 3T3 cells. Another transcription factor, p53, which is an indicator of the level of cell apoptosis and increases upon irradiation, is also reduced by S32A in combination with GA. The low-molecular-weight protein p21, which is a marker of cell senescence and whose production increases upon irradiation, is also normalized when S32A is used in combination with GA. In addition, the use of this combination of radioprotective drugs significantly reduces the stress response of 3T3 cells to X-ray irradiation., (© 2022. Pleiades Publishing, Ltd.)

Peroxiredoxin 6 (Prdx6) is an important antioxidant enzyme with multiple functions in the cell. Prdx6 neutralizes a wide range of hydroperoxides, participates in phospholipid metabolism and cell membrane repair, and in transmission of intracellular and intercellular signals. Disruption of normal Prdx6 expression in the cell leads to the development of pathological conditions. Decrease in the Prdx6 concentration leads to increase in oxidative damage to the cell. At the same time, hyperproduction of Prdx6 is associated with increase in antioxidant status, suppression of apoptosis, and carcinogenesis. Currently, mechanisms of carcinogenic action of peroxiredoxins are poorly understood. In this work we established that the 3-4-fold increase in Prdx6 production in mouse embryonic fibroblast 3T3 cells leads to the 4-5-fold decrease in the level of oncosuppressor p53. At the same time, hyperproduction of Prdx6 leads to the increased expression of RELA and HIF1A, which have oncogenic effects. The 3-4-fold increase in intracellular Prdx6 increases intensity of cell proliferation by 20-30%, promotes increase in antioxidant activity by 30-50%, and increases radioresistance of the transfected 3T3 cells by 30-40%. Increase of the level of intranuclear Prdx6 leads to the decrease in expression of the DNA repair genes in response to radiation, indicating decrease in the genomic DNA damage. This work discusses possible molecular mechanisms of p53 suppression during Prdx6 hyperproduction, which could be used in the development of new approaches in cancer therapy.

Pathways regulating cell senescence and cell cycle underlie many processes associated with ageing and age-related pathologies, and they also mediate cellular responses to exposure to stressors. Meanwhile, there are central mechanisms of the regulation of stress responses that induce/enhance or weaken the response of the whole organism, such as hormones of the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic and parasympathetic systems, thymic hormones, and the pineal hormone melatonin. Although there are many analyses considering relationships between the HPA axis and organism ageing, we found no systematic analyses of relationships between the neuroendocrine regulators of stress and inflammation and intracellular mechanisms controlling cell cycle, senescence, and apoptosis. Here, we provide a review of the effects of neuroendocrine regulators on these mechanisms. Our analysis allowed us to postulate a multilevel system of central regulators involving neurotransmitters, glucocorticoids, melatonin, and the thymic hormones. This system finely regulates the cell cycle and metabolic/catabolic processes depending on the level of systemic stress, stage of stress response, and energy capabilities of the body, shifting the balance between cell cycle progression, cell cycle stopping, senescence, and apoptosis. These processes and levels of regulation should be considered when studying the mechanisms of ageing and the proliferation on the level of the whole organism.

Although many different classes of antioxidants have been evaluated as radioprotectors, none of them are in widespread clinical use because of their low efficiency. The goal of our study was to evaluate the potential of the antioxidant protein peroxiredoxin 6 (Prdx6) to increase the radioresistance of 3T3 fibroblasts when Prdx6 was applied after exposure to 6 Gy X-ray. In the present study, we analyzed the mRNA expression profiles of genes associated with proliferation, apoptosis, cellular stress, senescence, and the production of corresponding proteins from biological samples after exposure of 3T3 cells to X-ray radiation and application of Prdx6. Our results suggested that Prdx6 treatment normalized p53 and NF-κB/p65 expression, p21 levels, DNA repair-associated genes (XRCC4, XRCC5, H2AX, Apex1), TLR expression, cytokine production (TNF-α and IL-6), and apoptosis, as evidenced by decreased caspase 3 level in irradiated 3T3 cells. In addition, Prdx6 treatment reduced senescence, as evidenced by the decreased percentage of SA-β-Gal positive cells in cultured 3T3 fibroblasts. Importantly, the activity of the NRF2 gene, an important regulator of the antioxidant cellular machinery, was completely suppressed by irradiation but was restored by post-irradiation Prdx6 treatment. These data support the radioprotective therapeutic efficacy of Prdx6.

The review discusses information on the development of type 1 diabetes mellitus (T1D) as a systemic autoimmune and inflammatory disease. Focus of the review is on the role of innate immune system, including activation of some signaling cascades, cytokine response, and activity of the Toll-like receptors in the development of T1D. Dysfunction of innate immunity is the cause of the attack of pancreatic beta cells by the host T-lymphocytes, which leads to the death of pancreatic beta cells that produce insulin. Lack of insulin causes hyperglycemia and the need for lifelong injections of insulin in patients with T1D, which, nevertheless, does not exclude damage to many organs and tissues, given particular vulnerability of the blood vessels under conditions of hyperglycemia. The review discusses the role of oxidative stress as a factor that plays a major role in damage of vascular system and pancreatic tissue during the development of T1D. Considering high sensitivity of pancreatic beta cells to the action of reactive oxygen species (ROS), the possibility of using antioxidants for reducing the level of pathological consequences in the course of T1D development is discussed. New information on anti-diabetic activity of the exogenous antioxidant enzyme peroxiredoxin 6, which is capable of penetrating cells, activating insulin production in beta cells, reducing ROS levels, as well as decreasing activation of some signaling cascades, production of pro-inflammatory cytokines, and expression of Toll-like receptors in beta cells and in immune cells during T1D development is discussed.

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with multi-substrate peroxidase and phospholipase activities that is involved in cell redox homeostasis and regulates intracellular processes. Previously, recombinant Prdx6 was shown to exert a radioprotective effect during whole-body exposure to a lethal dose of X-ray radiation. Moreover, a mutant form Prdx6-C47S, which lacks peroxidase activity, also had a radioprotective effect, and this indicates that the mechanism of radioprotection is unknown. The present study was aimed to test the hypothesis that the radioprotective effect of Prdx6 and Prdx6-C47S may be mediated through the TLR4/NF-κB signaling pathway. It was demonstrated that exogenously applied Prdx6 protected 3T3 fibroblast cells against LD50 X-ray radiation in vitro. Pretreatment with Prdx6 increased cell survival, stimulated proliferation, normalized the level of reactive oxygen species in culture, and suppressed apoptosis and necrosis. Wild-type Prdx6 and, to a lesser degree, the Prdx6-C47S mutant proteins promoted a significant increase in NF-κB activation in irradiated cells, which likely contributes to the antiapoptotic effect. Pretreatment with TLR4 inhibitors, especially those directed to the extracellular part of the receptor, significantly reduced the radioprotective effect, and this supports the role of TLR4 signaling in the protective effects of Prdx6. Therefore, the radioprotective effect of Prdx6 was related not only to its antioxidant properties, but also to its ability to trigger cellular defense mechanisms through interaction with the TLR4 receptor and subsequent activation of the NF-κB pathway. Recombinant Prdx6 may be useful for the development of a new class of safe radioprotective compounds that have a combination of antioxidant and immunomodulatory properties., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Protective effects of peroxiredoxin 6 (PRDX6) in RIN-m5F β-cells and of thymulin in mice with alloxan-induced diabetes were recently reported. The present work was aimed at studying the efficiency of thymulin and PRDX6 in a type 1 diabetes mellitus model induced by streptozotocin in mice. Effects of prolonged treatment with PRDX6 or thymic peptide thymulin on diabetes development were evaluated. We assessed the effects of the drugs on the physiological status of diabetic mice by measuring blood glucose, body weight, and cell counts in several organs, as well as effects of thymulin and PRDX6 on the immune status of diabetic mice measuring concentrations of pro-inflammatory cytokines in blood plasma (TNF-α, interleukin-5 and 17, and interferon-γ), activity of NF-κB and JNK pathways, and Hsp90α expression in immune cells. Both thymulin and PRDX6 reduced the physiological impairments in diabetic mice at various levels. Thymulin and PRDX6 provide beneficial effects in the model of diabetes via very different mechanisms. Taken together, the results of our study indicated that the thymic peptide and the antioxidant enzyme have anti-inflammatory functions. As increasing evidences show diabetes mellitus as a distinct comorbidity leading to acute respiratory distress syndrome and increased mortality in patients with COVID-19 having cytokine storm, thymulin, and PRDX6 might serve as a supporting anti-inflammatory treatment in the therapy of COVID 19 in diabetic patients.

Type 1 diabetes is associated with the destruction of pancreatic beta cells, which is mediated via an autoimmune mechanism and consequent inflammatory processes. In this article, we describe a beneficial effect of peroxiredoxin 6 (PRDX6) in a type 1 diabetes mouse model. The main idea of this study was based on the well-known data that oxidative stress plays an important role in pathogenesis of diabetes and its associated complications. We hypothesised that PRDX6, which is well known for its various biological functions, including antioxidant activity, may provide an antidiabetic effect. It was shown that PRDX6 prevented hyperglycemia, lowered the mortality rate, restored the plasma cytokine profile, reversed the splenic cell apoptosis, and reduced the β cell destruction in Langerhans islets in mice with a severe form of alloxan-induced diabetes. In addition, PRDX6 protected rat insulinoma RIN-m5F β cells, cultured with TNF- α and IL-1 β , against the cytokine-induced cytotoxicity and reduced the apoptotic cell death and production of ROS. Signal transduction studies showed that PRDX6 prevented the activation of NF- κ B and c-Jun N-terminal kinase signaling cascades in RIN-m5F β cells cultured with cytokines. In conclusion, there is a prospect for therapeutic application of PRDX6 to delay or even prevent β cell apoptosis in type 1 diabetes., Competing Interests: The authors report no conflict of interest., (Copyright © 2020 Elena G. Novoselova et al.)

The role of two heat shock proteins, Hsp70 and Hsp90α, on stress response in mice with severe diabetes mellitus induced by a high dose of alloxan (500 mg/kg body weight), as well as in RIN-m5F β cells cultured in the presence of cytokines (IL-1 and TNF-α) was studied. Our results showed that severe type 1 diabetes mellitus (T1D) caused a higher expression of Hsp90α, but not Hsp70. Moreover, injections of the peroxiredoxin 6 antioxidant enzyme (PRDX6) did not affect the expression of these chaperones. Conversely, pro-inflammatory cytokines added to β-cells caused a significant increase in the expression of Hsp90α and, substantially, Hsp70. Moreover, cells cultivated in the presence of PRDX6 were more susceptible to the cytokine effect. Thus, in the course of severe alloxan-induced T1D, no protective role of the heat shock proteins, was revealed, and their expression level was not increased by PRDX6. At the same time the protective potential of these proteins was shown in vitro with the use of RIN-m5F β cells. Thus, the system of heat shock proteins was unable to prevent the devastating effects of severe T1D accompanied by high animal mortality.

Relapsing-remitting experimental autoimmune encephalomyelitis (rEAE) in mice is a model that closely resembles relapsing-remitting multiple sclerosis in humans. This study aims to investigate a new approach to modulation of the inflammatory response in rEAE mice using a thymic peptide thymulin bound to polybutylcyanoacrylate (PBCA) nanoparticles. PBCA nanoparticles were used to prolong the presence of thymulin in the blood. Cytokine levels in blood were measured by ELISA; NF-κB and SAPK/JNK cascade activation, as well as Hsp72 and p53 protein expression, were measured by Western blotting. Animal health statuses were estimated using severity scores. Results showed that the cytokine response in rEAE was multi-staged: an early phase was accompanied by an increase in plasma interferon-γ, while the interleukin (IL)-17 response was markedly increased at a later stage. The stages were attributed to rEAE induction and maintenance phases. Thymulin significantly alleviated symptoms of rEAE and lowered plasma cytokine levels both in early and later stages of rEAE, and decreased NF-κB and SAPK/JNK cascade activation. Thymulin modulated NF-kappaB pathway activity via site-specific phosphorylation of RelA/p65 protein (at Ser276 and Ser536). The effect of nanoparticle-bound thymulin was more pronounced than the effect of free thymulin. Therefore, PBCA-thymulin can be considered a prospective treatment for this pathology.

Taking into account a special role of pancreatic β-cells in the development of diabetes mellitus, the effects of peroxiredoxin 6 (Prx6) on the viability and functional activity of rat insulinoma RIN-m5F β-cells were studied under diabetes-simulating conditions. For this purpose, the cells were cultured at elevated glucose concentrations or in the presence of pro-inflammatory cytokines (TNF-α and IL-1) known for their special role in the cytotoxic autoimmune response in diabetes. It was found that the increased glucose concentration of 23-43 mM caused death of 20-60% β-cells. Prx6 added to cells significantly reduced the level of reactive oxygen species and protected the RIN-m5F β-cells from hyperglycemia, reducing the death of these cells by several fold. A measurement of insulin secretion by the RIN-m5F β-cells showed a significant stimulatory effect of Prx6 on the insulin-producing activity of pancreatic β-cells. It should be noted that the stimulatory activity of Prx6 was detected during culturing the cells under both normal and unfavorable conditions. The regulation of the NF-κB signaling cascade could be one of the mechanisms of Prx6 action on β-cells, in particular, through activation of RelA/p65 phosphorylation at Ser536.

Aims: To initiate a state of artificial torpor we suggested a pharmacological multi-targeting strategy for simulation of the physiological pattern of natural hibernation including a significant reduction in heart rate, respiratory rate, body temperature and oxygen consumption as well as a decline in brain activity known as torpor., Materials and Methods: We have developed a composition which initiates a pharmacologically induced torpor-like state (PITS-composition), made up of eight therapeutic agents, inert gas xenon and lipid emulsion served as a drug vehicle., Key Findings: After a single intravenous injection to rats, PITS-composition causes a rapid decline in heart rate followed by a steady decrease in body temperature from about 38.5 °C to 31.5 °C, at ambient temperature of 22 °C-23 °C. The hypothermic state may continue on average for 16-17 h with the subsequent spontaneous return of heart rate and body temperature to the initial values. In the open field test at torpor the motility, rearing and grooming were suppressed but 4-8 days later they were restored., Significance: Suspended animation states, including natural hibernation or pharmacologically induced synthetic torpor are of special attention of medicine, since it may improve survival rate after cardiac arrest, brain hemorrhage and ischemia, and during long-term space traveling. The suggested here multi-targeting strategy made possible to develop the pharmacological composition able, after a single intravenous injection, to initiate long, stable and reversible hypothermia and torpor at room temperature. After the torpor, animals were able to spontaneously restore both physiological parameters, and behavioral reactions., (Copyright © 2019. Published by Elsevier Inc.)

Characteristics of the mouse model of relapsing-remitting experimental autoimmune encephalomyelitis (rEAE) closely resemble manifestations of multiple sclerosis in humans. In the present study, we investigated the mechanisms of inflammatory response, focusing on NF-κB pathway activation. Cytokine response in rEAE mice was multiphasic: the early phase was characterized by the increase in interferon-γ level in plasma. In the later stage, the level of interleukin-17, but not of interferon-γ, was increased. The early phase of rEAE was also accompanied by increased RelA/p65 phosphorylation at Ser276 in spleen cells, whereas the rEAE maintenance phase was characterized by RelA/p65 phosphorylation at Ser536 and IKK phosphorylation. The IKKα/β inhibitor reduced interleukin-17 and interferon-γ levels in plasma and alleviated rEAE symptoms. The IKKα/β inhibitor decreased IKK and p65(Ser536) phosphorylation, but doubled p65(Ser276) phosphorylation in rEAE mice. The increased RelA/p65(Ser276) phosphorylation coincided in time with the production of interferon-γ, Hsp72, and the early phase of IL-17 generation, whereas increased RelA/p65(Ser536) phosphorylation coincided with the activation of IKK, SAPK/JNK, and p53, as well as the late phase of IL-17 production, indicating the role of the RelA/p65 phosphorylation events in the induction and maintenance of rEAE., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We investigated the effects of weak combined magnetic fields (MFs) produced by superimposing a constant MF (in the range 30 - 150 µT) and an alternating MF (100 or 200 nT) on cytokine production in healthy Balb/C male mice exposed 2 h daily for 14 days. The alternating magnetic field was a sum of several frequencies (ranging from 2.5 - 17.5 Hz). The frequencies of the alternating magnetic field were calculated formally based on the cyclotron resonance of ions of free amino acids (glutamic and aspartic acids, arginine, lysine, histidine, and tyrosine). The selection of different intensity and frequency combinations of constant and alternating magnetic fields was performed to find the optimal characteristics for cytokine production stimulation in immune cells. MF with a constant component of 60 μT and an alternating component of 100 nT, which was a sum of six frequencies (from 5 to 7 Hz), was found to stimulate the production of tumor necrosis factor-α, interferon-gamma, interleukin-2, and interleukin-3 in healthy mouse cells and induce cytokine accumulation in blood plasma. Then, we studied the effect of this MF on tumor-bearing mice with solid tumors induced by Ehrlich ascite carcinoma cells by observing tumor development processes, including tumor size, mouse survival rate, and average lifespan. Tumor-bearing mice exposed to a combined constant magnetic field of 60 μT and an alternating magnetic field of 100 nT containing six frequencies showed a strong suppression of tumor growth with an increase in survival rate and enhancement of average lifespan.

In this study, we examined the effects of uridine on plasma cytokine levels, heat shock protein (HSP) 72 expression, and nuclear factor (NF)-κB signaling in spleen lymphocytes after exposure of male BALB/c mice to Escherichia coli lipopolysaccharide (LPS). Mice were treated with uridine (30 mg/kg body weight, intraperitoneal injection [i.p.]) or saline solution of LPS (2.5 mg/kg, i. p.). Endotoxin increased plasma levels of tumor necrosis factor-α, interferon-γ, interleukin (IL)-1, IL-2, and IL-6 by 2.1-, 1.9-, 1.7-, 1.6-, and 2.3-fold, respectively. Prior treatment with uridine prevented LPS-induced increases in all studied cytokines. In splenic lymphocytes, LPS treatment increased the expression of HSP 72 by 2.4-fold, whereas preliminary treatment with uridine completely prevented this effect. LPS also activated NF-κB signaling in splenic lymphocytes, and uridine decreased NF-κB pathway activity. Inhibitory analysis showed that the mechanism of uridine action was associated with the formation of the UDP-metabolic activator of the mitochondrial ATP-dependent potassium channel (mitoK ATP ) and the UTP-activator of glycogen synthesis in the tissues. A specific inhibitor of mitoK ATP , 5-hydroxydecanoate (5 mg/kg), and an inhibitor of glycogen synthesis, galactosamine (110 mg/kg), prevented the effects of uridine. Thus, uridine itself or uridine phosphates, which increased after uridine treatment, appeared to inhibit pro-inflammatory responses induced by LPS application. Overall, these findings demonstrated that the mechanisms mediating the effects of uridine were regulated by activation of glycogen synthesis and opening of the mitoK ATP , which in turn increased the energy potential of the cell and reduced oxidative stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.

In the present work, we aimed to study the effects of free and polybutylcyanoacrylate nanoparticle-bound thymulin on immune cell activity in mice with chronic inflammation. NF-κB, MAPK, and PKC-θ signaling pathway activity was assessed, alongside Hsp72, Hsp90-α, and TLR4 expression and levels of apoptosis. In addition, plasma cytokines and blood and brain melatonin and serotonin levels were measured. In mice treated with gradually raised doses of lipopolysaccharide, significant increases in the activity of the signaling pathways tested, heat-shock protein and TLR4 expression, lymphocyte apoptosis, and plasma proinflammatory cytokine levels were noted. Moreover, we observed significantly heightened serotonin concentrations in the plasma and especially the brains of mice with inflammation. In contrast, melatonin levels were reduced in the tissues examined, particularly so in the brain. Treatment of these mice with thymulin alleviated fever, reduced apoptosis, increased splenic cell number, and decreased cytokine production, Hsp72, Hsp90, and TLR4 expression, and the activity of the signaling pathways examined. In addition, thymulin partially restored brain and blood serotonin and melatonin levels. Thus, thymulin suppressed the proinflammatory response in LPS-treated mice, indicating the potential of thymulin co-therapy in the treatment of sepsis. Nanoparticle-bound thymulin was more effective in several respects., Competing Interests: The authors have declared that no competing interests exist.

The role of the p38 MAPK signaling cascade was studied in stress response of RAW 264.7 macrophages to extremely low-intensity centimeter microwaves. Irradiation stimulated production of a number of cytokines (IL-1, IL-6, TNF-α, INF-γ and IL-10), as well as induced activation of the signaling cascades NF- κB and p38 MAPK, and enhanced expression of Hsp72 heat shock protein. In the presence of the cascade p38 MAPK inhibitor (p38 MAP kinase inhibitor XI), the stimulating effects of electromagnetic waves were abrogated either completely (for NF-κB and Hsp72) or partially (for p38 MAPK and cytokines). The results obtained are indicative of a high sensitivity of the signaling cascade p38 MAPK to the effect of low-intensity physical fields.

Purpose: To clarify whether extremely low-level microwaves (MW) alone or in combination with p38 inhibitor affect immune cell responses to inhalation exposure of mice to low-level toluene., Materials and Methods: The cytokine profile, heat shock proteins expression, and the activity of several signal cascades, namely, NF-κB, SAPK/JNK, IRF-3, p38 MAPK, and TLR4 were measured in spleen lymphocytes of mice treated to air-delivered toluene (0.6 mg/m 3 ) or extremely low-level microwaves (8.15-18 GHz, 1μW/cm 2 , 1 Hz swinging frequency) or combined action of these two factors., Results: A single exposure to air-delivered low-level toluene induced activation of NF-κB, SAPK/JNK, IFR-3, p38 MAPK and TLR4 pathways. Furthermore, air toluene induced the expression of Hsp72 and enhanced IL-1, IL-6, and TNF-α in blood plasma, which is indicative of a pro-inflammatory response. Exposure to MW alone also resulted in the enhancement of the plasma cytokine values (e.g. IL-6, TNF-α, and IFN-γ) and activation of the NF-κB, MAPK p38, and especially the TLR4 pathways in splenic lymphocytes. Paradoxically, pre-exposure to MW partially recovered or normalized the lymphocyte parameters in the toluene-exposed mice, while the p38 inhibitor XI additionally increased protective activity of microwaves by down regulating MAPKs (JNK and p38), IKK, as well as expression of TLR4 and Hsp90-α., Conclusions: The results suggest that exposure to low-intensity MW at specific conditions may recover immune parameters in mice undergoing inhalation exposure to low-level toluene via mechanisms involving cellular signaling.

Thymic peptides are immune regulators produced mainly in the thymus. However, thymic peptides such as thymosin-α and thymopoietin have precursors widely expressed outside the thymus, localized in cell nuclei, and involved in vital nuclear functions. In stress-related conditions, they can relocalize. We hypothesized that another thymic peptide, thymulin, could be similarly produced by non-thymic cells during stress and have a precursor therein. Non-thymic cells, including macrophages and fibroblasts, were exposed to oxidative stress, heat, apoptosis, or necrosis. Extracellular thymulin was identified in media of both cell types 2 h after exposure to stress or lethal signals. Therefore, thymulin is released by non-thymic cells. To examine possible thymulin precursors in non-thymic cells, macrophage lysates were analyzed by western blotting. Bands stained with anti-thymulin antibody were detected in two locations, approximately 60 kDa and 10 kDa, which may be a possible precursor and intermediate. All of the exposures except for heat were effective for induction of the 10 kDa protein. BLAST search using thymulin sequence identified SPATS2L, an intranucleolar stress-response protein with molecular weight of 62 kDa, containing thymulin-like sequence. Comparisons of blots stained with anti-thymulin and anti-SPATS2L antibodies indicate that SPATS2L may be a possible candidate for the precursor of thymulin.

The aim of this study was to compare immune imbalances in "pre-diabetic" and diabetic mice and to evaluate the efficacy of several agents in improving the immunity of mice with type 1 diabetes. Pre-diabetic and diabetic models generated by a single or double alloxan injection were monitored for plasma glucose and pancreas immunohistochemistry. To study the immunity in pre-diabetic and diabetic Balb/C male mice; the levels of cytokines; synthesis of inducible heat shock proteins HSP72 and HSP90α; activity of the NF-κB, IFR3, SAPK/JNK, and TLR4 pathways; and apoptosis levels in thymuses were measured. Pre-diabetes resulted in a decrease in IL-4, IL-5 and IL-10 in plasma; in diabetic mice, plasma IFN-gamma, IL-6, TNF-alpha, and IL-10 were decreased. The NF-κB alternative pathway activity and TLR4 expression were significantly increased only in pre-diabetic mice, whereas SAPK/JNK activation was observed at both stages of diabetes. Other measured parameters also showed distinct altered patterns in the immunity of pre-diabetic and diabetic mice. Treatment with an inhibitor of NF-κB, thymulin, or a diet with an antioxidant improved or normalized the immune balance in diabetic mice and also notably decreased pancreatic cell damage in pre-diabetic mice., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To investigate some cellular and molecular aspects of the autoimmune response and anti-inflammatory efficiency of potential therapeutic agents in a severe form of experimental autoimmune encephalomyelitis (sEAE), an inhibitor of NF-kappaB signalling, IKK Inhibitor XII, and/or thymic peptide thymulin, were injected intraperitoneally at 1.8 and 0.15mg/kg e.o.d, respectively, to C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein and several adjuvants. The immunization induced high lethality in three weeks. The biphasic cytokine response observed in earlier and delayed phases was attributed to the activity of Th1 and Th17 cells, respectively. Phosphorylation of RelA protein from the NF-kappaB family increased during the earlier phase and decreased in the delayed phase. SAPK/JNK signalling protein and heat shock protein Hsp72 significantly increased in lymphocytes. Both the IKK Inhibitor XII and thymulin reduced disease severity, attenuated immune imbalance, and increased mouse life-span. Co-administration of the agents produced no additive effect. Both the inhibitor and thymulin reduced the Th1 response but not the Th17 response. Therefore, RelA-associated Th1 activation and RelA-independent Th17 activation occurred in sEAE. Thymulin and the inhibitor demonstrate similar patterns of activity, potentially through the RelA pathway inhibition, resulting in a partial therapeutic effect on the animals' health status., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: To investigate the role of the toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), and stress activated protein kinases/Jun N-terminal kinase (SAPK/JNK) signalling pathways in the responses of RAW 264.7 macrophages to low-intensity microwaves (MW)., Materials and Methods: Three inhibitors of TLR4, SAPK/JNK, and NF-κB signalling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before MW treatment., Results: MW exposure resulted in stimulation of RAW 264.7 cell activity manifested by increases in cytokine production and the stimulation of cell signalling. The blocking of a key kinase of the NF-κB pathway by IKK Inhibitor XII resulted in decreased MW-induced TLR4 expression and increased SAPK/JNK and NF-κB phosphorylation in irradiated cells. In addition, IKK Inhibitor XII significantly decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin 1α (IL-1α), interleukin 6 (IL-6), and interleukin 10 (IL-10) production in both exposed and unexposed RAW 264.7 macrophages. Inhibitor SP6000125 did not prevent an MW effect on signal proteins with the exception of decreased SAPK/JNK phosphorylation in RAW 264.7 cells. Cytokine production was markedly decreased in MW-exposed cells cultured with SP6000125. The inhibitor of TLR4, CLI-095, did not affect signal proteins and cytokine production changes in MW-exposed cells., Conclusions: The results suggest that low-intensity MW promotes macrophage activity via mechanisms involving cellular signalling, particularly the NF-κB pathway.

The role of casein kinase 2 (CK2) in the activation of the key NF-κB signaling cascade and other signaling and stress proteins during stress induced by exposure to nonthermal low-intensity electromagnetic radiation has been investigated. The ability of CK2 to regulate the activation of the NF-κB signaling cascade and upregulate the proinflammatory cytokine production and TLR4 receptor expression has been demonstrated. The existence of an alternative signaling pathway for NF-κB cascade activation directly involving protein kinase CK2 has been demonstrated using inhibition analysis in cells stressed by microwave irradiation.

The present study was designed to compare the anti-inflammatory effects of several agents applied in vivo, namely, a synthetic inhibitor of the NF-κB cascade, fat-soluble antioxidants, and the thymic peptide thymulin. Cytokine response in LPS-treated mice was analysed in tandem with the following parameters: the synthesis of inducible forms of the heat shock proteins HSP72 and HSP90α; activity of the NF-κB and SAPK/JNK signalling pathways; and TLR4 expression. Inflammation-bearing Balb/c male mice were pretreated with an inhibitor of IKK-α/β kinases (IKK Inhibitor XII); with thymulin; with dietary coenzyme Q9, α-tocopherol, and β-carotene; or with combinations of the inhibitor and peptide or antioxidants. Comparable anti-inflammatory effects were observed in inflammation-bearing mice treated separately with thymulin or with dietary antioxidants administered daily for two weeks before LPS treatment. When LPS-injected mice were treated with the inhibitor and antioxidants together, neither plasma cytokines, signal proteins, nor heat shock proteins recovered more efficiently than when mice were treated with these agents separately. In contrast to antioxidant diet, the thymulin was shown to increase the effect of IKK Inhibitor XII in preventing IKK activation in LPS-treated mice.

The present study was designed to examine and compare the effects of three suppressors on the cytokine response in tandem with examining: the synthesis of inducible forms of heat shock proteins; HSP72 and HSP90α; activities of NF-κB and SAPK/JNK signaling pathways; and TLR4 expression. Pre-treatment with inhibitors offers promise as protective means to lower the activity of these cascades, thereby circumventing the formation of excessive amounts of pro-inflammatory molecules. Three inhibitors of TLR4, SAPK/JNK, and NF-κB signaling, namely CLI-095, SP600125, and IKK Inhibitor XII, respectively, were added to cultured RAW 264.7 macrophages before the Escherichia coli lipopolysaccharide (LPS) application. Treatments of RAW 264.7 cells with each of the inhibitors resulted in a reduced response to LPS as was visualized by a decrease of TNF-α, IL-1, and IFN-γ production. In addition, inhibitors of the NF-κB and SAPK/JNK signaling reduced IL-6 production in LPS-treated cells, whereas the IKK inhibitor XII also decreased IL-10 production. Further, the NO production in LPS-stimulated macrophages was significantly reduced following application of CLI-095 or IKK inhibitor XII. The results also showed that the inhibitors suppressed TLR4 production and decreased phosphorylation of NF-κB and SAPK/JNK proteins, thereby preventing the activation NF-κB and SAPK/JNK signaling pathways in LPS-activated cells. In addition, the production of inducible heat shock proteins, HSP72 and HSP90-α, was reduced in LPS-stimulated RAW 264.7 cells pre-treated with inhibitors. These results suggest that inhibitors CLI-095, SP600125, and IKK inhibitor XII demonstrate potential effectiveness in the reduction of the inflammatory response by mechanisms involving both the cellular defense system and cellular signaling. In conclusion, suppressor of NF-κB cascade, IKK inhibitor XII, seems to be the most effective anti-toxic agent among studied inhibitors.

Modulation of autoimmune inflammation by the thymic peptides thymulin and thymopentin was studied in mice with acute experimental autoimmune encephalomyelitis (EAE), which resembles multiple sclerosis in humans. EAE was induced in NZW mice by a single immunisation with myelin basic protein coupled with adjuvants. Visible signs of pathology appeared on days 12-14 after the immunisation, peaked on days 20-25, were retained up to day 45, and then reverted. A biphasic cytokine response was also detected. In the "early" phase, which started at day 35, increased levels of interferon-gamma and interleukin-6 in the blood were observed; during the "delayed" phase, which started at day 48, the levels of plasma interleukin-17 and tumour necrosis factor-alpha were also raised. In addition, the phosphorylation of NF-kappaB signalling proteins and the production of heat shock protein Hsp72 were significantly increased in splenic lymphocytes from EAE-bearing mice. When applied intraperitoneally every other day for 30 days, either thymulin or thymopentin (15 μg per 100g of body weight) significantly reduced the disease severity compared to untreated EAE mice. The effect of thymulin but not thymopentin remained after its withdrawal. Thymulin reduced the cytokine response in both the early and the delayed phases, whereas thymopentin only reduced the "early phase cytokines" (IL-6 and interferon-gamma). Both peptides significantly reduced the level of phosphorylation of the NF-kappaB signalling protein IKK and the production of Hsp72 protein. The data presented here indicate the presence of time-dependent immune responses in EAE-bearing mice, which may be associated with the Th1 and Th17 subpopulations of T-cells. Thymulin and thymopentin demonstrated different patterns of activity, most likely via mechanisms involved in NF-kappa B signalling and Hsp72 expression., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Effects of four inhibitors of NF-kappaB, SAPK/JNK and TLR4 signaling, namely, inhibitor XII, SP600125, CLI-095 and Oxpapc on a macrophage response to low dose ammonium were studied in RAW 264.7 cells. Low dose ammonium induced pro-inflammatory response in cells as judged from enhanced production of TNF-alpha, IF-gamma, and IL-6, and by activation of signal cascades. The increase in production of cytokines, namely TNF, IFN, and IL-6, demonstrated that low-dose ammonium induced a pro-inflammatory cellular response. In addition, an activation of NF-kappaB and SAPK/JNK cascades, as well as enhancement of TLR4 expression was shown. Each of used inhibitors reduced to a variable degree the pro-inflammatory response of RAW 264.7 cells on chemical toxin by decreasing cytokine production. The inhibitor of NF-kappaB cascade, IKK Inhibitor XII, was more effective, and not only prevented the development of pro-inflammatory response induced by ammonium, but also decreased cytokine production below control values. The inhibitor of extra cellular domains of TLR2 and TLR4 (OxPAPC) had almost the same anti-inflammatory effect, and an addition of the inhibitor of JNK cascade (SP600125) to cell culture practically neutralized effect of ammonium ions by decreasing cytokine production to control level. Inhibitory analysis showed that activation of RAW 264.7 cells induced by chemical toxin coincide incompletely with intracellular signaling pathways that were early determined regarding macrophage's response to toxin from gram-negative bacteria. Nevertheless, application of the inhibitors defended RAW 264.7 from toxic effect of the low dose ammonium.

Objectives: The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation., Methods: The effects of two thymic peptides- thymulin and thymopentin were studied in cultured RAW 264.7 macrophages (lipopolysaccharide-stimulated or unstimulated) by measuring cytokine production and signal protein levels., Results: Both peptides increased proinflammatory cytokine secretion by unstimulated RAW 264.7 macrophages and these effects were blocked by the NF-κB cascade inhibitor, stress-activated protein kinase (SAPK)/JNK cascade inhibitor and, to a lesser extent, Toll-like 4 receptor activity inhibitor. In macrophages stimulated by bacterial lipopolysaccharide, peptides alone did not affect cytokine secretion, but significantly enhanced effects of each of the inhibitors. Thymopentin increased activation of both NF-κB and SAPK/JNK cascades in unstimulated macrophages, while thymulin significantly decreased activation of the SAPK/JNK but not NF-κB cascade in LPS-stimulated macrophages. Thymulin and thymopentin increased production of the heat shock protein HSP72 both in LPS-stimulated and unstimulated cells., Conclusions: Thymulin and thymopentin are effective anti-inflammatory modulators with direct actions on innate immune cells; the effects involve multiple signal cascades, including NF-κB and SAPK/JNK pathways. Since signaling cascades are now considered to be targets for new therapies, thymic peptides may be prospective modulators of signaling cascades, acting alone or in combination with other agents.

The involvement of heat shock protein Hsp90 in pro-inflammatory response in male NMRI mice under conditions of acute toxic stress, caused by lipopolysaccharide from Gram negative bacteria, was studied using geldanamycin, a specific blocker of the activity of this protein. It is shown that the introduction of geldanamycin lowers total intoxication of the organism upon acute toxic stress caused by endotoxin. Thus, a decrease in cytokine TNF-alpha, IFN-gamma, IL-1, and IL-10 concentrations in blood serum of the geldanamycin-treated animals with acute toxic stress was found along with normalization of functional activity of nitric oxide producing peritoneal macrophages. Studying expression of receptor protein Tlr-4 as well of proteins of two signal cascades, NF-kappaB and SAPK/JNK, has shown that mechanisms of the geldanamycin protective effect are realized at the level of inhibition of Tlr-4 receptor expression, which provides for endotoxin-to-cell binding, and due to lowering the endotoxin-stimulated activation of signal cascades NF-kappaB and SAPK/JNK. The results suggest Hsp90 might be a therapeutic target in diseases accompanied by acute toxic stress.

Effects of three chemical compounds: ammonia, diethyl ether, and acetic acid, known as common environmental contaminants in technogenic accidents, were investigated in vivo and in vitro in low concentrations. When added in cultivation media, each of the chemicals has affected peritoneal macrophages and spleen lymphocytes isolated from male NMRI mice and led to a rise in the production of several cytokines, particularly the tumor necrosis factor-alpha and interferon-gamma, as well as the expression of the inducible form of heat shock proteins (HSP72 and HSP90-alpha) and in the activation of signal cascades NF-kappaB and SAPK/JNK. The increase of the nitric oxide (NO) production in macrophages has been observed only when ammonia was added in cultivation media. Also, low concentrations of all compounds investigated led to the activation of the expression of receptor protein TLR4. When mice were exposed to airborne toxic contaminants in a hermetically sealed experimental chamber, an increase in the concentrations of cytokines, heat shock proteins, and signal proteins in immune cells was also observed in response to low concentrations of all chemicals investigated. Similarly to in vitro experiments, the NO production was augmented only in the presence of the airborne ammonia. The results indicate the environmental hazard of chemical contaminants even in rather low concentrations, which nevertheless lead to the stress response.

The effects of mixed dietary coenzyme Q(9), alpha-tocopherol, and beta-carotene on immune cell activity and blood cytokine profile were studied in peritoneal macrophages, spleen lymphocytes, and blood plasma from mice with acute inflammation induced by lipopolysaccharide (LPS). The activity of each fat-soluble antioxidant was also investigated separately in several model systems, both in vivo and in vitro. NMRI male mice were fed a diet supplemented with fat-soluble antioxidants for 15 days prior to LPS injection. LPS-induced inflammation resulted in induction of cellular production of pro-inflammatory cytokines such as TNF-alpha, IL-1alpha, IL-1beta, IL-2, IL-6, IFN-gamma, and also IL-10, an anti-inflammatory cytokine, and subsequent accumulation of these cytokines in blood plasma. In animals fed the antioxidant-rich diet, the inflammatory response to LPS injection was significantly reduced. The production of anti-inflammatory cytokine IL-10 in response to toxic stress and its accumulation in plasma were not modified by the diet. In addition, the expression of the inducible form of heat-shock protein 70 in mice treated with endotoxin was reduced in the animals pretreated with the antioxidant-rich diet. We showed that the diet suppressed phosphorylation of NF-kappaB, I kappaB kinase and SAPK/JNK proteins, thereby preventing the activation of the NF-kappaB kinase and SAPK/JNK signaling pathways in LPS-treated mice. In this report we demonstrate the potential effectiveness of naturally occurring antioxidant nutrients in the reduction of the inflammatory response. Therefore, it may be possible to develop novel therapeutic combinations, containing coenzyme Q(9), alpha-tocopherol, and beta-carotene, which promote immune stimulation.

The effect of low-intensity lases light (0.2 mW/cm2, 632.8 nm, exposure time 1 min) or centimeter waves (8.15-18 GHz, 1 W/cm2, exposure time 1 h) on PhosphoSAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes or cells incubated previously with geldanamycin, an inhibitor of heat shock protein 90 (HSP90), were used in the experiments. A significant stimulation of PhosphoSAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models. It was proposed that the activation of SAPK/JNK signal pathway plays one of the central roles in cellular stress response to low-power nonionizing radiation.

The effects of thymopentin, a synthetic analogue of the active center of the thymus hormone thymopoietin, on the immune status of mice with two different models of inflammation induced by injection of lipopolysaccaride (LPS) from gram-negative bacteria were studied. Acute inflammation was induced by a single injection of LPS in a dose of 250 microg/100 g of body weight, and chronic inflammation (sepsis) was modeled by a daily injection of LPS for 11 days with a gradual increase in the dose range from 25 to 250 microg/100 g of body weight. Under acute inflammation, a preliminary injection of thymopentin did not induce any additional stimulation in cytokine production increased by LPS. On the contrary, whereas the chronic introduction of LPS was characterized by a depressed production of several cytokines, thymopentin produced an immunostimulating effect. Thus, an increase in the production of nitric oxide, interferon-gamma, and Hsp70 was demonstrated. In addition, a more effective restoration of the number of thymus cells, as well as an increase in macrophage tumor necrosis factor-alpha production were observed after repeal of LPS + hormone injections. The results show that preliminary application of thymopentin promotes the regulation of immune cell activity under acute and chronic inflammation.

In vitro effects of low-level electromagnetic waves (8.18 GHz, frequency swings within 1 s, intensity 1 microW/cm, exposure for 1 h) and low-energy laser light (He-Ne laser with 632.8 nm, 0.2 mW/cm, dose 1.2 x 10(-2) J/cm2) on the expression of receptor protein TLR4, which is known as a part of the system for microbal toxin recognition, were studied in mouse lymphocytes. In addition, TLR4 expression was examined in situations when stress responses to low-level nonionizing radiation were modified by the antibiotic geldanamycin, which suppresses the activity of the heat shock protein Hsp90. It was found that low-level microwaves significantly raised the amount of TLR4; in contrast, laser light decreased the expression of the receptor in lymphocytes. In cells pretreated with geldanamycin, the TLR4 expression in irradiated cells was reduced to minimum levels, much lower than control values. The results showed that TLR4, which is involved in specific binding of toxin from gram-negative bacteria, can regulate cell responses to signals of other origin, in particular to nonionizig radiation, including low-level microwaves and laser light.

The effects of geldanamycin, which is known as inhibitor of heat shock protein 90 activities, on expression of several signal and heat shock proteins were studied by Western blot analysis in cultivated spleen lymphocytes isolated from male NMRI mice. It has been revealed that cultivating the cells with geldanamycin resulted in decrease in transcription factor NF-kappaB amount, as well as decrease in its phosphorylated form, pNF-kappaB, and lowering in its suppressor, IkappaB-alpha. Besides, cells cultivated with geldanamycin demonstrated significant decrease in the amount of protein kinase SAPK(JNK). The modifications in signal pathways, which had been induced by geldanamycin, pointed to direct influence of the antibiotics on cellular stress response to damaging impact. This assumption was examined with the model of cellular stress response induced by low-level laser radiation. It was proved that Hsp90-binding drug, geldanamycin, significantly decreased in vitro stress response to laser light via lowering the production of heat shock proteins, Hsp70 and Hsp25, both in irradiated lymphocytes and in theirs intracellular structures. These findings show the prospect for using of geldanamycin in various therapies that are compromised with objectionable side effects manifested as heightened stress response in immune cells.

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.

The effects of centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 10 days; MW) on the production of the tumor necrosis factor alpha, interleukin-lalpha, interleukin-1beta, interleukin-2, and the expression of interleukin-6, interleukin-10, interferon-gamma, nitric oxide and HSP27, HSP72 and HSP90alpha in mice irradiated before or after LPS injection were studied. An acute endotoxic model was produced by a single LPS injection. The effects of microwaves on nitric oxide, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were dependent on the functional status of exposed animals. Thus, an exposure of healthy mice to microwaves for 10 days was followed by a decrease in nitric oxide and interferon-gamma production, and an increase in the production of the tumor necrosis factor-alpha and interleukin-6. On the contrary, an exposure to MW before intoxication resulted in an increase in the synthesis of nitric oxide and interferon-gamma as well as a decrease in the concentration of the tumor necrosis factor-alpha and interleukin-6 in blood of mice in endotoxic shock. When microwave exposure was used after LPS injection, it did not provide any protective effect, and preliminary irradiation enhanced the resistance of the organism to endotoxic shock.

The effects of low-intensity laser radiation (LILR, 632.8 nm, 0.2 mW/cm2) and low-intensity electromagnetic waves (LIEW, 8.15 - 18 GHz, 1 MW/cm2) on the production of transcription factors in lymphocytes from NMRI male mice were examined. The total level of NF-KB and its phosphorylated metabolite Phospho-NF-kappaB, as well as the regulatory protein IkappaB-alpha were determined in spleen lymphocytes subjected to laser or microwave radiations. The proteins were determined by immunoblotting. Laser light induced a lowering in the level of NF-kappaB and IkappaB-alpha. By contrast, irradiation with electromagnetic waves resulted in a significant increase in the amount of NF-kappaB and IkappaB-alpha. The phosphorylated form of NF-kappaB did not noticeably change under either of the two kinds of radiation. The results showed that electromagnetic waves activate the production of both NF-kappaB and the regulatory protein IkappaB-alpha and these data confirm the stress character of the response of spleen lymphocytes to low-level microwaves of the centimeter range.

The effect of preliminary short-term irradiation with He-Ne laser light (632.8 nm, 0.2 mW/cm2) of the thymus zone projection of male NMRI mice subjected to acute toxic stress on the responses of immune cells was studied. Stress was modeled by lipopolysaccharide injection, 250 mg/100 g of body weight, which induced a significant increase in the production of several macrophage cytokines, IL-1alpha, IL-1beta, IL-6, IL-10 and TNF-alpha. A single irradiation with laser light did not provoke considerable variations in NO production in cells but induced an enhancement in the production of heat shock proteins Hsp25, Hsp70, and Hsp90. Nevertheless, when irradiation with red laser light was applied prior to toxic stress, considerable normalization of production of nearly all cytokines studied and nitric oxide was observed. Moreover, the normalization of production of heat shock proteins has been shown in these conditions. Thus, preliminary exposure of a small area of animal skin surface provoked a significant lowering in the toxic effect of lipopolysaccharide.

The effect of in vitro exposure to low-power laser light with a power density of 0.2 mW/cm2 and a wavelength of 632.8 nm induced by helium-neon laser on the functional activity of macrophages and splenic lymphocytes was studied. If the exposure period did not exceed 60 sec, the stimulation in interleukin-2 (IL-2) and nitric oxide (NO) production, as well as an increase in the activity of natural killer cells were observed. The increase of irradiation dose by prolongation of the exposure duration up to 180 s induced a significant decrease in NO production and natural killer cell activity, but IL-2 production was not different from the control level. A remarkable decrease in interferon-gamma (IFN-gamma) production was observed following laser light exposure of cells for 60 or 180 s, whereas under lower doses (exposure for 5 or 30 sec) IFN-gamma production increased. Irradiation of isolated macrophages induced a significant stimulation of cellular tumor necrosis factor-alpha (TNF- alpha) production at all dboes used, and, what is more important, an enhancement in both TNF-a phaand interleukin-6 (IL-6) production was revealed as early as after a 5-s exposure. In this case, more prolonged exposure periods, 60 and 180 s, either did not induce changes in IL-6 production (in macrophages) or decreased IL-6 production (in lymphocytes). Thus, upon in vitro exposure of cells to extremely low-power laser light, a basic tendency was observed: short-term irradiation predominantly induced stimulation in secretory activity of cells, whereas prolongation of exposure mainly induced immunosuppression. The only exception to the rule was a change in interleukin-3 (IL-3) production, which decreased after short-time exposure, but, on the opposite, increased when the cells were exposed for 180 s. In addition, a high sensitivity to extremely low-power laser light was supported by expression of the inducible heat shock protein, Hsp70, the effect being observed at all doses used, including the exposure for 5 s. At the same time, expression of another heat shock protein, Hsp90, was somewhat reduced after irradiation of cells with laser light.

Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-alpha, TNF-beta), interferon-gamma (IFN-gamma), interleukin-2, -3, -6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-alpha in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-alpha, TNF-beta, and NO in mouse cells. The production of other cytokines (interleukins and IFN-gamma) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.