Rat Peritoneal Macrophages Research Articles

Sesamol protects against LPS-induced inflammation in rat peritoneal macrophages by promoting SIRT1-induced repression of NF-κB.

Sesamol, a polyphenolic compound isolated from roasted sesame seeds exhibits significant anti-inflammatory effect, but the molecular mechanism is poorly understood. Peritoneal macrophages play a pivotal role in the control of infections and inflammatory pathologies and are also found in injured tissuesalong with resident macrophages. The present study aimed to examine the anti-inflammatory effectof sesamol and the molecular mechanisms involved, particularly the role of sesamol in modulating SIRT1- and SIRT1-mediated deacetylation of NF-κB p65 using in vivo activated peritoneal macrophages. Sprague Dawley rats were injected with LPS to induce inflammation and sesamol was intraperitoneally administered to study its anti-inflammatory effect. ELISA and real time PCR were used to study the expression of proinflammatory cytokines. Effects of sesamol on iNOS and COX-2 were studied with activity assays and ELISA. ICAM-1, MMP-9 and TIMP-1 expressions were analysed by ELISA, RT PCR and zymography. Western blot analysis was performed to determine p65 acetylation. Nuclear translocation of p65 was evaluated by ELISA. The gene and protein expression of SIRT1 was analysed with ELISA and real time PCR. Sesamol downregulated the expression of proinflammatory markers TNF-α, IL-6, iNOS, COX-2, TLR-4, ICAM-1 and MMP-9 in rat peritoneal macrophages. Additionally, sesamol upregulated SIRT1expression and attenuated the nuclear translocation of NF-κB p65 by promoting its deacetylation. Inhibition of SIRT1 by its specific inhibitor EX527 diminished the inhibitory effect of sesamol on TNF-α and IL-6. Moreover, EX527 reduced the suppressive impact of sesamol on p65 acetylation and subsequent nuclear translocation. Our findings suggest that the anti-inflammatory effect of sesamol involves upregulation of SIRT-1, leading to the downregulation of the nuclear translocation of NF-κB p65 through its deacetylation. Therefore, the dietary bioactive compound sesamol shows potential as a promising strategy for preventing inflammatory diseases by modulating SIRT1 expression.

The role of PI3K-Akt-mTOR axis in Warburg effect and its modification by specific protein kinase inhibitors in human and rat inflammatory macrophages

The Warburg effect occurs both in cancer cells and in inflammatory macrophages. The aim of our work was to demonstrate the role of PI3K-Akt-mTOR axis in the Warburg effect in HL-60 derived, rat peritoneal and human blood macrophages and to investigate the potential of selected inhibitors of this pathway to antagonize it. M1 polarization in HL-60-derived and human blood monocyte-derived macrophages was supported by the increased expression of NOS2 and inflammatory cytokines. All M1 polarized and inflammatory macrophages investigated expressed higher levels of HIF-1α and NOS2, which were reduced by selected kinase inhibitors, supporting the role of PI3K-Akt-mTOR axis. Using Seahorse XF plates, we found that in HL-60-derived and human blood-derived macrophages, glucose loading reduced oxygen consumption (OCR) and increased glycolysis (ECAR) in M1 polarization, which was antagonized by selected kinase inhibitors and by dichloroacetate. In rat peritoneal macrophages, the changes in oxidative and glycolytic metabolism were less marked and the NOS2 inhibitor decreased OCR and increased ECAR. Non-mitochondrial oxygen consumption and ROS production were likely due to NADPH oxidase, expressed in each macrophage type, independently of PI3K-Akt-mTOR axis. Our results suggest that inflammation changed the metabolism in each macrophage model, but a clear relationship between polarization and Warburg effect was confirmed only after glucose loading in HL-60 and human blood derived macrophages. The effect of kinase inhibitors on Warburg effect was variable in different cell types, whereas dichloroacetate caused a shift toward oxidative metabolism. Our findings suggest that these originally anti-cancer inhibitors may also be candidates for anti-inflammatory therapy.

Retraction: Functional mechanism of AMPK activation in mitochondrial regeneration of rat peritoneal macrophages mediated by uremic serum.

Retraction: Functional mechanism of AMPK activation in mitochondrial regeneration of rat peritoneal macrophages mediated by uremic serum.

Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway.

Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved. Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process. To assess the influence of interleukin-10 (IL-10) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) following their interaction with macrophages in an inflammatory environment. IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment. In this study, we investigated its impact on the proliferation, migration, and osteogenesis of BMSCs. The expression levels of signal transducer and activator of transcription 3 (STAT3) and its activated form, phosphorylated-STAT3, were examined in IL-10-stimulated macrophages. Subsequently, a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling. IL-10-stimulated macrophages underwent polarization to the M2 type through substitution, and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs. Mechanistically, STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages. Specifically, IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response, as evidenced by its diminished impact on the osteogenic differentiation of BMSCs. Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs. The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs' osteogenic differentiation.

АНТАГОНИСТ РЕЦЕПТОРОВ СИГМА-1, СОЕДИНЕНИЕ BD-1063, ПОДАВЛЯЕТ Са2+-ОТВЕТЫ, ВЫЗЫВАЕМЫЕ МОЛИКСАНОМ В МАКРОФАГАХ

Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Acting as chaperones, sigma-1 receptors modulate a wide range of cellular processes in health and disease, including Ca2+ signaling processes. The pharmacological analogue of oxidized glutathione, drug molixan®, is used as an immunomodulator and cytoprotector in the complex therapy of bacterial, viral and oncological diseases; effective in the prevention and treatment of coronavirus infection COVID-19. To elucidate the involvement of sigma-1 receptors in the effect of molixan on the intracellular Ca2+ concentration in macrophages and in the regulation of Ca2+ signaling processes in macrophages in general, the effect of the sigma-1 receptor selective antagonist, compound BD-1063, on Ca2+ responses induced by molixan in rat peritoneal macrophages was investigated. Using Fura-2AM microfluorimetry we have shown for the first time that compound BD-1063 significantly suppresses both Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by molixan in peritoneal macrophages. The data obtained indicate the involvement of sigma-1 receptors in the complex signaling cascade triggered by molixan and leading to intracellular Ca2+ concentration increase in macrophages. The results also suggest the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Novel trifluoromethyl ketone derivatives as oral cPLA2/COX-2 dual inhibitors for resolution of inflammation in rheumatoid arthritis

Thirty-five trifluoromethyl hydrazones and seventeen trifluoromethyl oxime esters were designed and synthesized via molecular hybridization. All the target compounds were initially screened for in vitro anti-inflammatory activity by assessing their inhibitory effect on NO release in LPS-stimulated RAW264.7 cells, and the optimal compound was finally identified as 2-(3-Methoxyphenyl)-N'-((6Z,9Z,12Z,15Z)-1,1,1-trifluorohenicosa-6,9,12,15-tetraen-2-ylidene)acetohydrazide (F26, IC50 = 4.55 ± 0.92 μM) with no cytotoxicity. Moreover, F26 potently reduced the production of PGE2 in LPS-stimulated RAW264.7 cells compared to indomethacin. The interaction of F26 with COX-2 and cPLA2 was directly verified by the CETSA technique. F26 was found to modulate the phosphorylation levels of p38 MAPK and NF-κB p65, as well as the protein expression of IκB, cPLA2, COX-2, and iNOS in LPS-stimulated rat peritoneal macrophages. Additionally, F26 was observed to prevent the nuclear translocation of NF-κB p65 in LPS-stimulated rat peritoneal macrophages by immunofluorescence localization. Therefore, the aforementioned in vitro experiments demonstrated that F26 blocked the p38 MAPK and NF-κB pathways by binding to COX-2 and cPLA2. In the adjuvant-induced arthritis model, F26 demonstrated a significant effect in preventing arthritis symptoms and inflammatory status in rats, exerting an immunomodulatory role by regulating the homeostasis between Th17 and Treg through inhibition of the p38 MAPK/cPLA2/COX-2/PGE2 and NF-κB pathways. Encouragingly, F26 caused less acute ulcerogenicity in rats at a dose of 50 mg/kg compared to indomethacin. Overall, F26 is a promising candidate worthy of further investigation for treating inflammation and associated pain with lesser gastrointestinal irritation, as well as other symptoms in which cPLA2 and COX-2 are implicated in the pathophysiology.

Ethyl Acetate Fraction of Momordica Charantia L. Fruits Induce the Phagocytosis Activity and Capacity of Rat Peritoneal Macrophages

Phagocytosis is one of the body's immune mechanisms in the elimination of antigens, including bacteria. Its mechanism is affected by many factors, such as the inducers. The present study aims to elaborate on the effect of ethyl acetate fraction of Momordica charantia L fruits on the activity and capacity of rat peritoneal macrophages on a non-A Staphylococcus aureus, including the white blood cell percentage. A series of M. charantia ethyl acetate extract concentrations of 25 mg/kg BW, 50 mg/kg BW, and 100 mg/kg BW were used to identify the phagocytosis activity and capacity, as well as the percentage of white blood cells (WBC). The result showed that the concentration of 50 mg/kg BW had the highest phagocytic activity and capacity compared to other concentrations (P < 0.05), while for the WBC percentage, there were no significant differences among the concentrations (P > 0.05). The conclusion of the present study is M. charantia ethyl acetate extract has the potential to be used as a natural immunostimulant.

Exosomes Derived From Cerulein-Stimulated Pancreatic Acinar Cells Mediate Peritoneal Macrophage M1 Polarization and Pyroptosis via an miR-24-3p/MARCH3/NLRP3 Axis in Acute Pancreatitis.

Acute pancreatitis (AP) has a high incidence of hospitalizations, morbidity, and mortality worldwide. A growing number of studies on AP pathogenesis are based on cerulein-induced experimental model, which simulates human AP in vivo. It has been demonstrated that both pancreatic acinar cells and peritoneal macrophages are involved in pancreatic inflammation and damage. However, their connection has not been well understood. A cerulein-induced AP model was established on the pancreatic acinar cell line AR42J. Rat macrophages were isolated from the peritoneal cavity. The effects of cerulein-induced pancreatic exosomes on the peritoneal macrophage and pancreas in vivo and in vitro were examined. The underlying molecular mechanism was investigated by exploring the regulatory role of downstream molecules. We found that exosomes derived from cerulein-treated AR42J cells induced rat peritoneal macrophage M1 polarization and pyroptosis. miR-24-3p was upregulated in cerulein-stimulated exosomes, whereas the miR-24-3p inhibitor counteracted the effect of pancreatic exosomes on peritoneal macrophage M1 polarization and pyroptosis. Furthermore, miR-24-3p inhibited March3 expression, whereas MARCH3 mediated NLRP3 ubiquitination in rat peritoneal macrophages, which, in turn, contributed to the apoptosis, reactive oxygen species production, and inflammation in AR42J cells. Exosomes derived from cerulein-stimulated pancreatic acinar cells mediate peritoneal macrophage M1 polarization and pyroptosis via an miR-24-3p/MARCH3/NLRP3 axis in AP.

Tricyclic antidepressant amitriptyline attenuates Ca<sup>2+</sup> responses in rat peritoneal macrophages

Amitriptyline is a tricyclic antidepressant widely used in clinical practice for the treatment of anxiety, depression and chronic pain. These drugs have a multifaceted effect on cellular processes. One of their targets is sigma-1 receptors. Sigma-1 receptors are molecular chaperones located in endoplasmic reticulum membrane; they are characterized by a unique structure and pharmacological profile. Sigma-1 receptors regulate many cellular processes in health and disease, including Ca2+ signaling. Using Fura-2AM microfluorimetry, it was shown for the first time that sigma-1 receptor agonist, antidepressant amitriptyline, significantly suppresses both Ca2+ mobilization from intracellular Ca2+-stores and subsequent store-dependent Ca2+ entry into cells, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, as well as disulfide-containing immunomodulators glutoxim and molixan, in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in a complex signaling cascade induced by glutoxim or molixan, leading to an increase of intracellular Ca2+ concentration in macrophages. The results also indicate the participation of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Antioxidative and anti-inflammatory activities of Erica spiculifolia extracts and fractions

There is little data on the phytochemical/pharmacological properties of Erica spiculifolia Salisb. (syn. Bruckentalia spiculifolia (Salisb.) Rchb.). This study examines the antioxidative and anti-inflammatory activities of different extracts and fractions of E. spiculifolia in vitro on isolated rat peritoneal macrophages, in the carrageenan-induced rat paw oedema test, BSA test, and two complementary antioxidant assays. Ethanolic extracts of leaves, flowers, and aboveground parts, and petroleum ether, ether, ethyl acetate, and water fractionations of the ethanol extract of E. spiculifolia applied at doses of 50–200 mg/kg p.o. exhibited dose-dependent anti-inflammatory activity comparable with indomethacin. All tested samples, except for the petroleum ether fraction, exerted excellent in vitro antioxidant activity, and all of them exhibited significant and similar inhibition of BSA denaturation comparable with diclofenac. Ethanolic extract of the aboveground parts obtained by percolation, ethyl acetate and water fractions had the highest efficiency, attenuating inflammation by more than 50% in the lowest applied concentration alongside exceptional radical scavenging activity.

How do different bile acid derivatives affect rat macrophage function – Friends or foes?

Due to an increased need for new immunomodulatory agents, many previously known molecules have been structurally modified in order to obtain new drugs, preserving at the same time some of the benevolent characteristics of the parent molecule. This study aimed to evaluate the immunomodulatory potential of a selected library of bile acid derivatives (BAD) using a broad spectrum of assays, evaluating rat peritoneal macrophages viability, cell membrane damage, lysosomal and adhesion function, and nitric oxide and cytokine production as a response to lipopolysaccharide stimulation. Also, in silico studies on two bile acid-activated receptors were conducted and the results were related to the observed in vitro effects. All tested BAD exerted significant toxicity in concentrations higher than 10 μM, which was determined based on mitochondria and cell membrane damage in a panel of assays. On the other hand, at lower concentrations, the tested BAD proved to be immunomodulatory since they affected lysosomal function, cell adhesion capacities and the ability to produce inflammatory cytokines in response to a stimulus. One of the compounds proved to exhibit significant toxicity toward macrophages, but also caused a concentration-dependent decrease in nitric oxide levels and was identified as a potential farnesoid X receptor agonist.

Haloperidol, a sigma-1 receptor antagonist, inhibits Ca<sup>2+</sup> responses in rat peritoneal macrophages

Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane and have a unique history, structure, and pharmacological profile. Sigma-1 receptors modulate a wide range of cellular processes in health and disease and Ca2+ signaling processes. Using the Ca2+ sensitive fluorescent ratiometric dye Fura-2AM, it has been shown that neuroleptic (haloperidol, an antagonist of sigma-1 receptors) significantly suppresses Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by thapsigargin and cyclopiazonic acid, as inhibitors of endoplasmic reticulum Ca2+-ATPases, and also by glutoxim and molixan, disulfide-containing immunomodulatory drugs, in rat peritoneal macrophages. Findings of the study revealed that sigma-1 receptors are involved in the complex signaling cascade triggered by glutoxim or molixan that leads to the increase in the intracellular Ca2+ concentration in macrophages and participate in regulation of store-dependent Ca2+ entry in macrophages.

Iristectorigenin C suppresses LPS-induced macrophages activation by regulating mPGES-1 expression and p38/JNK pathway

Ethnopharmacological relevanceNonsteroidal anti-inflammatory drugs (NSAIDs) have been used clinically to treat inflammatory diseases clinically. However, the adverse effects of NSAIDs cannot be ignored. Therefore, it is critical for us to find alternative anti-inflammatory drugs that can reduce adverse reactions to herbal medicine, such as Iris tectorum Maxim., which has therapeutic effects and can treat inflammatory diseases and liver-related diseases. Aim of the studyThis study aimed to isolate active compounds from I. tectorum and investigate their anti-inflammatory effects and action mechanisms. Materials and methodsFourteen compounds were isolated from I. tectorum using silica gel column chromatography, Sephadex LH-20, ODS and high performance liquid chromatography, and their structures were identified by examining physicochemical properties, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. Classical inflammatory cell models were established using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and rat primary peritoneal macrophages to examine the effect of these compounds. To examine the action mechanisms, the nitric oxide (NO) levels were measured by Griess reagent and the levels of inflammatory cytokines in the supernatant were measured by ELISA; The expressions of major proteins in prostaglandin E2 (PGE2) synthesis and the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were examined by Western blotting, and the mRNA expression levels were measured by quantitative real-time polymerase chain reaction; and the nuclear translocation of p65 was examined by high content imaging. Molecular docking was used to predict the binding of active compound to target protein. ResultsOur findings revealed that Iristectorigenin C (IT24) significantly inhibited the levels of NO and PGE2 without affecting cyclooxygenase (COX)-1/COX-2 expression in LPS-induced RAW264.7 cells and rat peritoneal macrophages. Furthermore, IT24 was shown to decrease the expression of microsomal prostaglandin synthetase-1 (mPGES-1) in LPS-induced rat peritoneal macrophages. IT24 did not suppress the phosphorylation and nuclear translocation of proteins in the NF-κB pathway, but it inhibited the phosphorylation of p38/JNK in LPS-stimulated RAW264.7 cells. Additionally, molecular docking analysis indicated that IT24 may directly bind to the mPGES-1 protein. ConclusionIT24 might inhibit mPGES-1 and the p38/JNK pathway to exert its anti-inflammatory effects and could be also developed as an inhibitor of mPGES-1 to prevent and treat mPGES-1-related diseases, such as inflammatory diseases, and holds promise for further research and drug development.

Ginsenosides enhance P2X7-dependent cytokine secretion from LPS-primed rodent macrophages

The activation of P2X7 is a well-known stimulus for the NLRP3-caspase 1 inflammasome and subsequent rapid IL-1β secretion from monocytes and macrophages. Here we show that positive allosteric modulators of P2X7, ginsenosides, can enhance the release of three important cytokines, IL-1β, IL-6 and TNF-α from LPS-primed rodent macrophages using the J774 mouse macrophage cell line and primary rat peritoneal macrophages. We compared the immediate P2X7 responses in un-primed and LPS-primed macrophages and found no difference in calcium response amplitude or kinetics. These results suggest that under inflammatory conditions positive allosteric modulators are capable of increasing cytokine secretion at lower concentrations of ATP, thus boosting the initial pro-inflammatory signal. This may be important in the control of intracellular infections.

The effect of hyperglycemia on the activation of peritoneal macrophages of albino rats.

Hyperglycemia is one of the main damaging factors of diabetes mellitus (DM). The severity of this disease is most clearly manifested under conditions of the inflammatory process. In this work, we have studied the activation features of rat peritoneal macrophages (MPs) under conditions of high glucose concentration in vitro. Comparison of the independent and combined effects of streptozotocin-induced DM and hyperglycemia on proliferation and accumulation of nitrites in the MPs culture medium revealed similarity of their effects. Elevated glucose levels and, to a lesser extent, DM decreased basal proliferation and NO production by MPs in vitro. The use of the protein kinase C (PKC) activator, phorbol ester (PMA), abolished the proinflammatory effect of thrombin on PMs. This suggests the involvement of PKC in the effects of the protease. At the same time, the effect of thrombin on the level of nitrites in the culture medium demonstrates a pronounced dose-dependence, which was not recognized during evaluation of proliferation. Proinflammatory activation of MPs is potentiated by hyperglycemia, one of the main pathological factors of diabetes. Despite the fact that high concentrations of glucose have a significant effect on proliferation and NO production, no statistically significant differences were found between the responses of MPs obtained from healthy animals and from animals with streptozotocin-induced DM. This ratio was observed for all parameters studied in the work, during analysis of cell proliferation and measurement of nitrites in the culture medium. Thus, the results obtained indicate the leading role of elevated glucose levels in the regulation of MPs activation, which is comparable to the effect of DM and even "masks" it.

Regulation of SIRT1-TLR2/TLR4 pathway in cell communication from macrophages to hepatic stellate cells contribute to alleviates hepatic fibrosis by Luteoloside

Regulating macrophage-hepatic stellate cells (HSCs) crosstalk through SIRT1-TLR2/TLR4 has contributed to the essence of new pharmacologic strategies to improve hepatic fibrosis. We investigated how Luteoloside (LUT), one of the flavonoid monomers isolated from Eclipta prostrata (L.) L., modulates macrophage-HSCs crosstalk during hepatic fibrosis. HSC-T6 or rat peritoneal macrophages were activated by TGF-β or LPS/ATP, and then treated with LUT or Sirtinol (SIRT1 inhibitor) for 6 h. Further, HSCs were cultured with the conditioned medium from the LPS/ATP activated peritoneal macrophages. In HSC-T6 or peritoneal macrophages, LUT could decrease the expressions of α-SMA, Collagen-I, the ratio of TIMP-1/MMP-13. LUT also significantly increased the expressions of SIRT1 and ERRα. And LUT significantly suppressed the releases of pro-inflammatory cytokines, including NLRP3, ASC, caspase-1, IL-1β, and regulated signaling TLR2/TLR4-MyD88 activation. The expressions of TLR2, TLR4, NLRP3, caspase-1, IL-1β, α-SMA were increased and the expression of ERRα was decreased by Sirtinol, indicated that LUT might mediate SIRT1 to regulate TLR4 expression and further alleviate inflammation and fibrosis. LUT could regulate SIRT1-mediated TLR4 and ECM in HSCs was reduced, when HSCs were cultured with conditioned medium. Hence, LUT could decrease the expressions of fibrosis markers, reduce the releases of inflammatory cytokines in activated HSCs or macrophages. In conclusion, LUT might be a promising candidate that regulating SIRT1-TLR2/TLR4 signaling in macrophages interacting with HSCs during hepatic fibrosis.

Decreasing effects of protein kinase inhibitors on the expression of NOS2 and inflammatory cytokines and on phagocytosis in rat peritoneal macrophages is partly related to repolarization

The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) pathway plays a pivotal role in macrophage polarization, but other signaling routes may also be involved. The aim of this study was to reveal the relationship of activation between rat peritoneal macrophages and their polarization, to detect the signaling routes involved, and find selective protein kinase inhibitors decreasing the production of inflammatory proteins in activated peritoneal macrophages. Rat macrophages were elicited with i.p. casein injection. CD80 and CD206 markers, NOS2 (Nitric oxide synthase 2), arginase, cytokines and phagocytosis were investigated by ELISA (Enzyme Linked Immunosorbent Assay), Western Blot, fluorescent microscopic and flow cytometry. Statistical methods were ANOVA (Analysis Of Variance) and Student t-tests. Resident and elicited cells expressed both CD80 and CD206 polarization markers. The involvement of MAPK (mitogen-activated protein kinases) and JAK/STAT pathways in the polarization was evidenced by a phosphorylation array, supported by Western blotting, by cytokine markers and by the inhibitory effects of kinase inhibitors. The expression of NOS2 and inflammatory cytokines was higher in elicited cells suggesting their M1 polarization. This effect was reduced by the inhibitors of MAPK and JAK/STAT pathways. Phagocytosis was also higher in elicited macrophages and decreased by these inhibitors. Nevertheless, they cannot change macrophage polarization unambiguously, as levels of CD80 and CD206 markers were not changed. For comparison, human blood macrophages were also studied. Similar effects and several differences were observed between the two types of macrophages, suggesting the role of the previous differentiation in defining their characteristics. Selected anti-cancer protein kinase inhibitors of p38, MAPK and JAK/STAT pathways are possible candidates for the therapy of inflammatory diseases.

Нейролептики подавляют Са2+-ответы, вызываемые глутоксимом и моликсаном в макрофагах

Haloperidol (butyrophenone derivative) and phenothiazine derivatives chlorpromazine and trifluoperazine belong to the typical first-generation neuroleptics and are widely used in clinical practice for treatment of schizophrenia and other mental diseases. Their multifaceted effect on cellular processes is well known. Thus, a high affinity of typical neuroleptics for sigma-1 receptors was revealed. Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Acting as chaperones, sigma-1 receptors modulate a wide range of cellular processes in health and disease, including Ca2+ signaling processes. To elucidate the involvement of sigma-1 receptors in Ca2+ signaling processes in macrophages, the effect of sigma-1 receptor ligands on Ca2+ responses induced by disulfide-containing immunomodulators glutoxim and molixan in rat peritoneal macrophages was investigated. Using Fura-2AM microfluorimetry we have shown that haloperidol, chlorpromazine and trifluoperazine significantly suppress both Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by glutoxim or molixan in peritoneal macrophages. The data obtained indicate the possible involvement of sigma-1 receptors in the complex signaling cascade triggered by glutoxim or molixan and leading to intracellular Ca2+ concentration increase in macrophages. The results also suggest the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

The involvement of peroxisome proliferator-activated receptor gamma (PPARγ) in anti-inflammatory activity of N-stearoylethanolamine

BackgroundN-stearoylethanolamine (NSE) is a bioactive lipid amine with a wide range of biological activities. Anti-inflammatory properties of NSE were previously confirmed on multiple animal models. However, the molecular mechanisms of anti-inflammatory action of NSE remain unclear. In the current study, we examined the involvement of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the NF-kB –dependent pathway of anti-inflammatory action of NSE using different methodological approaches. MethodsMolecular modeling calculated the possibility of NSE binding PPAR. Ex vivo experiment, using selective agonist of PPARα/γ - LY-171883 and antagonist of PPARγ - GW9662, examined the role of PPARα/γ in the NSE’s effect on nuclear NF-kB translocation in LPS-activated rat peritoneal macrophages. Finally, the NSE’s action on mRNA level of PPARγ-dependent genes was studied in the liver of insulin-resistant rats. ResultsThe results of molecular docking showed that NSE could bind to PPARγ and compete for the binding site with antagonist GW9662 and agonist LY-171883. These data was supported by in vitro study where pre-treatment with NSE prevented further LPS-induced NF-kB translocation into the nuclei of rat peritoneal macrophages. NSE treatment before GW9662 and LPS addition normalized the level of NF-kB translocation and IL-1β content. This finding confirmed a competitive binding of NSE with GW9662 for the ligand-binding domain of PPARγ. Additional in vivo study showed that NSE administration changed the mRNA expression of several PPARγ target genes, including SLC27A1 encoding fatty acid transport protein-1 and IL1RN - interleukin-1 receptor antagonist in insulin resistant rats. ConclusionNSE suppressed nuclear translocation of NF-κB in LPS-stimulated peritoneal macrophages via PPARγ and changed hepatic mRNA expression of PPARγ target genes (SLC27A1, IL1RN) in insulin resistant rats.

Depression Exacerbates Dextran Sulfate Sodium-Induced Colitis via IRF5-Mediated Macrophage Polarization.

Patients with inflammatory bowel disease (IBD) and concurrent depression are predisposed to severer disease activity and a worse prognosis. Macrophage polarization toward the M1 phenotype may contribute to the exacerbation of IBD with comorbid depression. Moreover, interferon regulatory factor 5 (IRF5) is involved in the pathogenesis of IBD. The aim of this study was to explore the role of IRF5 in macrophage polarization in the impact of depression upon colitis. Depressive-like behavior was induced by repeated forced swim stress. Colon length, disease activity index (DAI), colon morphology, histology, ultrastructure of epithelial barrier, lamina propria macrophage polarization, and expression of IRF5 were compared between DSS colitis rats with and without depressive-like behavior. IRF5 shRNA was constructed to affect the rat peritoneal macrophages polarization in vitro. After IRF5 shRNA lentivirus was introduced into colon by enema, the colitis severity, lamina propria macrophage polarization, and TNF-α, IL-1β, and IL-10 of colon tissues were measured. The study found severer colonic inflammation in depressed versus non-depressed DSS-colitis rats. Depressed DSS-colitis rats exhibited smaller subepithelial macrophages size and reduced intracellular granule diversity compared with nondepressed DSS-colitis rats. Increased polarization toward the M1 phenotype, elevated expression of IRF5, and co-expression of IRF5 with CD86 were found in depressed versus nondepressed DSS-colitis rats. Lentivirus-mediated shRNA interference with IRF5 expression switched rat peritoneal macrophage polarization from the M1 to the M2 phenotype, downregulated TNF-α, IL-1β expression to a greater extent in depressed versus nondepressed colitis rats. IRF5-mediated macrophage polarization may likely underlie the deterioration of DSS-induced colitis caused by depression.