Vaccinia virus expressing IL‐37 promotes antitumor immune responses in hepatocellular carcinoma

The aim of this study was to investigate the effect of vaccinia virus expressing IL‐37 (VV‐IL‐37) on cell proliferation, migration and invasion of hepatocellular carcinoma (HCC) and its possible underlying molecular mechanisms. In this study, we constructed a cancer‐targeted vaccinia virus carrying the IL‐37 gene knocked in the region of the viral thymidine kinase (TK) gene. Human HCC cell lines were assayed in vitro for cell proliferation, migration and invasion. Serum level, relative mRNA level and protein level of IL‐37 in HCC cell lines SMMC7721 and Bel7402 were tested by ELISA assay, qRT‐PCR and western blot, respectively. The levels of IL‐2, IFN‐γ and TNF‐α in HCC tumor tissues were also analyzed by ELISA. STAT3 and p‐STAT3 expression in tumor tissues were determined by western blot. Our results showed that VV‐IL‐37 efficiently infected and inhibited HCC cells proliferation, migration and invasion via decreasing STAT3 phosphorylation. In vivo, VV‐IL‐37 expressed IL‐37 at a high level in the transplanted tumor, reduced STAT3 activity, and eventually inhibited tumor growth. In conclusion, we demonstrate that VV‐IL‐37 promotes antitumor immune responses in HCC.     

IL‐27 Rα+ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Recently, emerging evidence strongly suggested that the activation of interleukin‐27 Receptor α (IL‐27Rα) could modulate different inflammatory diseases. However, whether IL‐27Rα affects allotransplantation rejection is not fully understood. Here, we investigated the role of IL‐27Rα on allorejection both in vivo and in vitro. The skin allotransplantation mice models were established, and the dynamic IL‐27Rα/IL‐27 expression was detected, and IL‐27Rα⁺ spleen cells adoptive transfer was performed. STAT1/3/5 phosphorylation, proliferation and apoptosis were investigated in mixed lymphocyte reaction (MLR) with recombinant IL‐27 (rIL‐27) stimulation. Finally, IFN‐γ/ IL‐10 in graft/serum from model mice was detected. Results showed higher IL‐27Rα/IL‐27 expression in allografted group compared that syngrafted group on day 10 (top point of allorejection). IL‐27Rα⁺ spleen cells accelerated allograft rejection in vivo. rIL‐27 significantly promoted proliferation, inhibited apoptosis and increased STAT1/3/5 phosphorylation of alloreactive splenocytes, and these effects of rIL‐27 could be almost totally blocked by JAK/ STAT inhibitor and anti‐IL‐27 p28 Ab. Finally, higher IL‐27Rα⁺IFN‐γ⁺ cells and lower IL‐27Rα⁺IL‐10⁺ cells within allografts, and high IFN‐γ/low IL‐10 in serum of allorejecting mice were detected. In conclusion, these data suggested that IL‐27Rα⁺ cells apparently promoted allograft rejection through enhancing alloreactive proliferation, inhibiting apoptosis and up‐regulating IFN‐γ via enhancing STAT pathway. Blocking IL‐27 pathway may favour to prevent allorejection, and IL‐27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.     

Interleukin‐15 Contributes to the Regulation of Murine Adipose Tissue and Human Adipocytes

An alarming global rise in the prevalence of obesity and its contribution to the development of chronic diseases is a serious health concern. Recently, obesity has been described as a chronic low‐grade inflammatory condition, influenced by both adipose tissue and immune cells suggesting proinflammatory cytokines may play a role in its etiology. Here we examined the effects of interleukin‐15 (IL‐15) on adipose tissue and its association with obesity. Over expression of IL‐15 (IL‐15tg) was associated with lean body condition whereas lack of IL‐15 (IL‐15^?/?) results in significant increase in weight gain without altering appetite. Interestingly, there were no differences in proinflammatory cytokines such as IL‐6 and tumor necrosis factor‐α (TNF‐α) in serum between the three strains of mice. In addition, there were significant numbers of natural killer (NK) cells in fat tissues from IL‐15tg and B6 compared to IL‐15^?/? mice. IL‐15 treatment results in significant weight loss in IL‐15^?/? knockout and diet‐induced obese mice independent of food intake. Fat pad cross‐sections show decreased pad size with over expression of IL‐15 is due to adipocyte shrinkage. IL‐15 induces weight loss without altering food consumption by affecting lipid deposition in adipocytes. Treatment of differentiated human adipocytes with recombinant human IL‐15 protein resulted in decreased lipid deposition. In addition, obese patients had significantly lower serum IL‐15 levels when compared to normal weight individuals. These results clearly suggest that IL‐15 may be involved in adipose tissue regulation and linked to obesity.     

The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2^?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2^?/?, and p27^?/?Skp2^?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2^?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2^?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27^?/?Skp2^?/? double knockout mice. The Skp2^?/? decrements in adipocyte number and fat pad mass were totally reversed in p27^?/?Skp2^?/? mice. Adipogenesis was inhibited in MEFs from Skp2^?/? vs. WT mice, and this inhibition was absent in MEFs from p27^?/?Skp2^?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.     

Decreased expression of microRNA‐21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis

The imbalance of Th17/Treg cell populations has been suggested to be involved in the regulation of rheumatoid arthritis (RA) pathogenesis; however, the mechanism behind this phenomenon remains unclear. Recent studies have shown how microRNAs (miRNAs) are important regulators of immune responses and are involved in the development of a variety of inflammatory diseases, including RA. In this study, we demonstrated that the frequencies of CD3⁺CD4⁺IL‐17⁺Th17 cells were significantly higher, and CD4⁺CD25⁺FOXP3⁺ Treg cells significantly lower in peripheral blood mononuclear cells from RA patients. Detection of cytokines from RA patients revealed an elevated panel of pro‐inflammatory cytokines, including IL‐17, IL‐6, IL‐1β, TNF‐α and IL‐22, which carry the inflammatory signature of RA and are crucial in the differentiation and maintenance of pathogenic Th17 cells and dysfunction of Treg cells. However, the level of miR‐21 was significantly lower in RA patients, accompanied by the increase in STAT3 expression and activation, and decrease in STAT5/pSTAT5 protein and Foxp3 mRNA levels. Furthermore, lipopolysaccharide stimulation up‐regulated miR‐21 expression from healthy controls, but down‐regulated miR‐21 expression from RA patients. Therefore, we speculate that miR‐21 may be part of a negative feedback loop in the normal setting. However, miR‐21 levels decrease significantly in RA patients, suggesting that this feedback loop is dysregulated and may contribute to the imbalance of Th17 and Treg cells. MiR‐21 may thus serve as a novel regulator in T‐cell differentiation and homoeostasis, and provides a new therapeutic target for the treatment of RA.     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.     

Protective role of mouse mast cell tryptase Mcpt6 in melanoma

Tryptase‐positive mast cells populate melanomas, but it is not known whether tryptase impacts on melanoma progression. Here we addressed this and show that melanoma growth is significantly higher in tryptase‐deficient (Mcpt6^?/?) versus wild‐type mice. Histochemical analysis showed that mast cells were frequent in the tumor stroma of both wild‐type and Mcpt6^?/? mice, and also revealed their presence within the tumor parenchyma. Confocal microscopy analysis revealed that tryptase was taken up by the tumor cells. Further, tryptase‐positive granules were released from mast cells and were widely distributed within the tumor tissue, suggesting that tryptase could impact on the tumor microenvironment. Indeed, gene expression analysis showed that the absence of Mcpt6 caused decreased expression of numerous genes, including Cxcl9, Tgtp2, and Gbp10, while the expression of 5p‐miR3098 was enhanced. The levels of CXCL9 were lower in serum from Mcpt6^?/? versus wild‐type mice. In further support of a functional impact of tryptase on melanoma, recombinant tryptase (Mcpt6) was taken up by cultured melanoma cells and caused reduced proliferation. Altogether, our results indicate a protective role of mast cell tryptase in melanoma growth.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype

Atherosclerosis (AS) is characterized as progressive arterial plaque, which is easy to rupture under low stability. Macrophage polarization and inflammation response plays an important role in regulating plaque stability. Ginsenoside Rb1 (Rb1), one of the main active principles of Panax Ginseng, has been found powerful potential in alleviating inflammatory response. However, whether Rb1 could exert protective effects on AS plaque stability remains unclear. This study investigated the role of Rb1 on macrophage polarization and atherosclerotic plaque stability using primary peritoneal macrophages isolated from C57BL/6 mice and AS model in ApoE^?/? mice. In vitro, Rb1 treatment promoted the expression of arginase‐I (Arg‐I) and macrophage mannose receptor (CD206), two classic M2 macrophages markers, while the expression of iNOS (M1 macrophages) was decreased. Rb1 increased interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13) secretion in supernatant and promoted STAT6 phosphorylation. IL‐4 and/or IL‐13 neutralizing antibodies and leflunomide, a STAT6 inhibitor attenuated the up‐regulation of M2 markers induced by Rb1. In vivo, the administration of Rb1 promoted atherosclerotic lesion stability, accompanied by increased M2 macrophage phenotype and reduced MMP‐9 staining. These data suggested that Rb1 enhanced atherosclerotic plaque stability through promoting anti‐inflammatory M2 macrophage polarization, which is achieved partly by increasing the production of IL‐4 and/or IL‐13 and STAT6 phosphorylation. Our study provides new evidence for possibility of Rb1 in prevention and treatment of atherosclerosis.     

Interleukin-17 deficiency reduced vascular inflammation and development of atherosclerosis in Western diet-induced apoE-deficient mice

ObjectiveSeveral reports describe the role of interleukin (IL)-17 in the development of atherosclerosis; however, its precise role remains controversial. We generated double-deficient mice for apolipoprotein E (apoE) and IL-17 (apoE^?/?IL-17^?/? mice) and investigated the effect of IL-17 deficiency on vascular inflammation and atherosclerosis.Methods and resultsAtherosclerotic plaque areas in apoE^?/?IL-17^?/? mice fed a Western diet (WD) were significantly reduced compared with those in apoE^?/? mice. No significant differences in plasma lipid profiles were observed between apoE^?/? and apoE^?/?IL-17^?/? mice. The number of infiltrated macrophages in the plaques was significantly decreased in WD-fed apoE^?/?IL-17^?/? mice compared with WD-fed apoE^?/? mice, whereas vascular smooth muscle cell content was not altered by IL-17 deficiency. Expression of inflammatory cytokines (MCP-1, IL-1β, IL-6, IFN-γ, and IL-12 p40) and scavenger receptors (Msr-1, Scarb1, and Olr1) in the plaques was inhibited in WD-fed apoE^?/?IL-17^?/? mice. Furthermore, expression of inducible nitric oxide (M1 marker) and arginase-1 (M2 marker) was inhibited in WD-fed apoE^?/?IL-17^?/? mice.ConclusionOur results indicate that IL-17 deficiency reduces vascular inflammation and atherosclerosis and that modulation of IL-17 could be a potential target for prevention and treatment of atherosclerosis.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Attenuation of inflammation and cellular stress‐related pathways maintains insulin sensitivity in obese type I interleukin‐1 receptor knockout mice on a high‐fat diet

The development of insulin resistance in the obese is associated with chronic, low‐grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin‐1 receptor knockout (IL‐1RI^?/?) mice, which are protected against diet‐induced insulin resistance. Mice were fed a high‐fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL‐1RI^?/? mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL‐1RI^?/? mice was associated with down‐regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid β‐oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid‐binding protein expression and decreased lipogenesis. Correlation analysis revealed down‐regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL‐1RI‐mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet‐induced insulin resistance, independent of obesity.     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.     

Chronic exercise decreases cytokine production in healthy rat skeletal muscle

Skeletal muscle is the source of pro‐ and anti‐inflammatory cytokines, and recently, it has been recognized as an important source of interleukin‐6 (IL‐6). Acute physical exercise is known to induce a pro‐inflammatory cytokine profile in the plasma. However, the effect of chronic physical exercise in the production of pro‐ and anti‐inflammatory cytokines by the skeletal muscle has never been examined. We assessed IL‐6, TNF‐α, IL‐1β and IL‐10 levels in the skeletal muscle of rats submitted to endurance training. Animals were randomly assigned to either a sedentary group (S, n = 7) or an endurance exercise trained group (T, n = 8). Trained rats ran on a treadmill for 5 days week^?1 for 8 weeks (60% VO_2max). Detection of IL‐6, TNF‐α, IL‐1β and IL‐10 protein expression was carried out by ELISA. We found decreased expression of IL‐1β, IL‐6, TNF‐α and IL‐10 (28%, 27%, 32% and 37%, respectively, p < 0.05) in the extensor digital longus (EDL) from T, when compared with S. In the soleus, IL‐1β, TNF‐α and IL‐10 protein levels were similarly decreased (34%, 42% and 50%, respectively, p < 0.05) in T in relation to S, while IL‐6 expression was not affected by the training protocol. In conclusion, exercise training induced decreased cytokine protein expression in the skeletal muscle. These data show that in healthy rats, 8‐week moderate‐intensity aerobic training down regulates skeletal muscle production of cytokines involved in the onset, maintenance and regulation of inflammation, and that the response is heterogeneous according to fibre composition. Copyright © 2009 John Wiley & Sons, Ltd.     

Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings

This study tested the hypothesis that melatonin (Mel) therapy preserved the brain architectural and functional integrity against ischaemic stroke (IS) dependently through suppressing the inflammatory/oxidative stress downstream signalling pathways. Adult male B6 (n = 6 per each B6 group) and TLR4 knockout (ie TLR4^?/?) (n = 6 per each TLR4^?/? group) mice were categorized into sham control (SC^B6), SC^TLR4?/?, IS^B6, IS^TLR4?/?, IS^B6 + Mel (i.p. daily administration) and IS^TLR4?/? + Mel (i.p. daily administration). By day 28 after IS, the protein expressions of inflammatory (HMBG1/TLR2/TLR4/MAL/MyD88/RAM TRIF/TRAF6/IKK‐α/p‐NF‐κB/nuclear‐NF‐κB/nuclear‐IRF‐3&7/IL‐1β/IL‐6/TNF‐α/IFN‐γ) and oxidative stress (NOX‐1/NOX‐2/ASK1/p‐MKK4&7/p‐JNK/p‐c‐JUN) downstream pathways as well as mitochondrial‐damaged markers (cytosolic cytochrome C/cyclophilin D/SRP1/autophagy) were highest in group IS^B6, lowest in groups SC^B6 and SC^TLR4?/?, lower in group IS^TLR4?/? + Mel than in groups IS^TLR4?/? and IS^B6 + Mel and lower in group IS^B6 + Mel than in group IS^TLR4?/? (all P < .0001). The brain infarct volume, brain infarct area and the number of inflammatory cells in brain (CD14/F4‐88) and in circulation (MPO+//Ly6C+/CD11b+//Ly6G+/CD11b+) exhibited an identical pattern, whereas the neurological function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.