Omega­3 polyunsaturated fatty acids inhibit IL­11/STAT3 signaling in hepatocytes during acetaminophen hepatotoxicity.

Omega­3 polyunsaturated fatty acids (n­3 PUFAs) exert a negative effect on IL­6 production in several liver disorders, including cirrhosis, acute liver failure and fatty liver disease. However, its effect on the production of IL­11, another important IL­6 family cytokine, remains unclear. IL­11 was found to be significantly elevated in acetaminophen (APAP)­induced liver damage. The aim of the present study was to investigate whether and how n­3 PUFAs modulate IL­11 production during APAP­induced liver injury. For that purpose, wild­type (WT) and fat­1 transgenic mice were intraperitoneally injected with APAP to induce liver injury. Serum was collected for ELISA and alanine aminotransferase assay. The hepatocytes of APAP­injected mice were isolated for reverse transcription­quantitative PCR and western blot analyses. For the in vitro study, primary hepatocytes isolated from WT or fat­1 mice were stimulated with APAP. The results revealed that both endogenous and exogenous n­3 PUFAs significantly aggravated APAP­induced liver damage via the downregulation of STAT3 signaling. Notably, n­3 PUFAs inhibited IL­11 expression, but not IL­6 expression in hepatocytes during the APAP challenge. Furthermore, it was demonstrated that limited phosphorylation of ERK1/2 and Fos­â€‹like­1 (Fra­1) expression are responsible for the n­3 PUFA­mediated inhibitory effect on IL­11 production in APAP­treated hepatocytes. It was concluded that n­3 PUFAs inhibit IL­11 production and further STAT3 activation in hepatocytes during APAP­induced liver injury. Therefore, ERK1/2­mediated Fra­1 expression is responsible for the effect of n­3 PUFAs on IL­11 expression.

Effects of Shen Cao Granules on Chemotherapy-Induced Thrombocytopenia in Gastrointestinal Cancer Patients: A Randomized Controlled Trial.

BACKGROUND: To observe clinical effects of Shen Cao granules on thrombocytopenia in patients with gastrointestinal cancer undergoing chemotherapy. PATIENTS AND METHODS: Patients under a FOLFIRI chemotherapy regimen (n = 92) were randomly divided into study and control groups (n = 46 for each group) and were given 10 g of Shen Cao granules and a placebo, respectively, once daily on chemotherapy treatment days. Platelet counts were measured every other day and any adverse reaction recorded during the study and at follow-up. RESULTS: The incidence of thrombocytopenia (grades II-IV) in the study group was significantly decreased, and the length of hospitalization significantly reduced compared with the control group (11.21 ± 2.46 vs 15.34 ± 3.68 days, P < .05). The minimum numbers of post-chemotherapy platelets and the values of platelet counts 21 days after chemotherapy were significantly increased ([100.65 ± 63.16] × 109/L vs [60.21 ±37.22] × 109/L, P < .05; [267.81 ± 81.32] × 109/L vs [146.42 ± 70.54] × 109/L, P < .001), and the duration of thrombocytopenia and treatment with recombinant human interleukin-11 was significantly decreased in the Shen Cao treatment compared with the control group. No serious adverse events were observed. CONCLUSIONS: Shen Cao granules were effective in decreasing chemotherapy-induced thrombocytopenia, shortened the duration of thrombocytopenia, and reduced the length of hospital stay and costs.

Neuroprotective effects of ellagic acid on cuprizone-induced acute demyelination through limitation of microgliosis, adjustment of CXCL12/IL-17/IL-11 axis and restriction of mature oligodendrocytes apoptosis.

CONTEXT: Ellagic acid (EA) is a natural phenol antioxidant with various therapeutic activities. However, the efficacy of EA has not been examined in neuropathologic conditions. OBJECTIVE: In vivo neuroprotective effects of EA on cuprizone (cup)-induced demyelination were evaluated. MATERIAL AND METHODS: C57BL/6 J mice were fed with chow containing 0.2% cup for 4 weeks to induce oligodendrocytes (OLGs) depletion predominantly in the corpus callosum (CC). EA was administered at different doses (40 or 80 mg/kg body weight/day/i.p.) from the first day of cup diet. Oligodendrocytes apoptosis [TUNEL assay and myelin oligodendrocyte glycoprotein (MOG+)/caspase-3+ cells), gliosis (H&E staining, glial fibrillary acidic protein (GFAP+) and macrophage-3 (Mac-3+) cells) and inflammatory markers (interleukin 17 (IL-17), interleukin 11 (IL-11) and stromal cell-derived factor 1 α (SDF-1α) or CXCL12] during cup intoxication were examined. RESULTS: High dose of EA (EA-80) increased mature oligodendrocytes population (MOG+ cells, p < 0.001), and decreased apoptosis (p < 0.05) compared with the cup mice. Treatment with both EA doses did not show any considerable effects on the expression of CXCL12, but significantly down-regulated the expression of IL-17 and up-regulated the expression of IL-11 in mRNA levels compared with the cup mice. Only treatment with EA-80 significantly decreased the population of active macrophage (MAC-3+ cells, p < 0.001) but not reactive astrocytes (GFAP+ cells) compared with the cup mice. DISCUSSION AND CONCLUSION: In this model, EA-80 effectively reduces lesions via reduction of neuroinflammation and toxic effects of cup on mature OLGs. EA is a suitable therapeutic agent for moderate brain damage in neurodegenerative diseases such as multiple sclerosis.

Dietary iron enhances colonic inflammation and IL-6/IL-11-Stat3 signaling promoting colonic tumor development in mice.

Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk.

The synergistic anti-inflammatory effect of the combination of sodium ferulate and oxymatrine and its modulation on inflammation-associated mediators in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait. was extensively used in traditional Chinese medicine to treat inflammatory diseases, such as acne, heart disease, and hepatitis. Sodium ferulate (SF) and oxymatrine (OMT) were effective component of Radix Angelicae sinensis (Oliv.) Diels and Radix Sophora flavescens Ait., respectively. AIM OF THE STUDY: In this study, we investigated the synergistic anti-inflammatory effect of the combination of SF and OMT, and its modulation on inflammation-associated mediators in RAW 264.7 cells. MATERIALS AND METHODS: In vivo, the anti-inflammatory effects of the combination of SF and OMT were evaluated with the xylene-induced mouse ear edema model and the carrageenan-induced rat paw edema model. In vitro, chemokines and cytokines mRNA expressions in lipopolysaccharide (LPS)-activated RAW 264.7 cells were determined by real-time PCR (RT-PCR) microarray analysis. The levels of interleukin-11 (IL-11), C-reactive protein (CRP) and interferon-γ (INF-γ) in the supernatant of LPS-stimulated RAW 264.7 cells were measured by enzyme-linked immune-sorbent assay (ELISA). RESULTS: The combination of SF and OMT could significantly inhibit the edema in the xylene-induced mouse ear edema and carrageenan-induced rat paw edema, but no effect was found when each drug was used alone according to above doses. The combination exhibited a better effect in down-regulating mRNA expressions of inflammation-associated mediators in LPS-stimulated RAW 264.7 cells than SF or OMT alone. The ELISA results showed that the combination synergistically inhibited LPS-induced IL-11, CRP and INF-γ production in a dose-dependent manner. CONCLUSION: The combination of SF and OMT showed synergistic anti-inflammatory effect, and the activity was probably related to its modulation on inflammation-associated mediators, especially IL-11, CRP and INF-γ.

Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation.

UNLABELLED: We determined that sFRP-1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP-1 neutralizing antibodies and siRNA complementary to sFRP-1 coding sequence enhanced, while recombinant sFRP-1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP-1, we found that sFRP-1 could bind recombinant RANKL. These results suggest potential cross-talk between Wnt and RANKL pathways. INTRODUCTION: Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage-colony-stimulating factor (M-CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled-related protein-1 (sFRP-1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast-derived factor that impinges on osteoclast formation and activity. MATERIALS AND METHODS: Differential display of mRNA from osteoblast lineage cell lines established sFRP-1 to be highly expressed in an osteoclast supporting cell line. sFRP-1 expression in bone was determined by in situ hybridization, and the effects of sFRP-1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP-1 and recombinant protein. RESULTS: In situ hybridization revealed sFRP-1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP-1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2 (PGE2) or interleukin (IL)-11, whereas many other osteotropic agents (e.g., IL-1, IL-6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP-1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP-1 enhanced TRACP+ mononuclear and multinuclear osteoclast formation (3- and 2-fold, respectively) in co-cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP-1 coding sequence significantly enhanced osteoclast formation in co-cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2 and 1,25(OH)2 vitamin D3. Recombinant sFRP-1 dose-dependently inhibited osteoclast formation in osteoblast/spleen co-cultures, RANKL + M-CSF-treated splenic cultures, and RANKL-treated RAW264.7 cell cultures, indicating a direct action of sFRP-1 on hematopoietic cells. Consistent with this, sFRP-1 was found to bind to RANKL in ELISAs. CONCLUSION: sFRP-1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP-1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP-1.

The hemoregulatory peptide pEEDCK may inhibit stem cell proliferation via hydropathic binding to antisense sequence motifs in interleukin-11 and other growth factors.

In undisturbed bone marrow, most hemopoietic stem cells are nonproliferating despite the presence of multiple growth factors. Endogenous inhibitory factors are responsible for maintenance of this quiescence. Previously we sequenced and synthesized the inhibitory pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK), which originally derives from granulocytes, and investigated the role of this peptide in stem cell quiescence. To provide some mechanistic insight, in the present work we studied the structural relationship of this peptide to specific growth-factor-derived sequence motifs. In the murine system in vivo as well as in long-term bone marrow, antiserum to pEEDCK produced a significant stimulation of formation of colony-forming units-granulocyte/macrophage. Binding of peptides to proteins often takes place at hydropathically complementary sites. Therefore, we searched for peptides corresponding to the complementary sequence to pEEDCK. We identified antisense sequences in the genes of various cytokines and cytokine receptors including interleukin-11. The corresponding peptide Val-Leu-Leu-Thre-Arg (VLLTR) and several other peptides hydropathically complementary to pEEDCK were synthesized. We found that pEEDCK binds specifically to these peptides as well as to complete interleukin-11. Dissociation constants were in the 10 microM range. The peptide hydropathically corresponding to pEEDCK (VLLTR) was found to stimulate colony-forming units-granulocyte/macrophage formation. Our data suggest that pEEDCK could exert a coordinating function in the hemopoietic cytokine network by binding to multiple regulatory proteins and modulating their activity.

In vitro secretion of cytokines by human bone marrow: effects of age and estrogen status.

It has been proposed that cytokines mediate the acceleration of bone loss following menopause. Because of the intimate relationship between bone marrow stromal cells and bone tissue, it is possible that marrow cells and their products contribute to the bone microenvironment and influence the regulation of bone cell differentiation and activity. We examined the production of cytokines by bone marrow stromal cells from a total of 37 women and 15 men undergoing total hip replacement for noninflammatory joint disease. Low-density mononuclear cells were isolated from bone marrow and were cultured in phenol red-free alpha MEM medium supplemented with 10% FBS and antibiotics. Constitutive secretion of interleukin-6 (IL-6) was positively correlated with age in a series of 8 women and 5 men measured by bioassay (r = 0.98; P < 0.01) and in a series of 18 women and 10 men measured by immunoassay (r = 0.56; P < 0.01). The pattern of cytokine production by bone marrow stromal cells was examined in detail in 23 postmenopausal women, aged 49-88 yr. Basal secretion of immunoreactive IL-6 and IL-11, but not granulocyte-macrophage colony-stimulating factor, increased with time in culture. Exogenous IL-1 beta stimulated secretion of IL-6 and IL-11 in a saturable, dose-dependent manner. Secretion of soluble IL-6 receptor was not correlated with secretion of IL-6, either constitutively or in the presence of IL-1 beta. In 4 of 14 samples, IL-1 beta also stimulated secretion of granulocyte-macrophage colony-stimulating factor. IL-1 beta was undetectable in 7 of 9 cultures during the 2-week culture period. IL-6 did not stimulate secretion of IL-1 beta in the 7 cultures tested. Cells were dependent upon serum for viability and growth and were not sustained by a serum substitute (1% insulin-transferrin-selenium-BSA). Cells grown in medium with 10% FBS and supplemented with 1% insulin-transferrin-selenium-BSA secreted 10-fold more IL-6 than cells grown in serum alone. Marrow from 7 women receiving estrogen replacement therapy showed lower constitutive secretion of IL-6 (75%; P < 0.006) and IL-11 (43%; P < 0.05) than marrow from age-matched controls and had blunted stimulation of IL-6 and IL-11 secretion by exogenous IL-1 beta. These data indicate distinct patterns of cytokine production by human marrow stromal cultures dependent upon age and estrogen status.

Comparative study of serum and synovial fluid interleukin-11 levels in patients with various arthritides.

OBJECTIVE: To determine the levels of serum and synovial fluid (SF) interleukin (IL)-11 in patients with various arthritides and estimate the contribution of IL-11 to acute phase response (APR). DESIGN AND METHODS: Serum and SF IL-11 were measured by ELISA in patients with rheumatoid arthritis (RA, n = 31), seronegative spondyloarthritis (SSA, n = 23), gout (GT, n = 14) and osteoarthritis (OA, n = 20) and were correlated with ESR and acute phase proteins as well as with cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF alpha. RESULTS: IL-11 was detected in both serum and SF in each group, with IL-11 being statistically higher in SF than serum in all groups, suggesting reduced catabolism or increased synthesis of IL-11 intra-articularly. Median SF IL-11 levels were higher in OA patients than in other groups and in the treated than in the untreated RA subgroup. Moreover, serum and SF IL-11 were correlated significantly with each other, and moderately with the other cytokines examined in RA, SSA, and GT, but not in OA patients, while a significant negative correlation was found with a few of the inflammatory markers examined in each group. CONCLUSIONS: Our findings provide evidence of extensive intra-articular expression of IL-11 in arthritides, especially in OA and treated RA patients, suggesting a protective role for IL-11 in joints, probably through the induction of tissue inhibitor of metalloproteinases.