Fermented Soybean Paste Attenuates Biogenic Amine-Induced Liver Damage in Obese Mice.

Biogenic amines are cellular components produced by the decarboxylation of amino acids; however, excessive biogenic amine production causes adverse health problems. The relationship between hepatic damage and biogenic amine levels in nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, mice were fed a high-fat diet (HFD) for 10 weeks to induce obesity, presenting early-stage of NAFLD. We administered histamine (20 mg/kg) + tyramine (100 mg/kg) via oral gavage for 6 days to mice with HFD-induced early-stage NAFLD. The results showed that combined histamine and tyramine administration increased cleaved PARP-1 and IL-1ß in the liver, as well as MAO-A, total MAO, CRP, and AST/ALT levels. In contrast, the survival rate decreased in HFD-induced NAFLD mice. Treatment with manufactured or traditional fermented soybean paste decreased biogenically elevated hepatic cleaved PARP-1 and IL-1ß expression and blood plasma MAO-A, CRP, and AST/ALT levels in HFD-induced NAFLD mice. Additionally, the biogenic amine-induced reduction in survival rate was alleviated by fermented soybean paste in HFD-induced NAFLD mice. These results show that biogenic amine-induced liver damage can be exacerbated by obesity and may adversely affect life conservation. However, fermented soybean paste can reduce biogenic amine-induced liver damage in NAFLD mice. These results suggest a beneficial effect of fermented soybean paste on biogenic amine-induced liver damage and provide a new research perspective on the relationship between biogenic amines and obesity.

S6K1 negatively regulates TAK1 activity in the toll-like receptor signaling pathway.

Transforming growth factor ß (TGF-ß)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1(-/-) and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1(-/-) mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.

Anti-inflammatory effects of apocynin, an inhibitor of NADPH oxidase, in airway inflammation.

Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-κB and AP-1, induced by pro-inflammatory stimuli, such as TNF-α, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-α. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity.

Reciprocal inhibition between the transforming growth factor-ß-activated kinase 1 (TAK1) and apoptosis signal-regulating kinase 1 (ASK1) mitogen-activated protein kinase kinase kinases and its suppression by TAK1-binding protein 2 (TAB2), an adapter protein for TAK1.

Mitogen-activated protein kinase kinase kinases (MAP3Ks) are activated by a wide spectrum of extracellular stimuli and are involved in various cellular events including proinflammatory and oxidative damage response through activations of two specific transcription factors, nuclear factor κB (NF-κB) and activator protein-1 (AP-1). Although members of the MAP3K family have both overlapping and distinct functions, the inter-regulatory mechanism of MAP3Ks remains largely unknown. In this study we demonstrated that transforming growth factor-ß-activated kinase 1 (TAK1)-TAK1-binding protein 1 (TAB1) complex negatively regulates ASK1-mediated signaling, and TAB2 reciprocally regulates TAK1-induced NF-κB and apoptosis signal-regulating kinase 1 (ASK1)-mediated AP-1 activations through the TAK1-TAB2 interaction and the interferences of TAK1-ASK1 interaction. TAK1 interacted with the N or C terminus of ASK1 through the C-terminal TAB2 binding domain of TAK1, with resultant inhibition of ASK1-induced AP-1 activation. Interestingly, the interaction between TAK1 and TAB2 significantly attenuated the ASK1-TAK1 interaction through the competitive interaction with ASK1 to TAK1 and resulted in the activations of TAK1-induced activations of NF-κB and AP-1. More interestingly, H(2)O(2)- and TNF-α-induced apoptosis in TAK1-deficient mouse embryo fibroblast cells were dramatically enhanced by overexpression of ASK1, whereas the apoptosis was markedly inhibited by the overexpression of TAK1. Overall, these results demonstrate that TAK1 and its adapter protein, TAB2, reciprocally regulate both TAK1- and ASK1-mediated signaling pathways to direct the activations of NF-κB and AP-1.

Notch-induced hIL-6 production facilitates the maintenance of self-renewal of hCD34+ cord blood cells through the activation of Jak-PI3K-STAT3 pathway.

Ex vivo expansion of CD34(+) stem cells in contact culture between hCD34(+)CD38(-)Lin(-) cord blood stem cells and human delta-like-expressing AFT024 feeder cells revealed increased amounts of stemness-related proteins such as HoxB4, GATA2, Bmi-1, and p21 and anti-apoptotic proteins such as Bcl-2, Bcl-xL, Mcl-1, and phospho-Bad, when compared with control or noncontact culture. Production of human IL-6 (hIL-6) was markedly elevated in the culture, but was profoundly inhibited by treatment with γ-secretase inhibitor. In addition, Notch-induced activation of STAT3 was directly involved in gene expression of hIL-6 and soluble hIL-6Rα, indicating the close linkage between Notch signaling and hIL-6 production. Furthermore, depletion of soluble hIL-6 (with hIL-6-specific antibodies) and inhibition of IL-6-mediated signals (with a Jak1 inhibitor and wortmannin) severely affected the maintenance of self-renewal of hCD34(+) cord blood cells. It was also observed that the ex vivo expanded CD34(+) cord blood cells were induced to reconstitute human immune cells in nonobese diabetic mice with severe combined immunodeficiency when compared with freshly isolated CD34(+) cord blood cells. Together, these results strongly demonstrate that Notch signaling in the "cell-to-cell contact" between hCD34(+) cord blood and delta-like-expressing AFT024 feeder cells facilitates maintenance of self-renewal of hCD34(+) cord blood cells through direct regulation of hIL-6 production.

Co-transplantation of fetal bone tissue facilitates the development and reconstitution in human B cells in humanized NOD/SCID/IL-2Rγnull (NSG) mice.

BACKGROUND: In terms of the function and reconstitution efficacy of human immune cells, co-transplantation of human fetal tissues, such as thymus and liver, with CD34(+) hematopoietic stem cells (HSCs) has potential advantages in the generation of humanized mice. OBJECTIVE AND METHODS: To examine the effects of bone tissues in the reconstitution of human immune cells, particularly in B cells, we generated a new humanized mice co-transplanted with human fetal thymus (hFT)/fetal bone (hFB) tissues and human fetal liver-derived CD34(+) cells. RESULTS: Humanized mice exhibited effective reconstitution of human immune cells earlier compared to control humanized mice. In terms of quantity, the number of immune cells, such as human T, B, and monocyte/macrophages was significantly increased. Furthermore, significant increase of B cell progenitors and immature/naïve B cells could be detected in the bone marrow and spleen of humanized mice. CONCLUSION: Our results demonstrate that co-transplantation of hFB tissue may facilitate the reconstitution of human B and T cells, and therefore the humanized model may be used to develop therapeutic human antibodies for clinical use.

Kir3.1 channel is functionally involved in TLR4-mediated signaling.

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1ß, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1(KD) THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.

Human T cell development in the liver of humanized NOD/SCID/IL-2Rγ(null)(NSG) mice generated by intrahepatic injection of CD34(+) human (h) cord blood (CB) cells.

In this study, we explore the possibility of human T cell development in the liver of humanized mice generated by intrahepatic injection of CD34(+) hCB cells into conditioned NOD/SCID/IL-2Rγ(null)(NSG) newborn mice. The intrahepatic injection of CD34(+) hCB cells led to effective reconstitution of human myeloid and lymphoid lineage cells. In contrast to the previously reported Rag2(-/-)γ(c)(-/-) humanized mice, interestingly, the thymus function of humanized NSG mice was markedly reduced in terms of its size and cell contents, whereas the livers of humanized NSG mice profoundly contained double-positive (DP), hCD4 and hCD8 single positive (SP), hCD34(+)hCD38(lo)hCD1a(-) (TSP), hCD34(+)hCD38(hi)hCD1a(-) (ETP), and hCD34(+)hCD38(+)hCD1a(+) (pre-T cells) cells. Furthermore, immunostaining of the liver revealed that human T cells were co-localized with hDCs. Taken together, our results demonstrate that the intrahepatic injection of hCD34(+) hCB cells can facilitate human T cell development in the livers of humanized NSG mice.

Preferential effects of leptin on CD4 T cells in central and peripheral immune system are critically linked to the expression of leptin receptor.

Leptin can enhance thymopoiesis and modulate the T-cell immune response. However, it remains controversial whether these effects correlate with the expression of leptin receptor, ObR. We herein addressed this issue by using in vivo animal models and in vitro culture systems. Leptin treatment in both ob/ob mice and normal young mice induced increases of CD4 SP thymocytes in thymus and CD4 T cells in the periphery. Interestingly, expression of the long form ObR was significantly restricted to DN, DP and CD4 SP, but not CD8 SP thymocytes. Moreover, in the reaggregated DP thymocyte cultures with leptin plus TSCs, leptin profoundly induced differentiation of CD4 SP but not CD8 SP thymocytes, suggesting that the effects of leptin on thymocyte differentiation might be closely related to the expression of leptin receptor in developing thymocytes. Surprisingly, ObR expression was markedly higher in peripheral CD4 T cells than that in CD8 T cells. Furthermore, leptin treatment with or without IL-2 and PHA had preferential effects on cell proliferation of CD4 T cells compared to that of CD8 T cells. Collectively, these data provide evidence that the effects of leptin on differentiation and proliferation of CD4 T cells might be closely related to the expression of leptin receptor.

H2O2-dependent hyperoxidation of peroxiredoxin 6 (Prdx6) plays a role in cellular toxicity via up-regulation of iPLA2 activity.

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase activity and Ca2+-independent phospholipase A2 (iPLA2) activity. Here, we report that H2O2-induced cellular toxicity acts through Prdx6 hyperoxidation. Under high concentrations of H2O2 (> 100 microm), Prdx6, and 2-Cys Prdxs were hyperoxidized. Contrary to hyperoxidation of 2-Cys Prdxs, hyperoxidation of Prdx6 was irreversible in vivo. Surprisingly, H2O2-induced cell cycle arrest at the G2/M transition correlated with hyperoxidation and increased iPLA2 activity of Prdx6. This arrest was also associated with up-regulation of p53 and p21 and with down-regulation of cyclin B1. Furthermore, the H2O2-mediated increase in iPLA2 activity was dramatically abolished in a hyperoxidation mutant (C47A), an iPLA2 mutant (S32A), and a double mutant (C47A/S32A) of Prdx6, demonstrating the essential requirement of Prdx6 C47 hyperoxidation for its iPLA2 activity. Together, our results demonstrate that H2O2-mediated hyperoxidation of Prdx6 induces cell cycle arrest at the G2/M transition through up-regulation of iPLA2 activity.

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo.

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.

A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway.

We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.