Mechanistic study of saikosaponin‐d (Ssd) on suppression of murine T lymphocyte activation

Saikosaponin‐d (Ssd) is a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae). Previous findings showed that Ssd exhibits a variety of pharmacological and immunomodulatory activities including anti‐inflammatory, anti‐bacterial, anti‐viral and anti‐cancer effects. In the current study we have investigated the effects of Ssd on activated mouse T lymphocytes through the NF‐κB, NF‐AT and AP‐1 signaling pathways, cytokine secretion, and IL‐2 receptor expression. The results demonstrated that Ssd not only suppressed OKT3/CD28‐costimulated human T cell proliferation, it also inhibited PMA, PMA/Ionomycin and Con A‐induced mouse T cell activation in vitro. The inhibitory effect of Ssd on PMA‐induced T cell activation was associated with down‐regulation of NF‐κB signaling through suppression of IKK and Akt activities. In addition, Ssd suppressed both DNA binding activity and the nuclear translocation of NF‐AT and activator protein 1 (AP‐1) of the PMA/Ionomycin‐stimulated T cells. The cell surface markers like IL‐2 receptor (CD25) were also down‐regulated together with decreased production of pro‐inflammatory cytokines of IL‐6, TNF‐α and IFN‐γ. These results indicate that the NF‐κB, NF‐AT and AP‐1 (c‐Fos) signaling pathways are involved in the T cell inhibition evoked by Ssd, so it can be a potential candidate for further study in treating T cell‐mediated autoimmune conditions. J. Cell. Biochem. 107: 303–315, 2009. © 2009 Wiley‐Liss, Inc.     

Chlorogenic acid: A potent molecule that protects cardiomyocytes from TNF‐α–induced injury via inhibiting NF‐κB and JNK signals

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.     

Embelin impact on paraquat‐induced lung injury through suppressing oxidative stress,inflammatory cascade,and MAPK/NF‐κB signaling pathway

The current examination was intended to observe the defensive impacts of embelin against paraquat‐incited lung damage in relationship with its antioxidant and anti‐inflammatory action. Oxidative stress marker, like malondialdehyde (MDA), antioxidative enzymes, for example, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px), inflammatory cytokines, such as interleukin‐1β (IL‐1β), tumor necrosis factor‐α, and IL‐6, histological examination, and nuclear factor kappa B/mitogen‐activated protein kinase (NF‐κB/MAPK) gene expression were evaluated in lung tissue. Embelin treatment significantly decreased MDA and increased SOD, CAT, and GSH Px. Embelin significantly reduced levels of inflammatory cytokines in paraquat‐administered and paraquat‐intoxicated rats. In addition, embelin suggestively decreased relative protein expression of nuclear NF‐κB p65, p‐NF‐κBp65, p38 MAPK, and p‐p38 MAPKs in paraquat‐intoxicated rats. The outcomes show the impact of embelin inhibitory action on NF‐κB and MAPK and inflammatory cytokines release, and the decrease of lung tissue damage caused by paraquat.     

NF‐κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor‐κB (NF‐κB) activity in myometrium which both up‐regulates contraction‐associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF‐κB p65, or IκB‐α between upper‐ or lower‐segment myometrium or before or after labour, there is nuclear localization of serine‐256‐phospho‐p65 and serine‐536‐phospho‐p65 in both upper‐ and lower‐segment myometrium both before and after the onset of labour at term. This shows that NF‐κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF‐κB we overexpressed p65 in myocytes in culture. This led to NF‐κB activation identical to that seen following interleukin (IL)‐1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF‐κB increased expression of 38 genes principally related to immunity and inflammation. IL‐1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL‐1β proving a central role for NF‐κB. We conclude that NF‐κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

Swiprosin‐1 is expressed in mast cells and up‐regulated through the protein kinase CβI/η pathway

Swiprosin‐1 exhibits the highest expression in CD8⁺ T cells and immature B cells and has been thought to play a role in lymphocyte physiology. Here we report that swiprosin‐1 is also expressed in mast cells and up‐regulated in both in vitro cultured mast cells by phorbol ester and in vivo model tissues of passive cutaneous anaphylaxis and atopic dermatitis. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC‐βI/η are involved in the expression of swiprosin‐1 in the human mast cell line HMC‐1. In contrast, down‐regulation of swiprosin‐1 by A23187 or ionomycin suggests that calcium‐signaling plays a negative role. The ectopic expression of swiprosin‐1 augmented PMA/A23187‐induced NF‐κB promoter activity, and resulted in increased expression of cytokines. Moreover, knock‐down of swiprosin‐1 attenuated PMA/A23187‐induced cytokine expression. Collectively, these results suggest that swiprosin‐1 is a PKC‐βI/η‐inducible gene and it modulates mast cell activation through NF‐κB‐dependent pathway. J. Cell. Biochem. 108: 705–715, 2009. © 2009 Wiley‐Liss, Inc.     

Sulfur mustard induced cytokine production and cell death: Investigating the potential roles of the p38, p53, and NF‐κB signaling pathways with RNA interference

Cutaneous and ocular injuries caused by sulfur mustard (SM; bis‐(2‐chloroethyl) sulfide) are characterized by severe inflammation and death of exposed cells. Given the known roles of p38MAPK and NF‐κB in inflammatory cytokine production, and the known roles of NF‐κB and p53 in cell fate, these pathways are of particular interest in the study of SM injury. In this study, we utilized inhibitory RNA (RNAi) targeted against p38α, the p50 subunit of NF‐κB, or p53 to characterize their role in SM‐induced inflammation and cell death in normal human epidermal keratinocytes (NHEK). Analysis of culture supernatant from 200 μM SM‐exposed cells showed that inflammatory cytokine production was inhibited by p38α RNAi but not by NF‐κB p50 RNAi. These findings further support a critical role for p38 in SM‐induced inflammatory cytokine production in NHEK and suggest that NF‐κB may not play a role in the SM‐induced inflammatory response of this cell type. Inhibition of NF‐κB by p50 RNAi did, however, partially inhibit SM‐induced cell death, suggesting a role for NF‐κB in SM‐induced apoptosis or necrosis. Interestingly, inhibition of p53 by RNAi potentiated SM‐induced cell death, suggesting that the role of p53 in SM injury, may be complex and not simply prodeath. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:155–164, 2010; Published online inWiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20321     

Corilagin suppresses RANKL‐induced osteoclastogenesis and inhibits oestrogen deficiency‐induced bone loss via the NF‐κB and PI3K/AKT signalling pathways

Over‐activated osteoclastogenesis, which is initiated by inflammation, has been implicated in osteoporosis. Corilagin, a natural compound extracted from various medicinal herbaceous plants, such as Cinnamomum cassia, has antioxidant and anti‐inflammatory activities. We found that Corilagin suppressed osteoclast differentiation in a dose‐dependent manner, significantly decreased osteoclast‐related gene expression and impaired bone resorption by osteoclasts. Moreover, phosphorylation of members of the nuclear factor‐kappaB (NF‐κB) and PI3K/AKT signalling pathways was reduced by Corilagin. In a murine model of osteoporosis, Corilagin inhibited osteoclast functions in vivo and restored oestrogen deficiency‐induced bone loss. In conclusion, our findings suggested that Corilagin inhibited osteoclastogenesis by down‐regulating the NF‐κB and PI3K/AKT signalling pathways, thus showing its potential possibility for the treatment of osteoporosis.     

Anti‐inflammatory function of Withangulatin A by targeted inhibiting COX‐2 expression via MAPK and NF‐κB pathways

Withangulatin A (WA), an active component isolated from Physalis angulata L., has been reported to possess anti‐tumor and trypanocidal activities in model systems via multiple biochemical mechanisms. The aim of this study is to investigate its anti‐inflammatory potential and the possible underlying mechanisms. In the current study, WA significantly suppressed mice T lymphocytes proliferation stimulated with LPS in a dose‐ and time‐dependent manner and inhibited pro‐inflammation cytokines (IL‐2, IFN‐γ, and IL‐6) dramatically. Moreover, WA targeted inhibited COX‐2 expression mediated by MAPKs and NF‐κB nuclear translocation pathways in mice T lymphocytes, and this result was further confirmed by the COX‐1/2 luciferase reporter assay. Intriguingly, administration of WA inhibited the extent of mice ear swelling and decreased pro‐inflammatory cytokines production in mice blood serum. Based on these evidences, WA influences the mice T lymphocytes function through targeted inhibiting COX‐2 expression via MAPKs and NF‐κB nuclear translocation signaling pathways, and this would make WA a strong candidate for further study as an anti‐inflammatory agent. J. Cell. Biochem. 109: 532–541, 2010. © 2009 Wiley‐Liss, Inc.     

Dauricine induces apoptosis,inhibits proliferation and invasion through inhibiting NF‐κB signaling pathway in colon cancer cells

Dauricine, a bioactive component of Asiatic Moonseed Rhizome, has been widely used to treat a large number of inflammatory diseases in traditional Chinese medicine. In our study, we demonstrated that dauricine inhibited colon cancer cell proliferation and invasion, and induced apoptosis by suppressing nuclear factor‐kappaB (NF‐κB) activation in a dose‐ and time‐dependent manner. Addition of dauricine inhibited the phosphorylation and degradation of IκBα, and the phosphorylation and translocation of p65. Moreover, dauricine down‐regulated the expression of various NF‐κB‐regulated genes, including genes involved cell proliferation (cyclinD1, COX2, and c‐Myc), anti‐apoptosis (survivin, Bcl‐2, XIAP, and IAP1), invasion (MMP‐9 and ICAM‐1), and angiogenesis (VEGF). In athymic nu/nu mouse model, we further demonstrated that dauricine significantly suppressed colonic tumor growth. Taken together, our results demonstrated that dauricine inhibited colon cancer cell proliferation, invasion, and induced cell apoptosis by suppressing NF‐κB activity and the expression profile of its downstream genes. These findings provide evidence for a novel role of dauricine in preventing or treating colon cancer through modulation of NF‐κB singling pathway. J. Cell. Physiol. 225: 266–275, 2010. © 2010 Wiley‐Liss, Inc.     

NF‐κB and estrogen receptor α interactions: Differential function in estrogen receptor‐negative and ‐positive hormone‐independent breast cancer cells

Estrogen receptor (ER)‐positive breast cancer cells have low levels of constitutive NF‐κB activity while ER negative (?) cells and hormone‐independent cells have relatively high constitutive levels of NF‐κB activity. In this study, we have examined the aspects of mutual repression between the ERα and NF‐κB proteins in ER+ and ER? hormone‐independent cells. Ectopic expression of the ERα reduced cell numbers in ER+ and ER? breast cancer cell lines while NF‐κB‐binding activity and the expression of several NF‐κB‐regulated proteins were reduced in ER? cells. ER overexpression in ER+/E2‐independent LCC1 cells only weakly inhibited the predominant p50 NF‐κB. GST‐ERα fusion protein pull downs and in vivo co‐immunoprecipitations of NF‐κB:ERα complexes showed that the ERα interacts with p50 and p65 in vitro and in vivo. Inhibition of NF‐κB increased the expression of diverse E2‐regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF‐7 cells by supershift analysis while p65 antibody reduced ERα:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF‐κB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF‐κB undergo mutual repression, which may explain, in part, why expression of the ERα in ER? cells does not confer growth signaling. Secondly, the acquisition of E2‐independence in ER+ cells is associated with predominantly p50:p50 NF‐κB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell. J. Cell. Biochem. 107: 448–459, 2009. © 2009 Wiley‐Liss, Inc.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.