Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase

An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2’, 7’-dichlorodihydrofluorescein diacetate assay. TNF-α-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-α-induced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NF-kappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes.     

Tumor Necrosis Factor (TNF)-alpha Promoter Polymorphisms in Ankylosing Spondylitis: Comment on the Article by Ji et al

TNF-α promoter polymorphisms may be associated with the severity rather than the susceptibility of AS. Further studies with a large sample size and precise methodology are needed. Interactions may already exist between gene and environment. All of these are helpful to determine the exact mechanisms of TNF-α promoter polymorphisms in AS.     

Azadirachta indica Modulates Electrical Properties and Type of Cell Death in NDEA-Induced Hepatic Tumors

Tissue electrical conductivity is an important indicator of tissue structure and composition. Present study demonstrates modulatory effect of Azadirachta indica on the electrical conductivity and cell death in hepatic tumors. Hepatic tumors were generated by intraperitoneal injection of N-nitrosodiethylamine (cumulative dose: 200 μg/g body mass) to male BALB/c mice. Aqueous A. indica leaf extract (AAILE) was administered orally at a dosage of 100 μg/g body mass till the termination of experiment. At the end of experiment, electrical conductivity of hepatic tumors was measured with four-pin electrode method. Tissues and tumors were then processed for TUNEL assay and DNA fragmentation analysis. The levels of TNF-α were also determined in the normal hepatic and tumor tissue. Hepatic tumors had higher electrical conductivity compared to normal liver tissue. An increased necrotic cell percentage along with elevated TNF-α was also observed. Although, AAILE co-treatment resulted in tumors with higher electrical conductivity compared to normal animals. However, the electrical conductivity was decreased significantly compared to untreated tumors. A significant increase in apoptotic cell percentage and concomitant decrease in necrotic cell percentage along with the increased TNF-α level was observed in these tumors. The results suggest that A. indica modulated mode of cell death in tumors and type of cell death had significant contribution in determining hepatic tumor electrical conductivity.     

Secretion of adipocytes and macrophages under conditions of inflammation and/or insulin resistance and effect of adipocytes on preadipocytes under these conditions

The purpose of the present study was to examine changes in preadipocytes following the coculture of preadipocytes and adipocytes and the effects on the secretion of adipocytes and macrophages following induction of inflammation and insulin resistance. Mature adipocytes and RAW264.7 macrophages were treated with lipopolysaccharide and insulin to establish models of inflammation and insulin resistance, respectively. The mRNA expression levels of IL-6, MCP-1, and TNF-α in all adipocyte treatment groups were significantly greater compared with the control, and that of adiponectin was less (P < 0.05). In the RAW264.7 macrophages, the mRNA expression levels of IL-6 and TNF-α were greater than those in the control group (P < 0.05). Moreover, the results of this study confirmed that adipocytes and macrophages increased the secretion of inflammatory factors under conditions of induced inflammation and insulin resistance. In addition, 3T3-L1 adipocytes inhibited the proliferation and differentiation of preadipocytes when cocultured with adipocytes under conditions of inflammation and/or insulin resistance, and the phenotype of preadipocytes did not change.     

In vitro isolation and cultivation of human chondrocytes for osteoarthritis renovation

The purpose of this study was to evaluate the repair effects of chondrocytes that were cultured in vitro on osteoarthritis (OA). Chondrocytes were isolated from fetal rabbits and cultured in Biosilon microcarriers. Sixty rabbits were randomly divided into three groups equally (blank group, model group, treatment group). The rabbit knee OA model was established by inducing papain. Rabbits in the treatment group were injected with the chondrocytes that were cultured in vitro. Hematoxylin-eosin (HE) staining and gross morphologic observation were conducted. Expression level of cytokines such as IL-1bβ, IL-6, and TNF-α in cartilage synovial cells was also analyzed by an ELISA assay. The cultured chondrocyte was validated by a positive stain of type II collagen and vimentin by immunofluorescence. Compared to the model group, the articular cartilage of the rabbit knee in the treatment group showed a normal color, smooth surface, and none of malacia and coloboma. HE staining indicated that the articular surface of the treatment group tended to be smooth and flat; the matrix stained tinge and the cartilage destruction and fiber hyperplasia of the synovia were lightened. The expression levels of IL-1bβ, IL-6, and TNF-α also declined in the treatment group. OA symptoms were improved by treating with chondrocytes. In summary, the animal experiment in the present study indicated that chondrocyte injection played an active effect on renovation of OA.     

TNF-α, IL-6, and Leptin Increase the Expression of miR-378, an Adipogenesis-Related microRNA in Human Adipocytes

Obesity has become a global public health problem associated with complications including type 2 diabetes, cardiovascular disease, and several cancers. Adipocyte differentiation (adipogenesis) plays an important role in obesity and energy homeostasis. Adipose tissue secretes multiple cytokines and adipokines which can cause the complications of obesity, especially insulin resistance. TNF-α, IL-6, leptin, and resistin have been identified as the main regulators of obesity and insulin activity. miR-378 is highly induced during adipogenesis and has been reported to be positively regulated in adipogenesis. In the current study, matured human adipocytes were treated with TNF-α, IL-6, leptin, or resistin on the 15th day after the induction of human pre-adipocyte differentiation. We demonstrated that TNF-α, IL-6, and leptin upregulated miR-378 expression indicating that miR-378 probably is a novel mediator in the development of insulin resistance related to obesity.     

Immunoregulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Xenogeneic Acellular Nerve Grafts Transplant

This study evaluated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with xenogeneic acellular nerve grafts (xANGs) would reduce the inflammation reaction of xANGs transplantation. BM-MSCs were extracted, separated, purified, and cultured from the bone marrow of rats. Then BM-MSCs were seeded into 5 mm xANGs as experimental group, while xANGs group was chosen as control. Subcutaneous implantation and nerve grafts transplantation were done in this study. Walking-track tests, electrophysiological tests, H&E staining, and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations, cytokine concentrations of IL-2, IL-10, IFN-γ and TNF-α in lymphocytes supernatants and serum of the two groups were evaluated. Walking-track tests and electrophysiological tests suggested the group of BM-MSCs with xANGs obtained better results than xANGs group (P < 0.05). H&E staining and immunostaining of CD4, CD8, and CD68 of subcutaneous implantations showed there were less inflammatory cells in the group of BM-MSCs when compared with the xANGs group. The cytokine concentrations of IL-2, IFN-γ, and TNF-α in BM-MSCs group were lower than xANGs group in lymphocytes supernatants and serum (P < 0.05). However, IL-10 concentrations in BM-MSCs group were higher than xANGs group (P < 0.05). xANG with BM-MSCs showed better nerve repair function when compared with xANG group. Furthermore, xANG with BM-MSCs showed less inflammatory reaction which might indicate the reason of its better nerve regeneration.     

Secretory expression of a bispecific antibody targeting tumor necrosis factor and ED-B fibronectin in Pichia pastoris and its functional analysis

Specific targeting of tumor necrosis factor (TNF)-α antagonist to the inflamed site could increase its efficacy and reduce side-effects. Here, we constructed a bispecific diabody (BsDb) that targets TNF-α and ED-B-containing fibronectin, a fibronectin isoform specifically expressed in the pannus of the inflamed synovium in rheumatoid arthritis. BsDb was secreted from Pichia pastoris as functional protein and was purified to homogeneity. BsDb could simultaneously bind to human TNF-α and B-FN and neutralize TNF-α action. Additionally, BsDb showed a significant gain both in the antigen-binding affinity and in TNF-α-neutralizing ability as compared to its original antibodies, L19 and anti-TNF-α scFv, which were produced in E. coli. BsDb was constructed and was endowed with enhanced bioactivities and improved production processing. Therefore, it holds great potential for in vivo applications.     

Kidney injury molecule-1 is involved in the chemotactic migration of mesenchymal stem cells

A better understanding of the organ specific factors that regulate the migration of mesenchymal stem cells (MSCs) into the target organ is essential for optimization of strategies to improve the repair after injury. In the present study, we showed that the kidney injury molecule-1 (KIM-1), a well-known kidney-specific biomarker, enhanced the in vitro migration capacity of MSCs as a potent kidney-specific chemo-attractant or an inducer. The in vitro roles were verified by migration assay using KIM1-PK1 cell lines, the mouse proximal tubular epithelial cells (mPTEs) and recombinant human KIM-1 proteins (rhKIM-1). Immunofluorescence staining displayed specific ectodomain binding of KIM-1 on the surface of MSCs. Upregulation of chemokine receptor type 4 (CXCR4) protein when treated with tumor necrosis factor alpha (TNF-α) was shown. The effect of KIM-1 on migration of MSCs was augmented by TNF-α pretreatment in a dose-dependent manner, and reduced by AMD3100, an antagonist of CXCR4. These results suggest that KIM-1 is a potential chemo-ligand of CXCR4 and may play an important role in kidney-specific migration of MSCs via interaction between KIM-1 and CXCR4.     

The Attenuation of Lung Ischemia Reperfusion Injury by Oxymatrine

To investigate the protective effects of oxymatrine (OMT) on lung ischemia reperfusion injury (LIRI) in rabbits, models of LIRI in rabbit were used. Thirty-two rabbits were randomly divided into four groups: control group (n = 8), ischemia reperfusion group (I/R group, n = 8), OMTl group (n = 8), OMT2 group (n = 8). Lung tissue samples were collected at 40, 80, 120 min time-points after lung ischemia reperfusion. TNF-α, 1I-8, IL-10, apoptosis index (AI), and index of quantitative assessment of histologic lung injury (IQA) were measured in each group. TNF-α and IL-8 in I/R group were significantly higher than those of the control group and OMT2 group (P < 0.01), but in OMT2 group they were significantly lower than those of OMTl group (P < 0.05). IL-10 in OMT2 group and OMTl group was significantly higher than that of I/R group (P < 0.01). But in OMTl group it was significantly lower than that of OMT2 group (P < 0.05). AI in I/R group was significantly higher than that of OMT2 group and the control group at 80 min after lung ischemia reperfusion (P < 0.01). IQA in OMTl group and OMT2 group was significantly lower than that of the I/R group (P < 0.01). Oxymatrine can protect against LIRI in rabbits by upregulating levels of IL-10 and downregulating levels of TNF-α and IL-8, inhibiting the alveolar cells apoptosis and inflammatory response, and attenuating the acute LIRI.     

Negative-Pressure Wound Therapy Enhances Local Inflammatory Responses in Acute Infected Soft-Tissue Wound

Clinical studies found that negative-pressure wound therapy (NPWT) displayed significant clinical benefits in the healing of infected wounds. However, the effect of NPWT on local inflammatory responses in acute infected soft-tissue wound has not been investigated thoroughly. The purpose of this study was to test the impact of NPWT on local expression of proinflammatory cytokines, amount of neutrophils, and bacterial bioburden in wound from acute infected soft-tissue wounds. Full-thickness wounds were created on the back of rabbits, and were inoculated with Staphylococcus aureus strain ATCC29213. The wounds were treated with sterile saline-moistened gauze dressings and NPWT with continuous negative pressure (?125 mmHg). Wound samples were harvested on days 0 (6 h after bacterial inoculation), 2, 4, 6, and 8 at the center of wound beds before irrigation for real-time PCR analysis of gene expression of IL-1β, IL-8, and TNF-α. Wound biopsies were examined histologically for neutrophil quantification in different layers of tissue. Quantitative bacterial cultures at the same time point were analyzed for bacterial clearance. Application of NPWT to acute infected wounds in rabbits was compared with treatment with sterile saline-moistened gauze, over an 8-day period. NPWT-treated wounds exhibited earlier and greater peaking of IL-1β and IL-8 expression and decrease in TNF-α expression over the early 4 days (P < 0.05). Furthermore, histologic examination revealed that significantly increased neutrophil count was observed in the shallow layer in wound biopsies of NPWT treatment at day 2 (P < 0.001). In addition, there was a statistically significant decrease of bacteria load from baseline (day 0) at days 2 and 8 in NPWT group (P < 0.05). In conclusion, this study demonstrates that NPWT of acute infected soft-tissue wounds leads to increased local IL-1β and IL-8 expression in early phase of inflammation, which may trigger accumulation of neutrophils and thus accelerate bacterial clearance. Meanwhile, the success of NPWT in the treatment of acute wounds can attenuate the expression of TNF-α, and the result may partly explain how NPWT can avoid significantly impairing wound healing.     

Neural Differentiation of Mesenchymal Stem Cells Influences Their Chemotactic Responses to Stromal Cell-Derived Factor-1α

Mesenchymal stem cells (MSCs) are proposed as a promising source for cell-based therapies in neural disease. Although increasing numbers of studies have been devoted to the delineation of factors involved in the migration of MSCs, the relationship between the chemotactic response and the differentiation status of these cells is still unclear. In the present study, we demonstrated that MSCs in varying neural differentiation states display various chemotactic responses to stromal cell-derived factor-1α (SDF-1α). The chemotactic responses of MSCs under different differentiation stages in response to SDF-1α were analyzed by Boyden chamber, and the results showed that cells of undifferentiation, 24-h preinduction, 5-h induction, and 18-h maintenance states displayed a stronger chemotactic response to SDF-1α, while 48-h maintenance did not. Further, we found that the phosphorylation levels of PI3K/Akt, ERK1/2, SAPK/JNK, and p38MAPK are closely related to the differentiation states of MSCs subjected to SDF-1α, and finally, inhibition of SAPK/JNK signaling significantly attenuates SDF-1α-stimulated transfilter migration of MSCs of undifferentiation, 24-h preinduction, 18-h maintenance, and 48-h maintenance, but not MSCs of 5-h induction. Meanwhile, interference with PI3K/Akt, p38MAPK, or ERK1/2 signaling prevents only cells at certain differentiation state from migrating in response to SDF-1α. Collectively, these results demonstrate that MSCs in varying neural differentiation states have different migratory capacities, thereby illuminating optimization of the therapeutic potential of MSCs to be used for neural regeneration after injury.     

Reducing the bioactivity of Tannerella forsythia lipopolysaccharide by Porphyromonas gingivalis

Tannerella forsythia is considered a pathogen of periodontitis and forms a biofilm with multi-species bacteria in oral cavity. Lipopolysaccharide is a powerful immunostimulator and induces inflammation and shock. The purpose of this study was to investigate the characteristics of T. forsythia LPS in its co-cultivation with Fusobacterium nucleatum or Porphyromonas gingivalis. T. forsythia was co-cultured in the presence and absence of F. nucleatum and P. gingivalis and then T. forsythia LPS was extracted. The extracts were analyzed by SDS-PAGE and NF-κB reporter CHO cell lines. THP-1 cells were treated with the LPS and evaluated induction of cytokine expression by real-time RT-PCR and ELISA. For analysis of the bioactivity of T. forsythia LPS, the binding assay on LPS-binding protein (LBP) and CD14 was processed. The extracts did not contaminate other molecules except LPS and showed TLR4 agonists. Co-cultured T. forsythia LPS with P. gingivalis exhibited a lower level of induction of TNF-α, IL-1β, and IL-6 expression than singleor co-cultured T. forsythia LPS with F. nucleatum in the conditions of human serum. However, the three T. forsythia LPS did not show difference of cytokine induction in the serum free conditions. Co-cultured T. forsythia LPS with P. gingivalis exhibited a lower affinity to LBP and CD14 as binding site of O-antigen and attached at a lower level to THP-1 cells compared to single- or co-cultured T. forsythia LPS with F. nucleatum. The virulence of T. forsythia LPS was decreased by co-culturing with P. gingivalis and their affinity to LBP and CD14 was reduced, which may due to modification of O-antigen chain by P. gingivalis.     

Evidence of endoplasmic reticulum stress and liver inflammation in the American mink Neovison vison with benign hepatic steatosis

We investigated the presence of inflammatory signs in the progression of fatty liver disease induced by fasting. Sixty standard black American mink (Neovison vison) were fasted for 0, 1, 3, 5, or 7 days and one group for 7 days followed by re-feeding for 28 days. Liver sections were evaluated histologically and liver mRNA levels indicating endoplasmic reticulum (ER) stress, adipogenic transformation, and inflammation were assessed by quantitative real-time PCR. After 3 days of fasting, the mink had developed moderate liver steatosis. Increased hyaluronan reactivity in lymphocytic foci but no Mallory–Denk bodies were seen in livers of the mink fasted for 5–7 days. Up-regulation of glucose-regulated protein, 78 kDa was observed on day 7 indicating ER stress, especially in the females. Liver lipoprotein lipase and monocyte chemoattractant protein 1 mRNA levels increased in response to 5–7 days of food deprivation, while tumor necrosis factor α (TNF-α) was the highest in the mink fasted for 5 days. The expression of the genes of interest, except for TNF-α, correlated with each other and with the liver fat content. The mRNA levels were found to change more rapidly below n-3/n-6 polyunsaturated fatty acid ratio threshold of 0.15. Following re-feeding, hepatocyte morphology and mRNA abundance returned to pre-fasting levels. Within the studied timeframe, evidence for ER stress, adipogenic transformation, and liver inflammation suggested incipient transition from steatosis to steatohepatitis with potential for development of more severe liver disease. This may present a possibility to influence disease progression before histologically observable steatohepatitis.     

Enhanced biotransformation of dehydroepiandrosterone to 3β,7α,15α-trihydroxy-5-androsten-17-one with Gibberella intermedia CA3-1 by natural oils addition

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3β,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2 %, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8 % compared with that of the control at DHEA concentration of 8.0 g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates.     

Expression status of let-7a and miR-335 among breast tumors in patients with and without germ-line BRCA mutations

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation (P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC.     

Dietary l-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine

This study was conducted to determine effects of dietary supplementation with 1 % l-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.