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.     

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.     

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.     

In vitro study of the anti-inflammatory activity of some medicinal and edible plants growing in Russia

The effects of the ethanolic extracts of 105 plants used in Russian traditional medicine, 26 vegetables and fruits and 2 mushrooms on the release of tumor necrosis factor α (TNF-α) and prostaglandin E₂ (PGE₂) in lipopolysaccharide (LPS)-stimulated differentiated human acute monocytic leukemia THP-1 cells were studied using TNF-α and PGE₂ assays, respectively. We found that 16 plant extracts inhibited TNF-α production and 15 extracts decreased PGE₂ release in the cells in a concentration-dependent manner. Guilder rose, tansy, shrubby cinquefoil, wintergreen, and prince’s pine, the last two of which belong to the Pyrolaceae family, notably inhibited the levels of both of the inflammatory mediators.     

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.     

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.     

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.     

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.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.     

Anti-influenza effect of Cordyceps militaris through immunomodulation in a DBA/2 mouse model

The immune-modulatory as well as anti-influenza effects of Cordyceps extract were investigated using a DBA/2 mouse model. Three different concentrations of Cordyceps extract, red ginseng extract, or drinking water were orally administered to mice for seven days, and then the mice were intranasally infected with 2009 pandemic influenza H1N1 virus. Body weight changes and survival rate were measured daily post-infection. Plasma IL-12, TNF-α, and the frequency of natural killer (NK) cells were measured on day 4 post-infection. The DBA/2 strain was highly susceptible to H1N1 virus infection. We also found that Cordyceps extract had an antiinfluenza effect that was associated with stable body weight and reduced mortality. The anti-viral effect of Cordyceps extract on influenza infection was mediated presumably by increased IL-12 expression and greater number of NK cells. However, high TNF-α expression after infection of H1N1 virus in mice not receiving treatment with Cordyceps extract suggested a two-sided effect of the extract on host immune regulation.     

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.     

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.     

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.     

The oxidation of HSP70 is associated with functional impairment and lack of stimulatory capacity

Expression of intracellular HSP70 is associated with cytoprotective effects against a wide range of stressful stimuli, such as inflammation, oxidative stress, hypoxia, endotoxins, infections, and fever. This cytoprotective effect is mainly attributed to their ability to stabilize protein structures through chaperone-like reversible interactions. HSP70 was recently detected in the extracellular medium, and its presence in serum is commonly associated with pathological situations, where it exerts modulatory effects on cells of the immune system. Previously, we have described the relationship between serum HSP70 levels, oxidant status, and clinical outcome of septic patients; the group of patients with higher prooxidant status and higher serum HSP70 had also higher mortality. To investigate the possible association between oxidized HSP70 and cytoprotection or cell death, we incubated RAW 264.7 macrophages with oxidized HSP70 and evaluated nitrite production, cell proliferation, cell viability, TNF-α release, and phagocytic activity. We also evaluated structural modifications caused by oxidation in purified HSP70. Oxidation of HSP70 altered its protein structure; besides, the modulatory effect of oxidized HSP70 on RAW264.7 cells was different from that of native HSP70. Macrophages treated with oxidized HSP70 presented lower proliferation and viability, lower phagocytic activity, and lower TNF-α release. These results indicate that oxidation of extracellular HSP70 modified its signaling properties, causing alterations on its modulatory effects on macrophage function and viability.     

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.     

Utilization of Extracorporeal Membrane Oxygenation Alleviates Intestinal Ischemia–Reperfusion Injury in Prolonged Hemorrhagic Shock Animal Model

Intestinal ischemia–reperfusion injury is one of the main factors leading to multiple organ failure after resuscitation of prolonged hemorrhagic shock; however, the current conventional fluid resuscitation still cannot effectively reduce intestinal injury caused by prolonged hemorrhagic shock. To investigate the effect of ECMO resuscitation on alleviating intestinal ischemia–reperfusion injury in a prolonged hemorrhagic shock rabbit model. Thirty New Zealand white rabbits were randomly divided into three groups: control group, conventional fluid resuscitation group, and ECMO resuscitation group. The prolonged hemorrhagic shock model was established by keeping the arterial blood pressure from 31 to 40 mmHg for 3 h through the femoral artery bleeding, and performing the resuscitation for 2 h by conventional fluid resuscitation and ECMO resuscitation, respectively. Chiu’s score of intestinal injury, serum lactate and TNF-α levels, intestinal mucosamyeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM-1), and Claudin-1expression were detected. The mean arterial blood pressure in Group 2 was significantly higher after resuscitation than in Group 1, but serum lactate and inflammatory cytokines TNF-α level were significantly lower. And Chiu’s score of intestinal injury and myeloperoxidase (MPO) activity level and ICAM-1 expression were significantly lower in the ECMO resuscitation group, in which the Claudin-1 levels were significantly increased. ECMO resuscitation for the prolonged hemorrhagic shock improves tissue perfusion and reduces the systemic inflammation, and thus alleviates intestinal damage caused by prolonged hemorrhagic shock.     

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.