Different Concentrations Of TNF-α Research Articles

Cooperative induction of CXCL chemokines by inflammatory cytokines and oxidized phospholipids.

Inflammation is initiated and driven by a mixture of mediators, which modify effects of each other. This study analysed in vitro pro-inflammatory activity of inflammatory cytokines (TNFα and IL-1β) in a combination with a lipid DAMP molecule, oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC). The study was performed on endothelial and monocytic cell lines. The cells were treated with different concentrations of TNFα or IL-1β, OxPAPC and their combinations, either in the presence or absence of drugs regulating inflammation. Pro-inflammatory effects of TNFα/IL-1β and OxPAPC were estimated by analysis of chemokines CXCL8, CXCL2 and CXCL3 by ELISA and RT-PCR. Toxicity was determined by analysis of metabolic activity. Statistical significance was estimated by ANOVA and Dunnett's test. OxPAPC was a much weaker chemokine inducer as compared to TNFα or IL-1β. However, OxPAPC and TNFα/IL-1β together induced effects that were significantly stronger than the arithmetical sum of individual effects. This cooperative action of OxPAPC and TNFαwas reversed by inhibitors of p38 MAPK. We hypothesise that the boosting of TNFα and IL-1β effects by OxPAPC may be more pathologically important than the action of the lipid alone. Inhibitors of p38 MAPK may become a tool for analysis of pathological role of oxidized phospholipids.

Tumor Necrosis Factor-Alpha Disrupts Cx43-Mediated Corneal Endothelial Gap Junction Intercellular Communication.

Connexin43 (Cx43)-mediated gap junctions are vital in maintaining corneal endothelium homeostasis. Tumor necrosis factor-alpha (TNF-α) is among the most important inflammatory factors which cause corneal endothelial dysfunction in various eye diseases. However, the effect of TNF-α on Cx43-mediated gap junctions of the corneal endothelium remains undefined. In the current research, we determined the effect of TNF-α on gap junction intercellular communication (GJIC) in rabbit corneal endothelium. To evaluate alterations of GJIC, if any, we treated ex vivo cultured rabbit corneal endothelium with different concentrations of TNF-α (2-20 ng/ml). The localization of Cx43 was analyzed by immunostaining, while RT-qPCR and western blot were used to profile the expression of Cx43 and zonula occludens-1 (ZO-1). The association between ZO-1 and Cx43 was evaluated using immunoprecipitation and double staining. GJIC activity was determined by the scrap loading and dye transfer assay (SLDT). Our data demonstrated that a high concentration of TNF-α (10 ng/ml and 20 ng/ml) disrupts the Cx43 mediated gap junction distribution in rabbit corneal endothelium and suppresses the expression of Cx43 protein. Furthermore, rabbit corneal endothelial GJIC was inhibited due to the decreased association between the ZO-1 and Cx43 proteins. Current results demonstrate that TNF-α inhibits corneal endothelial GJIC via decreasing the association between ZO-1 and Cx43, disrupting the distribution of Cx43, and downregulating the expression of Cx43 protein. This study offers a new theoretical foundation for diagnosing and treating corneal endothelial cell decompensation induced by elevated TNF-α in various eye diseases.

An in-vitro analysis of L-Carnitine mediated rescue of TNF-? induced apoptosis in mice oocytes

: Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine implicated in various physiological and pathological events. Carnitine is a quaternary amine which plays a significant role in fatty acid oxidation and is reported to produce antiapoptotic effects. Aim of this work was to study the effect of L-Carnitine (LC) on TNF-α induced apoptosis in mice oocytes. : Oocytes were isolated from super ovulated Swiss Albino mice and treated with different concentrations of TNF-α (0.1ng/ml, 1ng/ml, 10ng/ml, 100ng/ml) and LC (0.1mg/ml, 0.3mg/ml, 0.5mg/ml, 1.0 mg/ml). TUNEL Assay was done for the biochemical assessment of apoptosis.: Apoptotic indices with different doses of TNF-α (0.1ng/ml, 1ng/ml, 10ng/ml, 100 ng/ml) were 28.5%, 71.4%, 100%, 42.8% respectively. The concentration of TNF-α that produced the highest apoptotic index was 10ng/ml. LC alone in different doses (0.1mg/ml, 0.3mg/ml, 0.5mg/ml, 1.0 mg/ml) did not elicit any apoptotic signal. Further LC was added in different doses with 10ng/ml TNF-α to study the rate of apoptosis in mice oocytes. Apoptotic index with 10 ng/ml TNF-α and different doses of LC (0.1mg/ml, 0.3mg/ml, 0.5mg/ml, 1 mg/ml) were 25%, 37.5%, 50%, 62.5% respectively. The concentration of LC that reduced the apoptotic index to the maximum was 0.1mg/ml.: Present study could demonstrate the anti-apoptotic effect of LC against apoptotic effects of TNF-α in mice oocytes. The study presents preliminary data suggesting a possible therapeutic role of LC in inflammatory etiologies such as ovarian failure.

Role of immune-related lncRNAs--PRKCQ-AS1 and EGOT in the regulation of IL-1β, IL-6 and IL-8 expression in human gingival fibroblasts with TNF-α stimulation

Background/purposeIt was reported that lncRNAs have an effect on immune-related diseases, however, their roles in periodontitis remain to be investigated. The aim of this study was to look for immune-related lncRNAs in periodontitis, and to preliminarily explore their function in vitro. Materials and methodsCIBERSORT was used to analyze abundance of immune cell in the periodontal tissue. Correlation between the expression profile of lncRNAs and abundance of immune cell was calculated and immune-related lncRNAs were identified. The expressions of immune-related lncRNAs identified were validated by RT-qPCR with 15 periodontitis and 15 healthy gingival tissues. The expressions of PRKCQ-AS1 and EGOT in HGFs were detected under the stimulation of different concentrations of TNF-α (0, 10, 15, 20, 30 ng/mL) and different duration (0, 12, 24 and 48 h). Then, siRNA was used to silence PRKCQ-AS1 and EGOT in HGFs. The expression level of IL-1β, IL-6, IL-8 of the HGFs after stimulated by 15 ng/mL TNF-α, and the activation of NF-κB pathway was observed. ResultsPRKCQ-AS1 and EGOT were identified as top 2 immune-related lncRNAs in periodontal tissues. The expressions of PRKCQ-AS1 and EGOT were significantly up-regulated in inflamed periodontal tissue and in HGFs under TNF-α stimulation. After knock-down of PRKCQ-AS1 and EGOT, expression level of IL-1β, IL-6, and IL-8 in HGFs with TNF-α stimulation were decreased, and activation of NF-κB pathway was inhibited. ConclusionPRKCQ-AS1 and EGOT were firstly identified as immune-related lncRNAs in periodontal tissue, and they regulate the expression of IL-1β, IL-6, and IL-8 of HGFs through the NF-κB pathway.

TNF-α induces AQP4 overexpression in astrocytes through the NF-κB pathway causing cellular edema and apoptosis

Aquaporin 4 (AQP4) is highly expressed on astrocytes and is critical for controlling brain water transport in neurological diseases. Tumor necrosis factor (TNF)-α is a common cytokine found in disease microenvironment. The aim of the present study was to determine whether TNF-α can regulate the expression of AQP4 in astrocytes. Primary astrocyte cultures were treated with different concentrations of TNF-α and the cell viability was assessed through cell counting kit-8 (CCK-8) assay and AQP4 expression was detected by qPCR, Western blots, and immunofluorescence assays. The activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway was detected by Western blot. Further, dual-luciferase reporting system and chromatin immunoprecipitation (ChIP) were used to detect the transcriptional regulation of AQP4 by p65. These experiments demonstrated that treatment with TNF-α can lead to astrocyte edema and an increase in AQP4 expression. Following TNF-α treatment, the expression levels of P-IKKα/β-IκBα and P-p65 increased significantly over time. The results of the dual-luciferase reporter system and ChIP assays revealed that p65 protein and AQP4 promoter had a robust binding effect after TNF-α treatment, and the NF-κB pathway inhibitor, BAY 11-7082 could inhibit these effects of TNF-α. The expression level of AQP4 was significantly decreased upon p65 interference, while the astrocyte viability was significantly increased compared with that in the TNF-α only group. In conclusion, TNF-α activated NF-κB pathway, which promoted the binding of p65 to the AQP4 gene promoter region, and enhanced AQP4 expression, ultimately reducing astrocyte viability and causing cell edema.

TNFα regulates intestinal organoids from mice with both defined and conventional microbiota

Cytokines are key factors affecting the fate of intestinal stem cells (ISCs) and effective reagents to manipulate ISCs for research purpose. Tumor necrosis factor alpha (TNFα) is a cytokine produced primarily by monocytes and macrophages. It can induce apoptotic cell death and inflammation, and to inhibit tumorigenesis and viral replication. Additionally, TNFα has been shown to play a critical role in the pathogenesis of inflammatory bowel disease (IBD). It is therefore important to identify the mechanism by which individual cytokines affect particular cell types. For this purpose, we used both conventional (CONV) and altered Schaedler flora (ASF) C3H/HeN mice to elucidate the effect of different microbial populations (complex versus defined) on growth of miniguts derived from two different intestinal environments. Furthermore, we studied the effects of different concentrations of TNFα extracted from the lymph and spleen on the growth and viability of ISCs recovered from mice bearing the ASF or CONV microbiota. The effect of TNFα on miniguts growth depends not only on the source and concentration, but also on the intestinal microenvironment from which the ISCs were derived. The findings suggest that TNFα influences the proliferation of miniguts derived from ISCs and, therefore, modulates mucosal homeostasis of the host.

Relationship of Circulating Tumor Necrosis Factor Alpha (TNF-α) and Insulin Secretion and Resistance in Euglycaemic Dogs

Background: Insulin resistance is a state that is characterized with reduced sensitivity of peripheral tissues to insulin. It can be related with increased level of tumor necrosis factor alpha (TNF-α) in dogs. Insulin resistance can be evaluated by homeostasis model assessment (HOMA-IR, HOMA-β). The aim of this study was to determine correlation of circulating TNF-α level with insulin production and insulin resistance indexes in euglycaemic dogs. Materials, Methods & Results: Seventydogs of normal body score were included in this study. After blood sampling levels of glucose, insulin and TNF-α were determined and indexes HOMA-IR and HOMA-β were calculated. Three groups in accordance to TNF-α levels were formed: the first-TNF-α 0-2.0 pg/mL, the second-TNF-α below median (2.1-17.0 pg/mL) and the third-TNF-α above median (17.1-51.8 pg/mL). Differences in insulin and glucose levels, HOMA-IR and HOMA-β were determined in all three groups. ANOVA and posthock LSD analyses were used. Correlation between HOMA-IR and HOMA-β was determined. Linear regression between HOMA-β/HOMA-IR ratio and glucose concentration was calculated. SPSS statistical program was used (IBM). Highest insulin level was detected in the second group and the lowest was detected in the third group. The lowest glucose level was detected in the first group. The highest value of HOMA-β index was noted in the first group and it decreases with TNF-α increase. The highest HOMA-IR value was detected in the second group and the lowest was in the third group. Positive correlation was noted between HOMA-IR and HOMA-β. Significant linear correlation was noted between glucose levels in function of HOMA-β/HOMA-IR (R2= 0.51-0.78, P = 0.0007). The first group showed the minor change of glucose level (b= 0.29 mmol/L). In the third group the greatest change of glucose level in function of HOMA-β/HOMA-IR was noted (b= 0.52 mmol/L). In the third group the highest increase of glucose level followed by decrease in HOMA-β/HOMA-IR ratio was noted. Discussion: Increase of TNF-α followed by increase of insulin was noted in the second group. That indicates reduced insulin action and compensatory increase in his concentrations in order to achieve the same effect. TNF-α induces reduction in expression of glucose transporter 4 that is insulin-regulating hormone. Serine phosphorylation of insulin receptor substrate-1 that is induced by TNF-α causes inhibition of insulin receptors. These actions cause insulin resistance and compensatory increase in insulin secretion. Increased tissue resistance is reflected in increased HOMA-IR index which is directly associated with insulin and glucose level. Increase of insulin value and HOMA-IR index were noted in second group. This indicates the influence of TNF-α on insulin resistance. The lowest insulin level was noted in the third group of dogs. Decrease in production and secretion leads to reduction in circulating insulin and can be evaluated by HOMA-β index. The highest value of this index was noted in first group and decreases with TNF-α increase. This can be related with apoptotic effect that TNF-α has on beta- pancreatic cells. Increase of HOMA-β index causes increase of HOMA-IR. This is indication of increased peripheral resistance. Compensatory mechanism for that state is increased insulin secretion. Glucose level will increase more during increase of HOMA-IR and decrease of HOMA-β. This indicates that TNF-α regulates glucose level directly and explains differences in glucose concentrations in dogs with different concentrations of TNF-α showed. Values of insulin resistance indexes, glucose and insulin were affected by circulating concentration of TNF-α. The most unfavorable change in glucose concentration based on insulin production and tissue resistance was founded in dogs with the highest circulating TNF-α concentration in blood.

MiR-21 may Act as a Potential Mediator Between Inflammation and Abnormal Bone Formation in Ankylosing Spondylitis Based on TNF-α Concentration-Dependent Manner Through the JAK2/STAT3 Pathway.

Objective:To explore the role of microRNA (miR-21) in new bone formation in ankylosing spondylitis (AS) as mediated by different concentration of tumor necrosis factor-α (TNF-α).Methods:Fibroblasts isolated from the hips of patients with AS were induced to osteogenesis. These cells were then stimulated with varying concentrations of TNF-α. MicroRNA-21 expressions were evaluated using reverse transcription–polymerase chain reaction (RT-PCR) and osteogenesis was detected via Alizarin Red S (ARS) staining and measurement of alkaline phosphatase (ALP) activity. Relative expressions of p-STAT3, Nuclear STAT3, cytoplasm STAT3, Runx2, BMP2, osteopontin, osteocalcin, and LC3B in AS fibroblasts were measured after exposure to different concentrations of TNF-α. The STAT3-inhibiting small interfering RNA allowed further exploration on its impact on miR-21 and primary miR-21 expressions. A proteoglycan-induced arthritis (PGIA) Balb/c mouse model was established in order to monitor sacroiliac joint (SIJ) inflammation and subsequent damage through magnetic resonance image. Serum miR-21 and TNF-α expressions were evaluated using RT-PCR and enzyme-linked immunosorbent assay. At week 16, mice models were transfected intravenously with miR-21 overexpressing agomir and miR-21 inhibiting antagomir for 7 successive days. The rate of abnormal bone formation at SIJ was evaluated using microcomputed tomography and hematoxylin and eosin staining at week 24. Western blot analysis enabled quantification of STAT-3, JAK-2, and interleukin (IL)-17A expressions present in the SIJ.Results:The in vitro miR-21 expression and osteogenesis activity were noted to be augmented in the setting of low TNF-α concentrations (0.01-0.1 ng/mL) while they were depressed in settings with higher TNF-α concentrations (1-10 ng/mL). Samples with the most distinct ARS manifestation and ALP activity as well as the highest miR-21 expressions were those who received 0.1 ng/mL of TNF-α. Primary miR-21 was found to be notable raised by Si-STAT3, while the converse effect was seen in mature miR-21 expressions. Intravenous injection of exogenous miR-21 contributed to new bone formation and significantly elevated expressions of STAT3, JAK2, and IL-17 in PGIA mice.Conclusions:The results revealed that miR-21 may act as a potential mediator between new bone formation and inflammation in AS.

Hydroxysafflor Yellow A Inhibits TNF-α-Induced Inflammation of Human Fetal Lung Fibroblasts via NF-κB Signaling Pathway.

Objective Hydroxysafflor yellow A (HSYA), an effective ingredient of the Chinese herb Carthamus tinctorius L, attenuated bleomycin-induced pulmonary fibrosis in mice. This study is to investigate the effect of HSYA on the proliferation and inflammatory level of human fetal lung fibroblasts (MRC-5 cells) induced by tumor necrosis factor-α (TNF-α) and explore the underlying mechanisms. Methods MRC-5 cells were treated with different concentrations of TNF-α, HSYA, or/and etanercept (ENCP, TNF-α receptor (TNFR1) antagonist, 500 ng/mL) before cell proliferation was detected. The laser confocal microscope was used to observe the role of HSYA in binding of TNF-α and its receptor. Co-immunoprecipitation was used to detect the binding of TNFR1 and TAK1-TAB2 complex. Real-time quantitative RT-PCR and western blot were used to detect the expressions of inflammation-related cytokines and proteins related with the NF-κB pathway. Luciferase reporter gene assay and chromatin coprecipitation method were used to detect the interaction between AP-1 and TGF-β1 promoter. Results TNF-α (5 ng/mL) was used to induce inflammation and proliferation in MRC-5 cells. HSYA can partially suppress the stimulation of TNF-α on proliferation and inflammatory response of MRC-5 cells. HSYA could compete with TNF-α to bind with TNFR1 and hamper the binding of TNFR1 to TAK1-TAB2 complex. In addition, HSYA could also inhibit the activation of the NF-κB signal pathway and suppress the binding of TGF-β1 promoter with AP-1. Conclusion Evidence in this study suggested that HSYA affects TNF-α-induced proliferation and inflammatory response of MRC-5 cells through the NF-κB/AP-1 signaling pathway, which may provide theoretical basis for HSYA treatment in pulmonary fibrosis.

Effects of Tumor Necrosis Factor Alpha on the Expression of Programmed Cell Death Factor 5 in Arthritis.

ObjectiveTo investigate the effect of tumor necrosis factor alpha (TNF‐α) on the proliferation of fibroblast‐like synoviocytes (FLS) and the expression of programmed cell death factor 5 (PDCD5) in an inflammatory microenvironment, for the further understanding of the mechanism of action of TNF‐α in promoting the proliferation of synovial cells and the apoptosis of the chondrocytes.MethodsArticular carriage specimens were obtained from 21 cases with osteoarthritis and 12 cases with femoral neck fractures as healthy controls during arthroplasties. The expression of PDCD5 was evaluated by immunofluorescence analyzed by mean option density (MOD) detected using the software ImagePro Plus. Real‐time PCR was performed to evaluate the transcriptions of PDCD5 and TNF‐α in synovium. FLS cells derived from rheumatoid arthritis patients were cultured in vitro and incubated with different concentrations of TNF‐α. The effects of TNF‐α at different concentrations on the proliferation of FLS cells were detected by Cell Counting Kit‐8 (CCK‐8) assay to evaluate the cell proliferation rate. After incubation with the absence or presence of recombinant human TNF‐α at different concentrations, the FLS cells were isolated for detection of PDCD5 protein and PDCD5 gene. The expression of PDCD5 protein was detected by western‐blot and the transcription of PDCD5 gene from the cells was detected by real‐time quantitative PCR.ResultsThe MOD of PDCD5 as well as TNF‐α of osteoarthritis cartilage sections were significantly increased compared with those of the controls, and in synovium there was a positive correlation between transcriptions of their mRNA. When the concentration of TNF‐α was 1 ng/mL, the cell proliferation rate was not significantly different from that of the control group (P = 0.592), while the proliferation of FLS cells was significantly promoted when the concentration of TNF‐α was 5, 10, 15, or 20 ng/mL, and the proliferation‐promoting rates were 35.64% ± 6.96%, 48.72% ± 7.69%, 45.60% ± 8.85%, and 39.32% ± 6.18%, respectively (P < 0.01). The transcription of PDCD5 gene was significantly downregulated, which was 80.44% ± 4.07% and 84.30% ± 5.48%, respectively (P < 0.05), in the FLS cells incubated with TNF‐α at the concentration of 10 and 15 ng/mL for 24 h. When the concentration of TNF‐α was 1, 5, or 20 ng/mL, the transcription of PDCD5 mRNA in FLS cells was not significantly different from that in the control group (P > 0.05). The expression of PDCD5 protein was only significantly downregulated when the concentration of TNF‐α was 10 ng/mL (P < 0.01), while the expression of PDCD5 protein in FLS cells was not significantly different from that in the control group (P > 0.05).ConclusionThe expression of PDCD5 as well as TNF‐α in osteoarthritis cartilage and synovium was significantly higher than in healthy tissues, and TNF‐α can promote the proliferation of FLS cells in patients with rheumatoid arthritis, and inhibit the expression of PDCD5. PDCD5 may be involved in the abnormal proliferation of synoviocytes and the degeneration of chondrocytes stimulated by TNF‐α.

TNF-alpha increases angiogenic potential in a co-culture system of dental pulp cells and endothelial cells.

We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Thursday, September 27, 2018 2:05 PM–3:05 PM Section on Biologics and Basic Research Abstract Presentations: 124. NF-κB dependent unfolded protein response in the survival and proliferation of nucleus pulposus cells under TNF-α stimulation

BACKGROUND CONTEXT Intervertebral disc (IVD) degeneration is a degenerative disease closely related to inflammation of nucleus pulposus (NP) cells. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine expressed by NP cells (NPCs) of degenerate IVD, which induces NPCs apoptosis and extracellular matrix (ECM) degradation, and accelerate IVD degeneration (IDD). The endoplasmic reticulum serves several important cell functions, which are essential for normal cell function and survival. Nuclear factor-kappaB (NF-κB) is important for genes involved in cell survival, adhesion, differentiation, and proliferation. However, the roles of ER stress and NF-κB in IDD remain to be elucidated. PURPOSE This study aims to clarify the roles of NF-κB and ER stress related unfolded protein response (UPR) in TNF-α-induced biological changes in rat NPCs and IVD degeneration. METHODS In vivo, different doses of exogenous TNF-α were injected into rat tail discs. Magnetic resonance imaging (MRI) yielding T2-weighted images were performed. Then, the discs were stained by Hematoxylin and Eosin, Masson's trichrome to visualize the histocytological changes, immunohistochemical staining was performed to evaluate the expression of ECM including aggrecan and collagen II. In vitro, we cultured rat NPCs with different concentrations of TNF-α, with or without UPR and NF-κB pathway small interfering RNA (siRNA). The mRNA expression and protein levels of UPR markers (XBP1s and p-eIF2α) and p-p65 were measured by immunofluorescence staining, qRT-PCR and western blot analysis respectively and were used to monitor UPR and NF-κB. Cell proliferation was evaluated by CCK-8 assay, cell-cycle analysis and cyclin D1 expression. Apoptosis was detected by flowcytometry and Western blot analyses. All the data were expressed as mean ± SD and from multiple independent experiments. The results were compared among different groups by using unpaired student-test. P-values RESULTS In vivo, after one month, a significant decrease in T2 value calculated from T2-mapping was observed in TNF-α injection groups along with clearly decrease in disc height and nucleus volumes in comparison to the control discs. Histological analysis showed degenerative changes with NPCs and ECM loss, while no degenerative changes were observed in control groups. In vitro, TNF-α induced the apoptosis of some NPCs in the early stage and then accelerated the proliferation of surviving cells. In addition, TNF-α stimulus up-regulated XBP1s, p-eIF2α and p-p65 at the protein level, which indicated that TNF-α activated UPR and NF-κB signals in rat NP cells. However, these effects could be reversed by UPR and NF-κB siRNA, and UPR interference decreased the expression of p-p65 notably. In parallel, both UPR and NF-κB interference reduced cell proliferation and enhanced apoptosis. CONCLUSIONS Our study demonstrated that TNF-α is an important early pathogenetic factor in disc degeneration. UPR reinforces the survival and proliferation of NPCs in TNF-α stimulus by activating NF-κB signaling, which could be an important therapeutic target in the future. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Endoplasmic Reticulum Stress Facilitates the Survival and Proliferation of Nucleus Pulposus Cells in TNF-α Stimulus by Activating Unfolded Protein Response.

Intervertebral disc (IVD) degeneration is closely related to inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α). The endoplasmic reticulum (ER) serves several important cell functions, which are essential for normal cell metabolism and survival. This study aims to clarify the role of ER stress and unfolded protein response (UPR) in TNF-α-induced biological changes in rat nucleus pulposus cells (NPCs) and IVD degeneration. In our research, rat NPCs were cultured with different concentrations of TNF-α in the presence or absence of ER stress inhibitors. Related genes and proteins were measured by immunofluorescence staining, quantitative real-time PCR, and Western blot analyses to monitor ER stress. Cell proliferation was evaluated by CCK-8 assay and cyclin D1 expression. Apoptosis was detected by flow cytometry and Western blot analyses. Our results showed that TNF-α induced the apoptosis of some NPCs in the early stage and then accelerated the proliferation of surviving cells. In addition, TNF-α stimulus upregulated ER stress markers and initiated UPR. However, these effects could be reversed by inhibitors, thereby reducing cell proliferation and enhancing apoptosis. In conclusion, ER stress reinforces the survival and proliferation of NPCs in TNF-α stimulus by activating UPR signaling, which could be an important therapeutic target in the future.

Ganglioside GM1 contributes to extracellular/intracellular regulation of insulin resistance, impairment of insulin signaling and down-stream eNOS activation, in human aortic endothelial cells after short- or long-term exposure to TNFα.

Vascular insulin resistance induced by inflammatory cytokines leads to the initiation and development of vascular diseases. In humans, circulating TNFα levels are increased during aging, suggesting a correlation between vascular insulin resistance and plasma TNFα levels. Currently, the precise molecular mechanisms of vascular insulin resistance mediated by TNFα are not well characterized. We aimed at clarifying whether glycosphingolipids contribute to vascular insulin resistance after inflammatory stimulation. In this study, we examined vascular insulin resistance using human aortic endothelial cells after treatment with different concentrations of TNFα for different time intervals for mimicking in vivo acute or chronic inflammatory situations. We show that ganglioside GM1 levels on cell membranes change depending on time of exposure to TNFα and its concentration and that the GM1 expression is associated with specific extracellular/intracellular regulation of the insulin signaling cascade. Furthermore, we provide evidence that factors such as aging and senescence affect the regulation of insulin resistance. Our data suggest that GM1 is a key player in the induction of vascular insulin resistance after short- or long-term exposure to TNFα and is a good extracellular target for prevention and cure of vascular diseases.

The inflammatory cytokine TNF-\u03b1 promotes the premature senescence of rat nucleus pulposus cells via the PI3K/Akt signaling pathway

Premature senescence of nucleus pulposus (NP) cells and inflammation are two common features of degenerated discs. This study investigated the effects of the inflammatory cytokine TNF-α on the premature senescence of NP cells and the molecular mechanism behind this process. Rat NP cells were cultured with or without different concentrations of TNF-α for 1 and 3 days. The inhibitor LY294002 was used to determine the role of the PI3K/Akt pathway. NP cells that were incubated with TNF-α for 3 days followed by 3 days of recovery in the control medium were used to analyze cellular senescence. Results showed that TNF-α promoted premature senescence of NP cells, as indicated by decreased cell proliferation, decreased telomerase activity, increased SA-β-gal staining, the fraction of cells arrested in the G1 phase of the cell cycle, the attenuated ability to synthesize matrix proteins and the up-regulated expression of the senescence marker p16 and p53. Moreover, a high TNF-α concentration produced greater effects than a low TNF-α concentration on day 3 of the experiment. Further analysis indicated that the inhibition of the PI3K/Akt pathway attenuated the TNF-α-induced premature senescence of NP cells. Additionally, TNF-α-induced NP cell senescence did not recover after TNF-α was withdrawn. In conclusion, TNF-α promotes the premature senescence of NP cells, and activation of the PI3K/Akt pathway is involved in this process.

The effects in vitro of TNF-α and its antagonist ‘etanercept’ on ejaculated human sperm

Tumour necrosis factor (TNF)-α is primarily involved in the regulation of cell proliferation and apoptosis; in addition it possesses pro-inflammatory properties. Anti-TNF-α strategies involve either administration of anti-TNF-α antibody or soluble TNF receptor to mop up circulating TNF-α. Etanercept, a recombinant human TNF-α receptor, was found to be effective in the treatment of rheumatoid arthritis. The impact of TNF-α inhibitors on human fertility is of notable interest. This in vitro study investigated the effect of different concentrations of TNF-α and etanercept used alone or in combination on sperm viability, motility, mitochondrial function, percentage of apoptosis and chromatin integrity in swim-up selected human spermatozoa. A negative effect of TNF-α (300 and 500ng mL-1) and etanercept (from 800µg mL-1 to 2000µg mL-1) individually on sperm viability, motility, mitochondrial function, percentage of apoptotic spermatozoa and sperm DNA integrity was demonstrated. However, at concentrations of 100 and 200µg mL-1, etanercept can block, in a significant way, the toxic effects of TNF-α (500ng mL-1) on studied sperm characteristics. Our results confirm that TNF-α has a detrimental effect on sperm function and suggest, for the first time, that etanercept may counteract the in vitro toxic action of TNF-α. This data appears to be quite promising, although further studies, both in vivo and in vitro, are needed to understand the exact mechanism of action of TNF-α and TNF-α antagonists on sperm function.

Correlation between nutritional status and expression of intestinal dipeptide transporter 1 protein in gastric cancer patients

Objective To study the expression of intestinal dipeptide transporter 1(PEPT1) protein in gastric cancer patients at different nutritional status, and to explore the possible regulatory mechanism. Methods According to Nutritional Risk Screening 2002 (NRS 2002) score, a total of 60 gastric cancer patients were divided into nutritional risk group (NRS 2002 score ≥ 3, n=18) and non-nutritional risk group (NRS 2002 score <3, n=42). With specimens of the small intestinal mucosa taken during operation, the expression of intestinal PEPT1 protein was detected using Western blot. The serum concentration of tumor necrosis factor (TNF-α) were measured using enzyme-linked immunosorbent assay. The expression of PEPT1 in Caco-2 cells treated with different concentrations of TNF-α (20, 50, 100 μg/L) were detected using Western blot at different time points (24, 48, 72 hours). Results The expression of intestinal PEPT1 (0.63 vs. 0.23, P=0.000) and serum TNF-α concentration (0.23 μg/L vs. 0.17 μg/L, P=0.001) in the nutritional risk group were significantly higher than those in the non-nutritional risk group. In Caco-2 cells, those treated with different concentrations of TNF-α (20, 50, 100 μg/L) for 24 hours had significantly higher PEPT1 expression than the blank group did (0.68 vs. 0.54, P=0.005; 0.72 vs. 0.54, P=0.001; 0.78 vs. 0.54, P=0.000). The Caco-2 cells treated with 50 μg/L TNF-α for 24, 48, and 72 hours had significantly higher PEPT1 expression compared with the cells in the blank group (0.57 vs. 0.52, P=0.004; 0.75 vs. 0.52, P=0.000; 0.77 vs. 0.52, P=0.000). Conclusion The expression of intestinal PEPT1 protein was increased in gastric cancer patients with nutritional risk, which was probably attributed to the regulation of TNF-α. Key words: Dipeptide transporter 1; Tumor necrosis factor; Gastric cancer; Nutritional Risk Screening 2002

Inhibition of gap junctions relieves the hepatotoxicity of TNF-α

The aim of this study was to observe the influence of gap junction (GJ) functional changes on the hepatotoxicity of TNF-α. Three different methods were employed to study functional effects of the GJ inhibition: 1) pretreatment with a GJ inhibitor; 2) inoculation of cells at high and low densities; and 3) inhibition of the expression of connexin 32 (Cx32) by small inhibitory RNA transfection. We then observed the influence of these treatments on hepatotoxicity following treatment with different concentrations of TNF-α for various duration. The hepatotoxicity of TNF-α was observed to occur in a dose- and time-dependent manner; after pretreatment inhibition, the hepatotoxicity of TNF-α was significantly reduced (P < 0.01). The hepatotoxicity of TNF-α was also found to be remarkably lower in cells that had been inoculated at low density (as measured by the amount of GJ formation among cells) than in those inoculated at density (P < 0.01). In addition, following Cx32 inhibition, the hepatotoxicity of TNF-α was significantly decreased (P < 0.01) as well. Together, these results suggest that inhibition of GJ function or of its component Cx32 significantly decreases the hepatotoxicity of TNF-α, and that the expression of Cx32 plays an important role in the hepatotoxicity of TNF-α.

Activity of matrix metalloproteinases 2 and 9 in cultured rabbit corneal epithelium cells stimulated by tumor necrosis factor alpha.

We studied the activity of matrix metalloproteinases (MMP) 2 and 9 generated by cultured rabbit corneal epithelium cells that had been stimulated with tumor necrosis factor alpha (TNF-α), to investigate the possible regulative mechanisms of MMP-2/9 and their potential effect on corneal inflammatory diseases. The rabbit corneal epithelium cells were cultured in vitro and incubated with different concentrations of TNF-α (0, 1, 10, and 100 ng/mL) for 24 h. The activity of MMP-2/9 was examined using gelatin zymography. The results were analyzed by computer image analysis and statistical tests. TNF-α stimulated the secretion of MMP-2/9 in a dose-dependent manner, and MMP-2 was activated by TNF-α. Inflammatory factors such as TNF-α can stimulate MMP-2/9 activity in corneal epithelium cells. This may be a potential manipulating mechanism of MMP expression in the pathogenesis of corneal diseases, and could play an important role in the prevention and treatment of corneal inflammatory diseases.

Green Tea Polyphenol Epigallocatechin-3-Gallate Attenuates TNF-α-Induced Intercellular Adhesion Molecule-1 Expression and Monocyte Adhesion to Retinal Pigment Epithelial Cells

Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea (Camellia sinensis) and demonstrates anti-oxidant, anticancer and anti-inflammatory properties. EGCG has been shown to protect retinal pigment epithelium (RPE) against oxidative stress-induced cell death. The pathogenesis of diseases in the retina is usually initiated by local inflammation at the RPE cell layer, and inflammation is mostly associated with leukocyte migration and the secretion of pro-inflammatory cytokines. Whether EGCG can modulate the cytokine-induced inflammatory response of RPE, particularly leukocyte migration, has not been clearly elucidated, and was therefore the objective of this study. ARPE-19 cells were cultured with different concentrations of TNF-α in the presence or absence of EGCG to different time points. Intracellular reactive oxygen species (ROS) levels were determined. Intercellular adhesion molecule (ICAM)-1 and phosphor-NF-κB and IκB expression were determined by Western blot analysis. Phosphor-NF-κB nuclear translocation and monocyte-RPE adhesion were investigated using immunofluorescence confocal laser scanning microscopy. Scanning electron microscopy (SEM) was carried out to further determine the ultrastructure of monocyte-RPE adhesion. The results demonstrated that TNF-α modulated inflammatory effects in ARPE-19 by induction of ROS and up-regulation of ICAM-1 expression. Moreover, TNF-α-induced phosphor-NF-κB nuclear translocation, increased phosphor-NF-κB expression and IκB degradation, and increased the degree of monocyte-RPE adhesion. Pretreating the cells with EGCG ameliorated the inflammatory effects of TNF-α. The results indicated that EGCG significantly exerts anti-inflammatory effects in ARPE-19 cells, partly as a suppressor of TNF-α signaling and that the inhibition was mediated via the NF-κB pathway.