Inhibition Of TNF-alpha Expression Research Articles

Effect of Topical Application of Black Seed Oil (Nigella Sativa) on Wound Healing in Diabetic Rat Ulcer Model

Objective : This study aims to evaluate the use of topical black seed oil on wound healing in a diabetic rat model. Method:10 experimental rats were induced with streptozotocin at a dose of 55 mg/kg BW. Diabetic rats were anesthetized and then the wound was made with a biopsy push with a diameter of 10 mm. The rats were divided into 2 groups, namely the untreated group and the topical treatment group with 1 ml of 100% Black Seed Oil every day for 14 days. Wound healing evaluation was carried out on days 7 and 14. Gene expression analysis was performed using molecular technique RT PCR an histopathologically using He staining. Data analysis used T test. Result: On day 7 there was a decrease in TNF expression with p <0.05, while on day 14 it also showed a decrease in TNF alpha expression with p <0.01. Histopathological showed positive development by showing better wound healing with complete epithelialization, granulation with lower inflammatory cells, higher fibroblasts and collagen compared to the control group. Conclusion :Black seed oil can increase the speed of wound healing in diabetic rat models by inhibiting TNF alpha expression.

Lenalidomide Attenuates High Fat Diet Induced‐Cyclooxygenase‐2 Levels in Human Vascular Smooth Muscle Cells

Poor diet and nutrition, e.g. consumption of excess saturated fatty acids, play a large role in the development of obesity, a significant risk factor for cardiovascular disease. Experimental and clinical studies suggest that obesity is a low‐grade systemic inflammatory disease. Along with inflammation, obesity can also trigger systemic oxidative stress, and together can initiate pathological mechanisms that lead to obesity‐associated organ damage such as endothelial dysfunction and vascular smooth muscle inflammation. Lenalidomide is a derivative of thalidomide and is currently used as a drug to treat multiple myeloma cancer. Lenalidomide exhibits potent cytokine modulatory capacity by inhibition of TNFalpha expression during conditions of cellular stress and in an experimental rodent model of obesity. The impact of lenalidomide on modulating inflammation and oxidative stress in the human vasculature has not been investigated. Therefore, the present study investigated the effects of lenalidomide on cyclooxygenase‐2 (COX‐2) levels (inflammation) and ROS (oxidative stress) in primary human vascular smooth muscle cells treated with high fat exposure. Our previous studies have demonstrated that exposure to palmitic acid, a saturated and high‐density fat, results in oxidative stress and an inflammatory response. Others have demonstrated that increased oxidative stress and inflammation occurs as a result of enhanced production of ROS. In this study, primary male aortic vascular smooth muscle cells at passage 7 were treated with vehicle or palmitate (100 μM) for 18 h with lenalidomide (0.5, 1.0, or 5 μM) or vehicle added at the 15 hour time point to investigate a reversal effect. Following treatment, cells were isolated, homogenized, and analyzed for COX‐2 levels using western analysis. Consistent with previous data, palmitate dose dependently increased basal COX‐2 protein levels; the highest dose of palmitate concomitantly increased production of prostaglandin E2. Lenalidomide reversed palmitate‐induced COX‐2, but only at the highest dose. Palmitate do not alter TNFalpha levels; however, lenalidomide elicited an increase in TNFalpha levels independent of palmitate. Superoxide production was analyzed using a 96‐well total cellular superoxide detection assay kit (Abcam). Lenalidomide, but not palmitate, increased oxidative stress and this response was dose dependent. In conclusion, high fat exposure elicits an inflammatory response in cultured primary human vascular smooth muscle, but this response appears to be independent of local cytokine or ROS production. Lenalidomide, although effective at reversing palmitate‐induced COX‐2, alone augments the pro‐inflammatory mediators, COX‐2 and TNFalpha, as well as promotes oxidative stress independent of high fat exposure in human vascular smooth muscle cells.Support or Funding InformationValley Research Partnership P2 VRP27Award (RJG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of tumor necrosis factor-alpha in the anti-HBV activity of tetracycline

To study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV). The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR. The TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA. A Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

The potential additional benefit of infliximab in patients with chronic pulmonary sarcoidosis already receiving corticosteroids: A retrospective analysis from a randomized clinical trial

Infliximab, a TNF-alpha antagonist, has shown efficacy in the treatment of sarcoidosis. Since corticosteroids inhibit TNF-alpha expression, we postulated that sarcoidosis patients receiving a sufficient corticosteroid dose may have an attenuated response to the addition of infliximab. We analyzed data from a previous randomized double blind prospective trial of infliximab versus placebo for chronic pulmonary sarcoidosis. The effect of the maintenance corticosteroid dose on the change in FVC % predicted between 0 and 24 weeks (ΔFVC%pred0-24) was analyzed in two ways. First, the mean ΔFVC%pred0-24 was calculated for the placebo and infliximab groups using three different daily prednisone equivalent dose thresholds: a) <10mg versus ≥10mg; b) <15mg versus ≥15mg; c) <20mg versus ≥20mg. Second, in both the placebo and infliximab groups, a correlation coefficient was calculated between the maintenance corticosteroid dose and ΔFVC%pred0-24. Both the group that received infliximab and either a maintenance daily dose of <10mg of prednisone and the group receiving ≥10mg had a significant increase in FVC%pred0-24. However, both the groups that received infliximab and a corticosteroid dose of >15mg of prednisone and ≥20mg of prednisone did not demonstrate a significant ΔFVC%pred0-24. For the placebo group, there was no significant correlation between the corticosteroid dose and the ΔFVC%pred0-24. For the infliximab group, there was a significant correlation (p=0.0097) between higher corticosteroid dose and less improvement in FVC%pred0-24. Our results suggest that infliximab adds minimal potential benefit to corticosteroids for pulmonary sarcoidosis at doses above 15-20mg/day of prednisone.

Effects of Yinchenhao Decoction ( ) for Non-alcoholic Steatohepatitis in Rats and Study of the Mechanism

To observe the effects of Yinchenhao Decoction (see text) for non-alcoholic steatohepatitis (NASH) in rats and study the mechanism. Total 18 male SD rats were randomly divided into a normal control group, a model group and a treatment group, 6 rats in each group. Rats in the model and treatment groups were fed with high-fat forage for 10 weeks to prepare the NASH model, and the rats in the treatment group were administrated with Yinchenhao Decoction from the 6th week for 5 weeks. All rats were sacrificed at the end of the 10th week and the samples were collected. Serum alanine aminotransferase (ALT) activity, tumor necrosis factor-alpha (TNF-alpha) level, and hepatic triglyceride (TG) and free fatty acid (FFA) contents were determined. Hepatic pathological changes were detected by HE staining. Serum ALT activity, TNF-alpha level, hepatic TG and FFA contents, and the fatty deposition in hepatocytes were significantly reduced in the rats of the treatment group. Yinchenhao Decoction has good therapeutic effects for NASH, protecting the liver function and reducing the fatty deposition in liver, which are possibly related with reduction of FFA content and inhibition of TNF-alpha expression.

Cathelicidin Modulates Clostridium difficile Toxin a-Mediated Enteritis

Background and Aims: Clostridium difficile infection is the major cause of nosocomial antibiotic-associated colitis. C. difficile produces toxin A, a proinflammatory enterotoxin involved in the pathophysiology of C. difficile infection. Cathelicidin is an endogenous antimicrobial peptide that protects the host from infections. Recent findings also support the ability of cathelicidin to modulate innate immunity and alter the development of intestinal inflammation. Here we explored whether toxin Amodifies the expression of cathelicidin inmouse ileum and cultured human colonocytes. We also determined the anti-inflammatory mechanism of cathelicidin in toxin A-mediated intestinal inflammation using a mouse model of enteritis and human primary peripheral blood monocytes. Methods: Ileal loops were prepared in anesthetized mice and injected with either toxin A (10 microg/20g mouse) or saline (control) (n= 6 loops per group). A separate group was treated with cathelicidin (200 microg/mouse) administered into the ileal loops. After 4 hrs, levels of cathelicidin and TNFalpha and mucosal histological changes of ileal loops were evaluated. Results: Toxin A significantly increased cathelicidin mRNA and protein expression (up to 5 fold, P=0.005) in mouse ileum. In human colonic epithelial NCM460 cells, toxin A induced cathelicidin protein (by ~2 fold, P 50%, P 50%, P 80%, P 50%, P<0.001) from human primary peripheral blood monocytes and mouse RAW 264.3 macrophages. This anti-inflammatory pathway was NFkappaB dependent since cathelicidin-induced inhibition of TNFalpha expression was augmented by the NF-kappaB inhibitor CAPE and reversed by the NF-kappaB activator PMA. In addition, cathelicidin (5 microM) reduced toxin A mediated NF-kappaB p65 phosphorylation in human monocytes, indicating that cathelicidin exerts its anti-inflammatory effects by inhibiting NF-kappaB. Conclusion: Toxin A induces cathelicidin mRNA and protein expression inmouse intestine and only protein expression in human colonocytes. Administration of exogenous cathelicidin reduces toxin Amediated intestinal inflammation, suggesting that this approach may be beneficial in controlling C. difficile-associated diarrhea and colitis. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, a Career Development Award from Crohn's and Colitis Foundation of America and K01DK084256 from National Institute of Health to HWK.

Increased Expression of Autotaxin / Lysophospholipase D on Intestinal Vessels Involves in Aggravation of Intestinal Damage Through Lymphocytes Migration

Background and Aims: Clostridium difficile infection is the major cause of nosocomial antibiotic-associated colitis. C. difficile produces toxin A, a proinflammatory enterotoxin involved in the pathophysiology of C. difficile infection. Cathelicidin is an endogenous antimicrobial peptide that protects the host from infections. Recent findings also support the ability of cathelicidin to modulate innate immunity and alter the development of intestinal inflammation. Here we explored whether toxin Amodifies the expression of cathelicidin inmouse ileum and cultured human colonocytes. We also determined the anti-inflammatory mechanism of cathelicidin in toxin A-mediated intestinal inflammation using a mouse model of enteritis and human primary peripheral blood monocytes. Methods: Ileal loops were prepared in anesthetized mice and injected with either toxin A (10 microg/20g mouse) or saline (control) (n= 6 loops per group). A separate group was treated with cathelicidin (200 microg/mouse) administered into the ileal loops. After 4 hrs, levels of cathelicidin and TNFalpha and mucosal histological changes of ileal loops were evaluated. Results: Toxin A significantly increased cathelicidin mRNA and protein expression (up to 5 fold, P=0.005) in mouse ileum. In human colonic epithelial NCM460 cells, toxin A induced cathelicidin protein (by ~2 fold, P 50%, P 50%, P 80%, P 50%, P<0.001) from human primary peripheral blood monocytes and mouse RAW 264.3 macrophages. This anti-inflammatory pathway was NFkappaB dependent since cathelicidin-induced inhibition of TNFalpha expression was augmented by the NF-kappaB inhibitor CAPE and reversed by the NF-kappaB activator PMA. In addition, cathelicidin (5 microM) reduced toxin A mediated NF-kappaB p65 phosphorylation in human monocytes, indicating that cathelicidin exerts its anti-inflammatory effects by inhibiting NF-kappaB. Conclusion: Toxin A induces cathelicidin mRNA and protein expression inmouse intestine and only protein expression in human colonocytes. Administration of exogenous cathelicidin reduces toxin Amediated intestinal inflammation, suggesting that this approach may be beneficial in controlling C. difficile-associated diarrhea and colitis. Supported by a Pilot and Feasibility Study grant from UCLA-CURE Center, a Career Development Award from Crohn's and Colitis Foundation of America and K01DK084256 from National Institute of Health to HWK.

Mechanisms underlying the neuroprotective effects of fisetin and ibuprofen in the MPTP model of Parkinson's disease

Several studies mentioned the association of oxidative stress and neuroinflammation with Parkinson's disease (PD). In the MPTP‐injured PC12 cells, a rat model of PD, MPTP induces cell death through complex I inhibition in the mitochondria, increases the production of nitric oxide (NO) and pro‐inflammatory mediators such as IL‐1beta and TNF‐alpha. This study, investigated the mechanisms underlying the neuroprotective effect of two anti‐inflammatory drugs, Fisetin and Ibuprofen in MPTP‐induced PC12. Cell viability was measured by MTT assay, IL‐1beta levels were quantified by ELISA, TNF‐alpha expression was assessed by western blot, and NO levels were also evaluated. PC12 cells were pretreated with varying doses of fisetin, ibuprofen, or both drugs in combination for 2 hours, prior to MPTP treatment. Pretreatment with fisetin produced a dose‐dependent increase in cell viability, and a dose‐dependent decrease in the expression of TNF‐alpha. Ibuprofen at 2 μg/ml also decreased TNF‐alpha expression in MPTP treated PC12 cells. The combination of both drugs showed a synergistic effect in the inhibition of TNF‐alpha expression. Fisetin also suppressed the production of NO and IL‐1beta in a dose‐dependent manner, and that both drugs combined showed a synergistic effect in suppressing the NO and IL‐1beta production. The present study indicates that fisetin and ibuprofen may provide a promising approach for the treatment of neuroinflammation associated with PD.

Effects of Bawei Xilei San on mice with oxazolone-induced colitis and the mechanisms

To investigate the therapeutic effects of Bawei Xilei San (BWXLS), a compound traditional Chinese herbal medicine, on mice with oxazolone-induced colitis and to explore the mechanisms. Thirty-two BALB/c mice were randomly divided into 4 groups (8 for each): normal control group, untreated group, hydrocortisone group and BWXLS group. Except for the mice in the normal control group, all mice were intrarectally administered with 3.0% oxazolone to induce colitis. Then the mice in the normal control group and untreated group were administered with 0.9% carboxymethyl cellulose sodium solution. Mice in the BWXLS group were intrarectally administered with 0.2 mg/g BWXLS and hydrocortisone group with 0.02 mg/g respectively for 5 days. The body weight and stool consistency and occult or gross blood were recorded to calculate the disease activity index (DAI). The mice were sacrificed at the 6th day. The macroscopic and histological changes of the colon were evaluated. The expressions of Toll-like receptor 4 (TLR4), nuclear factor-kappaB (NF-kappaB), and epithelial tight junction protein occludin were assessed by immunohistochemical method. The level of tumor necrosis factor-alpha (TNF-alpha) in colonic mucosa was evaluated by enzyme-linked immunosorbent assay. The DAI, and macroscopic and histological changes in the BWXLS group were improved as compared with those in the untreated group (P<0.05) but were similar to those in the hydrocortisone group. The expression of occludin was significantly increased (P<0.05) while the expressions of TLR4, NF-kappaB and TNF-alpha were significantly decreased in the BWXLS group as compared with the untreated group, and were similar to those in the hydrocortisone group (P>0.05). Up-regulating the expression of occludin and down-regulating the expressions of TLR4 and NF-kappaB, and hence inhibiting TNF-alpha expression and improving the mucosa barrier function may be part of the mechanisms of BWXLS in treating oxazolone-induced colitis in mice.

Avian leukosis virus p27 inhibits tumor necrosis factor α expression in RAW264.7 macrophages after stimulation with lipopolysaccharide

Avian leukosis is a widely distributed disease caused by Avian leukosis virus (ALV). ALV p27 is a capsid protein and group specific antigen, whose role in host immune response is poorly understood. To explore ALV p27, we developed a RAW264.7 macrophage cell line stably expressing p27-GFP fusion protein. The cells of this line and control cell line expressing GFP protein only were compared in production of tumor necrosis factor alpha (TNF-alpha), interleukins IL-1beta, IL-6, IL-12, and in proliferative activity stimulated by the lipopolysaccharide (LPS). It was found that the ALV p27 expression markedly reduced the production of TNF-alpha, but did not affect the production of IL-1beta, IL-6, and 12, and cell proliferative activity. These results demonstrate that ALV p27 specifically inhibits TNF-alpha expression in the macrophages stimulated by LPS, suggesting a possible contribution of ALV p27 to immunosuppression detected in ALV-infected hosts. Avian leukosis virus; p27; tumor necrosis factor alpha; interleukins; macrophages; lipopolysaccharide.

The Flow‐mediated Dilation Response to Acute Exercise in Overweight Active and Inactive Men

Inflammation has been found to play a role in the etiology of cardiovascular disease as well as provoke endothelial dysfunction. Inflammatory cytokines associated with endothelial function are interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). IL-6 is exercise intensity dependent and has been shown to inhibit TNF-alpha expression directly. The aim of this study was to investigate the interaction of IL-6 and TNF-alpha on endothelial function in response to acute exercise in overweight men exhibiting different physical activity profiles. Using a randomized mixed factorial design, 16 overweight men (8 active, maximal exercise capacity (VO(2)peak) = 34.2 +/- 1.7, BMI = 27.4 +/- 0.7 and 8 inactive, VO(2)peak = 30.9 +/- 1.2, BMI = 29.3 +/- 1.0) performed three different intensity acute exercise treatments. Brachial artery flow-mediated dilation (FMD) and subsequent blood samples were taken pre-exercise and 1 h following the cessation of exercise. Independent of exercise intensity, the active group displayed a 24% increase (P = 0.034) in FMD following acute exercise compared to a 32% decrease (P = 0.010) in the inactive group. Elevated (P < 0.001) concentrations of IL-6 following moderate (50% VO(2)) and high (75% VO(2)) intensity acute exercise were observed in both groups; however, concentrations of TNF-alpha were unchanged in response to acute exercise (P = 0.584). The FMD response to acute exercise is enhanced in active men who are overweight, whereas inactive men who are overweight exhibit an attenuated response. The interaction of IL-6 and TNF-alpha did not provide insight into the physiological mechanisms associated with the disparity of FMD observed between groups.

Neostigmine and pilocarpine attenuated tumour necrosis factor α expression and cardiac hypertrophy in the heart with pressure overload

The inflammatory cytokine tumour necrosis factor alpha (TNF alpha) is known to be a major factor contributing to cardiac remodelling and dysfunction. Parasympathetic nervous system cholinergic function can inhibit TNF alpha expression during systemic infection. In the present study, we tested the effects of a cholinesterase inhibitor, neostigmine, and a muscarinic cholinergic agonist, pilocarpine, on cardiac hypertrophy and TNF alpha levels during pressure overload. Rats with transverse aortic constriction exhibited elevated TNF alpha protein levels in the heart, increased heart weight to body weight ratios (an index of cardiac hypertrophy) and decreased left ventricular diastolic function. Two weeks of infusion with neostigmine (6 microg kg(-1) day(-1)) or pilocarpine (0.3 mg kg(-1) day(-1)) significantly reduced cardiac hypertrophy, reduced TNF alpha levels and elevated interleukin-10 levels in heart tissues, and improved ventricular function in rats with transverse aortic constriction. Neither of these treatments significantly changed ventricular pressure load. Furthermore, in primary cultured neonatal cardiac cells, treatment with pilocarpine attenuated adrenergic agonist phenylephrine-induced increased TNF alpha expression and [3H]leucine (a marker of protein synthesis) incorporation in the cells. Collectively, both cholinergic agents decreased TNF alpha levels and attenuated cardiac hypertrophy. Since both agents potentially enhanced cholinergic function, the anti-inflammatory action may be involved in the cardioprotective effect of the treatments with these agents.

A derivative of the plasma protease inhibitor α2‐macroglobulin regulates the response to peripheral nerve injury

Peripheral nerve injury induces endoneural inflammation, controlled by diverse cytokines and extracellular mediators. Although inflammation is coupled to axonal regeneration, fulminant inflammation may increase nerve damage and neuropathic pain. alpha(2)-Macroglobulin (alpha2M) is a plasma protease inhibitor, cytokine carrier, and ligand for cell-signaling receptors, which exists in two well-characterized conformations and in less well-characterized intermediate states. Previously, we generated an alpha2M derivative (alpha(2)-macroglobulin activated for cytokine binding; MAC) similar in structure to alpha(2)M conformational intermediates, which binds tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and inhibits endotoxin toxicity. In this study, we report that the continuum of cytokines that bind to MAC includes IL-6 and IL-18. MAC inhibited TNF-alpha-induced p38 mitogen-activated protein kinase activation and cell death in cultured Schwann cells. When administered by i.p. injection to mice with sciatic nerve crush injury, MAC decreased inflammation and preserved axons. Macrophage infiltration and TNF-alpha expression also are decreased. MAC inhibited TNF-alpha expression in the chronic constriction injury model of nerve injury. When MAC was prepared using a mutated recombinant alpha2M, which does not bind to the alpha2M receptor, low-density lipoprotein receptor-related protein-1, activity in the chronic constriction injury model was blocked. These studies demonstrate that an alpha2M derivative is capable of regulating the response to peripheral nerve injury by a mechanism that requires low-density lipoprotein receptor-related protein-1.

Ginkgo biloba extract protects against alcohol‐induced liver injury in rats

Ginkgo biloba extract has been shown to be hepatoprotective. However, its benefits when used in alcoholic liver injury have not been demonstrated. This study investigated the effects of Ginkgo biloba extract on alcohol-induced liver injury in a chronic alcohol plus fish oil gavage model in rats. Liver injury was evaluated histologically and by serum alanine aminotransferase (ALT). Liver tumor necrosis factor-alpha (TNF-alpha) protein levels, malondialdehyde (MDA) and glutathione (GSH) contents were determined. TNF-alpha mRNA expression in the liver was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Rats given fish oil plus alcohol developed macrovesicular and microvesicular steatosis, spotty necrosis and mild inflammation in the liver and elevated serum ALT levels, which were accompanied by increased MDA contents, reduced GSH contents and elevated TNF-alpha protein and mRNA expressions in the liver. Supplementation with Ginkgo biloba extract (200 mg/kg, orally) improved the liver injury, blunted the rises of MDA contents and TNF-alpha expression, and restored the GSH content in the liver. Ginkgo biloba extract protects against alcohol-induced liver injury, and the mechanism may involve a reduction of lipid peroxidation, prevention of GSH depletion and inhibition of TNF-alpha expression in the liver.

RETRACTED: The anti-inflammatory activity of 1,25-dihydroxyvitamin D3 in macrophages

In a previous study we demonstrated a down-regulatory effect of vitamin D active metabolite (1,25(OH)(2)D(3)) and its vitamin D(2) analog (1,24(OH)(2)D(2)) on TNFalpha expression in macrophages. We also found an inhibitory effect in the physiological concentration (10(-10)M) of 1,25(OH)(2)D(3) which was dose-dependent. This down-regulation, caused by the decrease in NFkappaB activity by 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2), was demonstrated in P388D1 cells transfected with NFkappaB reporter gene (p NFkappaB-Luc) and by EMSA. In our present study we investigated the processes leading to reduced NFkappaB activity on P388D1 cells. A decrease in nuclei NFkappaB-p65 and an increase in cytosolic NFkappaB-p65, were measured, while no changes in total NFkappaB-p65 mRNA and protein levels were observed. Simultaneously, a significant increase in both mRNA and protein levels of the NFkappaB-cytosolic inhibitor, IkappaBalpha, were determined. The half-life of IkappaBalpha-mRNA increased, with a parallel decrease in the phosphorylation of its protein, as the first step of ubiquitinization and degradation. The present results demonstrate that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) inhibit TNFalpha expression in macrophages, by increasing IkappaBalpha and decreasing NFkappaB activity. Since NFkappaB is a major transcription factor for TNFalpha and other inflammatory mediators, these findings suggest that 1,25(OH)(2)D(3) and 1,24(OH)(2)D(2) may be used therapeutically as anti-inflammatory agents.

Effects of Qinggan Huoxue Recipe and its decomposed formulas on CD14, Toll like receptor 4 and nuclear factor-κB expressed by Kupffer cells

To investigate the effects of Qinggan Huoxue Recipe (QGHXR), a compound Chinese herbal medicine, and its decomposed formulas Qinggan Recipe (QGR) and Huoxue Recipe (HXR) on expressions of CD14, Toll like receptor 4 (TLR(4)) and nuclear factor-kappaB (NF-kappaB) in Kupffer cells. The isolated primary rat Kupffer cells were treated with lipopolysaccharide (LPS) for a certain period of time, a series of concentrations of drug-containing serums of QGHXR and its decomposed formulas were added, the expressions of NF-kappaB, tumor necrosis factor-alpha (TNF-alpha), CD14, and TLR(4) of the Kupffer cells were detected in different culture conditions by using Western blot, real-time reverse transcription polymerase chain reaction (RT-PCR) and ELISA methods respectively. QGR could down-regulate the expression of membrane receptor CD14, but the expression of NF-kappaB and TNF-alpha were not significantly decreased after QGR treatment. HXR could down-regulate the expression of membrane receptor TLR4 and inhibit the expressions of NF-kappaB and TNF-alpha. QGHXR could down regulate the expressions of membrane receptors CD14 and TLR(4) and inhibit the expressions of NF-kappaB and TNF-alpha. QGHXR can protect liver cells by down regulating the expressions of CD14, TLR(4) and NF-kappaB and inhibiting TNF-alpha expression.

TNFα up‐regulates apelin expression in human and mouse adipose tissue

We have recently identified apelin as a novel adipokine up-regulated by insulin and obesity. Since obesity and insulin resistance are associated with chronically elevated levels of both insulin and TNFalpha, the present study was performed to investigate a putative regulation of apelin expression in adipocytes by TNFalpha. Herein, we report a tight correlation between apelin and TNFalpha expression in adipose tissue of lean and obese humans. Apelin regulation by TNFalpha was further studied in cultured explants of human adipose tissue. The endogenous expression of TNFalpha in adipocytes isolated from the explants was accompanied by a 6-9 h subsequent increase of apelin expression in adipocytes. This increase was reversed by inhibiting TNFalpha expression with 100 microM isobutylmethylxanthine. In different mouse models of obesity, expression of both TNFalpha and apelin was also significantly increased in adipocytes of obese mice. Furthermore, short-term exposure to an i.p. injection of TNFalpha in C57Bl6/J mice induced an increase of apelin expression in adipose tissue as well as apelin plasma levels. Finally, a direct positive effect of TNFalpha has been shown in differentiated 3T3F442A adipocytes on apelin expression and secretion. The signaling pathways of TNFalpha for the induction of apelin were dependent of PI3-kinase, c-Jun NH2-terminal kinase (JNK), and MAPK but not PKC activation. All together, these findings suggest that apelin might be a candidate to better understand potential links between obesity and associated disorders such as inflammation and insulin resistance.

PPARδ modulates lipopolysaccharide-induced TNFα inflammation signaling in cultured cardiomyocytes

Peroxisome proliferator activated receptors (PPARs: PPARalpha, gamma and delta) regulate fatty acid metabolism, glucose homeostasis, cell proliferation, differentiation and inflammation. Tumor necrosis factor alpha (TNFalpha) is one of the important pathological factors in inflammatory responses during the pathological progression of myocardial ischemic/reperfusion and hypertrophy. Accumulating evidence shows that synthetic ligands of PPARalpha and PPARgamma suppress myocardial inflammatory responses, such as the production of TNFalpha, thus exerting beneficial effects in animals who had undergone ischemia/reperfusion injury or cardiac hypertrophy. However, it remains obscured if PPARdelta and its ligands exert any effect on the production of TNFalpha, thus influencing cardiac inflammatory responses. In this study, we investigated the effects of PPARdelta and its synthetic ligand GW0742 on TNFalpha production in cultured cardiomyocytes. Our studies indicate that a PPARdelta-selective ligand inhibited lipopolysaccharide (LPS)-induced TNFalpha production from cardiomyocytes. Adenoviral-mediated overexpression of PPARdelta substantially inhibited TNFalpha expression in cultured cardiomyocytes compared to controls, whereas overexpression of a PPARdelta mutant with ablated ligand binding domain did not show similar effect. Conversely, absence of PPARdelta in cardiomyocytes further exaggerated LPS-induced TNFalpha production. Moreover, activation of PPARdelta abrogated LPS-induced degradation of IkappaBs, thus suppressing LPS-induced nuclear factor kappaB (NF-kappaB) activities. Therefore, PPARdelta is an important determinant of TNFalpha expression via the NF-kappaB signaling pathway, thus serving as therapeutic targets to attenuate inflammation that are involved in cardiac pathological progression.

Mechanism of oligochitosan-induced macrophage activation

To study the mechanism of oligochitosan-induced macrophage activation. Oligochitosan was chemically modified with fluorophore 2-aminoacridone (2-AMAC). The cellular events of 2-AMAC-oligochitosan-macrophage interaction were analyzed with confocal laser microscopy and the fluorescence intensity of cells was analyzed by BD LSR flow cytometer. The mechanism of oligochitosan uptake by macrophages was studied by competitive inhibition test and the effect of calcium, trypsin and colchicine on oligochitosan recognition and internalization were also determined. RT-PCR was performed to investigate the level of TNF-alpha secretion. Macrophage could bind and uptake oligochitosan, which was dependent on the temperature: the uptake proceeded rapidly at 37 degrees C and at 4 degrees C macrophage could only bind oligochitosan. EDTA decreased oligochitosan uptake. Trypsin treatment significantly reduced the internalization, and uptake was recovered by trypsin termination. Colchicine significantly inhibited the internalization process and was dose dependent. 0.1 mol/L mannose inhibited TNF-alpha expression induced by oligochitosan. Macrophage could uptake oligochitosan via mannose receptor mediated pinocytosis. Mannose receptor is crucial for the oligochitosan-induced macrophages activation.

Erythropoietin reduces Schwann cell TNF‐α, Wallerian degeneration and pain‐related behaviors after peripheral nerve injury

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-alpha has been implicated in the development of pain-related behaviors, we measured TNF-alpha mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-alpha mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-alpha in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-alpha immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-alpha in CCI by blocking expression of TNF-alpha in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-alpha expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-alphain vitro. These results indicated that rhEpo regulates TNF-alpha by multiple mechanisms; rhEpo regulates TNF-alpha mRNA expression by Schwann cells but also may directly counteract TNF-alpha signaling pathways that lead to injury, chronic pain and/or death.