Investigation of the Effects of Glutens on Serum Interleukin-1 Beta and Tumor Necrosis Factor-Alpha Levels and the Immunohistochemical Distribution of CD3 and CD8 Receptors in the Small Intestine in Male Rats

Abstract While the role of cytokines in celiac disease has been investigated in detail, cytokine release in the event of the exposure of healthy subjects to glutens has only recently been studied. This study was aimed at determining the effects of corn and wheat glutens, incorporated as protein sources into the diet, on serum interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels and the immunohistochemical distribution of CD3 and CD8 receptors in the small intestine in male rats. The study material comprised 24 twenty-day-old male Wistar albino rats, which were randomly assigned in equal numbers to three groups (2 rats/replicate and 4 replicates/group). The feed rations provided to all three groups contained high levels of proteins, which were soybean meal, corn gluten and wheat gluten in the control, corn and wheat groups, respectively. The in Control, Corn and Wheat groups serum IL-1 beta and TNF-alpha levels respectively 55.83 - 46.37; 81.65 - 61.95 and 81.65-61.31 was determined but these differences were statistically insignificant. Furthermore, immunohistochemical examination demonstrated a mathematical increase to have occurred in the distribution of the CD3 and CD8 receptors in the duodenum, jejunum and ileum samples of the corn and wheat groups. In result, based on the findings obtained in this study, we suggest that the long-term feeding of rats on high levels of gluten causes systemic adverse effects.

Variable beige adipocyte morphology in obese rats by different times of melatonin administration / Variación de la morfología del adipocito beige en ratas obesas según los diferentes tiempos de administración de melatonina

This study aimed to evaluate changes in beige adipocytes at different times of melatonin administration, in the morning (ZT01) or in the evening (ZT11), at 30 mg/kg daily by gavage for 7 weeks or continuously with drinking water in the term of high-calorie diet-induced obesity (HCD). Melatonin received at ZT11 or with drinking water resulted in an increased area of the browning zone in the subcutaneous white adipose tissue (sWAT), even in rats with HCD (compared with Control or HCD, respectively). The beige adipocyte and lipid droplet area after melatonin use were reduced compared to those with HCD and Control, in all administration modes (group ZT01 showed smaller changes compared to ZT11 or with drinking water groups). The fibrosis level decreased and significantly differed in HCD ZT01, HCD ZT11, and HCD water compared to that in HCD; moreover, the lowest value determined in HCD water, reached the control parameters. Furthermore, the IL-1b and IL-8 level was decreased in the HCD groups under melatonin treatment at ZT11 or with drinking water compared to that in HCD. The obtained results suggest that melatonin promotes sWAT browning in rats with diet-induced obesity and influences morphological signs of normal rats depending on the time of administration. Different functional activity of beige adipocytes was observed after melatonin was used depending on the time of administration, resulting in heat production and lipolysis (the relative mass of visceral fat was likewise diminished). More rapid browning was observed when melatonin treatment was performed at 1 h before lights-off (ZT11) or continuously via drinking water. Melatonin acted on beige adipocytes of obese rats through changing some parameters such as the area of adipocytes and lipid drops, the number of lipid drops, the relative area browning of sWAT, and the level of tissue fibrosis.

Este estudio tuvo como objetivo evaluar los cambios en los adipocitos beige en diferentes momentos de la administración de melatonina, en la mañana (ZT01) o por la noche (ZT11). Se administraron 30 mg/kg diariamente por sonda durante 7 semanas o continuamente con agua potable durante el periodo de obesidad inducida por una dieta alta en calorías (HCD). La melatonina recibida en ZT11 o con agua potable resultó en un aumento de área dorada en tejido adiposo blanco subcutáneo (sWAT), incluso en ratas con HCD (en comparación con Control o HCD, respectivamente). El área de gotas de lípidos y adipocitos de color beige después del uso de melatonina se redujo en comparación con aquellos con HCD y Control, en todos los modos de administración (el grupo ZT01 mostró cambios más pequeños en comparación con ZT11 o con grupos de agua potable). El nivel de fibrosis disminuyó y difirió significativamente en HCD ZT01, HCD ZT11 y agua HCD, en comparación con el HCD; además, el valor más bajo determinado en agua HCD alcanzó los parámetros de control. Además, el nivel de IL-1b e IL-8 disminuyó en los grupos HCD bajo tratamiento con melatonina en ZT11 o con agua potable en comparación con el de HCD. Los resultados obtenidos sugieren que la melatonina promueve el dorado sWAT en ratas con obesidad inducida por la dieta e influye en los signos morfológicos de las ratas normales dependiendo del momento de la administración. Se observó una actividad funcional diferente de los adipocitos de color beige después de usar melatonina dependiendo del tiempo de administración, dando como resultado la producción de calor y lipólisis (la masa relativa de grasa visceral también disminuyó). Se observó un ennegrecimiento más rápido cuando el tratamiento con melatonina se realizó 1 h antes de apagar las luces (ZT11) o de forma continua en grupos de agua potable. La melatonina actuó en los adipocitos beige de ratas obesas al cambiar algunos parámetros, como el área de adipocitos y gotas de lípidos, el número de gotas de lípidos, el área relativa de ennegrecimiento de sWAT y el nivel de fibrosis tisular.

Dexmedetomidine attenuates P2X4 and NLRP3 expression in the spine of rats with diabetic neuropathic pain

Abstract Purpose: To evaluate the effects of Dexmedetomidine (Dex) on spinal pathology and inflammatory factor in a rat model of Diabetic neuropathic pain (DNP). Methods: The rats were divided into 3 groups (eight in each group): normal group (N group), diabetic neuropathic pain model group (DNP group), and DNP model with dexmedetomidine (Dex group). The rat model of diabetes was established with intraperitoneal streptozotocin (STZ) injections. Nerve cell ultrastructure was evaluated with transmission electron microscopy (TEM). The mechanical withdrawal threshold (MWT) and motor nerve conduction velocity (MNCV) tests documented that DNP rat model was characterized by a decreased pain threshold and nerve conduction velocity. Results: Dex restored the phenotype of neurocytes, reduced the extent of demyelination and improved MWT and MNCV of DNP-treated rats (P=0.01, P=0.038, respectively). The expression of three pain-and inflammation-associated factors (P2X4, NLRP3, and IL-IP) was significantly upregulated at the protein level in DNP rats, and this change was reversed by Dex administration (P=0.0022, P=0.0092, P=0.0028, respectively). Conclusion: The P2X4/NLRP3 signaling pathway is implicated in the development and presence of DNP in vivo, and Dex protects from this disorder.

Ginsenoside Rd inhibits IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1β, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1β by suppressing the increase in IL-1β, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1β-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1β to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1β-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.

Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

The effect of hippophae rhamnoides extract on oral mucositis induced in rats with methotrexate

ABSTRACT Objective: To investigate the effect of HRE (Hippophae rhamnoides extract) on oral mucositis induced in rats with MTX. Material and Methods: Experimental animals were divided into groups as healthy (HG), HRE+MTX (HMTX), and control group, which received MTX (MTXC). HMTX group received 50 mg/kg HRE while MTXC and HG groups received equivolume distilled water with gavage once a day. After one hour of HRE and distilled water administration, HMTX and MTXC groups received a single dose of oral MTX 5 mg/ kg. This procedure was repeated for one month. Results: The levels of MDA, IL-1β, and TNF-α were found to be significantly higher in the cheek, lower lip, and tongue tissue of the animals receiving MTX, compared with HG and HMTX groups; however, these parameters were lower in the cheek and low lip tissue, and a milder damage ocurred in these tissues, compared with the tongue tissue in MTXC group. No histopathologic damage was observed in the cheek, lower lip, and tongue tissues of the rats treated with HRE. Conclusion: This findings indicate that HRE as a natural product is an important advantage compared with synthetic drugs for prophylaxis of oral mucositis developed due to MTX.

Effect of ozone on colon anastomoses in rat peritonitis model

PURPOSE: To investigate the effects of medical ozone theraphy on the colon anastomosis of peritonitis model in rats. METHODS: Eighteen rats were randomly assigned into three equal groups; control, cecal punctuation and colon anastomosis and ozone theraphy. Sepsis was performed with a cecal punctuation in groups 2 and 3. The medical ozone theraphy was administered intraperitonealy for three weeks in group 3 while the other rats received saline injection. At the twenty second day serum were obtained for TNF-α and IL-1β, the colonic burst pressures were measured and colonic tissue samples were obtained for MDA and MPO levels. Histolopatological examination was evaluated with H&E stain, and Ki-67, IL-1β and the VEGF immunostaining densities were also compared. RESULTS: Intraperitoneal ozone administration reversed TNF-α, IL-1β, MDA and MPO levels and the colonic burst pressures. There was also a significant difference at immunostaining densities of histopathological examination. CONCLUSION: Medical ozone therapy may contribute to tissue healing by affecting the proliferation and the vascularization thus has benefits on colonic anastomosis at peritonitis in rats.

Human amnion mesenchymal cells inhibit lipopolysaccharide-induced TNF-α and IL-1ß production in THP-1 cells

BACKGROUND: Human amnion mesenchymal cells (hAMCs), isolated from the amniotic membrane of human placenta, are a unique population of mesenchymal stem cells. Recent studies demonstrated that hAMCs could inhibit the activities and functions of several immune cells. However, their effect on inflammatory macrophages is largely unknown. This study investigated the effect of hAMCs on expression of inflammatory cytokines and mitogen-activated protein kinases (MAPKs)/NF-kB pathway in human THP-1 macrophages induced by lipopolysaccharide (LPS). RESULTS: The levels of TNF-α and IL-1ß secreted by LPS- stimulated THP-1 cells were increased significantly compared with those in the control group. After co-culture with different numbers of hAMCs, the levels of TNF-α and IL-1ß in LPS-stimulated THP-1 cells were significantly reduced compared with the LPS group. The mRNA expression of TNF-α and IL-1ß were also markedly inhibited. Moreover, treating LPS-stimulated THP-1 cells with hAMCs supernatants could also suppress TNF-α and IL-1ß production in THP-1 cells. Important signaling pathways involved in the production of TNF-α and IL-1ß were affected by hAMCs co-culture: hAMCs remarkably suppressed NF-kB activation and down-regulated the phosphorylation of ERK and JNK in LPS- stimulated THP-1 cells. CONCLUSIONS: Human amnion mesenchymal cells inhibited the production of TNF-α and IL-1ß secreted by LPS-stimulated THP-1 cells, partly through the suppression of NF-kB activation and ERK and JNK phosphorylation.

Effects of Er-Miao-San extracts on TNF-alpha-indueed MMP-1 expression in human dermal fibroblasts

BACKGROUND: Various health benefits have been attributed to Er-Miao-San (EMS), a traditional Chinese herbal formulation that contains equal amounts of cortex phellodendri (Phellodendron amurense Ruprecht) and rhizoma atractylodis (Atractylodes lancea D.C). However, its effect on the anti-inflammatory activity in human dermal fibroblasts (HDFs) and the mechanism underlying this effect are unknown. RESULTS: This study investigated the effects of EMS on TNF-α-induced MMP-1 expression in HDFs. Our data show that EMS inhibited TNF-α-induced MMP-1 expression in a concentration-dependent manner. Interestingly, EMS maintained IkB content without inhibiting the phosphorylation of MAPKs, which are well-established upstream kinases of NF-kB. Moreover, EMS reduced the level of nuclear p65 protein in HDFs. Luciferase assay revealed that EMS inhibits the transcriptional activity of NF-kBbystabilizing IkB. Our results show that EMS exerts its anti-inflammatory effect by inhibiting NF-kB-regulated genes such as IL-1ß and IL-8. Moreover, EMS effectively inhibited TNF-α-induced expression of MMP-1 via the NF-kBpathway. CONCLUSIONS: Taken together, our data suggest that EMS could potentially be used as an anti-inflammatory and anti-aging treatment.

Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus

OBJECTIVE: Refractory status epilepticus is one of the most life-threatening neurological emergencies and is characterized by high morbidity and mortality. Additionally, the use of anti-inflammatory drugs during this period is very controversial. Thus, this study has been designed to analyze the effect of a low dose of indomethacin (a COX inhibitor) on the expression of inflammatory molecules. METHOD: The hippocampus of rats submitted to pilocarpine-induced long-lasting status epilepticus was analyzed to determine the expression of inflammatory molecules with RT-PCR and immunohistochemistry. RESULTS: Compared with controls, reduced levels of the kinin B2 receptors IL1β and TNFα were found in the hippocampus of rats submitted to long-lasting status epilepticus and treated with indomethacin. CONCLUSIONS: These data show that low doses of indomethacin could be employed to minimize inflammation during long-lasting status epilepticus. .

Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats / Efeitos da eritromicina sobre a γ-glutamil-cisteína-sintetase e a interleucina-1β no tecido pulmonar exposto à hiperóxia de ratos recém-nascidos prematuros

Objective: To explore the effect of erythromycin on hyperoxia-induced lung injury. Methods: One-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: Compared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). Conclusions: Changes in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect. .

Objetivo: Explorar o efeito da eritromicina sobre lesões pulmonares induzidas por hiperóxia. Métodos: Uma prole de ratos Sprague-Dawley (SD) prematuros com um dia de vida foi dividida aleatoriamente em quatro grupos: grupo 1 ar + cloreto de sódio, grupo 2 ar + eritromicina, grupo 3 hiperóxia + cloreto de sódio e grupo 4 hiperóxia + eritromicina. Com um, sete e 14 dias de exposição, foram detectadas Glutationa (GSH) e Interleucina-1 beta (IL-1 beta) pelo ensaio imunossorvente ligado à enzima (ELISA), e o ácido bicinconinico (BCA) foi utilizado para detectar a proteína GSH. O mRNA da γ-glutamil-cisteina-sintetase (γ-GCS) foi detectado por reação em cadeia da polimerase via transcriptase reversa (RT-PCR). Resultados: Comparadas ao grupo 1, as expressões do mRNA da GSH e da γ-GCS no grupo 3 aumentaram significativamente com um e sete dias de exposição (p < 0,05), porém a expressão de mRNA da γ-GCS diminuiu significativamente aos 14 dias; a expressão de IL-1 beta no grupo 3 aumentou significativamente aos 7 dias de exposição (p < 0,05) e diminuiu significativamente aos 14 dias. Comparadas ao grupo 3, as expressões do mRNA da GSH e da γ-GCS no grupo 4 aumentaram significativamente com um, sete e 14 dias de exposição (p < 0,05), porém as expressões de GSH mostraram uma tendência de queda aos 14 dias; a expressão de IL-1 beta no grupo 4 foi reduzida significativamente com um e sete dias de exposição (p < 0,05). Conclusões: As variações de IL-1 beta e GSH mediadas por oxidantes estão envolvidas no desenvolvimento de lesão pulmonar induzida por hiperóxia. A eritromicina poderá regular positivamente a atividade da γ-GCS, aumentando a expressão de GSH, inibindo os níveis de interleucina-1beta mediada por ...

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.

Down-regulation of ATP-binding cassette transporter G1 expression by unmethylated CpG oligodeoxynucleotides in RAW 264.7 macrophages

We have investigated the effect of various forms of phosphodiester cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) on the production of pro-inflammatory cytokines and related genes in RAW 264.7 macrophages. Treatment with the CpG ODNs increased the expression of tumor necrosis factor alpha (TNF-alpha), IL-6, and inducible nitric oxide synthase but not interleukin-1beta (IL-1beta). We also investigated the effect of CpG ODNs on the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) genes which are known to facilitate cholesterol efflux from macrophages for anti-atherosclerosis. CpG 2006 significantly reduced the levels of ABCG1 mRNA as determined by real-time polymerase chain reaction, whereas ABCA1 mRNA level was not changed. Western blot analysis further confirmed the reduction of ABCG1 protein expression by CpG 2006. In addition, we also determined the protein level of peroxisome proliferator activated receptor gamma (PPARgamma), which is recognized as a transcriptional activator of ABC transporters, was also reduced by CpG 2006. Thus, these results suggest that ABCG1 is specifically down-regulated by CpG 2006 in a PPARgamma-dependent manner in macrophages.

Antipyretic and antioxidant activities of 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles in rats

The objective of this study was to determine the effect of eight 5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-carboxyamidepyrazoles (TFDPs) on rat body temperature and baker’s yeast-induced fever. TFDPs or vehicle (5 percent Tween 80 in 0.9 percent NaCl, 5 mL/kg) were injected subcutaneously and rectal temperature was measured as a function of time in 28-day-old male Wistar rats (N = 5-12 per group). Antipyretic activity was determined in feverish animals injected with baker’s yeast (Saccharomyces cerevisiae suspension, 0.135 mg/kg, 10 mL/kg, ip). 3-Ethyl- and 3-propyl-TFDP (140 and 200 μmol/kg, respectively, 4 h after yeast injection) attenuated baker’s yeast-induced fever by 61 and 82 percent, respectively. These two effective antipyretics were selected for subsequent analysis of putative mechanisms of action. We then determined the effects on cyclooxygenase-1 and -2 (COX-1 and COX-2) activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) oxidation in vitro, on tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels and on leukocyte counts in the washes of peritoneal cavities of rats injected with baker’s yeast. While 3-ethyl- and 3-propyl-TFDP did not reduce baker’s yeast-induced increases of IL-1β or TNF-α levels, 3-ethyl-TFDP caused a 42 percent reduction in peritoneal leukocyte count. 3-Ethyl- and 3-propyl-TFDP did not alter COX-1 or COX-2 activities in vitro, but presented antioxidant activity in the DPPH assay with an IC50 of 39 mM (25-62) and 163 mM (136-196), respectively. The data indicate that mechanisms of action of these two novel antipyretic pyrazole derivatives do not involve the classic inhibition of the COX pathway or pyrogenic cytokine release.