High dose gabapentin does not alter tumor growth in mice but reduces arginase activity and increases superoxide dismutase, IL-6 and MCP-1 levels in Ehrlich ascites.

OBJECTIVES: The purpose of this study was to evaluate the effect of gabapentin on Ehrlich tumor growth in Swiss mice, a highly aggressive and inflammatory tumor model. Mice were grouped into sets of 5 animals and treated from days 2 to 8 with gabapentin 30 mg/kg body weight (G30) or 100 mg/kg body weight (G100), or normal sterile saline (control). RESULTS: The mice were euthanized on day 10. Tumor growth, tumoricidal agents and inflammatory cytokines levels were assessed. At day 10, G30 and G100 mice gained weight, but there were no differences in tumor cell count or in ascites volume. In G100, there was a reduction in arginase and an increase in SOD activities. There was an increase in IL-6 and MCP-1 levels, especially in G100, but no alterations in TNF-α. There was no direct evidence of tumor induction by gabapentin. However, the findings suggest that its use modulates immune response to a more effector and less deleterious profile, with increase in activity of anti-oxidant enzymes and in cytokines that favor activation of macrophages, which could improve the general status of the tumor host.

Propofol attenuates sepsis-induced acute kidney injury by regulating miR-290-5p/CCL-2 signaling pathway.

Previous studies have indicated that propofol has immunomodulatory and antioxidative properties. However, the renoprotection effect and the precise mechanisms of propofol in sepsis-induced renal injury remain unclear. The purpose of the present study was to investigate the role of miR-290-5p/CCL-2 signaling in septic mice treatment with propofol. Mice were treated with propofol (50 mg/kg) twice within 24 h. Survival outcome was monitored within 48 h. The mRNA and protein levels were assayed by qRT-PCR and western blotting, respectively. Mouse podocytes (MPC5) were treated with lipopolysaccharide (LPS) to establish the cell model in vitro. The proliferation of MPC5 was monitored using the MTS assay. Cell apoptosis was analyzed by flow cytometry. Propofol improved survival outcome and alleviated acute kidney injury in cecal ligation and puncture-operated mice. Propofol increased miR-290-5p expression and decreased CCL-2 and inflammatory cytokines levels in the kidney for septic mice. We found that miR-290-5p was a direct regulator of CCL-2 in MPC5. Propofol could abrogate LPS-induced growth inhibition and apoptosis in MPC5. Meanwhile, propofol inhibited CCL-2 expression in LPS-treated MPC5, however, knockdown of miR-290-5p abrogated the inhibitory effect propofol on the mRNA and protein expressions of CCL-2. Propofol could serve as an effective therapeutic medication to suppress sepsis-induced renal injury in vivo and in vitro by regulating the miR-290-5p/CCL-2 signaling pathway.

Short-term insulin intensive therapy decreases MCP-1 and NF-κB expression of peripheral blood monocyte and the serum MCP-1 concentration in newlydiagnosed type 2 diabetics.

OBJECTIVE: To observe the effect of short-term insulin intensive treatment on the monocyte chemoattractant protein-1 (MCP-1) as well as on the nuclear factor-kappa B (NF-κB) expression of peripheral blood monocyte. This is also in addition to observing the serum MCP-1 level in newlydiagnosed type 2 diabetic patients and probing its anti-inflammation effects. SUBJECTS AND METHODS: Twenty newly-diagnosed type 2 diabetic patients were treated with an insulin intensive treatment for 2 weeks. MCP-1 and NF-κB expression on the monocyte surface were measured with flow cytometry, the serum MCP-1 level was measured by enzyme linked immunosorbent assay (ELISA) during pretreatment and post-treatment. RESULTS: After 2 weeks of the treatment, MCP-1 and NF-κB protein expression of peripheral blood monocyte and serum MCP-1 levels decreased significantly compared with those of pre-treatment, which were (0.50 ± 0.18)% vs (0.89 ± 0.26)% (12.22 ± 2.80)% vs (15.53 ± 2.49)% and (44.53 ± 3.97) pg/mL vs (49.53 ± 3.47) pg/mL, respectively (P < 0.01). The MCP-1 expression on monocyte surface had a significant positive relationship with serum MCP-1 levels (r = 0.47, P < 0.01). CONCLUSIONS: Short-term insulin intensive therapy plays a role in alleviating the increased inflammation reaction in type 2 diabetics.

Short-term insulin intensive therapy decreases MCP-1 and NF-κB expression of peripheral blood monocyte and the serum MCP-1 concentration in newlydiagnosed type 2 diabetics

ABSTRACT Objective To observe the effect of short-term insulin intensive treatment on the monocyte chemoattractant protein-1 (MCP-1) as well as on the nuclear factor-kappa B (NF-κB) expression of peripheral blood monocyte. This is also in addition to observing the serum MCP-1 level in newlydiagnosed type 2 diabetic patients and probing its anti-inflammation effects. Subjects and methods Twenty newly-diagnosed type 2 diabetic patients were treated with an insulin intensive treatment for 2 weeks. MCP-1 and NF-κB expression on the monocyte surface were measured with flow cytometry, the serum MCP-1 level was measured by enzyme linked immunosorbent assay (ELISA) during pretreatment and post-treatment. Results After 2 weeks of the treatment, MCP-1 and NF-κB protein expression of peripheral blood monocyte and serum MCP-1 levels decreased significantly compared with those of pre-treatment, which were (0.50 ± 0.18)% vs (0.89 ± 0.26)% (12.22 ± 2.80)% vs (15.53 ± 2.49)% and (44.53 ± 3.97) pg/mL vs (49.53 ± 3.47) pg/mL, respectively (P < 0.01). The MCP-1 expression on monocyte surface had a significant positive relationship with serum MCP-1 levels (r = 0.47, P < 0.01). Conclusions Short-term insulin intensive therapy plays a role in alleviating the increased inflammation reaction in type 2 diabetics.

Propofol attenuates sepsis-induced acute kidney injury by regulating miR-290-5p/CCL-2 signaling pathway

Previous studies have indicated that propofol has immunomodulatory and antioxidative properties. However, the renoprotection effect and the precise mechanisms of propofol in sepsis-induced renal injury remain unclear. The purpose of the present study was to investigate the role of miR-290-5p/CCL-2 signaling in septic mice treatment with propofol. Mice were treated with propofol (50 mg/kg) twice within 24 h. Survival outcome was monitored within 48 h. The mRNA and protein levels were assayed by qRT-PCR and western blotting, respectively. Mouse podocytes (MPC5) were treated with lipopolysaccharide (LPS) to establish the cell model in vitro. The proliferation of MPC5 was monitored using the MTS assay. Cell apoptosis was analyzed by flow cytometry. Propofol improved survival outcome and alleviated acute kidney injury in cecal ligation and puncture-operated mice. Propofol increased miR-290-5p expression and decreased CCL-2 and inflammatory cytokines levels in the kidney for septic mice. We found that miR-290-5p was a direct regulator of CCL-2 in MPC5. Propofol could abrogate LPS-induced growth inhibition and apoptosis in MPC5. Meanwhile, propofol inhibited CCL-2 expression in LPS-treated MPC5, however, knockdown of miR-290-5p abrogated the inhibitory effect propofol on the mRNA and protein expressions of CCL-2. Propofol could serve as an effective therapeutic medication to suppress sepsis-induced renal injury in vivo and in vitro by regulating the miR-290-5p/CCL-2 signaling pathway.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines.

PURPOSE:: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. METHODS:: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. RESULTS:: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. CONCLUSIONS:: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines / Avaliação da nicotinamida como agente anti-inflamatório e anti-angiogênico em linhas celulares de melanoma uveal

ABSTRACT Purpose: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. Methods: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. Results: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. Conclusions: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

RESUMO Objetivo: Acredita-se que a nicotinamida (NIC) seja capaz de diminuir a angiogênese induzida pelo fator de crescimento endotelial vascular (VEGF). Investigar os efeitos da nicotinamida sobre a secreção de citocinas pró-angiogênicas e pró-inflamatórias em linhagens de células de melanoma uveal humano (UM). Métodos: Duas linhagens de células humanas de UM (92,1 e OCM-1) foram tratadas com NIC (10 mmol/L) ou apenas com meio de cultura por 48 horas. O sobrenadante das culturas obtido após a administração de nicotinamida foi comparado com o sobrenadante das culturas controle quanto à expressão de 20 fatores pró-angiogênicos e pró-inflamatórios, pela técnica de enzyme-linked immunosorbent assay (ELISA). Resultados: Sete citocinas pró-angiogênicas foram detectadas nas condições de controle em ambas as linhagens de células de UM. O tratamento com nicotinamida promoveu uma redução significativa da secreção das seguintes citocinas angiogênicas: Angiogenina, ANG2, EGF e VEGF-A em células 92.1; bFGF em células OCM-1; PIGF em ambas as linhagens celulares. Quanto às proteínas pró-inflamatórias, a expressão de MCP-1 e IL-8 foi significativamente reduzida com a administração de nicotinamida em relação às culturas de células que não receberam o tratamento. Conclusões: Nicotinamida apresenta propriedades anti-inflamatórias e anti-angiogênicas em modelo experimental in vitro. Tais efeitos sugerem a possibilidade de utilizar esta substância na quimioprevenção do UM. Entretanto, estudos com modelos experimentais in vivo são necessários para melhor avaliar o benefício do tratamento do UM com nicotinamida.

p-cresol but not p-cresyl sulfate stimulate MCP-1 production via NF-κB p65 in human vascular smooth muscle cells. / p-cresol mas não p-cresil sulfato estimulam a produção de MCP-1 via NF-κB p65 em células vasculares musculares lisas humanas.

INTRODUCTION: p-cresol (PC) and p-cresyl sulfate (PCS) are responsible for many of the uremia clinical consequences, such as atherosclerosis in Chronic Kidney Disease (CKD) patients. OBJECTIVES: We investigate the in vitro impact of PC and PCS on monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC. METHODS: PCS was synthesized by PC sulfatation. VSMC were extracted by enzymatic digestion of umbilical cord vein and characterized by immunofluorescence against α-actin antibody. The cells were treated with PC and PCS at their normal (n), uremic (u) and maximum uremic concentrations (m). Cell viability was assessed by MTT. MCP-1 expression was investigated by ELISA in cells supernatants after toxins treatment with or without the NF-κB p65 inhibitor. RESULTS: There was no significant difference in cell viability after toxins treatment for all concentrations tested. There was a significant increase in MCP-1 expression in cells treated with PCu and PCm (p < 0.001) and PCSn, PCSu and PCSm (p < 0.001), compared with the control. When VSMC were treated with the NF-κB p65 inhibitor plus PCu and PCm, there was a significant decrease in MCP-1 production (p < 0.005). This effect was not observed with PCS. CONCLUSIONS: VSMC are involved in atherosclerosis lesion formation and production of MCP-1, which contributes to the inflammatory response initiation. Our results suggest that PC mediates MCP-1 production in VSMC, probably through NF-κB p65 pathway, although we hypothesize that PCS acts through a different subunit pathway since NF-κB p65 inhibitor was not able to inhibit MCP-1 production.

p-cresol mas não p-cresil sulfato estimulam a produção de MCP-1 via NF-κB p65 em células vasculares musculares lisas humanas / p-cresol but not p-cresyl sulfate stimulate MCP-1 production via NF-κB p65 in human vascular smooth muscle cells

RESUMO Introdução: p-cresol (PC) e p-cresil sulfato (PCS) são responsáveis por muitas das consequências clínicas uremia, tais como a aterosclerose em pacientes com Doença Renal Crônica (DRC). Objetivos: No presente trabalho, investigamos in vitro o impacto de PC e PCS na expressão da quimiocina monocyte chemoattractant protein-1 (MCP-1) via NF-kappa B (NF-κB) p65 em VSMC. Métodos: O PCS foi sintetizado por sulfatação do PC. As VSMC foram extraídas por digestão enzimática da veia do cordão umbilical e caracterizadas por imunofluorescência através do anticorpo α-actina. As células foram tratadas com PC e PCS em suas concentrações normal (n), urêmica (u) e urêmica máxima (m). A viabilidade celular foi avaliada pelo ensaio de MTT. A expressão de MCP-1 foi investigada por ELISA em sobrenadantes de células após o tratamento com as toxinas, com ou sem o inibidor de NF-κB p65. Resultados: Não houve diferença significativa na viabilidade das células após o tratamento com toxinas para todas as concentrações testadas. Houve um aumento significativo na expressão de MCP-1 em células tratadas com PCu e PCm (p < 0,001) e PCSn, PCSu e PCSm (p < 0,001), em comparação com o controle. Quando as VSMC foram tratadas com o inibidor de NF-κB p65 mais PCu e PCm, houve uma diminuição significativa na produção de MCP-1 (p < 0,005). Este efeito não foi observado com PCS. Conclusões: VSMC estão envolvidas na formação da lesão aterosclerótica e produção de MCP-1, o que contribui para o início da resposta inflamatória. Os nossos resultados sugerem que a PC medeia a produção de MCP-1 em VSMC, provavelmente através da via NF-κB p65 e que PCS atue através de uma subunidade diferente da via, uma vez que o inibidor da porção p65 não foi capaz de inibir a produção de MCP-1.

ABSTRACT Introduction: p-cresol (PC) and p-cresyl sulfate (PCS) are responsible for many of the uremia clinical consequences, such as atherosclerosis in Chronic Kidney Disease (CKD) patients. Objectives: We investigate the in vitro impact of PC and PCS on monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC. Methods: PCS was synthesized by PC sulfatation. VSMC were extracted by enzymatic digestion of umbilical cord vein and characterized by immunofluorescence against α-actin antibody. The cells were treated with PC and PCS at their normal (n), uremic (u) and maximum uremic concentrations (m). Cell viability was assessed by MTT. MCP-1 expression was investigated by ELISA in cells supernatants after toxins treatment with or without the NF-κB p65 inhibitor. Results: There was no significant difference in cell viability after toxins treatment for all concentrations tested. There was a significant increase in MCP-1 expression in cells treated with PCu and PCm (p < 0.001) and PCSn, PCSu and PCSm (p < 0.001), compared with the control. When VSMC were treated with the NF-κB p65 inhibitor plus PCu and PCm, there was a significant decrease in MCP-1 production (p < 0.005). This effect was not observed with PCS. Conclusions: VSMC are involved in atherosclerosis lesion formation and production of MCP-1, which contributes to the inflammatory response initiation. Our results suggest that PC mediates MCP-1 production in VSMC, probably through NF-κB p65 pathway, although we hypothesize that PCS acts through a different subunit pathway since NF-κB p65 inhibitor was not able to inhibit MCP-1 production.

Morphine decreases early peritoneal innate immunity responses in Swiss-Webster and C57BL6/J mice through the inhibition of mast cell TNF-α release.

OBJECTIVE: To characterize immunosuppressive effects of morphine on the early innate immunity response of cytokine production in peritoneal cavity after LPS challenge. METHODS: The effects of a single i.p. administration of morphine (3.1 or 31 mg/kg) on LPS-induced tumor necrosis factor α (TNF-α) and monocyte chemoattractant protein-1 (CCL2) intraperitoneal release was tested in Swiss-Webster, C57BL/6J, mast cell deficient Kit(Wsh/Wsh) (W-sh) and mast cell reconstituted (W-sh-rec) mice. RESULTS: Morphine was found to inhibit LPS-induced TNF-α but not CCL2 release in the peritoneal cavity. Studies on mast cell deficient and reconstituted mice indicate that resident mast cells mediate selective morphine immunosuppression in the peritoneal cavity.

Anti-inflammatory and immunomodulatory effects of RDV-8 [C18H22N2O2S (ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydropyrimidine-5-carboxylate)] in a rat model of induced pleurisy and in vitro lymphoproliferation.

RDV-8 [C(18)H(22)N(2)O(2)S (ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydropyrimidine-5-carboxylate)] is derived from the 4-thioxopyrimidine, and presents important clinical effects. The present study explored the RDV-8 effects in the proliferation of human peripheral blood mononuclear cells (PBMCs), as well as in a pleurisy-induced rat model. PBMCs were directly plated in four different RDV-8 concentrations (0.0125, 0.025, 0.05 and 0.1 mg/mL). RDV-8 decreased cell proliferation and monocyte chemotactic protein 1 synthesis. The interleukin 1 levels and the cytotoxic effect were not significantly affected by RDV-8 treatment. In the carrageenan-induced pleurisy model, the RDV-8 (3 mg/kg) treatment induced a significant reduction in the exudate volume, in the polymorphonuclear leukocyte migration and in the pleural exudate NO levels. The results indicate that RDV-8 may have an immunomodulatory effect, as well as anti-inflammatory actions suggesting that it could represent a new strategy in the inflammatory response modulation.

Angiotensin II stimulates MCP-1 production in rat glomerular endothelial cells via NAD(P)H oxidase-dependent nuclear factor-kappa B signaling.

Angiotensin II (Ang II) plays a crucial role in the pathogenesis of renal diseases. The objective of the present study was to investigate the possible inflammatory effect of Ang II on glomerular endothelial cells and the underlying mechanism. We isolated and characterized primary cultures of rat glomerular endothelial cells (GECs) and observed that Ang II induced the synthesis of monocyte chemoattractant protein-1 (MCP-1) in GECs as demonstrated by Western blot. Ang II stimulation, at concentrations ranging from 0.1 to 10 microm, of rat GECs induced a rapid increase in the generation of reactive oxygen species as indicated by laser fluoroscopy. The level of p47phox protein, an NAD(P)H oxidase subunit, was also increased by Ang II treatment. These effects of Ang II on GECs were all reduced by diphenyleneiodonium (1.0 microm), an NAD(P)H oxidase inhibitor. Ang II stimulation also promoted the activation of nuclear factor-kappa B (NF-kappaB). Telmisartan (1.0 microm), an AT1 receptor blocker, blocked all the effects of Ang II on rat GECs. These data suggest that the inhibition of NAD(P)H oxidase-dependent NF-kappaB signaling reduces the increase in MCP-1 production by GECs induced by Ang II. This may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with chronic renal disease.

Effect of treatment on mononuclear cell migration in cervical cancer patients.

AIMS AND BACKGROUND: Our aim was to evaluate the effect of treatment on the in vitro migration of circulating mononuclear cells in cervical cancer patients at different stages. METHODS: We prospectively investigated 24 patients with cervical neoplasia, without prior treatment, submitted to surgery or chemotherapy as therapeutic conduct. Controls were healthy volunteer women (n = 23). Mononuclear cells were isolated from peripheral venous blood before and after treatment, and their migration capacity was evaluated in a microchemotaxis chamber assay towards the chemotactic stimuli fMLP, MCP-1 and RANTES, compared to basal migration. Serum levels of nitric oxide metabolites were assayed by the Griess reaction. RESULTS: Increased mononuclear cell migration in response to the chemotactic stimuli, compared to basal migration, was observed in controls and patients, without differences between them. After treatment (n = 14), mononuclear cell migration in response to MCP-1 and RANTES was increased compared to pre-treatment. Serum levels of nitric oxide metabolites were more elevated in patients (n = 19) than in controls (n = 17), but decreased after treatment (n = 15). CONCLUSIONS: The results suggest that the production of soluble circulating factors by tumor cells could interfere with the functional activity of blood mononuclear cells.

beta-Sitosterol modulation of monocyte-endothelial cell interaction: a comparison to female hormones.

OBJECTIVE: To investigate the effect of beta-sitosterol, 17beta-estradiol and progesterone on oxidized LDL (oxLDL)-stimulated human umbilical venous endothelial cell (HUVEC) expression of intercellular adhesion molecule-1 (ICAM-1), THP-1 monocyte chemotactic activity, migration and adhesion of THP-1 cells co-cultured with HUVECs. METHODS: ICAM-1 expression was determined by immunofluorescence in HUVEC monolayers treated with LDL or oxLDL and 17beta-estradiol, progesterone or beta-sitosterol. Monocyte chemotactic activity was performed in Transwell chambers by culturing HUVECs with different stimuli and steroids, THP-1 cells labeled with [(3)H] thymidine were added to the upper chamber and the radioactivity was measured. Migration assays were performed using Transwell chambers but monocytes were labeled with BCECF-AM and THP-1 cells adhered to HUVECs were visualized by fluorescence microscopy. MCP-1 was quantified by ELISA. RESULTS: ICAM-1 expression was inhibited by beta-sitosterol alone, when combined with 17beta-estradiol or progesterone, or with both hormones. It was shown that 7.5 microM beta-sitosterol decreased migration and adhesion of THP-1 cells to HUVECs cultured in the presence of oxLDL. This effect was also observed in HUVEC cultures in the presence of beta-sitosterol, the 17beta-estradiol and progesterone mixture, and in the presence of the two hormones. It was shown that 7.5 microM beta-sitosterol significantly inhibited chemotaxis of [(3)H] thymidine labeled THP-1 cells in oxLDL-stimulated HUVEC cultures. MCP-1 concentrations in the supernatants of oxLDL-stimulated HUVEC cultures were inhibited by 7.5 microM beta-sitosterol as well as by progesterone and the mixture of the two female hormones.