Systemic and local antiinflammatory effect of magnesium chloride in experimental arthritis

Abstract Objective Despite some knowledge gaps in scientific evidence, MgCl2 is largely used for pain relief in musculoskeletal diseases. Mg salts were shown to provide analgesia postoperatively in orthopedic surgery and low Mg levels were linked to arthritis development and severity. We determined the anti-inflammatory activity of MgCl2 in an acute arthritis model. Methods Mice received 0.1 mg/25μL Zymosan (Zy) or saline into the knees. Joint pain was evaluated using von Frey test; cell influx, and interleukin (IL)-1 level were assessed in joint lavage at 6 h. Synovia were excised for histopathology and analysis of immunoexpression of nuclear factor kappa B (NFκB) and tumor necrosis factor (TNF)-α. Groups (n = 6/group) received either 90 mg/kg MgCl2/100 μL or saline per os (systemic) or 500 μg/25 μL MgCl2 or saline intra-articularly (i.a.) 30 min prior to Zy. Results MgCl2 given either systemically or locally significantly reduced cell influx (p = 0.0012 and p = 0.0269, respectively), pain (p = 0.0005 and p = 0.0038, respectively), and intra-articular IL-1 level (p = 0.0391), as compared to saline. Systemic MgCl2 significantly decreased NFκB (p < 0.05) immmunoexpression, as compared to saline. Conclusion MgCl2 given systemically or locally displayed anti-inflammatory activity in a severe acute arthritis model reducing cell influx, pain, and cytokine release. MgCl2 operates at least partially via inhibiting NFκB activation. This is the first in vivo demonstration that MgCl2 decreases cytokine release in arthritis, prompting reduction of inflammation and pain relief.

Oral administration of eucalyptol reduces cell migration and pain-like behavior in zymosan-induced arthritis mice

Abstract Rheumatoid arthritis (RA) is an inflammatory disease that utilizes nonbiologic and biologic drugs for appropriate disease management. However, high cost, adverse effects, reduced effectiveness, and risk of infection have stimulated the search for safer and more efficacious therapeutic strategies. In the present study, we aimed to evaluate the anti-inflammatory and analgesic properties of eucalyptol in an experimental model of arthritis. Mice were administered zymosan or saline intra-articularly. One hour before the zymosan administration, the mice were treated with oral eucalyptol (200-400 mg/kg) and vehicle. Cell influx, neutrophils, lymphocytes, and monocytes were measured in joint exudates. Joint pain was assessed using paw-pressure tests. Orally administered eucalyptol (200 and 400 mg/kg) significantly reduced cell influx, as well as neutrophils, lymphocytes, and monocytes, when compared with the control. Eucalyptol at a dose of 400 mg/kg significantly reversed joint pain and demonstrated analgesic activity (60%); however, 200 mg/kg failed to alter joint pain. These results indicate that oral eucalyptol promotes anti-inflammatory and analgesic activity in mice subjected to zymosan-induced arthritis.

Advances in Toll-like Receptor Signaling Pathway Induced by Pyrogens / 中国药师

Lipopolysaccharide(LPS),lipteichoic acid(LTA) and zymosan can be recognized by Toll-like receptors (TLRs) ex-pressing in the inflammatory cells membrane,such as macrophages,monocytes and endothelial cells,and then induce the secretion of pyrogenic cytokines followed by triggering inflammatory reaction. The signaling pathway mechanisms were summarized, including the extracellular signaling cascades from pyrogens to individual TLRs,and intracellular signaling cascades from TLRs to nucleus.

Effect of Cholecalciterol Cholesterol Emulsion on Liver of Zymosan-induced Multiple Organ Failure in Mice / 中国医科大学学报

Objective To study the effect of cholecalciterol cholesterol emulsion(CCE)in the zymosan(Z)-induced acute hepatic injury. Meth-ods A total of 36 C57 BL/6 mice were randomly divided into 4 groups,namely negative control(CON)group,CCE group,Z group and CCE+Z group,respectively. There were 9 mice in each group. Mice from CON group and Z group were fed with pure water. Mice from CCE group and CCE+Z group were fed with cholecalciterol cholesterol emulsion 20μL dissolved in 200 mL pure water which was kept in darkness. After 14 days, Z group and CCE+Z group were injected with zymosan at a dose of 500 mg/kg. After 18 hours,all the mice in each group were sacrificed. The liver tissues were harvested for histopathological examination. The serum ALT levels were determined. The molecular expression of IL-6 and IL-18 in liv-er tissue of mice were evaluated by Western blotting and real-time quantitative PCR method. Results The results of histopathological examination showed that liver tissue damage in CCE+Z group was lighter than that of Z group ,and heavier than that of the CON group. Compared to the CON group,Z group had the highest serum ALT level,followed by CCE+Z group,while in Z group was significantly lower than that in CON group(all P<0.05). The expression of IL-6 and IL-18 protein and mRNA showed level of Z group was apparently higher than those of CON group and CCE+Z group(all P<0.05). Conclusion Cholecalciterol cholesterol emulsion can play certain protective effect on zymosan-induced liver injury in mice.

The effects of agmatine on acute peritoneal inflammatory injury and neutrophil infiltration induced by zymosan in mice / 中华危重病急救医学

Objective To investigate the protective effect of agmatine (AGM) against peritoneal inflammatory response and neutrophil (PMN) infiltration induced by zymosan (ZYM) in mice. Methods Thirty-six adult male C57BL/6 mice were randomly divided into sham group, model group, and AGM treatment group. Peritonitis model was reproduced by intra-peritoneal injection of 1 mg/mL ZYM (0.5 mL), while equivalent phosphate buffer saline (PBS) was given to sham group. 200 mg/kg AGM was injected into peritoneal cavity after ZYM challenge in AGM treatment group. Six mice in each group were sacrificed at 2 hours and 6 hours, respectively, after reproduction of the model. Blood sample and peritoneal lavage fluid (PLF) were collected. The levels of keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor-α (TNF-α), interleukins-6 (IL-6) in serum and PLF were determined by enzyme linked immunosorbent assay (ELISA). The number of leukocytes and PMN in PLF were determined by hemocytometer and flow cytometry, respectively. Results Compared with sham group, all serum and PLF levels of KC, MIP-2, TNF-α and IL-6 were greatly elevated at 2 hours after ZYM injection in model group, while AGM treatment could dramatically reduce the levels of the above-mentioned cytokines in serum and PLF as compared with those of the model group [serum KC (ng/L): 990.7±137.9 vs. 2 053.2±262.7, MIP-2 (ng/L): 642.2±124.4 vs. 1 369.7±146.5, TNF-α (ng/L): 608.6±38.1 vs. 1 044.7±101.0, IL-6 (ng/L): 1 058.2±129.1 vs. 1 443.3±190.1; PLF KC (ng/L): 7 462.3±839.6 vs. 12 723.5±1 515.7, MIP-2 (ng/L): 1 570.8±193.4 vs. 3 471.4±384.7, TNF-α (ng/L): 1 115.8±156.7 vs. 1 499.2±231.2, IL-6 (ng/L): 2 646.5±223.2 vs. 3 126.7±291.4; all P < 0.05]. The expressions of KC, MIP-2 and TNF-α at 6 hours were significantly lower than those at 2 hours in model group and AGM treatment group, but IL-6 levels were further increased. The levels of KC and MIP-2 in serum and PLF at 6 hours were decreased to the levels of sham group. At 6 hours after the reproduction of the model, the number of total inflammatory cells and PMN of PLF in the model group was significantly higher than those of the sham group. In contrast, AGM notably lowered the number of inflammatory cells and PMN in peritoneal fluid after ZYM attack [total inflammatory cells (×109/L): 14.7±1.1 vs. 2.0±0.4, 10.1±1.2 vs. 14.7±1.1; PMN (×109/L): 11.37±1.22 vs. 0.18±0.05, 7.69±0.57 vs. 11.37±1.22, all P < 0.05]. Conclusion AGM can effectively alleviate acute peritoneal inflammatory injury induced by ZYM, mainly through reducing the secretion of inflammatory mediators and chemokines, and inhibiting the infiltration of leukocytes and neutrophils.

Oral administration of curcumin (Curcuma longa) can attenuate the neutrophil inflammatory response in zymosan-induced arthritis in rats

PURPOSE: To evaluate the effect of curcumin in the acute phase of zymosan-induced arthritis. METHODS: Twenty-eight male rats were subjected to intra-articular infiltration of zymosan of both knees and, in four the infiltration was made with saline. The animals were divided into five groups second received every six hours by gavage: corn oil by (positive and negative control); curcumin (100 mg/kg); prednisone 1 mg/kg/day; prednisone 8 mg/kg. All animals were sacrificed after six, 12, 24 and 48 hours of the infiltration. The knees were removed for evaluation of neutrophil infiltration. The number of neutrophils was counted by computer-assisted analysis of the images. The neutrophil infiltrate was stratified into four grades: 0 = normal; + = mild; ++/+++ = moderate; > ++++ = severe. The results were compared using the Mann-Whitney test and the variance by Kruskal-Wallis test adopting a significance level of 5% (p<0.05). RESULTS: Curcumin reduces inflammatory activity in the first six hours after zymosan-induced arthritis when compared to saline (p<0.01). This was also observed in animals subjected to administration of prednisone (1 mg/kg) and those treated with prednisone (8 mg/kg). Curcumin was more effective than lower doses of prednisone in the first six hours after induction of the arthritis. After 12, 24 and 48 hours, curcumin does not have the same anti-inflammatory effects when compared to prednisone. After 48 hours, prednisone is more effective than curcumin in reducing the inflammatory infiltrate regardless of the dose of prednisone used. CONCLUSION: Oral administration of curcumin reduces inflammation in the first six hours after experimentally zymosan-induced arthritis. .

A novel rat model of atherosclerosis induced by inflammation and the therapeutic effect of Rb1 / 中国实验动物学报

Objective To establish a novel atherosclerosis model by inflammation in rats and investigate the anti-atherosclerotic effect of Rb1.Methods Healthy male SD rats were randomly divided into three groups, namely the control group, model group (using zymosan A to induce inflammation) and Rb1-treated group (12 rats in each group).The rats were administered liquid paraffin (i.p.), zymosan A (20 mg/kg, i.p., once every 4 days) or zymosan A and Rb1 (40 mg/kg, i.p., once daily), respectively.All animals were fed a high-fat diet for 10 weeks.At scheduled time points, pathological changes in the aorta were observed using Sudan IV staining and transmission electron microscopy.White blood cell count was used to assess the inflammation.The expression of NFκB, TNFα, IL6 was evaluated by real time PCR, im-munohistochemistry and ELISA, respectively.Results Typical atherosclerotic changes such as fatty streaks, plaque, foam cells in the rats following zymosan A induction were alleviated by Rb1 treatment.In the Rb1-treated group, there was a markedly decreased expression of NFκB, TNFα, and IL6.Conclusion The model of atherosclerosis can be established by inflammation based on high-fat diet in rats.Rb1 inhibits atherosclerosis through anti-inflammatory effect.

PD-L1 blockade ameliorates suppression of tolerogenic dendritic cells on immunoactivity of T lymphocytes in spleen of zymosan-injured mice / 临床与实验病理学杂志

Purpose To investigate the effect of tolerogenic dendritic cells ( DC) on T lymphocytes in the spleen during the develop-ment of zymosan-induced sepsis in mice, and to explore whether PD-L1 blockade could alleviate the immunosuppressive effect of tolero-genic DC on T lymphocytes. Methods Mice sepsis model was established by intraperitoneal injection of zymosan. Splenic DC and T lymphocytes were isolated respectively by using anti-CD11c and anti-CD3 magnetic beads. The expressions of PD-L1, PD-1 and PIR-B on splenic DC were measured, and IL-12 and IL-10 secreted from DC were determined. Mitogen-induced T lymphocyte proliferation and IL-2 secretion were assessed. Anti- PD-L1 antibody was added into mixed culture of tolerogenic DCs with normal Tcells. T cell proliferation and IL-2, IL-12 and IL-10 concentrations in the supernatant of mixed culture were determined. Results At 5 days and 12 days after zymosan injection, the expressions of PD-L1, PD-1 and PIR-B on splenic DC increased greatly, secretion of IL-12p70 re-duced and that of IL-12p40 and IL-10 augmented in DC, which were associated with decrease of T cells proliferation and IL-2 secre-tion. Administrating anti-PD-L1 antibody into the mixed culture of tolerogenic DC and Tcell could alleviate the suppression of DC on T lymphocyte proliferation and secretion of IL-2, and ameliorate the ability of DC secreting IL-12 and IL-10 as well. Conclusions At late stage of zymosan-induced sepsis, the formation of splenic tolerogenic DC resulted in immunosuppression of T lymphocytes. Anti-PD-L1 antibody could improve the immunoactivity of DC and T lymphocyte through intervening PD-L1/PD-1 pathway.

Efeito da nicotina sobre fagócitos ativados / Effect of the nicotine on actived phagocytes

Indivíduos fumantes apresentam aumento na proporção de leucócitos polimorfonucleares (LPMN), como por exemplo, no tecido pulmonar, resultando em aumento nos níveis de enzimas proteolíticas e espécies reativas de oxigênio, que ocasionam efeito destrutivo na matriz celular e contribuem para a progressão da doença pulmonar, além de favorecer o aparecimento de infecções microbianas, e determinam que as células fagocíticas estejam em frequente estado de ativação (fagocitose). Neste trabalho, avaliou-se a influência da nicotina (NIC) sobre a viabilidade de LPMN e macrófagos ativados ou não, pelos estímulos zymosan e acetato de forbol miristato. Os resultados indicaram que a NIC promove aumento na viabilidade de LPMN e macrófagos ativados em relação a essas células ativadas sem a presença de NIC, avaliada 'ex vivo' pelo teste de exclusão do azul de trypan. Esse efeito foi significativamente mais pronunciado sobre LPMN que sobre os macrófagos. Essa redução na citotoxicidade favorece a sobrevida da célula, podendo exacerbar os seus efeitos deletérios, especialmente em no seu estado ativado, pela maior produção de espécies reativas de oxigênio.

Smokers show increased rates of polymorphonuclear leukocytes (PMNL), including in pulmonary tissue, resulting increased levels of proteolytic enzymes and reactive oxygen species, which have a destructive effect on the cellular matrix and contribute to the progression of pulmonary disease, in addition to promoting the onset of microbial infections which cause phagocytic cells to be in a state of frequent activation (phagocytosis). This work evaluated the influence of nicotine (NIC) on the viability of PMNL and macrophages (activated or not), by stimuli zymosan and phorbol myristate acetate. The results indicated that NIC led to increased viability of PMNL and activated macrophages compared to these cells activated without NIC, measured ex vivo by trypan blue exclusion test. This effect was significantly higher on PMNL than on macrophages. This reduction in cytotoxicity favors cell survival, and may exacerbate its deleterious effect, especially in the active state, due to increased production of reactive oxygen species.

EFFECT OF MAXIMAL EXERCISE ON NADPH OXIDASE ACTIVITY OF NEUTROPHILS AND RELATION TO SUPEROXIDE-GENERATING ACTIVITY BY CELL-SYSTEM IN HUMAN PERIPHERAL BLOOD / 体力科学

The purpose of this study was to examine reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, - a generated superoxide - of neutrophils in human peripheral blood after maximal exercise. Ten healthy male college students (20.2 ± 0.4 yr, 170.5 ± 1.3 cm, 62.8 ± 1.9 kg) participated after giving written informed consent. They performed an incremental exercise to volitional exhaustion using a bicycle ergometer. Peripheral blood was collected before exercise (Pre), just after exercise (Post) and 1-hour after exercise (Post-1h). Phorbol 12-myristate 13-acetate (PMA)-stimulated and opsonaized zymosan (OZ)-stimulated superoxide-generating activity of neutrophils was measured by the cytochrome c reduction assay. NADPH oxidase activity was measured by a cell-free system. NADPH oxidase activity significantly decreased in Post-1h compared with Pre and Post. A similar tendency was seen in PMA-stimulated activity, but not in OZ-stimulated activity. A strong positive relationship between NADPH oxidase activity and PMA-stimulated activity was found in Pre and this relationship attenuated after exercise. NADPH oxidase activity was not related to OZ-stimulated activity at any time points. We concluded that NADPH oxidase activity decreased after exhaustive maximal exercise in human peripheral neutrophils, and suggest that PMA-stimulated activity, relatively - speaking, reflects NADPH oxidase activity; but OZ-stimulated activity is independent of NADPH oxidase activity.

Innovative technique for the study of pneumoperitoneum in the evaluation of abdominal pain after chemical irritation with zymozan / Técnica original para o estudo da influência do pneumoperitônio na avaliação da intensidade da dor abdominal após irritação química com zimozan

PURPOSE: To assess the influence of pneumoperitoneum in mice submitted to peritoneal irritation provoked by the biological agent Saccharomyces cerevisae, by counting the number of abdominal contractions elicited. METHODS: To study the effects of pneumoperitoneum analgesic action, 60 mice were divided into two groups: the experimental group, subjected to pneumoperitoneum; and the control group, without pneumoperitoneum. The both groups received intraperitoneal injection of zymosan at a dose of 1mg/0,2ml/mouse. RESULTS: The sum of the number of abdominal contractions of the experimental group (with pneumoperitoneum) was significantly lower than that of the control group (without pneumoperitoneum). In the experimental group, a lower number of contractions occurred in each min compared to the control. CONCLUSION: The observation of the analgesic effect of pneumoperitoneum using CO2 in mice submitted to peritoneal irritation by zymosan was verified.

OBJETIVO: Avaliar a influência do pneumoperitônio em animais submetidos à irritação peritoneal provocada pelo agente biológico Saccharomyces cerevisae mediante a contagem do número de contrações abdominais. MÉTODOS: Para o estudo do efeito da ação analgésica do pneumoperitônio os 60 camundongos foram divididos em dois grupos, grupo experimento (com pneumoperitôneo) e controle (sem pneumoperitôneo). Os dois grupos receberam injeção intraperitoneal de zimosan na dose de 1mg/0,2ml/camundongo. RESULTADOS: O somatório do número de contrações abdominais do grupo experimento (com pneumoperitôneo) foi significativamente menor que no grupo controle (sem pneumoperitôneo). O número médio de contrações no grupo controle foi significativamente maior quando comparado com o grupo experimento. CONCLUSÃO: Observou-se efeito analgésico do pneumoperitônio com CO2 em animais submetidos à irritação peritoneal pelo zimosan.

Animal model of non-bacterial multiple organ dysfunction syndrome in the elderly / 老年心脏病学杂志（英文版）

Objective To establish a model of multiple organ dysfunction syndrome in the elderly (MODSE) by intraperitoneal injection of different doses of zymosan, and to compare the multiple organ dysfunction syndrome (MODS) in adult and in the elderly rats. Methods Adult and senile rats, injected with different doses of zymosan intraperitoneally were examined for the changes in the function and morphology of the vital organs, including heart, liver, brain, lungs, and kidneys using blood gas and biochemistry analysis and histopathological examination methods. Results Compared with the normal controls of the adult and the elderly rats, the blood gas and blood biochemistry changed in different degrees in the different dosed zymosan groups. Pathological changes were also found in the vital organs including lungs, heart, liver, brain, kidneys, erc in the experimental groups. Under the same concentrations of zymosan, the reductions in respiratory, cardiac and renal functions in the senile groups were much more severe than those in the corresponding adult group. In the similar degree of model duplication, the senile rats had the tendency to die later than the adult rats. Conclusions Zymosan can be used in both elderly and adult rats to induce MODS model, and the best dosage for MODSE was 0.Sg/kg injected peritoneally. The model would hopefully be used in the study of mechanisms and the therapeutics on MODSE.

Role of inflammation in and effects of saponin of Panax Notoginseng on formation of foam cells / 第三军医大学学报

Objective To study the role of zymosan in and the effects of total saponin of Panax notoginseng (PNS) on the formation of foam cells originating from peritoneal macrophages. Methods The peritoneal macrophages of mice were cultured with zymosan, PNS and oxidized low density lipoprotein (ox-LDL) for 48 h. The shape of the foam cells was shown with the staining of red oil O dye. The content of total cholesterol (TC) in the foam cells was determined. The concentration of TNF? and IL-1 in the supernatant of the cell culture was measured. Results The TC content and the concentration of TNF-? and IL-1 were significantly higher in the zymosan group than in the PNS group. Conclusion Inflammation can accelerate the formation of foam cells through the pathway of inflammatory cytokines but PNS can reduce the formation with the inhibition of inflammation.

Phagocytosis of serum-and IgG-opsonized zymos an particles induces apoptosis through superoxide but not nitric oxide in macrophage J774A.1

Phagocytosis of serum- and IgG-opsonized zymosan (SOZ and IOZ, respectively) particles into J774A.1 macrophages induced apoptosis of the cells, accompanied by the expression of p21(WAF1), one of cyclin-dependent protein kinase (CDK) inhibitors. Furthermore, phagocytosis of SOZ and IOZ particles into macophages induced superoxide formation. Tat-superoxide dismutase (SOD), which is readily transduced into the cells using Tat-domain, protected the cells from the apoptosis induced by phagocytosis of SOZ and IOZ particles. lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) also caused the apoptosis of the cells. However, Tat-SOD could not protect the cells from LPS/IFN-gamma induced apoptosis, suggesting that apoptosis mechanisms involved are different from each other. In the present study, we determined the amounts of nitric oxide (NO) produced by SOZ, IOZ, and LPS/IFN-gamma, and found that SOZ and IOZ did not induce the generation of NO in macrophages, whereas LPS/ IFN-gamma did. The apoptosis due to phagocytosis was accompanied with the release of cytochrome c from mitochondrial membrane to cytosolic fraction. Furthermore, SOZ and IOZ induced the cleavage of procasapase-3 (35 kDa) to give rise to an active caspase-3 (20 kDa), which was blocked by Tat- SOD but not by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. On the other hand, LPS/IFN-gamma caused the activation of procaspase-3, which was blocked by PTIO but not by Tat-SOD. Taken together, phagocytosis of SOZ and IOZ particles induced apoptosis through superoxide but not NO in macrophages, accompanied with the release of cytochrome c and the activation of caspase-3.

Experimental study on the loss of PMN's bactericidal capacity induced by bypass-activated complement / 第三军医大学学报

In order to verify that bypass-activated complement can enable polymorphonuclear neutrophils (PMN) to lose their bactericidal capacity,3 experiments were performed as follows:(DA monolayer culture of human PMN was added with zymosan-activated human serum (ZAHS).then the superoxide ion (O2-,the specific granules,and the intracellular bactericidal activity of the cultured PMNs were signigicantly decreased and reached the lowest point in the 6th hour after the adding of ZAHS.0.05 ml of ZAHS showed the maximal effects on the PMNs.(2)Mice were injected intravenously with 0.5 ml of ZAHS and were killed 6 hours later.All the 3 parameters mentioned above dropped markedly in the PMNs isolated from the blood and the lungs of the mice.Pathologically,the lungs of the rats showed acute interstitial inflammation,focal edema,hemorrhage and atelectasis.and subcellular damages on the pulmonary blood-barrier.(3)ZAHS,after neutralization in vitro with antiserum against human C3 and C5,lost most of its harmful effects mentioned above.The findings suggest that the harmful effects of ZAHS originate from the fragments of C3 and Cs and they are most effective when its dosage is suitable and there must be a sufficient length of time to react before the phagocytosis of PMNs.The possible mechanism of the origination of the harmful effects of ZAHS is as follows:Before PMNs undertake phagocytosis,they releas the bactericidal and inflamnagenic (O2-and specific granules extra-cellularly.So the intracellular bactericidal activity of PMNs is decreased.The accumulation of PMNs in the pulmonary tissues can damage the PMNs themselves and the adjacent pulmonary blood-barrier,which will leads to the occurrence of infection and multiple organ failure.