Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica / Leukotriene-induced intracellular mechanisms during osteogenic differentiation

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.(AU)

Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNFα in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.(AU)

Association study between serum level of 5-LO and Hcy and carotid atherosclerotic plaque stability / 重庆医学

Objective To explore the correlation between 5-LO,Hcy and the stability of carotid artery arteriosclerosis (CAS).Methods A total of 176 patients diagnosis as CAS were assigned as study group then subdivided into stable plaques SP (group) and instable plaques IP(group).108 healthy volunteers were assigned as control group.The serum levels of 5-LO and Hcy were measured and the relationship between the two groups were analyzed.The risk factors of CAS were investigated by Logistic regression analysis and 5-LO and Hcy were used to predict the stability of carotid atherosclerotic plaque by drawing ROC curve.Results The levels of 5-LO and Hcy in the IP group were higher than those in the SP group and the control group(P＜0.05).The level of Hcy in SP group was higher than that in the control group (P＜0.05),while there is no statistical significance between SP and control group in the level of serum 5-LO(P＞0.05).Logistic regression analysis showed that 5-LO,Hcy and diabetes were the risk factors of CAS (P＜0.05).The ROC curve indicate that the optimal cut-off concentration of 5-LO was 232.89 pg/mL for discriminating the IP from SP,the sensibility and specificity were 84.4％ and 81.8％ respectively.And the optimal cut-off concentration of Hcy was 12.53 μmol/L and the sensibility and specificity were 70.1％ and 66.7％ respectively.Conclusion Serum 5-LO and Hcy are risk factors for predicting the stability of CAS plaques;regulating both levels may be a potential target for clinically stable CAS.

Enhanced Effect of Combinatorial Inhibition of Cyclooxygenase-2 and 5-Lipoxygenase on Head and Neck Cancer Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: It has recently been found that arachidonate 5-lipoxygenase (5-lipoxygenase, 5-LO or ALOX5), another molecule capable of arachidonic acid (AA) metabolism, might promote cancer cell viability through unique mechanisms. Interestingly, 5-LO appears to have similar mechanisms to prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2, COX-2) in the regulation of cell viability, although it often utilizes different signaling pathways. We found that not only COX-2 expression but also the expression of 5-LO is up-regulated in some of head and neck squamous cell carcinoma (HNSCC) cell lines. From these findings, we hypothesized that the combined inhibition of these pathways would be likely to be a more effective anti-cancer modality with less side-effect in HNSCC. MATERIALS AND METHOD: In HNSCC cell lines, we investigated the expression of COX-2 and 5-LO by western blotting and checked the levels of prostaglandin E2, leukotrien B4 and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. We performed MTT assay to analyze the growth-inhibitory effect of COX-2 and 5-LO inhibition. RESULTS: Actually, combined knock-down of COX-2 and 5-LO resulted in an enhanced inhibitory effect in cell proliferation of HNSCC than a single inhibition of COX-2. Furthermore, we observed that VEGF production was blocked more effectively by combined treatment of COX-2 and 5-LO small interfering RNA (siRNA) in all tested cell lines. CONCLUSION: Therefore, the combined inhibition of COX-2 and 5-LO may be one way to overcome low efficacy of single inhibition of COX-2 in cancer cells with both COX-2 and 5-LO overexpression.

Protective effect of caffeic acid on damage induced by aluminum-overload in primary cultured rat hippocampal neuron / 中国药理学通报

Aim In order to study the protective effect of caffeic acid on damage induced by aluminum-overload in primary cultured rat hippocampal neuron.Methods Primary cell cultures were obtained from the cerebral hippocampus of Newly born SD rats within 24 h.On the d 7 of neuronal culture, the immunohistory of NSE was used to identify the purity of neuron.There were 5 experimental groups,NaCl(200 μmol·L~(-1))-treated group, AlCl_3-treated group(200 μmol·L~(-1)),and aluminum+caffeic acid(10~(-6) mol·L~(-1),10~(-7) mol·L~(-1) and 10~(-8) mol·L~(-1))-treated groups. HE staining was used to observe the change of neuronal pathomorphology. The cell viability was measured by MTT assay. SOD activity, LDH leakage and MDA contents were also detected.Results The purity of neurons was more than 95%. Aluminum administration induced loss of neurons and damage to dendrite and axon.Compared with that of the control group,the decreased viability of neurons,increased leakage of LDH, decreased activity of SOD and increased contents of MDA were observed in aluminum-treated groups.Compared with that of the model group, the administration of Caffeic acid could significantly blunt the death of the primary cultured hippocampal neurons, and blunt the decrease of neuronal viability and SOD activity and the increase of LDH leakage and MDA contents.Conclusions These results suggest that caffeic acid has an obvious protective effect against neuronal damage induced by aluminum overload in primary cultured neurons. The mechanism of protection might involve the anti-inflammation and anti-oxidative effects of caffeic acid.