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 of the ALOX5AP gene SG13S114 A/T polymorphism with atherosclerosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the polymorphism of SG13S114 A/T in the 5-lipoxygenase-activating protein (ALOX5AP) gene and the stability of carotid atherosclerosis.</p><p><b>METHODS</b>Polymorphism of SG13S114 A/T in the ALOX5AP gene was analyzed in 152 cases of acute infarction with stable plaque, and 132 cases of acute infarction with vulnerable plaques, by using polymerase chain reaction and restriction fragment length polymorphism. Carotid artery plaque was analyzed by carotid artery color ultrasound.</p><p><b>RESULTS</b>The frequencies of SG13S114 AA genotype and the A allele in the vulnerable plaque group were higher than that in the stable plaque group (P< 0.01).</p><p><b>CONCLUSION</b>The polymorphism of SG13S114 A/T in the ALOX5AP gene may be associated with the instability of atherosclerosis. And the SG13S114 A allele may be a risk factor of vulnerable plaques.</p>

Molecular and cellular bases of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCRABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.

Effect of concanavalin A on expression of MAPEG mRNA in Balb/c mouse brain / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the gene expression of MAPEG in the cortex of concanavalin A (Con A)-induced mouse immune inflammatory model and the effect of cyclosporine A (Cs A).</p><p><b>METHODS</b>Male Balb/c mouse immune inflammation model was developed by intravenous injection of Con A (20 mg/kg). Cs A (150 mg/kg) was intravenously infected prior to Con A administration. The MAPEG expressions were determined by RT-PCR.</p><p><b>RESULT</b>mGST1, mGST3, LTC(4)S, FLAP and mPGES-1 were detected by RT-PCR but not mGST2. Eight hours after Con A treatment, mGST1 level was up-regulated to 1.2 approximately 1.5 folds of control with or without Cs A treatment. mGST3ìLTC(4)S, FLAP and mPGES-1 mRNA levels were not influenced by Con A administration.</p><p><b>CONCLUSION</b>Immune mechanism may be not involved in mGST1 up-regulation in this model and Con A does not alter arachidonic acid metabolism in cortex.</p>

Relationship of polymorphism of SG13S114A/T in ALOX5AP gene with atherosclerotic cerebral infarction / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the relationship of the polymorphism of SG13S114A/T in ALOX5AP gene with atherosclerotic cerebral infarction (ACI).</p><p><b>METHODS</b>By polymerase chain reaction and restriction fragment length polymorphism, polymorphism of SG13S114A/T in ALOX5AP gene in 412 cases with ACI and 368 non-ACI controls were analyzed.</p><p><b>RESULT</b>There were no statistically significant differences in the ALOX5AP gene SG13S114 AA genotype and A allele frequencies between ACI group and control group (P>0.05).</p><p><b>CONCLUSION</b>The results do not support genotype SG13S114 A allele as the risk gene for ACI.control group.</p>

Association between mRNA level of Pde4d and Alox5ap and hypertensive stroke as well as hypertension in rats / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate whether mRNA levels of Pde4d and Alox5ap were associated with hypertensive stroke and hypertension in stroke-prone renovascular hypertensive rats (RHRSP) which could simulate human being's hypertensive cerebral stroke.</p><p><b>METHODS</b>Five groups were established: normotensive group, gradient hypertensive groups I, II and III(with contractive pressure of 140-159 mmHg, 160-179 mmHg and 180-199 mmHg respectively) and spontaneous stroke group. RNA from leukocytes in peripheral blood of each rat underwent real time PCR after reversed.</p><p><b>RESULTS</b>The mRNA levels of Pde4d and Alox5ap of spontaneous stroke group were statistically higher than that of the other groups. Expression of Pde4d of hypertensive group I was a bit higher than that of normotensive group and hypertensive groups II and III; as for Alox5ap, there was no statistical difference between normotensive group and all gradient hypertensive groups.</p><p><b>CONCLUSION</b>Animal experiments come to conclusions that over-expression of Pde4d and Alox5ap are associated with hypertensive stroke but not with hypertension. Therefore, the two genes confer the risk of hypertensive stroke independent of traditional risk factors. It is speculated that over-expression of Pde4d and Alox5ap can motivate onset of hypertensive cerebral stroke by participating in inflammation of arterial walls.</p>

Effects of among compositions of Herba Ephedrae decoction on genic xpression of 5-lipoxygenase activating protein, IL-4 and leukotriene C4 in asthmatic mice / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the regularity of recipe composition by observing inhibitory effects on the genic expression of 5-lipoxygenase activating protein, IL-4 and the leukotriene C4 in asthmatic mice.</p><p><b>METHOD</b>The mice were challenged with OVA and administered ig with the Herba Ephedrae decoction (HED), separated compositions (2500 mg x kg(-1), calculated by Herba Ephedrae) and dexamethasone (2 mg x kg(-1)) respectively once daily for seven days. The real-time fluorescence quantitative PCR method was employed to measure the contents of FLAP mRNA and IL-4 mRNA expressions in lung and the ELISA method was used to determine the content of LTC4 in the washing solution of pulmonary alveolus and bronchi.</p><p><b>RESULT</b>In the lung of asthma mice, the expressions of FLAP and IL-4 and the content of LTC4 were significantly augmented compared with the control group. The HED and the separated compositions could suppress the expressions of FLAP and IL-4 and LTC4 release to a great extent in mice.</p><p><b>CONCLUSION</b>The HED had the remarkable effects of antianaphylaxis asthma and the original formula HED worked best. These results confirmed the rationality and scientific level of HED.</p>