Effect of a Novel Dihydroartemisinin Dimer Containing Nitrogen Atoms SM 1044 on Apoptosis of Human Leukemia Cell Line NB4-R1 / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of a water-soluble novel dihydroartemisinin dimer containing nitrogen atoms SM 1044 on the apoptosis of all-trans retinoic acid (ATRA) resistant acute promyelocytic leukemia (APL) NB4-R1 cells and its potential mechanism.@*METHODS@#The effects of SM 1044 on cell apoptosis, mitochondrial transmembrane potential, and the level of reactive oxygen species (ROS) were assessed by flow cytometry. Expressions of apoptosis-related proteins were determined by Western blot. The effects of SM 1044 on MAPK (ERK, JNK) signaling pathway, PML/RARα fusion protein, and expressions of apoptosis-related proteins were detected by Western blot.@*RESULTS@#SM 1044 could significantly induce apoptosis and the loss of mitochondrial transmembrane potential in NB4-R1 cells, and activate apoptosis-related proteins caspase-3, caspase-8, caspase-9 and poly (ADP-ribose) polymerase (PARP). SM 1044 could also induce NB4-R1 cells to produce ROS. Western blot showed that SM 1044 activated the phosphorylation of MAPK (ERK, JNK) signaling pathway and down-regulated the expression of PML/RARα fusion protein.@*CONCLUSION@#SM 1044 can induce apoptosis of ATRA resistant APL NB4-R1 cells, which may be related to ROS/ERK and ROS/JNK signaling pathway, and can also induce by down-regulating PML/RARα fusion protein.

Loss of RBFOX2 inhibits meiotic initiation in male mice / 生物工程学报

Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.

Multi-omics analysis of regulating effects of hyperoside on lipid metabolism in high-fat diet mice / 生理学报

The aim of this study was to explore the regulating effects of hyperoside (Hyp) on lipid metabolism in high-fat diet mice. The high-fat diet mouse model was established by high-fat diet induction. After 5 weeks of Hyp intragastric administration in high-fat diet mice, the serum lipid levels before and after Hyp administration were measured by the corresponding kits. The tissue structure of mouse liver was observed by HE staining before and after Hyp administration. The changes of intestinal flora and transcriptome were measured by Illumina platforms. Liquid chromatography-mass spectrometry (LC-MS) was used to determine non-targeted metabolites. The results showed that Hyp significantly reduced lipid levels in the high-fat diet mice and effectively restored the external morphology and internal structure of liver tissue. Hyp changed the species composition of the intestinal flora in high-fat diet mice, increased the abundance of beneficial flora such as Ruminococcus, and decreased the abundance of harmful flora such as Sutterella. Combined multi-omics analysis revealed that the effect of retinoic acid on lipid metabolism was significant in the high-fat diet mice treated with Hyp, while the increase of retinoic acid content was significantly negatively correlated with the expression of genes such as cyp1a2 and ugt1a6b, positively correlated with AF12 abundance, and significantly negatively correlated with unidentified_Desulfovibrionaceae abundance. These results suggest that Hyp may modulate the abundance of AF12, unidentified_Desulfovibrionaceae and inhibit the expression of genes such as cyp1a2 and ugt1a6b, thus increasing the content of retinoic acid and regulating lipid metabolism in the high-fat diet mice.

Effect of PADI4 on the Expression of Inflammatory Cytokines During NB4 Cells Differentiation / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of peptidylarginine deiminase 4 (PADI4) during the process of differentiation into granulocyte of NB4 cells induced by all-trans-retinoic acid (ATRA) and whether PADI4 is involved in the inflammatory cytokines expression.@*METHODS@#Granulocyte differentiation model of NB4 cells induced by ATRA was established. The cell morphology changes were observed by Wright-Giemsa staining. The expression of cell differentiation marker CD11b was analyzed by flow cytometry. The mRNA and protein expression of PADI4 was detected by RT-PCR and Western blot, respectively. The expression of tumor necrosis factor (TNF) α and interleukin (IL) 1β was analyzed by ELISA, and also examined with the knockdown of PADI4 expression by siRNA.@*RESULTS@#After NB4 cells induced by ATRA, the cytoplasm increased and the ratio of nuclear to cytoplasmic was reduced. Nuclear dented, and rod-shaped nucleus, lobulated phenomenon increased (P<0.05). Flow cytometry analysis results showed that the cell surface molecule CD11b expression increased (P<0.01). RT-PCR and Western blot showed the expression of PADI4 increased at both transcriptional and translational levels during the process of the differentiation. ELISA showed TNF-α and IL-1β secretion increased in differentiated macrophages, while they could be inhibited by PADI4-specific siRNA.@*CONCLUSION@#During the differentiation into granulocyte of NB4 cells induced by ATRA, PADI4 expression increased. Furthermore, PADI4 appeared to play a critical role in inflammatory cytokines secretion.

Effects of ATRA on diabetic rats with renal ischemia-reperfusion injury

Abstract Purpose To explore the role of all-trans retinoic acid (ATRA) in renal ischemia/reperfusion injury of diabetic rats. Methods Sixty adult male rats were randomly divided into 6 groups, including sham group (S group), ischemia-reperfusion group (I/R group), ischemia-reperfusion+ATRA group (A group), diabetic group (D group), diabetic ischemia-reperfusion group (DI/R group), diabetic ischemia-reperfusion +ATRA group (DA group). The levels of creatinine (Cr), cystatin C (Cys-C) and β2-microglobulin (β2-MG) were measured. Morphology of renal tissue was observed under light microscope. Results DJ-1, Nrf2, HO-1 and caspase-3 were detected by western blot. DJ-1, Nrf2, HO-1 and caspase-3 in I/R group, D group and DI/R group was higher than that in S group. Compared with I/R group, Nrf2 and HO-1 in A group was decreased, but caspase-3 was increased. However, Nrf2 in DA group was higher than that in DI/R group, HO-1 and caspase-3 in DA group were lower than that in DI/R group. Compared with group S, Cr, Cys-C and β2-MG in I/R group, A group, D group, and DI/R group were higher. Whereas the levels of Cr, Cys-C, β2-MG and renal injury score in DA group were lower than those in DI/R group. Conclusion ATRA has a protective effect on renal ischemia-reperfusion injury in diabetic rats, maybe relating to DJ/Nrf2 pathway.

Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

All-trans-retinoic acid generation is an antidotal clearance pathway for all-trans-retinal in the retina / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4-/-RDH8-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4-/-RDH8-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.

Effects of all-trans retinoic acid on goat dermal papilla cells cultured in vitro

Background: All-trans retinoic acid (ATRA), a vitamin A-derived active metabolite, exerts important functions in hair biology. Previous studies indicated that excess ATRA hampered hair follicle morphogenesis and cyclic regeneration in adulthood, but other studies stated that ATRA promoted hair growth. Dermal papilla (DP), a cluster of specialized fibroblasts, plays pivotal roles in controlling development and regeneration of hair follicle. Several lines of evidence indicated that DP might be the target cells of ATRA in the hair follicle. To confirm this hypothesis, the present study was performed to explore the biological effects of ATRA on goat dermal papilla cells (DPCs) and clarify the roles of ATRA in hair biology. Results: Our experimental results indicated that key signaling transducers of ATRA were dynamically expressed in distinct stages of goat cashmere growth cycle, and high-dose ATRA treatment (10-5 M) significantly impaired the viability of goat DPCs and lowered the ratio of proliferating cells. Otherwise, goat DPCs were stimulated to enter apoptosis and their cell cycle progression was severely blocked by ATRA. Moreover, the expression of fibroblast growth factor 7 (Fgf7), one of the potent hair growth stimulators secreted by DPCs, was transcriptionally repressed following ATRA treatment. Conclusion: DPCs are the targets of ATRA in the hair follicle, and ATRA negatively regulates hair growth by the targeted suppression of cell viability and growth factor expression of goat DPCs. Through these observations, we offer a new mechanistic insight into the roles of ATRA in hair biology.

Effects of isotretinoin on the olfactory function in patients with acne

Abstract: Background: Isotretinoin is a synthetic analog of vitamin A. Recent studies support a role for retinoic acid in the recovery of olfactory function following injury in mice. Objective: This study aimed at determining the effect of isotretinoin on olfactory function in patients who have acne and are otherwise healthy. Methods: Forty-five patients (aged 25-40 years) with acne were included in the study. All patients underwent a rhinological examination. Olfactory function was assessed by the Sniffin' Sticks Test. The test was assessed at baseline and in the third month of isotretinoin treatment. Results: Isotretinoin improved the performance of patients in the olfactory test. The SST score increased from 8.7±1.09 to 9.5±1.19 (p<0.001), prevalence of hyposmia decreased from 40% to 24% and normosmia increased from 60% to 75% (p=0.059). The percentage of patients whose olfactory function was categorized as "good" increased from 6% to 21.3%. This increase was statistically significant (p<0.05). Study limitations: Absence of a control group is one of the limitations of this study. Also, we did not evaluate patients with smell test after stopping isotretinoin treatment. Conclusion: We examined the effect of systemic isotretinoin on olfactory function. It can be concluded from the present investigation that isotretinoin therapy improves the sense of smell.

Retinoic acid protects from experimental cerebral infarction by upregulating GAP-43 expression

The aim of this study was to investigate whether exogenous retinoic acid (RA) can upregulate the mRNA and protein expression of growth-associated protein 43 (GAP-43), thereby promoting brain functional recovery in a rat distal middle cerebral artery occlusion (MCAO) model of ischemia. A total of 216 male Sprague Dawley rats weighing 300–320 g were divided into 3 groups: sham-operated group, MCAO+vehicle group and MCAO+RA group. Focal cortical infarction was induced with a distal MCAO model. The expression of GAP-43 mRNA and protein in the ipsilateral perifocal region was assessed using qPCR and immunocytochemistry at 1, 3, 7, 14, 21, and 28 days after distal MCAO. In addition, an intraperitoneal injection of RA was given 12 h before MCAO and continued every day until the animal was sacrificed. Following ischemia, the expression of GAP-43 first increased considerably and then decreased. Administration of RA reduced infarction volume, promoted neurological functional recovery and upregulated expression of GAP-43. Administration of RA can ameliorate neuronal damage and promote nerve regeneration by upregulating the expression of GAP-43 in the perifocal region after distal MCAO.

[Effect of combination of Melatonin and All-Trans retinoic acid on maturation, fertilization and embryonic development of immature mouse oocytes]

Background and Objective: With respect to the antioxidant role of melatonin and retinoic acid, it seems to be effective both in the maturation and embryonic development. This study was done to investigate the effect of combination of melatonin and All-Trans retinoic acid [RA] on maturation, fertilization and embryonic development of immature mouse oocytes

Methods: In this experimental study, cumulus - oocyte complex [COCs] were recovered from 4-6 week old female mice NMRI and were divided into 6 maturation medium groups including control, sham, experiment 1[melatonin 100 nM, 1 and 2 microM], experiment 2 [retinoic acid 1, 2, 4, 6 microM], experiment 3 [melatonin 2 microM+RA 4 microM], experiment 4 [Mel 100nM + retinoic acid 4 microM]. The maturation rate was recorded after 24 hours of culture in a humidified atmosphere of 5% CO2 at 37°C. The matured oocytes were fertilized with sperm. Fertilization and embryonic development rates to the blastocyst stage were recorded

Results: Maturation rate in the control and sham groups were 50.6% and 49.4%, respectively. Maturation rate were 54.3%, 54.8%, 59.9% in melatonin group with concentrations of 100 nM, 1 and 2 microM, respectively. Maturation rate were 51.6%, 51%, 59% and 49.6% in t-RA group with concentrations of 1, 2, 4, 6 microM. Maturation rate were 60.4% and 54.2% in the experiment 3 and 4 groups, respectively. The maturation rates in the melatonin 2 microM, retinoic acid 4 microM and experiment 3 significantly increased in compare to control [P<0.05]. The embryonic development rate in the melatonin with 100nM concentration and 4 microM of retinoic acid increased significantly compared to controls [P<0.05]. Although, embryonic development rate in experiment 3 was higher than control, but lower in compare to melatonin 100 nM and the retinoic acid 4 microM. The embryonic development rate in experiment 4 significantly increased in compare to control [P<0.05]

Conclusion: Combination of melatonin and All-Trans retinoic acid in medium culture increase maturation rate and improved embryonic development in dose dependent manner

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Phenotypic and Functional Analysis of HL-60 Cells Used in Opsonophagocytic-Killing Assay for Streptococcus pneumoniae

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1alpha, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

All-Trans Retinoic Acid Has a Potential Therapeutic Role for Diabetic Nephropathy

PURPOSE: The aim of this study was to examine the effects of all-trans retinoic acid (ATRA) on diabetic nephropathy. MATERIALS AND METHODS: We measured amounts of urinary albumin excretion (UAE) after administrating ATRA to Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In order to understand the mechanism of action for ATRA, we administrated ATRA to examine its inhibitory action on the production of transforming growth factor-beta1 (TGF-beta1), protein kinase C (PKC), and reactive oxidative stress (ROS) in cultured rat mesangial cells (RMCs). RESULTS: After 16 weeks of treatment, UAE was lower in the ATRA-treated OLETF rats than in the non-treated OLETF rats (0.07+/-0.03 mg/mgCr vs. 0.17+/-0.15 mg/mgCr, p<0.01). After incubation of RMCs in media containing 30 or 5 mM of glucose, treatment with ATRA showed time- and dose-dependent decreases in TGF-beta1 levels and ROS. Moreover, ATRA treatment showed a dose-dependent decrease in PKC expression. CONCLUSION: ATRA treatment suppressed UAE and TGF-beta1 synthesis, which was mediated by significant reductions in PKC activity and ROS production. Our results suggest that ATRA has a potential therapeutic role for diabetic nephropathy.

Efecto de la administración de ácido retinoico sobre el desarrollo del esqueleto axial en embriones de ratón Mus musculus / Effect of administration of retinoic acid on the development of the axial skeleton in mouse embryos Mus musculus

El déficit y exceso de vitamina A provoca malformaciones congénitas que afectan distintos órganos y sistemas. El objetivo de este estudio fue determinar el efecto que causa la administración de ácido retinoico a distintas dosis sobre la morfogénesis ósea del esqueleto axial en embriones de ratón Mus musculus. Mediante aleatorización simple se distribuyeron hembras recién preñadas en 4 categorías: A, B, C y D. El día 8 post fecundación (p.f), se administró 40 mg/kg de peso de ácido retinoico al grupo A, 20 mg/kg de peso de esta solución al grupo B, 1 ml/kg de peso de dimetil sulfóxido al grupo C, y el grupo D es grupo control. El día 17 de la gestación las hembras y sus fetos fueron anestesiadas y eutanasiadas con sobredosis de pentotal sódico intraperitoneal. Los fetos de cada camada fueron procesados mediante diafanización y tinción con azul de Alcian para destacar cartílago hialino y alizarina para observar tejido óseo. Los resultados se expresaron en porcentajes de malformaciones en los siguientes tres segmentos: 1) cráneo-columna cervical, 2) segmento torácico y abdominal y 3) cintura pélvica, considerándose un 100% cuando la totalidad de los elementos óseos se encontraban comprometidos. Se utilizó la prueba de Fisher para la comparación de frecuencias de malformaciones y se consideró estadísticamente significativo cuando p<0,05. En el grupo A se evidenciaron malformaciones mayores como ausencia de huesos frontales y parietales, exencefalia, defectos en el número de vértebras, y fusiones de costillas; y en el grupo B se observaron malformaciones menores como alteraciones numéricas y fusiones de costillas, existiendo diferencias significativas entre ambos grupos. En los grupos C y D no se consignaron malformaciones. El ácido retinoico administrado intraperitonealmente el dìa 8 p.f en dosis de 40 y 20 mg/kg de peso se comporta como un teratógeno en los embriones de ratón, existiendo además diferencias significativas entre las malformaciones generadas por ambas dosis de ácido retinoico. La primera concentración afecta los huesos de los tres segmentos estudiados (cráneo-cervical, toracoabdominal, y pélvico) y la segunda concentración sólo afecta a dos segmentos (cráneo-cervical y toracoabdominal). Ambos tratamientos afectan los segmentos en una gradiente céfalo caudal, independiente del origen embrionario de las estructuras. Esto se debería a que los cambios en las gradientes de ácido retinoico alteran el comportamiento de células de la cresta neural craneal y el orden de la expresión de genes Hox.

The deficit and excess of vitamin A causes birth defects affecting different organ systems. The objectives of this study are to determine the effect caused by the administration of different doses of retinoic acid on bone morphogenesis of the axial skeleton in embryonic mouse Mus musculus. By simple randomization newly pregnant females were distributed into 4 categories: A, B, C and D. On day 8 post fertilization, 40 mg/kg was administered by weight of retinoic acid to the group A, 20 mg/kg body weight of the group B solution 1 ml/kg body weight of dimethyl sulfoxide and group C. Group D is the control group. On day 17 of gestation the females and their fetuses were anesthetized and euthanized with an overdose of intraperitoneal sodium pentothal. Fetuses from each litter were processed using diaphanization and Alcian blue staining to hyaline cartilage and alizarin to observe bone tissue. The results are expressed as percentages of malformations in the following three segments: 1) cranio-cervical spine, 2) thoracic and abdominal segment and 3) pelvic segment, considering 100% when all the bony elements were compromised. Fisher's exact test for comparison of frequencies of malformations was used and considered statistically significant when p<0.05. In group A, major malformations and defects were evident in the indemnity of the cranial vault, exencephaly, defects in the number of vertebrae, and fusion of ribs. In group B minor malformations as numerical alterations and rib fusions were observed. Significant differences were found between both groups. In groups C and D no malformations were recorded. Retinoic acid administered intraperitoneally at doses of 40 and 20 mg/kg acts as a teratogen in mouse embryos. There are significant differences between the defects induced by concentrations of 40 mg/k and 20 mg/k of retinoic acid. Both concentrations affect the bones of the three segments studied (cranio cervical, thoraco-abdominal and pelvic) in a cephalo caudal gradient, independent of the embryonic origin of the structures. Changes in retinoic acid concentration alter the behavior of cranial neural crest and changing the order of the HOX gene expression in the axial skeleton.

Triple nanoemulsion potentiates the effects of topical treatments with microencapsulated retinol and modulates biological processes related to skin aging / Tripla nanoemulsao potencializa os efeitos do tratamento topico com retinol microencapsulado e modula processos biologicos relacionados ao envelhecimento da pele

BACKGROUND: The sum of environmental and genetic factors affects the appearance and function of the skin as it ages. The identification of molecular changes that take place during skin aging provides biomarkers and possible targets for therapeutic intervention. Retinoic acid in different formulations has emerged as an alternative to prevent and repair age-related skin damage. OBJECTIVES: To understand the effects of different retinoid formulations on the expression of genes associated with biological processes that undergo changes during skin aging. METHODS: Ex-vivo skin samples were treated topically with different retinoid formulations. The modulation of biological processes associated with skin aging was measured by Reverse Transcription quantitative PCR (RT-qPCR). RESULTS: A formulation containing microencapsulated retinol and a blend of active ingredients prepared as a triple nanoemulsion provided the best results for the modulation of biological, process-related genes that are usually affected during skin aging. CONCLUSION: This association proved to be therapeutically more effective than tretinoin or microencapsulated retinol used singly. .

FUNDAMENTOS: A soma de fatores genéticos e ambientais afeta a aparência e a funcionalidade da pele ao longo do envelhecimento. O conhecimento a respeito das mudanças moleculares durante o envelhecimento fornece biomarcadores e possíveis alvos para intervenções terapêuticas. O ácido retinoico em diferentes formulações surgiu como uma alternativa para prevenir e reparar os danos da pele associados ao envelhecimento. OBJETIVOS: Avaliar comparativamente os efeitos de diferentes formulações contendo retinoides na expressão de genes associados a processos biológicos que são alterados com o envelhecimento da pele. MÉTODOS: Peles ex vivo foram topicamente tratadas com diferentes retinoides, micro e nanoencapsulados. A modulação dos processos biológicos associados ao envelhecimento da pele foi medida por PCR quantitativa, precedida de transcrição reversa (RT-qPCR). RESULTADOS: A formulação contendo uma mistura de princípios ativos incorporados em uma tripla nanoemulsão e retinol microencapsulado apresentou os melhores resultados de modulação de genes relacionados a processos biológicos que são normalmente alterados durante o envelhecimento da pele. CONCLUSÃO: Essa associação demonstrou uma maior eficácia terapêutica quando comparada ao uso isolado de tretinoína ou retinol microencapsulado. .

Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

Effect of all-trans retinoic acid [ATRA] on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells

All-trans retinoic acid [ATRA], as an active metabolite of vitamin A, has been shown to affect immune cells. This study was performed to evaluate the effect of ATRA on viability, proliferation, activation and lineage-specific transcription factors of CD4+ T cells. CD4+ T cells were separated from heparinized blood of healthy donors and were cultured in conditions, some with, some without ATRA. Viability was assessed by PI flowcytometry and proliferation was measured by MTT assay. CD69 expression was determined by flowcytometry as a measure of cell activation. Lineage-specific transcription factors [FOXP3, RORgammat and T-bet] were examined by intracellular staining and flowcytometry. High doses of ATRA [0.1-1 mM] caused extensive cell death in both PBMCs and CD4+ T cells. Doses of ATRA equal to or lower than 10 microM did not adversely affect cell viability and proliferation in comparison to culture medium without ATRA. Doses of ATRA between 10 microM and InM significantly increased cell activation when compared to culture medium without ATRA. ATRA could increase FOXP3+ and also FOXP3+RORgammat+ T cells while it decreased RORgammat+ and T-bet+ T cells. This study showed that doses of ATRA up to 10 microM are safe when using with CD4+ T cells in terms of cell viability, proliferation and activation. We could also show that ATRA diverts the human immune response in neutral conditions [without adding polarizing cytokines] by increasing FOXP3+ cells and decreasing RORgammat+ cells. ATRA could be regarded as a potential therapy in inflammatory conditions and autoimmunities

Desenvolvimento e validação de método analítico em CLAE-UV para a quantificação de ácido retinóico em microcápsulas de alginato e quitosana / Development and validation of analytical method on HPLC-UV for quantification of retinoic acid in microcapsules of alginate and chitosan

O ácido retinóico (AR) tem sido utilizado para o tratamento de acne severa, rugas, estrias e celulite, no entanto, provoca irritação na pele e sofre rápida degradação quando exposto à luz e ao calor. Métodos analíticos rápidos para quantificação do AR são, portanto, necessários para ensaios de cinética de liberação in vitro. Nesse contexto, o objetivo deste trabalho foi desenvolver e validar um método rápido e sensível para o doseamento do AR em microcápsulas de alginato/quitosana contendo óleo de babaçu dispersas em gel natrosol® por cromatografia líquida de alta eficiência associada à espectroscopia UV e aplicá-lo na avaliação do perfil de liberação in vitro dessas formulações. As análises foram realizadas em modo isocrático utilizando coluna C18 de fase reversa 150 x 4,6 mm (5 μm) com detecção a 350 nm. A fase móvel foi constituída de metanol e ácido acético 1 por cento (85:15 v/v) com vazão de 1,8 mL/minuto. A faixa de linearidade do método foi de 0,5 a 60 μg/mL (r² = 0,999). O método validado mostrou-se sensível, específico, exato, preciso, de baixo custo e o tempo de retenção do AR foi de 5,8 ± 0,4 minutos sendo, desta forma, mais rápido do que os relatados na literatura.

Retinoic acid (RA) has been used in the treatment of severe acne, wrinkles and cellulite. However, it induces skin irritation and rapidly suffers degradation under light and high temperate exposure. Rapid analytical methods to quantify retinoic acid are therefore mandatory for in vitro drug release studies. In this framework, the aim of this study was to develop and validate a rapid and responsive method to quantify the RA in microcapsules of chitosan and alginate containing babassu oil dispersed in natrosol® hydrogel using high performance liquid chromatography (HPLC). Furthermore this method was used to quantify in vitro release kinetics of RA from microcapsules. The analyses have been carried through an isocratic HPLC-UV method using a reversed phase 150 x 4.6 mm C18 (5μm) column, a mobile phase constituted of methanol and 1 percent acetic acid (85:15) at a flow rate of 1.8 mL/min and detection at 350 nm. The linearity range was 0.5-60 μg/mL (r² = 0.999). The validated HPLC-UV method was responsive, specific, accurate, precise, and economic and the RA retention time was 5.8 ± 0.4 minutes, being therefore, faster than that previously reported.

effects of all-trans retinoic acid on blood cells in rat's embryo

All-trans retinoic acid [ATRA] has beneficial and teratogenicity effects when used in a variety conditions. The objectives of this study were to determine the effects of ATRA on the Progenitors of red blood cell and platelets in rat's embryo. Single oral dose [100 mg/kg] of ATRA was administered to rat on gestation day [GD] 10 and fetuses were observed on GD 18 and compared with untreated group. In the experimental embryos of GD 18, the mean number of red blood cells [RBC, 10.5%] and platelets number [15%] were decreased. There was a significant relationship in RBC and platelets count. The mean diameter of RBC and nucleated red blood [NRBC] were compared in two groups. There was no significant relationship between experimental and control groups, except in NRBC diameter. Thus, the present data shows that ATRA may have negative effects on proliferation, differentiation and maturation of erythroid cells and platelets, without having any deleterious effects on the dimenation of RBC