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This study aimed to explore the mediation effects of one-carbon metabolism (OCM) related nutrients on the association between MTHFR rs1801133 polymorphism and gestational diabetes mellitus (GDM). Folate, vitamin B12 and homocysteine (Hcy) were measured in the serum of 1254 pregnant women. Linear and logistic regressions were used to estimate the associations of OCM nutrients and MTHFR rs1801133 polymorphism with blood glucose levels and GDM risk. Mediation analysis was applied to test the mediation effects of folate, vitamin B12 and Hcy on the association of MTHFR rs1801133 polymorphism with blood glucose concentrations and GDM. Pregnant women with MTHFR rs1801133 CC genotype had higher serum folate (10·75 v. 8·90 and 9·40 ng/ml) and lower serum Hcy (4·84 v. 4·93 and 5·20 µmol/l) than those with CT and TT genotypes. Folate concentrations were positively associated with fasting plasma glucose (FPG), 1-h plasma glucose (1-h PG), 2-h plasma glucose (2-h PG) and GDM risk. Vitamin B12 levels were negatively correlated with FPG and GDM. Although no direct association was found between MTHFR rs1801133 genotypes and GDM, there were significant indirect effects of MTHFR rs1801133 CC genotype on FPG (ß: 0·005; 95 % CI: 0·001, 0·013), 1-h PG (ß: 0·006; 95 % CI: 0·001, 0·014), 2-h PG (ß: 0·007; 95 % CI: 0·001, 0·015) and GDM (ß: 0·006; 95 % CI: 0·001, 0·014) via folate. In conclusion, serum folate mediates the effect of MTHFR rs1801133 on blood glucose levels and GDM. Our findings potentially provide a feasible GDM prevention strategy via individualised folate supplementation according to the MTHFR genotypes.
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Diabetes Gestacional , Ácido Fólico , Feminino , Humanos , Gravidez , Glicemia/análise , Diabetes Gestacional/sangue , Diabetes Gestacional/genética , População do Leste Asiático , Ácido Fólico/genética , Genótipo , Homocisteína , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Vitamina B 12 , VitaminasRESUMO
This study aimed to investigate the relationship between depression and outcome of percutaneous endoscopic lumbar discectomy (PELD) in patients with lumbar disc herniation. We examined 268 patients who underwent PELD for lumbar disc herniation and were followed for five years. Patients were grouped according to mood: normal mood (159 patients) and continuous depression (109 patients). Depressive symptoms were assessed using the 21-item Beck Depression Inventory. Back and leg pain were assessed using the visual analogue scale. Subjective disability was measured using the Oswestry Disability Index. Neurological function and physical disability were assessed using the Japanese Orthopaedic Association score. Disc-height ratio and intervertebral instability were measured to assess lumbar stability. Clinical and radiological data were recorded before surgery and at the 3-month, 6-month, 1-year, 2-year, and 5-year follow-ups. Although the Japanese Orthopaedic Association, visual analogue scale, and Oswestry Disability Index scores did not significantly differ between groups before surgery, all three scores significantly differed between groups at all follow-up time points after PELD (p < 0.05). Measurements of disc-height ratio and intervertebral instability did not significantly differ between the groups before surgery nor at any point after surgery (P > 0.05). Patients with continuous depression exhibited less improvement in symptom severity and disability score after PELD at all time points in the five years after surgery. Depression had little effect on lumbar vertebral stability after PELD. Interventions to detect and treat depression should be performed before and after surgery.
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Discotomia Percutânea , Deslocamento do Disco Intervertebral , Humanos , Seguimentos , Discotomia Percutânea/efeitos adversos , Deslocamento do Disco Intervertebral/epidemiologia , Deslocamento do Disco Intervertebral/cirurgia , Deslocamento do Disco Intervertebral/etiologia , Depressão/epidemiologia , Resultado do Tratamento , Estudos Retrospectivos , Vértebras Lombares/cirurgia , DiscotomiaRESUMO
BACKGROUND: Osteosarcoma is the most common bone tumor that occurs in children. METHODS: To identify co-expression modules and pathways correlated with osteosarcoma and its clinical characteristics, we performed weighted gene co-expression network analysis (WGCNA) on RNA-seq data of osteosarcoma with 52 samples. Then we performed pathway enrichment analysis on genes from significant modules. RESULTS: A total of 5471 genes were included in WGCNA, and 16 modules were identified. Module-trait analysis identified that a module involved in microtubule bundle formation, drug metabolism-cytochrome P450, and IL-17 signaling pathway was negatively correlated with osteosarcoma and positively correlated with metastasis; a module involved in DNA replication was positively correlated with osteosarcoma; a module involved in cell junction was positively correlated with metastasis; and a module involved in heparin binding negatively correlated with osteosarcoma. Moreover, expression levels in four of the top ten differentially expressed genes were validated in another independent dataset. CONCLUSIONS: Our analysis might provide insight for molecular mechanisms of osteosarcoma.
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Neoplasias Ósseas/genética , Perfilação da Expressão Gênica , Osteossarcoma/genética , Osteossarcoma/secundário , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Neoplásica , Osteossarcoma/metabolismo , PrognósticoRESUMO
BACKGROUND: Dairy product consumption may affect the risk of hip fracture, but previous studies have reported inconsistent findings. The primary aim of our meta-analysis was to examine and quantify the potential association of dairy product consumption with risk of hip fracture. METHODS: We searched the databases of PubMed and EMBASE for relevant articles from their inception through April 17, 2017. The final analysis included 10 cohort studies and 8 case-control studies. Random-effects models were used to estimate the pooled risk. Subgroup and dose-response analyses were conducted to explore the relationships between the consumption of milk and the risk of hip fracture. RESULTS: After pooling the data from the included studies, the summary relative risk (RR) for hip fracture for highest versus lowest consumption were 0.91 (95% CI: 0.74-1.12), 0.75 (95% CI: 0.66-0.86), 0.68 (95% CI: 0.61-0. 77), 1.02 (95% CI: 0.93-1.12) for milk, yogurt, cheese, and total dairy products in cohort studies, respectively. Higher milk consumption [Odds ratio (OR), 0.71, 95% CI: 0.55-0. 91] was associated with lower risk of hip fracture for highest versus lowest consumption in case-control studies. After quantifying the specific dose of milk, the summary RR/OR for an increased milk consumption of 200 g/day was 1.00 (95% CI: 0.94-1.07), and 0.89 (95%CI: 0.64-1.24) with significant heterogeneity for cohort and case-control studies, respectively; There was a nonlinear association between milk consumption and hip fracture risk in cohort, and case-control studies. CONCLUSIONS: Our findings indicate that consumption of yogurt and cheese was associated with lower risk of hip fracture in cohort studies. However, the consumption of total dairy products and cream was not significantly associated with the risk of hip fracture. There was insufficient evidence to deduce the association between milk consumption and risk of hip fracture. A lower threshold of 200 g/day milk intake may have beneficial effects, whereas the effects of a higher threshold of milk intake are unclear.
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Laticínios/estatística & dados numéricos , Dieta/estatística & dados numéricos , Fraturas do Quadril/epidemiologia , Humanos , RiscoRESUMO
OBJECTIVE: The upper facet joint en bloc resection is the key step to open the intervertebral foramina for achieving the intervertebral fusion in transforaminal lumbar interbody fusion (TLIF) surgery. Our purpose is to introduce a upper facet joint resection technique which can avoid injuring the nerve root termed "two layers and two tunnels strategy" in TLIF surgery and to evaluate its clinical effects and neurological safety. METHODS: All 108 patients who underwent TLIF surgery using two layers and two tunnels strategy between December 2015 and January 2019 were analyzed for postoperative clinical treatment parameter. The visual analogue scale (VAS) method, Oswestry disability index (ODI), Japanese Orthopaedic Association (JOA) scores and the Macnab scoring system were used to evaluate the clinical effects during post-operative check-ups at 7 days, 3 months, 6 months, and the last follow-up visit. Data were represented by mean and standard deviation, and repeated measures analysis of variance was performed to make comparison. RESULTS: The result noted that, the VAS scores for back pain decreased by 30.13% at 7 days post-operation (3.64 ± 0.86), 63.15% at 3 months (1.92 ± 0.55), 72.17% at 6 months (1.45 ± 0.61) and 70.44% at the last follow-up (1.54 ± 0.62) compared with pre-operation (5.21 ± 0.93). The VAS scores for lower limb pain decreased by 44.22% at 7 days (3.86 ± 0.90), 61.42% at 3 months (2.67 ± 0.72), 66.62% at 6 months (2.31 ± 0.79) and 66.47% at the last follow-up (2.32 ± 0.72) compared with pre-operation (6.92 ± 1.04). The ODI scores decreased by 49.08% at 7 days (32.19 ± 5.13), 67.92% at 3 months (20.28 ± 5.50), 74.00% at 6 months (16.44 ± 4.21) and 75.42% at the last follow-up (15.54 ± 3.85) compared with pre-operation (63.22 ± 7.58). The JOA scores increased by 51.41% at 7 days (18.49 ± 1.48), 69.26% at 3 months (22.35 ± 1.44), 73.28% at 6 months (23.22 ± 1.18) and 77.53% at the last follow-up (24.14 ± 0.99) compared with pre-operation (7.37 ± 1.71). Among 108 cases, there is no neurological complications. CONCLUSION: Two layers and two tunnels strategy is an effective and safe procedure that can certainly avoid nerve root injury to reduce neurological complication and increase safety of TLIF surgery.
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Fusão Vertebral , Articulação Zigapofisária , Humanos , Articulação Zigapofisária/cirurgia , Fusão Vertebral/métodos , Vértebras Lombares/cirurgia , Resultado do Tratamento , Procedimentos Cirúrgicos Minimamente Invasivos , Estudos RetrospectivosRESUMO
The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. Here, we investigated whether PNS could promote osteogenesis of bone marrow stromal cells (BMSCs) through modulating the MAPK signaling pathways, which are implicated in BMSC osteogenesis. We found that PNS markedly increased the mineralization of BMSCs by alizarin red S assays and stimulate alkaline phosphatase activity of these cells. Additionally, PNS significantly increased the mRNA levels of alkaline phosphatase, core-binding factor a1, and bone sialoprotein while decreasing PPARγ2 mRNA levels. Furthermore, inhibitors of ERK, PD98059, and p38, SB203580 inhibited the osteogenesis-potentiating effects by PNS. PNS stimulated the activation of ERK and p38 as evidenced by increased phosphorylation of these proteins, which was inhibited by PD98059 and SB203580. Our findings indicate that PNS could promote BMSC osteogenesis by activating the ERK and p38 signaling pathways.
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Osteogênese/efeitos dos fármacos , Panax notoginseng/química , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Imidazóis/farmacologia , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Células Estromais/citologia , Células Estromais/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To compare the preventive effects of teriparatide and alendronate on the progression of vertebral body collapse in postmenopausal single-level Kümmell's disease (KD). METHODS: From March 2013 to December 2020, the medical records for 53 postmenopausal single-level KD patients who received conservative treatment with teriparatide (25 patients, teriparatide group) or alendronate (28 patients, alendronate group) were retrospectively reviewed. Midsagittal computed tomography (CT) images were analyzed by ImageJ to assess the intravertebral bone formation (mineralized bone) by calculating the ratio of area of intravertebral mineralized bone (AIMB) to the area of fractured vertebral body (AFVB). The changes in radiological parameters of the fractured vertebral body including kyphosis angle (KA), anterior and posterior border heights (ABH and PBH) and spinal canal diameter (SCD), bone turnover biomarkers (BTMs), and bone mineral density (BMD) were analyzed to evaluate the therapeutic effect. RESULTS: At month 12, the ratio of AIMB to AFVB was significantly greater in teriparatide group (54.28% ± 15.30%) than in alendronate group (35.57% ± 17.61%) (P < 0.001). Sagittal CT substantiated the formation of bone bridge in 16 patients in teriparatide group. No bone bridge was detected in alendronate group. The KA was significantly smaller and the ABH, PBH, and SCD was greater in teriparatide group than in alendronate group (all P < 0.001). The KA increments were significantly smaller in teriparatide group (3.98° ± 1.30°) than in alendronate group (11.43° ± 3.73°) (P < 0.001). The ABH and PBH decrement were significantly lower in teriparatide group (11.96% ± 1.93% and 2.80% ± 2.52%) than in alendronate group (37.04% ± 8.00% and 19.50% ± 8.22%) (both P < 0.001). The BTMs and BMD were significantly greater in the teriparatide group than in the alendronate group. In teriparatide group, KA increment was negatively correlated with the change in PINP (r = -0.781, P < 0.001) and the ratio of AIMB to AFVB (r = -0.592, P = 0.002) from baseline to month 12. The ABH decrement was negatively correlated with the change in PINP (r = -0.612, P = 0.001) and the ratio of AIMB to AFVB (r = -0.806, P < 0.001) from baseline to month 12. CONCLUSIONS: In postmenopausal single-level KD patients, conservative treatment with teriparatide was better than alendronate at preventing the progressive vertebral collapse.
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Alendronato/uso terapêutico , Fraturas por Osteoporose/prevenção & controle , Fraturas da Coluna Vertebral/prevenção & controle , Teriparatida/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Conservadores da Densidade Óssea/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Fraturas por Osteoporose/diagnóstico por imagem , Pós-Menopausa , Estudos Retrospectivos , Fraturas da Coluna Vertebral/diagnóstico por imagemRESUMO
Background: Current treatment of osteosarcoma is limited in part by side effects and low tolerability, problems generally avoided with traditional Chinese medicine. Ganoderma lucidum, a traditional Chinese medicine with antitumor effects, offers a potential alternative, but little is known about its molecular mechanisms in osteosarcoma cells. Objective: To investigate the effect of G lucidum on osteosarcoma cells and its mechanism. Methods: Osteosarcoma MG63 and U2-OS cells were treated with G lucidum, followed by assays for cell proliferation (Cell Counting Kit-8), colony formation, and apoptosis (Alexa Fluor 647-Annexin V/propidium iodide, flow cytometry). Migration and invasion of cells were assessed by wound healing and Transwell invasion assays, and the effect of G lucidum on Wnt/ß-catenin signal transduction was studied by real-time quantitative polymerase chain reaction, western blot, and dual-luciferase assay. Results:G lucidum inhibited the proliferation, migration, and invasion, and induced apoptosis of human osteosarcoma MG63 and U2-OS cells. Dual-luciferase assay showed that G lucidum suppressed the transcriptional activity of T-cell factor/lymphocyte enhancer factor in the Wnt/ß-catenin signaling pathway. Moreover, G lucidum blocked Wnt/ß-catenin signaling by inhibiting the Wnt co-receptor LRP5 and Wnt-related target genes, such as ß-catenin, cyclin D1, C-Myc, MMP-2, and MMP-9. At the same time, when Wnt/ß-catenin was inhibited, the expression of E-cadherin was upregulated. Conclusions: Our results suggest that G lucidum broadly suppresses osteosarcoma cell growth by inhibiting Wnt/ß-catenin signaling.
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Produtos Biológicos/farmacologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Reishi/química , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
Recent studies have indicated that ATRA inhibits chondrogenesis and can lead to congenital clubfoot (CCF). The molecular mechanism of ATRA-induced chondrogenesis is not clear. As RhoA/ROCK and SDF-1/CXCR4 signaling play important molecular roles for a variety of cellular processes, we hypothesized that RhoA/ROCK2 and SDF-1/CXCR4 signaling are involved in ATRA-induced chondrogenesis in rat embryo hind limb bud mesenchymal cells (rEHBMCs). We found that ATRA dose-dependently inhibits proliferation and expression of chondrogenic transcription factors (SOX9 and COL2A1) in rEHBMCs. In contrast, ATRA increases the expression of ROCK2, SDF-1 and CXCR4. Pharmacological inhibition of ROCK signaling and SDF-1/CXCR4 signaling by Y27632 and AMD3100, respectively, resulted in elevated expression of SOX9 and COL2A1. In addition, we found that disturbing SDF-1/CXCR4 signaling by AMD3100 decreases ATRA-induced ROCK2 expression. In vivo studies we also confirm that SOX9 expression of early-stage cartilage progenitors in the proliferative zone and COL2A1 expression in prehypertrophic chondrocytes are decreased in ATRA-treated rat embryo hind limb. Together, these results show that ATRA activates SDF-1/CXCR4/ROCK2 signaling to inhibit chondrogenesis to lead to CCF by suppressing differentiation through down-regulation of SOX9 and COL2A1 expression in rat embryo hind limb bud mesenchymal cells.
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In this study, a facile and versatile urea-assisted approach was proposed to synthesize Chinese rose-like NiO, pinecone-like ZnO and sponge-like CoO adsorbents. The presence of urea during syntheses endowed these adsorbents with high concentration of surface hydroxyl groups, which was estimated as 1.83, 1.32 and 4.19 mmol [OH-] g-1 for NiO, ZnO and CoO adsorbents, respectively. These surface hydroxyl groups would facilitate the adsorption of Cr(vi) species (e.g. HCrO4-, Cr2O72- and CrO42-) from wastewater by exchanging with hydroxyl protons or hydroxide ions, and hence result in extremely high maximum adsorbed amounts of Cr(vi), being 2974, 14 256 and 408 mg g-1 for NiO, ZnO and CoO adsorbents in the pH range of 5.02-5.66 at 298 K, respectively. More strikingly, the maximum adsorbed amounts of Cr(vi) would be greatly enhanced as the adsorbing temperature is increased, and even amount to 23 411 mg g-1 for ZnO adsorbents at 323 K. Based on the kinetics and equilibrium studies of adsorptive removal of Cr(vi) from wastewater, our synthetic route will greatly improve the adsorptivity of the as-synthesized metal-oxide adsorbents, and hence it will shed new light on the development of high-performance adsorbents.
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Despite the well-established role of all-trans-retinoic acid (ATRA) in congenital clubfoot (CCF)-like deformities in in vivo models, the essential cellular and molecular targets and the signaling mechanisms for ATRA-induced CCF-like deformities remain to be elucidated. Recent studies have demonstrated that p53 and p21, expressed in the hindlimb bud mesenchyme, regulate cellular proliferation and differentiation, contributing to a significant proportion of embryonic CCF-like abnormalities. The objective of the present study was to investigate the mechanisms for ATRA-induced CCF, by assessing ATRA-regulated chondrogenesis in rat embryo hindlimb bud mesenchymal cells (rEHBMCs) in vitro. The experimental study was based on varying concentrations of ATRA exposure on embryonic day 12.5 rEHBMCs in vitro. The present study demonstrated that ATRA inhibited the proliferation of cells by stimulating apoptotic cell death of rEHBMCs. It was also observed that ATRA induced a dose-dependent reduction of cartilage nodules compared with the control group. Reverse transcription-polymerase chain reaction and western blotting assays revealed that the mRNA and protein expression of cartilage-specific molecules, including aggrecan, Sox9 and collagen, type II, α 1 (Col2a1), were downregulated by ATRA in a dose-dependent manner; the mRNA levels of p53 and p21 were dose-dependently upregulated from 16 to 20 h of incubation with ATRA, but dose-dependently downregulated from 24 to 48 h. Of note, p53 and p21 were regulated at the translational level in parallel with the transcription with rEHBMCs treated with ATRA. Furthermore, the immunofluorescent microscopy assays indicated that proteins of p53 and p21 were predominantly expressed in the cartilage nodules. The present study demonstrated that ATRA decreases the chondrogenesis of rEHBMCs by inhibiting cartilage-specific molecules, including aggrecan, Sox9 and Col2al, via regulating the expression of p53 and p21.
Assuntos
Condrogênese/efeitos dos fármacos , Botões de Extremidades/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Tretinoína/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Agrecanas/genética , Agrecanas/metabolismo , Animais , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Membro Posterior , Botões de Extremidades/citologia , Botões de Extremidades/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Cultura Primária de Células , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Despite frequently well-established role of all-trans-retinoid acid (ATRA) in congenital limb deformities, its mechanism of action, thus far, is still ambiguous. Pitx1, which is expressed in the hindlimb bud mesenchyme, or its pathways may be etiologically responsible for the increased incidence of clubfoot. Here, we sought to investigate the mechanisms whereby Pitx1 regulated chondrogenesis of hindlimb bud mesenchymal cells in vitro. E12.5 embryonic rat hind limb bud mesenchymal cells were treated with ATRA at appropriate concentrations. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Hematoxylin-safranin-O-fast-green staining assays were used to observe cartilage nodules, and Pitx1 expression was examined by immunofluorescent microscopy. Real-time quantitative PCR and immunoblotting assays were applied to determine the mRNA expressions of Pitx1, Sox9 and type II collagen (Col2al), respectively. The results showed that ATRA inhibited the proliferation of hind limb bud cells dose-dependently. ATRA also induced a dose-dependent reduction in the number of cartilage nodules and the area of cartilage nodules compared with controls. Our real-time quantitative RT-PCR assays revealed that the mRNA expression of Pitx1, Sox9 and Col2al were significantly downregulated by ATRA. Furthermore, our immunofluorescent microscopy and Western blotting assays indicated that Pitx1 was mainly expressed in the cartilage nodules and the levels of Pitx1, Sox9 and Col2al were also downregulated by ATRA dose-dependently. The results indicated that ATRA may decrease chondrogenesis of hind limb bud mesenchymal cells by inhibiting cartilage-specific molecules, such as Sox9 and Col2al, via downregulating Pitx1 expression.
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Condrogênese/efeitos dos fármacos , Fatores de Transcrição Box Pareados/genética , Tretinoína/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo II/antagonistas & inibidores , Colágeno Tipo II/genética , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Membro Posterior/embriologia , Botões de Extremidades/citologia , Fatores de Transcrição Box Pareados/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição SOX9/antagonistas & inibidores , Fatores de Transcrição SOX9/genéticaRESUMO
P63 null mice have no or truncated limbs and mutations in human p63 cause several skeletal syndromes that also show limb and digit abnormalities, suggesting its essential role in bone development. In the current study, we investigated the effect of ATRA on chondrogenesis using mesenchymal cells from rat hind limb bud and further examined the mRNA and protein expression of Sox9 and Col2a1 and p63 in rat hind limb bud cells. Limb buds were isolated from embryos from euthanized female rats. Growth of hind limb bud mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays. Formation of cartilage nodules was examined by Alcian blue-nuclear fast red staining. The expression of Sox9, Col2al and p63 was determined by Real-time RT-PCR and immunoblotting assays, respectively. Our MTT assays revealed that ATRA at 1 and 10µM significantly suppressed the growth of mesenchymal cells from rat hind limb bud at 24 and 48h (P<0.01 vs. controls). Alcian blue staining further showed that ATRA caused a significant dose-dependent reduction in the area of cartilage nodules (P<0.05 in all vs. controls). At 1µM ATRA, the area of cartilage nodules from hind limb bud cells was reduced to 0.05±0.03mm from 0.15±0.01mm in controls. Real-time RT-PCR assays further indicated that 1 and 10µM ATRA markedly reduced the mRNA expression of Sox9, Col2al and p63 in hind limb bud cells (P<0.05 in all vs. controls). In addition, ATRA time-dependently inhibits the mRNA expression of p63, Sox9 and Col2al. Western blotting assays additionally showed that ATRA dose-dependently reduced the expression of Sox9, Col2al and p63 (P<0.05 in all vs. controls). Together, our results suggest that ATRA suppresses chondrogenesis by modulating the expression of Sox9, Col2al and p63 in primary hind limb bud mesenchymal cells.