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BACKGROUND: The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine. Stem cells can self-organise into microsized organ units, partially modelling tissue function and regeneration. Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases. However, the lack of vasculature limits the utility of dental pulp organoids. AIM: To improve survival and aid in recovery after stem cell transplantation, we demonstrated the three-dimensional (3D) self-assembly of adult stem cell-human dental pulp stem cells (hDPSCs) and endothelial cells (ECs) into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium (CM). METHODS: During culture, primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM. The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids. The biological characteristics of the organoids were analysed, and the regulatory pathways associated with angiogenesis were studied. RESULTS: The combination of these two agents resulted in prevascularized human dental pulp organoids (Vorganoids) that more closely resembled dental pulp tissue in terms of morphology and function. Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis. The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids. CONCLUSION: In this innovative study, we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration, facilitating the development of clinical treatment strategies.
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Background: Low back pain or sciatic pain because of lumbar intervertebral disc herniation (LDH) is caused by mechanical compression and/or an inflammatory component on the nerve root. However, it is difficult to define to what extent each component contributes to the pain. This study attempted to explore the effects of macrophage polarization on clinical symptoms in patients experiencing LDH after surgery, and investigated the association between macrophage cell percentages and clinical efficacy. Methods: This study retrospectively harvested nucleus pulposus (NP) tissue samples from 117 patients. Clinical symptoms and efficacy using the visual analog scale (VAS) and Oswestry Disability Index (ODI) were evaluated at different time points preoperatively and postoperatively. CD68, CCR7, CD163, and CD206 were selected as macrophage phenotypic markers. Results: Seventy-six samples showed positive expression of macrophage markers in NP samples of patients with LDH, whereas 41 patients displayed negative results. No significant differences were detected between the two groups, involvement of several demographic data, and preoperative clinical findings. With respect to the macrophage-positive group, no significant correlation was detected between the positive rate of the four markers and the VAS score or ODI after surgery. However, patients with NP samples positive for CD68 and CCR7 expression showed significantly lower VAS scores 1 week after surgery compared with those in the negative group. Moreover, the improvement in VAS score showed a strong positive correlation with CD68- and CCR7-positive cell percentages. Conclusions: Our results indicated that pro-inflammatory M1 macrophages may be associated with the reduction of chronic pain after surgery. Therefore, these findings contribute to better personalized pharmacological interventions for patients with LDH, considering the heterogeneity of pain.
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Macrophage infiltration and polarization during lumbar intervertebral disc herniation (LDH) have attracted increased attention but their role remains unclear. To explore macrophage polarization in herniated nucleus pulposus (NP) tissue of patients with LDH and investigate the association between cell frequency and different clinical characteristics or symptoms, we conducted a retrospective study by analyzing NP tissue samples from 79 patients. Clinical features and symptoms, using the visual analog scale (VAS) and Oswestry disability index (ODI), were collected. The macrophage markers CD68, CCR7, CD163, and CD206; pro-inflammatory cytokine TNF-α; and anti-inflammatory factor IL-4 were analyzed by immunohistochemistry. The frequency of polarized macrophages and positivity rate of pro- and anti-inflammatory cytokines showed significant differences in some of clinical characteristics. Specifically, higher CCR7+ and TNF-α + proportions were identified in the high-intensity zone (HIZ) and the type of extrusion and sequestration NP tissue than in non-HIZ and protrude NP tissue. Higher CD206+ and IL-4+ proportion were detected in Modic changes. However, no differences in gender, age, smoking status, Pfirrmann grade, analgesic use, leg pain duration, and segments were found between groups. CD68+ , CCR7+ , and CD206+ cell proportions, and TNF-α and IL-4 showed positive associations with VAS scores preoperation. Associations between ODI and the macrophages markers were weak/insignificant. Our results indicated that macrophage polarization or macrophage-like cells contribute to LDH pathological features. Macrophage populations displaying significant associations with VAS score reflected continuous M1/M2 transition contributing to pain during LDH. These findings may contribute to enhanced/personalized pharmacological interventions for patients with LDH considering pain heterogeneity.
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Degeneração do Disco Intervertebral , Deslocamento do Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Humanos , Deslocamento do Disco Intervertebral/patologia , Estudos Retrospectivos , Núcleo Pulposo/patologia , Interleucina-4/metabolismo , Receptores CCR7/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Dor , Vértebras Lombares/cirurgia , Macrófagos/metabolismo , Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/patologiaRESUMO
OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.
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Doenças Ósseas Metabólicas , Reabsorção Óssea , Osteoporose Pós-Menopausa , Osteoporose , Animais , Diferenciação Celular , Quimiocina CCL2 , Quimiocina CCL7 , Citocinas/metabolismo , Feminino , Humanos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/farmacologia , Indazóis , Camundongos , NF-kappa B/metabolismo , Osteoclastos , Osteogênese , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Ovariectomia , Propionatos , RNA Mensageiro/metabolismo , Transdução de Sinais , Microtomografia por Raio-XRESUMO
This study aims to investigate the effect of enriched plasma cells on the production of inflammatory cytokines and development of osteoarthritis (OA) in mice with B-cell-specific conditional deletion of the tuberous sclerosis 1 gene (TSC1). OA was induced by destabilization of the medial meniscus (DMM) in mice with TSC1 disruption in B cells (CD19-TSC1) and in littermate control mice (CON). The effects of DMM and incidence of OA were evaluated histologically, mRNA levels of inflammatory cytokines were detected by polymerase chain reaction, and serum cytokine levels were detected by enzyme-linked immunosorbent assay. Deletion of TSC1 caused constitutive activation of mechanistic target of rapamycin complex 1 mTORC1 in B cells. CON mice subjected to DMM exhibited a severe OA phenotype with increased inflammatory cytokines in B cells, serum, and the synovial membrane. Importantly, inflammatory cytokine production was also increased in B cells from the spleen of CD19-TSC1 conditional KO mice, but the OA phenotype was significantly elevated in conditional KO mice after DMM surgery compared with CON mice, as indicated by more severe articular cartilage destruction, increased protein expression of matrix metalloproteinase-13 and mRNA of type X collagen in the articular cartilage, decreased mRNA expression of type II collagen in the articular cartilage, and increased inflammatory cytokines in serum and the synovial membrane. The results demonstrate that inflammatory cytokine synthesis by B cells was enriched in CD19-TSC1 conditional KO mice, and this enhanced synthesis of inflammatory cytokines accelerated the incidence of OA.
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Linfócitos B/imunologia , Cartilagem Articular/patologia , Osteoartrite/imunologia , Plasmócitos/imunologia , Membrana Sinovial/imunologia , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Antígenos CD19 , Células Cultivadas , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Modelos Animais de Doenças , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , Proteína 1 do Complexo Esclerose Tuberosa/genéticaRESUMO
OBJECTIVE: To investigate the effect of estradiol on the expression of antioxidant enzymes in osteoblasts and its role in postmenopausal osteoporosis. METHODS: Rat models of osteoporosis established by ovariectomy were treated with estradiol for 3 months, and the changes in serum levels of reactive oxygen species (H2O2) and antioxidant enzymes (γ -GCS, GSH-ST and GSH-px) were detected. The effects of estradiol on the expression of γ -GCS mRNA and protein in osteoblast-like cells MC3T3-E1, MG63 and OB were examined with PCR and Western blotting. Using a mRNA microarray, we analyzed the changes in the expressions of 84 antioxidant enzymes in the osteoblast cell line MC3T3-E1 following estradiol treatment, and the enzymes with significant changes were verified by PCR. CCK-8 kit was used to evaluate the effect of estradiol and antioxidant NAC on the proliferation of MC3T3-E1 cells. RESULTS: Rat models of osteoporosis were successfully established with ovariectomy. The osteoporotic rats showed significantly increased serum level of reactive oxygen species (H2O2) and decreased levels of antioxidant enzymes. Estrogen treatment of the osteoporotic rats obviously reversed the phenotype of osteoporosis, lowered serum level of reactive oxygen species, and increased the level of γ -GCS. In MC3T3-E1, MG63 and OB cells, estradiol treatment significantly upregulated the expression levels of γ -GCS mRNA and protein. In MC3T3-E1 cells treated with estrogen, the mRNA chip identified 6 upregulated antioxidant enzymes (Gpx6, Gstk1, Nos2, Prdx2, Ngb and Ccs), and the results of PCR verified that estradiol upregulated Ccs and Ngb mRNAs in MC3T3-E1, MG63 and OB cells. Estradiol and antioxidant NAC obviously promoted the proliferation of MC3T3-E1 cells. CONCLUSION: Estradiol significantly increases the expression of antioxidase γ -Gcs, Ccs and Ngb in osteoblasts in vitro. Postmenopausal osteoporosis is closely related with the increase of reactive oxygen species and the decrease of antioxidant levels. In osteoblasts, estrogen deficiency may increase the level of reactive oxygen species, decrease the level of antioxidant enzymes, activate the oxidative stress cascade, and consequently inhibit the proliferation of osteoblasts to aggravate the condition of osteoporosis.
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Antioxidantes/metabolismo , Estradiol/farmacologia , Osteoblastos/enzimologia , Osteoporose/enzimologia , Estresse Oxidativo , Células 3T3 , Animais , Diferenciação Celular , Feminino , Peróxido de Hidrogênio/metabolismo , Camundongos , Osteoblastos/efeitos dos fármacos , Ovariectomia , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The small GTPase ras homolog enriched in brain (Rheb) is a downstream target of tuberous sclerosis complex 1/2 (TSC1/2) and an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), the emerging essential modulator of M1/M2 balance in macrophages. However, the role and regulatory mechanisms of Rheb in macrophage polarization and allergic asthma are not known. In the present study, we utilized a mouse model with myeloid cell-specific deletion of the Rheb1 gene and an ovalbumin (OVA)-induced allergic asthma model to investigate the role of Rheb1 in allergic asthma and macrophage polarization. Increased activity of Rheb1 and mTORC1 was observed in myeloid cells of C57BL/6 mice with OVA-induced asthma. In an OVA-induced asthma model, Rheb1-KO mice demonstrated a more serious inflammatory response, more mucus production, enhanced airway hyper-responsiveness, and greater eosinophil numbers in bronchoalveolar lavage fluid (BALF). They also showed increased numbers of bone marrow macrophages and BALF myeloid cells, elevated M2 polarization and reduced M1 polarization of macrophages. Thus, we have established that Rheb1 is critical for the polarization of macrophages and inhibition of allergic asthma. Deletion of Rheb1 enhances M2 polarization but decreases M1 polarization in alveolar macrophages, leading to the aggravation of OVA-induced allergic asthma.
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Deleção de Genes , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Ovalbumina/imunologia , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Alérgenos/imunologia , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Hipersensibilidade/patologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Transdução de Sinais , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro. METHODS: HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy. RESULTS: Compared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm. CONCLUSION: DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.
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Aflatoxina B1/farmacologia , Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Neoplasias Hepáticas/patologia , Ciclo Celular , Complexo de Golgi , Células Hep G2 , Humanos , Mitocôndrias , Invasividade NeoplásicaRESUMO
The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers, including colorectal cancer (CRC). DEPTOR is an mTOR inhibitor whose expression is negatively regulated by mTOR. However, the role of DEPTOR in the development of CRC is not known. The aim of this study was to investigate the expression of DEPTOR and mTORC1 activity (P-S6) in a subset of CRC patients and determine their relation to tumor differentiation, invasion, nodal metastasis and disease-free survival. Here, Immunohistochemical expression of P-S6 (S235/236) and DEPTOR were evaluated in 1.5 mm tumor cores from 90 CRC patients and in 90 samples of adjacent normal mucosa by tissue microarray. The expression of P-S6 (S235/236) was upregulated in CRC, with the positive rate of P-S6 (S235/236) in CRC (63.3%) significantly higher than that in control tissues (36.7%, 30%) (p<0.05). P-S6 (S235/236) also correlated with high tumor histologic grade (p=0.002), and positive nodal metastasis (p=0.002). In contrast, the expression level of DEPTOR was correlated with low tumor histological grade (p=0.006), and negative nodal metastasis (p=0.001). Interestingly, P-S6 (S235/236) expression showed a significant negative association with the expression of DEPTOR in CRC (p=0.011, R= -0.279). However, upregulation of P-S6 (S235/236) (p=0.693) and downregulation of DEPTOR (p=0.331) in CRC were not significantly associated with overall survival. Thus, we conclude that expression of DEPTOR negatively correlates with mTORC1 activity and tumor progression in CRC. DEPTOR is a potential marker for prognostic evaluation and a target for the treatment of CRC.
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Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular/genética , Neoplasias Colorretais/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Regulação para Baixo/genética , Feminino , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Prognóstico , Regulação para Cima/genética , Adulto JovemRESUMO
AIM: To investigate the effect of endogenous n-3 polyunsaturated fatty acids (PUFAs) on bone marrow adipogenesis under osteoporosis conditions. METHODS: A mouse osteoporosis model overexpressing the FAT1 gene from Caenorhabditis elegans and converting n-6 PUFAs to n-3 PUFAs endogenously was used. RESULTS: The mice presented significantly lower bone marrow adiposity (adipocyte volume/tissue volume, mean adipocyte number) but increased the bone parameters (bone mineral density, bone mineral content, bone volume/total volume) in the distal femoral metaphysis. CONCLUSION: Endogenous n-3 PUFAs protect bone marrow adipogenesis, which provides a novel drug target.
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Adipogenia , Medula Óssea/metabolismo , Caderinas/fisiologia , Ácidos Graxos Ômega-3/fisiologia , Osteoporose/prevenção & controle , Ovariectomia , Adiposidade , Animais , Caderinas/genética , Subunidade alfa 1 de Fator de Ligação ao Core/análise , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , PPAR gama/análiseRESUMO
BACKGROUND: Osteoporosis is accompanied by an increase in bone marrow adipose tissue. Bone marrow adipogenesis has emerged as a therapeutic target for prevention of bone loss. Amino-bisphosphonates have been widely used for treatment of osteoporosis, but the mechanism through which amino-bisphosphonates inhibit osteoporosis remains unclear. The purpose of this study is to investigate the effects of bisphosphonates on bone marrow adipogenesis and the pro-osteoclastic factors produced by adipocytes in bone marrow microenvironment. MATERIALS AND METHODS: Human mesenchymal stem cells were obtained and purified from six volunteer donors. Each sample of cells was treated by increasing concentrations of risedronate with or without adipogenic induction for 14 d, and then droplets of the differentiated adipocytes were analyzed. The level of receptor activator of nuclear factor-κB ligand and osteoprotegerin, as well as pro-osteoclastic inflammatory factors interleukin-1, interleukin-6, and tumor necrosis factor α produced by adipocytes were evaluated by Western blot and ELISA assay. Moreover, the effect of risedronate on the activity of mammalian target of rapamycin complex 1, a key Ser/Thr kinase for initiation of adipocyte differentiation, was investigated. RESULTS: Risedronate not only dose-dependently inhibited the bone marrow adipogenesis from human mesenchymal stem cells but also suppressed receptor activator of nuclear factor-κB ligand, not osteoprotegerin, expression in differentiated adipocytes, as well as pro-osteoclastic inflammatory factors. Furthermore, the activity of mammalian target of rapamycin complex 1 was suppressed by risedronate. CONCLUSION: Our findings that risedronate influences the crosstalk between bone marrow adipocyte-osteoclast represent a novel mechanism for the anti-osteoporotic effects of risedronate.
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Adipogenia/efeitos dos fármacos , Conservadores da Densidade Óssea/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Ácido Etidrônico/análogos & derivados , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoprotegerina/análise , Ligante RANK/análise , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ácido Etidrônico/farmacologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Células-Tronco Mesenquimais/citologia , Complexos Multiproteicos/antagonistas & inibidores , Osteoclastos/citologia , Ácido Risedrônico , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Osteoarthritis (OA) is a gradually progressive degenerative disease characterized by gradual inflammatory loss of articular cartilage caused by increased proteolytic catabolism, mediated by interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), matrix metalloproteinase (MMPs), aggrecanases and other proteinases, and reduced anabolism of cartilage components, contributed by interleukin-4 (IL-4), interleukin-10 (IL-10), insulin-like growth factor 1 (IGF-1), transforming growth factor ß (TGF-ß), and bone morphogenetic proteins (BMPs). Substantial studies showed n-3 polyunsaturated fatty acids (n-3 PUFAs) exhibit a powerful anti-inflammatory effects in and ex vivo through reducing the production of IL-1 and TNF-α and increasing the expression of IL-4, IL-10, TGF-ß and IGF-1 in OA. Meanwhile, more convincing results are observed in the fat-1 transgenic mice, which are exogenously inserted in a fat-1 gene from Caenorhabditis elegans, which can endogenously convert n-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. Taken together, it has long been realized that dietary supplementation with fish oils that are plentiful of n-3 PUFAs can bring benefits in the treatment of osteoarthritis. Previously two phase I human studies based on in vitro transfer of the cDNA via lentivirus to arthritic joints have confirmed its feasibility and safety in human subjects. Consequently, we hypothesis that directly infect the chondrocytes and synoviocytes with lentivirus carrying the fat-1 gene could be a well therapeutic strategy for OA in humans.
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Caderinas/genética , Terapia Genética , Lentivirus/genética , Osteoartrite/prevenção & controle , Humanos , Osteoartrite/genéticaRESUMO
OBJECTIVE: To evaluate the effect of metformin on the apoptosis of renal cell carcinoma (RCC) cells in vitro and its mechanisms. METHODS: Fluorescent microscopy and flow cytometry were used to examine the changes in the apoptosis of 786-O cells after metformin treatment. The possible signaling molecules involved in this process were analyzed by immunoblot analysis of AMP-activated protein kinase (AMPK) signaling and caspase 9. RESULTS: Metformin induced apoptosis and caspase 9 activation in 786-O cells in low-serum medium but not in normal-serum medium. Metformin also induced AMPK activation in 786-O cells, but this activation was not associated with the cell proliferation inhibition or apoptosis-inducing effect of metformin. CONCLUSION: Metformin can induce apoptosis of RCC cells in vitro, suggesting its potential as a therapeutic agent for RCC.
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Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Metformina/farmacologia , Apoptose/efeitos dos fármacos , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , HumanosRESUMO
It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP-activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1-50 mM of metformin in a dose- and time-dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666-1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time-dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (T37/46) and S6 (S235/236) in C666-1 cells. In a summary, metformin prevents proliferation of C666-1 cells by down-regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK-mediated inhibition of mTORC1 signaling may be involved in this process.
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Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Metformina/farmacologia , Neoplasias Nasofaríngeas/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Fatores de TempoRESUMO
Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.
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Metformina/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Osteoporose/prevenção & controle , Ovariectomia , Ligante RANK/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Crânio/citologia , Tíbia/metabolismoRESUMO
OBJECTIVE: To investigate the effects of inhibiting phospholipase C gamma1 signaling pathway on the apoptosis of human colorectal carcinoma cells. METHODS: SW620 cells were treated with U73122 in vitro to inhibit the phospholipase C gamma1 signalling pathway and examined under light microscope and transmission electron microscope for analyzing changes in apoptotic behavior of the cells. MTT assay was used to evaluate the cell killing effects, and the percentage of apoptotic cells analyzed using flow cytometry. RESULTS: After inhibition of the phospholipase C gamma1 signaling pathway by U73122, SW620 cells exhibited obvious apoptotic morphology, the viable cells decreased dramatically, and the percentage of apoptotic cells rose to above 50%. CONCLUSION: Inhibition of phospholipase C gamma1 signaling pathway can induce apoptosis of human colorectal carcinoma cells.
Assuntos
Apoptose/fisiologia , Neoplasias Colorretais/patologia , Estrenos/farmacologia , Fosfolipase C gama/antagonistas & inibidores , Pirrolidinonas/farmacologia , Transdução de Sinais , Humanos , Inibidores de Fosfodiesterase/farmacologia , Células Tumorais CultivadasRESUMO
It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Reabsorção Óssea , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Primers do DNA/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Citometria de Fluxo , Células HeLa , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Humanos , Peróxido de Hidrogênio/farmacologia , Ligantes , Camundongos , Osteoclastos/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Ligante RANK , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Regulação para CimaRESUMO
Many anticarcinogenic drugs kill tumour cells by inducing apoptosis. We examined the effects of hydrogen peroxide (H(2)O(2)) on arsenic trioxide (As(2)O(3))-induced cell killing. Low concentrations of H(2)O(2) (200 micromol/l) inhibited the ability of As(2)O(3) to induce apoptosis in the Burkitt's lymphoma cell line Raji. H(2)O(2) altered the form of cell death from apoptosis to pyknosis/necrosis and also lowered the degree of cell killing by As(2)O(3). H(2)O(2) was capable of preventing caspase-3 activation induced by As(2)O(3) in Raji cells. Incubation of cells with a phosphoinositide-3 kinase (PI-3K) inhibitor, wortmannin (100 nmol/l), blocked the effects of H(2)O(2) on As(2)O(3)-induced caspase-3 activation. In addition, the PI-3K inhibitor partially blocked the effects of H(2)O(2) on up-regulation of Bcl-2 and Bcl-X(L) protein expression, down-regulation of Bax protein expression, and phosphorylation of Bcl-2 and IkappaBalpha. This investigation demonstrated for the first time that low concentrations of H(2)O(2) provide protection against the in vivo of As(2)O(3)-induced apoptosis. PI-3K plays a crucial role in enhancing cell survival during H(2)O(2), inhibiting As(2)O(3)-induced apoptosis in the Burkitt's lymphoma cells. As(2)O(3)-induced cancer cell apoptosis may be enhanced by certain antioxidants in the treatment protocol.
Assuntos
Antineoplásicos/uso terapêutico , Arsenicais/uso terapêutico , Linfoma de Burkitt/enzimologia , Peróxido de Hidrogênio/metabolismo , Óxidos/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Androstadienos/farmacologia , Apoptose , Trióxido de Arsênio , Linfoma de Burkitt/tratamento farmacológico , Caspase 3 , Caspases/metabolismo , Morte Celular , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-bcl-2/genética , Wortmanina , Proteína bcl-XRESUMO
OBJECTIVE: To examine the effects of hydrogen peroxide (H2O2) on arsenic trioxide (As2O3)-induced apoptosis of human Burkitt lymphoma cells and evaluate the value of fluorescence microscope in analyzing cellular apoptosis. METHODS: As2O3 was used to induce apoptosis in human Burkitt lymphoma cells BJAB, and the cellular changes were analyzed quantitatively using transmission electron microscope and fluorescence microscope after staining with Hoechst 33342/ propidium iodide (PI). RESULTS: Under fluorescence microscope and electron microscope, BJAB cells displayed chromatin aggregation, nuclear margination and fragmentation in response to As2O3 treatment. In the presence of relatively low levels of H2O2 (200 micromol/L), the cell death resulting from apoptosis was inhibited with pyknosis and necrosis becoming the major pathway, while As2O3 failed to induce cell apoptosis. The cells showed none of the typical markers of apoptosis nor any characteristic necrotic changes, and the nuclei exhibited condensation instead of swelling. In addition, the rate of cell death induced by As2O3 was decreased in the presence of H2O2. CONCLUSIONS: Low levels of H2O2 (200 micromol/L) inhibits As2O3-induced (at clinically achievable concentrations) apoptosis of the human malignant lymphoma cells. Fluorescence microscopy makes possible quantitative analysis of apoptosis, capable of distinguishing not only cell apoptosis from necrosis, but also membrane-intact from membrane-permeable apoptotic cells.
Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/antagonistas & inibidores , Linfoma de Burkitt/tratamento farmacológico , Peróxido de Hidrogênio/farmacologia , Óxidos/antagonistas & inibidores , Trióxido de Arsênio , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura , Microscopia de FluorescênciaRESUMO
Signaling pathways involved in oxidative stress-induced inhibition of osteoblast differentiation are not known. We showed in this report that H(2)O(2) (0.1-0.2mM)-induced oxidative stress suppressed the osteoblastic differentiation process of primary rabbit bone marrow stromal cells (BMSC) and calvarial osteoblasts, manifested by a reduction of differentiation markers including alkaline phosphatase (ALP), type I collagen, colony-forming unit-osteoprogenitor (CFU-O) formation, and nuclear phosphorylation of Runx2. H(2)O(2) treatment stimulated phospholipase C-gamma1 (PLC-gamma1), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-kappaB signaling but inhibited p38 mitogen-activated protein kinase (MAPK) activation. In the presence of 20microM PD98059 or 50microM caffeic acid phenethyl ester (CAPE), specific inhibitor for ERKs or NF-kappaB, respectively, could significantly reverse the decrease of above-mentioned osteoblastic differentiation markers elicited by H(2)O(2) (0.1mM). Furthermore, PD98059 also suppressed H(2)O(2)-stimulated NF-kappaB signaling in this process. These data suggest that ERK and ERK-dependent NF-kappaB activation is required for oxidative stress-induced inhibition of osteoblastic differentiation in rabbit BMSC and calvarial osteoblasts.