RESUMO
Recently, there has been an increasing interest in finding new approaches to manage oral wound healing. Although resveratrol (RSV) exhibited many biological properties, such as antioxidant and anti-inflammatory activities, its use as a drug is limited by unfavorable bioavailability. This study aimed to investigate a series of RSV derivatives (1a-j) with better pharmacokinetic profiles. At first, their cytocompatibility at different concentrations was tested on gingival fibroblasts (HGFs). Among them, derivatives 1d and 1h significantly increased cell viability compared to the reference compound RSV. Thus, 1d and 1h were investigated for cytotoxicity, proliferation, and gene expression in HGFs, endothelial cells (HUVECs), and oral osteoblasts (HOBs), which are the main cells involved in oral wound healing. For HUVECs and HGFs, the morphology was also evaluated, while for HOBs ALP and mineralization were observed. The results showed that both 1d and 1h did not exert negative effects on cell viability, and at a lower concentration (5 µM) both even significantly enhanced the proliferative rate, compared to RSV. The morphology observations pointed out that the density of HUVECs and HGFs was promoted by 1d and 1h (5 µM) and mineralization was promoted in HOBs. Moreover, 1d and 1h (5 µM) induced a higher eNOS mRNA level in HUVECs, higher COL1 mRNA in HGFs, and higher OCN in HOBs, compared to RSV. The appreciable physicochemical properties and good enzymatic and chemical stability of 1d and 1h, along with their promising biological properties, provide the scientific basis for further studies leading to the development of RSV-based agents useful in oral tissue repair.
Assuntos
Células Endoteliais , Fibroblastos , Resveratrol/farmacologia , Células Cultivadas , Fibroblastos/metabolismo , Cicatrização , RNA Mensageiro/metabolismoRESUMO
Snail slime (SS) is a viscous secretion obtained from different snail species. SS composition is variable according to factors such as the extraction method. Even if several papers have been published regarding this topic, the molecular mechanisms at the base of SS biological effects remain unexplored. Thus, the aim of this study is to evaluate the capability of SS, extracted with the cruelty-free Muller method, to promote viability and angiogenesis processes and, in parallel, to counteract inflammation occurrence on skin cell populations. SS was administered to keratinocytes, macrophages and fibroblasts, then cell viability, through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, cytotoxicity by lactate dehydrogenase (LDH) assay, morphology by haematoxylin-eosin staining, gene and protein expression through real-time polymerase chain reaction (PCR) and Western blot, cell cycle phases by flow cytometry, and collagen secretion using an enzyme-linked immunosorbent assay (ELISA) test, were measured. Our results evidence SS capability to promote fibroblast viability and to trigger recovery mechanisms by activating the Erk protein. Moreover, an appreciable anti-inflammatory effect due to the significant reduction in cyclooxygenase-2 expression, and a positive modulation of new blood vessel formation demonstrated by increased Angiopoietin 1 gene expression and a higher matrix deposition (evidenced by the augmented amount of released collagen I) can be identified. This evidence led us to assume that the Muller method extracted-SS represents a valuable and promising natural product suitable for cosmetic and skin care formulations.
Assuntos
Colágeno Tipo I , Colágeno , Animais , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Caramujos , Inflamação/metabolismo , Fibroblastos/metabolismo , Sobrevivência CelularRESUMO
Nitric oxide (NO) is a key messenger in physiological and pathological processes in mammals. An excessive NO production is associated with pathological conditions underlying the inflammation response as a trigger. Among others, dental pulp inflammation results from the invasion of dentin by pathogenic bacteria. Vital functions of pulp mesenchymal stem cells (DPSCs, dental pulp stem cells), such as mineralization, might be affected by the inducible NOS (iNOS) upregulation. In this context, the iNOS selective inhibition can be considered an innovative therapeutic strategy to counteract inflammation and to promote the regeneration of the dentin-pulp complex. The present work aims at evaluating two acetamidines structurally related to the selective iNOS inhibitor 1400W, namely CM544 and FAB1020, in a model of LPS-stimulated primary DPSCs. Our data reveal that CM544 and even more FAB1020 are promising anti-inflammatory compounds, decreasing IL-6 secretion by enhancing CD73 expression-levels, a protein involved in innate immunity processes and thus confirming an immunomodulatory role of DPSCs. In parallel, cell mineralization potential is retained in the presence of compounds as well as VEGF secretion, and thus their angiogenetic potential. Data presented lay the ground for further investigation on the anti-inflammatory potential of acetamidines selectively targeting iNOS in a clinical context.
Assuntos
Inflamação , Óxido Nítrico Sintase Tipo II , Óxido Nítrico , Células-Tronco , Humanos , Amidinas , Polpa Dentária/citologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Células-Tronco/citologia , Calcificação FisiológicaRESUMO
Human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms IX and XII are overexpressed in solid hypoxic tumors, and they are considered as prognostic tools and therapeutic targets for cancer. Based on a molecular simplification of the well-known coumarin scaffold, we developed a new series of derivatives of the pyran-2-one core. The new compounds are endowed with potent and selective inhibitory activity against the tumor-related hCA isoforms IX and XII, in the low nanomolar range, whereas they are inactive against the two cytosolic off-targets hCA I and II. The compounds exhibiting the best hCA inhibition were further investigated against the breast adenocarcinoma cell line (MCF7) in hypoxic conditions, evaluating their ability to eventually synergize with doxorubicin. The compounds' biocompatibility on healthy cells was also tested and confirmed on Human Gingival Fibroblasts (HGFs). Furthermore, the possible binding mode of all compounds to the active site of the tumor-associated human CA IX was investigated by computational techniques which predicted the binding conformations and the persistency of binding poses within the active site of the enzyme, furnishing relevant data for the design of tight binding inhibitors.
Assuntos
Anidrases Carbônicas , Neoplasias , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/metabolismo , Humanos , Estrutura Molecular , Neoplasias/metabolismo , Pironas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Eg5 is a kinesin essential in bipolar spindle formation, overexpressed in tumours, thus representing a new target in cancer therapy. We aimed at evaluating the anti-cancer activity of Eg5 thiadiazoline inhibitors 2 and 41 on gastric adenocarcinoma cells (AGS), focusing on the modulation of angiogenic signalling. Docking studies confirmed a similar interaction with Eg5 to that of the parent compound K858. Thiadiazolines were also tested in combination with Hesperidin (HSD). Cell cycle analysis reveals a reduction of G1 and S phase percentages when 41 is administered as well as HSD in combination with K858. Western blot reveals Eg5 inhibitors capability to reduce PI3K, p-AKT/Akt and p-Erk/Erk expressions; p-Akt/Akt ratio is even more decreased in HSD+2 sample than the p-Erk/Erk ratio in HSD+41 or K858. VEGF expression is reduced when HSD+2 and HSD+41 are administered with respect to compounds alone, after 72 h. ANGPT2 gene expression increases in cells treated with 41 and HSD+2 compared to K858. The wound-healing assay highlights a reduction in the cut in HSD+2 sample compared to 2 and HSD. Thus, Eg5 inhibitors appear to modulate angiogenic signalling by controlling VEGF activity even better if combined with HSD. Overall, Eg5 inhibitors can represent a promising starting point to develop innovative anti-cancer strategies.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Cinesinas/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/patologia , Regulação Alostérica , Ciclo Celular , Proliferação de Células , Humanos , Técnicas In Vitro , Neovascularização Patológica/patologia , Neoplasias Gástricas/patologia , Células Tumorais CultivadasRESUMO
Gliomas are malignant brain tumors characterized by rapid spread and growth into neighboring tissues and graded I-IV by the World Health Organization. Glioblastoma is the fastest growing and most devastating IV glioma. The aim of this paper is to evaluate the biological effects of two potent and selective Monoamine Oxidase B (MAO-B) inhibitors, Cmp3 and Cmp5, in C6 glioma cells and in CTX/TNA2 astrocytes in terms of cell proliferation, apoptosis occurrence, inflammatory events and cell migration. These compounds decrease C6 glioma cells viability sparing normal astrocytes. Cell cycle analysis, the Mitochondrial Membrane Potential (MMP) and Reactive Oxygen Species (ROS) production were detected, revealing that Cmp3 and Cmp5 induce a G1 or G2/M cell cycle arrest, as well as a MMP depolarization and an overproduction of ROS; moreover, they inhibit the expression level of inducible nitric oxide synthase 2, thus contributing to fatal drug-induced oxidative stress. Cmp5 notably reduces glioma cell migration via down-regulating Matrix Metalloproteinases 2 and 9. This study demonstrated that our novel MAO-B inhibitors increase the oxidative stress level resulting in a cell cycle arrest and markedly reduces glioma cells migration thus reinforcing the hypothesis of a critical role-played by MAO-B in mediating oncogenesis in high-grade gliomas.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores da Monoaminoxidase/uso terapêutico , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Monoaminoxidase/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The Kinesins are proteins involved in several biological processes such as mitosis, intracellular transport, and microtubule movement. The mitotic process is allowed by the correct formation of the mitotic spindle which consists of microtubules originating from the spindle poles. In recent years, kinesin Eg5 inhibitors were studied as new chemotherapeutic drugs, due to the lack of side effects and resistance mechanisms. The aim of this work was to investigate the molecular signaling underlying the administration of novel kinesis Eg5 inhibitors in an in vitro model of gastric adenocarcinoma. Data obtained from analogues of K858 led us to select compounds 2 and 41, due to their lower IC50 values. The ability of kinesin inhibitors to induce apoptosis was investigated by evaluating Bax and Caspase-3 protein expression, evidencing that compound 41 and K858 markedly raise Bax expression, while only compounds 2 and 41 co-administrated with K858 trigger Caspase-3 activation. The inhibition of mitotic spindle was measured by ß-tubulin immunofluorescence analysis revealing monopolar spindles formation in gastric cancer cells treated with compounds 2, 41, and K858. Nitric Oxide Synthase (NOS-2) and Matrix Metalloproteinase 9 (MMP-9) expression levels were measured finding a NOS-2-mediated downregulation of MMP-9 when compound 41 and K858 are co-administered. However, this is in contrast to what was reported by migration assay in which both novel compounds and K858 in monotherapy markedly reduce cell migration. This work remarks the importance of understanding and exploring the biological effects of different novel Eg5 kinesin inhibitors administered in monotherapy and in combination with K858 as potential strategy to counteract gastric cancer.
Assuntos
Antineoplásicos/uso terapêutico , Cinesinas/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Cinesinas/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Modelos Moleculares , Neoplasias Gástricas/patologia , Tiadiazóis/química , Tiadiazóis/farmacologia , Tubulina (Proteína)/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
OBJECTIVES: Bisphosphonates (BPs) are drugs clinically used in resorptive diseases. It was already proved that some clinically relevant BPs can inhibit a class of enzymes called matrix metalloproteinases (MMPs), required during tissue remodelling. Combining the arylsulfonamide function with the bisphosphonic group, several compounds were synthesized to obtain selective inhibitors of MMPs. The aim of the present study was to compare the effect of zoledronic acid (ZA), the most potent bisphosphonate available as therapy, with new sulfonamide containing BPs in an in vitro model of human gingival fibroblasts (HGFs). MATERIALS AND METHODS: Western blot was used to measure procollagen I, ß1 integrin MMP-8 and MMP-9, phase contrast and MTT for cell viability; L-lactate-dehydrogenase (LDH) measurement was performed for toxicity evaluation and ELISA for prostaglandin E2 (PGE2) secretion assessment. RESULTS: When compared with ZA, the treatment with the newly synthesized compounds shows increasing viability, procollagen I expression and decreased expression of ß1 integrin in HGFs. Higher levels of released LDH, PGE2 and MMP-9 expression are recorded in ZA-treated HGFs. Increased levels of MMP-8 are recorded in newly synthesized compounds-treated samples. CONCLUSIONS: These findings allowed to conclude that new tested BPs did not affect HGFs viability and adhesion, did not induce cellular toxicity, were not responsible for inflammatory event induction and could preserve the physiological matrix turnover. CLINICAL RELEVANCE: It could be hypothesized that the new molecules were better tolerated by soft tissues, resulting in lesser side effects.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Difosfonatos/farmacologia , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Imidazóis/farmacologia , Adulto , Biomarcadores/análise , Western Blotting , Conservadores da Densidade Óssea/síntese química , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Difosfonatos/síntese química , Ensaio de Imunoadsorção Enzimática , Humanos , Imidazóis/síntese química , Técnicas In Vitro , Inflamação , Dente Serotino , Ácido ZoledrônicoRESUMO
The carotid body is a neural-crest-derived organ devoted to respiratory homeostasis through sensing changes in blood oxygen levels. The sensory units are the glomeruli composed of clusters of neuronal-like (type I) cells surrounded by glial-like (type II) cells. During chronic hypoxia, the carotid body shows growth, with increasing neuronal-like cell numbers. We are interested in the signals involved in the mechanisms that underlie such response, because they are not well understood and described. Considering that, in literature, galanin is involved in neurotrophic or neuroprotective role in cell proliferation and is expressed in animal carotid body, we investigated its expression in human. Here, we have shown the expression and localisation of galanin in the human carotid body.
Assuntos
Corpo Carotídeo/química , Galanina/análise , Neurônios/química , Adulto , Idoso , Corpo Carotídeo/citologia , Corpo Carotídeo/fisiologia , Galanina/fisiologia , Humanos , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: This study aimed to check the effect of zoledronic acid (ZA) at subtoxic dose on human osteoblasts (HOs) in terms of cell viability, apoptosis occurrence, and differentiation induction. ZA belongs to the family of bisphosphonates (BPs), largely used in the clinical practice for the treatment of bone diseases, often associated with jaw osteonecrosis onset. Their pharmacological action consists in the direct block of the osteoclast-mediated bone resorption along with indirect action on osteoblasts. MATERIALS AND METHODS: HOs were treated choosing the highest limit concentration (10(-5) M) which does not induce toxic effects. Live/dead staining, flow cytometry, mitochondrial membrane potential assay, osteocalcin western blotting, gp38 RT-PCR, collagen type I, PGE2, and IL-6 ELISA assays were performed. RESULTS: Similar viability level between control and ZA-treated samples is found along with no significant increase of apoptotic and necrotic cells in ZA-treated sample. To establish if an early apoptotic pathway was triggered, Bax expression and mitochondrial membrane potential were evaluated finding a higher protein expression in control sample and a good integrity of mitochondrial membrane in both experimental points. Type I collagen secretion and alkaline phosphatase (ALP) activity appear increased in ZA-treated sample, osteocalcin expression level is reduced in ZA-treated cells, whereas no modifications of gp38 mRNA level are evidenced. No statistical differences are identified in PGE2 secretion level whereas IL-6 secretion is lower in ZA-treated HOs with respect to control ones. CONCLUSIONS: These results highlight that ZA, delaying the osteoblastic differentiation process versus the osteocytic lineage, strengthens its pharmacological activity enhancing bone density. CLINICAL RELEVANCE: The knowledge of ZA effects on osteoblasts at subtoxic dose allows to improve therapeutic protocols in order to strengthen drug pharmacological activity through a combined action on both osteoclastic and osteoblastic cells.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Difosfonatos/farmacologia , Imidazóis/farmacologia , Osteoblastos/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Western Blotting , Densidade Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Interleucina-6/metabolismo , Potencial da Membrana Mitocondrial , Reação em Cadeia da Polimerase em Tempo Real , Ácido ZoledrônicoRESUMO
OBJECTIVES: The aim of our work was to evaluate the role of nitric oxide (NO) in the in vitro response of human gingival fibroblasts (HGFs) to 1, 5, 10, and 100 µM doses of zoledronic acid (ZA), a bisphosphonate largely used in the clinical practice and for which several adverse effects are reported. MATERIALS AND METHODS: Phase contrast microscopy and live/dead staining were used to evaluate HGFs morphology; cell viability, collagen type I and interleukin 6 (IL-6) secretion were evaluated by 3-[4,5-dimethyl-thiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA) assays. Reactive oxygen species (ROS) production and mitochondrial membrane potential were evaluated by flow cytometry; NO production and NOS activity by spectrophotometric analysis; endothelial NOS (eNOS) and neuronal NOS (nNOS) expression by immunofluorescence. RESULTS: Viable fibroblasts are evidenced in control sample while floating dead cells and cells close to detachment phase in ZA-treated sample, in agreement with decreased level of collagen type I. Control sample shows higher number of viable cells respect to ZA-treated one and ROS production increases when ZA is added. Released NO in ZA-treated sample appears higher and NO overproduction is related to increased nNOS activity. IL-6 secretion level is higher in ZA-treated sample than in control one. CONCLUSIONS: Our results suggest ROS involvement in NO overproduction, due to nNOS recruitment, both at low and high doses. In turn, NO release seems to be able to trigger the inflammatory response only when high doses are administered, thus confirming the ZA cytotoxic effect on HGFs. CLINICAL RELEVANCE: The knowledge of ZA-mediated cytotoxicity mechanisms on HGFs allows to better understand drug pharmacological activity.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Difosfonatos/farmacologia , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Imidazóis/farmacologia , Óxido Nítrico/fisiologia , Adulto , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Interleucina-6/metabolismo , Ácido ZoledrônicoRESUMO
OBJECTIVE: The aim of this study was to evaluate in vitro the behavior and the biocompatibility of primary human osteoblasts (HOs) grown onto different implant surface. METHODS AND MATERIALS: HOs were cultured onto sandblasted/acid-etched (control group) and sandblasted/acid-etched followed by coating with inorganic ions (test group) experimental titanium discs. At established times, SEM analysis, LDH assay, MTT assay, and enzyme-linked immunosorbent assay for type 1 collagen, interleukin (IL)-6, and PGE2 secretion were performed. RESULTS: Both surfaces promote HOs adhesion and proliferation. After 21 days, cells on test surfaces are well spread, flattened, and attached by cellular extensions, whereas cells on control discs appear mainly elongated. Lower LDH levels and higher values of MTT assay are recorded for cells on test respect to control surfaces at each experimental time. Type 1 collagen release increases until 14 days, significantly decreasing at day 21 in cells grown on both surfaces. IL-6 and PGE2 secretion shows a peak in control group samples at day 7, whereas their levels do not significantly modify in both groups at days 14 and 21. CONCLUSION: Results indicate that the test group surface is more biocompatible, well tolerated, and suitable for supporting osteoblasts growth and proliferation.
Assuntos
Implantação Dentária Endóssea/métodos , Implantes Dentários , Osseointegração/fisiologia , Osteoblastos/citologia , Propriedades de Superfície , Fosfatase Alcalina/biossíntese , Materiais Biocompatíveis , Adesão Celular , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/biossíntese , Dinoprostona/metabolismo , Humanos , Interleucina-6/biossíntese , Titânio/químicaRESUMO
BACKGROUND: We previously demonstrated that the exposure to hyperbaric hyperoxia increased respiratory system elastance and both the "ohmic" and viscoelastic components of inspiratory resistances, probably because of increased oxygen tension toxic effects. We presently investigated the possible consequences of a single exposure to 2.5-atmospheres absolute air (hyperbarism) lasting 90 min. METHODS: We used the end-inflation occlusion method on anesthetized rats after about 15 min from previous exposure to hyperbarism. The method allows the measurements of respiratory system elastance and of the ohmic and viscoelastic components of airway resistance, which respectively depend on the Newtonian pressure dissipation due to the ohmic airway resistance to airflow and on the viscoelastic pressure dissipation caused by respiratory system tissue stress relaxation. The expressions of inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) in the lung's tissues were also investigated, together with the histological characteristics of lung tissue. Data were compared with those obtained in control animals and in previously studied animals exposed to hyperoxic hyperbarism. RESULTS: Unlike with hyperoxic hyperbarism, hyperbarism per se did not change significantly the parameters of respiratory mechanics in the control animals (respiratory system elastance and ohmic and viscoelastic resistances were 2.01 ± 0.17 vs. 1.74 ± 0.08 cm H(2)O/ml, and 0.13 ± 0.02 vs. 0.13 ± 0.03 and 0.425 ± 0.04 vs. 0.33 ± 0.03 cm H(2)O/ml s(-1) in control vs. experimental animals, respectively, none significantly different), nor did it induce evident effects on lung histology. An increment of both iNOS and eNOS expressions was documented instead (0.50 ± 0.05 vs. 0.75 ± 0.07 and 1.04 ± 0.1 and 1.4 ± 0.15, respectively). CONCLUSION: Our results indicate that, at variance with hyperoxic hyperbarism, the acute exposure to only hyperbarism does not affect either the elastic or the resistive respiratory system properties, or lung histology.
Assuntos
Oxigenoterapia Hiperbárica/métodos , Pulmão/patologia , Pulmão/fisiopatologia , Mecânica Respiratória , Resistência das Vias Respiratórias , Animais , Elasticidade , Feminino , Oxigenoterapia Hiperbárica/efeitos adversos , Pulmão/metabolismo , Masculino , Modelos Animais , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pressão , Ratos Wistar , Medição de Risco , Fatores de Tempo , ViscosidadeRESUMO
The aim of this work was to investigate the morphological structure and the expression of vascular endothelial growth factor (VEGF) after maxillary sinus augmentation through equine and porcine bone substitutes in humans. Ten patients showing edentulous posterior maxilla underwent maxillary sinus augmentation through particulate equine bone substitute and 10 patients through particulate porcine bone substitute. At the moment of implants insertion, 6 months after grafting, bone specimens were withdrawn and processed for morphological and immunohistochemical analyses. Notwithstanding the almost comparable clinical performances of both bone substitutes, histological results showed a better integration when an equine bone substitute was used compared to a porcine one. In particular, evident signs of particles resorption were observed in equine bone substitute group specimens compared to porcine ones. Immunohistochemical analysis showed a statistically significant increase of VEGF expression in equine compared to porcine bone substitute group specimens. These results showed both bone substitutes to achieve comparable clinical performance, indicating their successful use for bone regenerative procedures. However, in the same experimental time, equine group specimens showed evident resorption phenomena, whereas no or little signs of resorption were evident in the porcine group specimens. However, a more rapid and intense vascularization was achieved in equine bone substitute group, as demonstrated by immunohistochemical analysis for VEGF expression. Even if differences in vascularization significantly affect the clinical performance of a heterologous bone substitute, its ability to be resorbed is also very important in influencing long-term integration and long-term predictability of implant-prosthetic rehabilitation in regenerated sites.
Assuntos
Substitutos Ósseos/uso terapêutico , Colágeno/uso terapêutico , Levantamento do Assoalho do Seio Maxilar/métodos , Fator A de Crescimento do Endotélio Vascular/análise , Animais , Densidade Óssea/fisiologia , Regeneração Óssea/fisiologia , Reabsorção Óssea/etiologia , Substitutos Ósseos/química , Colágeno/química , Implantação Dentária Endóssea/métodos , Implantes Dentários , Feminino , Seguimentos , Cavalos , Humanos , Imuno-Histoquímica , Arcada Edêntula/patologia , Arcada Edêntula/cirurgia , Masculino , Maxila/patologia , Maxila/cirurgia , Pessoa de Meia-Idade , Osteócitos/patologia , Osteogênese/fisiologia , Piezocirurgia/métodos , SuínosRESUMO
Eg5 is a protein encoded by KIF11 gene and is primarily involved in correct mitotic cell division. It is also involved in nonmitotic processes such as polypeptide synthesis, protein transport, and angiogenesis. The scientific literature sheds light on the ubiquitous functions of KIF11 and its involvement in the onset and progression of different pathologies. This review focuses attention on two main points: (1) the correlation between Eg5 and cancer and (2) the involvement of Eg5 in noncancerous conditions. Regarding the first point, several tumors revealed an overexpression of this kinesin, thus pushing to look for new Eg5 inhibitors for clinical practice. In addition, the evaluation of Eg5 expression represents a crucial step, as its overexpression could predict a poor prognosis for cancer patients. Referring to the second point, in specific pathological conditions, the reduced activity of Eg5 can be one of the causes of pathological onset. This is the case of Alzheimer's disease (AD), in which Aß and Tau work as Eg5 inhibitors, or in acquired immune deficiency syndrome (AIDS), in which Tat-mediated Eg5 determines the loss of CD4+ T-lymphocytes. Reduced Eg5 activity, due to mutations of KIF11 gene, is also responsible for pathological conditions such as microcephaly with or without chorioretinopathy, lymphedema, or intellectual disability (MCLRI) and familial exudative vitreous retinopathy (FEVR). In conclusion, this review highlights the double impact that overexpression or loss of function of Eg5 could have in the onset and progression of different pathological situations. This emphasizes, on one hand, a possible role of Eg5 as a potential biomarker and new target in cancer and, on the other hand, the promotion of Eg5 expression/activity as a new therapeutic strategy in different noncancerous diseases.
RESUMO
Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5'-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[l,5-a]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. Our data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFß1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. Our data demonstrate that pyrazolo[l,5-a]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[l,5-a]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages.
Assuntos
5'-Nucleotidase , Inflamação , Macrófagos , Fator 2 Relacionado a NF-E2 , Animais , Humanos , Camundongos , 5'-Nucleotidase/efeitos dos fármacos , 5'-Nucleotidase/metabolismo , Anti-Inflamatórios/farmacologia , Inflamação/metabolismo , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/imunologia , Fator 2 Relacionado a NF-E2/metabolismo , Pirimidinas/farmacologia , Pirimidinonas/farmacologia , Células RAW 264.7RESUMO
Cryogels represent a valid strategy as scaffolds for tissue engineering. In order to adequately support adhesion and proliferation of anchorage-dependent cells, different polymers need to be combined within the same scaffold trying to mimic the complex features of a natural extracellular matrix (ECM). For this reason, in this work, gelatin (Gel) and chondroitin sulfate (CS), both functionalized with methacrylic groups to produce CSMA and GelMA derivatives, were selected to prepare cryogel networks. Both homopolymer and heteropolymer cryogels were produced, via radical crosslinking reactions carried out at -12 °C for 2 h. All the scaffolds were characterized for their mechanical, swelling and morphological properties, before and after autoclave sterilization. Moreover, they were evaluated for their biocompatibility and ability to support the adhesion of human gingival fibroblasts and tenocytes. GelMA-based homopolymer networks better withstood the autoclave sterilization process, compared to CSMA cryogels. Indeed, GelMA cryogels showed a decrease in stiffness of approximately 30%, whereas CSMA cryogels of approximately 80%. When GelMA and CSMA were blended in the same network, an intermediate outcome was observed. However, the hybrid scaffolds showed a general worsening of the biological performance. Indeed, despite their ability to withstand autoclave sterilization with limited modification of the mechanical and morphological properties, the hybrid cryogels exhibited poor cell adhesion and high LDH leakage. Therefore, not only do network components need to be properly selected, but also their combination and ability to withstand effective sterilization process should be carefully evaluated for the development of efficient scaffolds designed for tissue engineering purposes.
RESUMO
New dihydro-pyrrol-2-one compounds, featuring dual sulfonamide groups, were synthesized through a one-pot, three-component approach utilizing trifluoroacetic acid as a catalyst. Computational analysis using density functional theory (DFT) and condensed Fukui function explored the structure-reactivity relationship. Evaluation against human carbonic anhydrase isoforms (hCA I, II, IX, XII) revealed potent inhibition. The widely expressed cytosolic hCA I was inhibited across a range of concentrations (KI 3.9-870.9 nM). hCA II, also cytosolic, exhibited good inhibition as well. Notably, all compounds effectively inhibited tumor-associated hCA IX (KI 1.9-211.2 nM) and hCA XII (low nanomolar). Biological assessments on MCF7 cancer cells highlighted the compounds' ability, in conjunction with doxorubicin, to significantly impact tumor cell viability. These findings underscore the potential therapeutic relevance of the synthesized compounds in cancer treatment.
Assuntos
Anidrases Carbônicas , Neoplasias , Humanos , Inibidores da Anidrase Carbônica/farmacologia , Anidrase Carbônica IX , Relação Estrutura-Atividade , Anidrases Carbônicas/metabolismo , Isoformas de Proteínas , Estrutura Molecular , Antígenos de NeoplasiasRESUMO
Premature newborns are frequently exposed to hyperoxia ventilation and some literature data indicate the possibility of hyperoxia-induced myocardial damage. Since nuclear factor κB (NF-κB) is a crucial signaling molecule involved in physiological response to hyperoxia in different cell types as well as in various tissues, our attention has been focused on the role played by NF-κB pathway in response to moderate and severe hyperoxia exposure in rat neonatal heart tissue. Akt and IκBα levels, involved in NF-κB activation, along with the balance between apoptotic and survival pathways have also been investigated. Experimental design of the study has involved exposure of newborn rats to room air (controls), 60 % O2 (moderate hyperoxia), or 95 % O2 (severe hyperoxia) for the first two postnatal weeks. Morphological analysis shows a less compact tissue in rat heart exposed to moderate hyperoxia and a decreased number of nuclei in samples exposed to severe hyperoxia. A significant increase of NF-κB positive nuclei percentage and p-IκBα expression in samples exposed to 95 % hyperoxia compared to control and to 60 % hyperoxia is evidenced; in parallel, an increase of pAkt/Akt ratio in both samples exposed to 95 and 60 % hyperoxia is shown. Furthermore, a more evident cytochrome c/Apaf-1 immunocomplex and a decreased Bcl2 expression in 95 % hyperoxia-exposed sample compared to 60 % exposed one is evidenced. In conclusion, our findings suggest the involvement of the NF-κB pathway and Akt signaling in the mechanisms of myocardial hyperoxic damage in the newborns, with particular reference to the induction of oxidative stress-related apoptosis.
Assuntos
Coração , Hiperóxia/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , NF-kappa B/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Modelos Animais de Doenças , Feminino , Hiperóxia/patologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
BACKGROUND: Alzheimer's disease (AD) is a frequent form of senile dementia. Neuroglobin (Ngb) has a neuroprotective role and decreases Aß peptide levels. Ngb, promoting Akt phosphorylation, activates cell survival involving cyclic-nucleotide response element-binding protein (CREB). A new molecule (IBU-LA) was synthetized and administered to an AD rat model to counteract AD progression. OBJECTIVE: The aim of this study was to investigate the IBU-LA-mediated induction of Ngb neuroprotective and antiapoptotic activities. METHODS: Brain morphology was analyzed through Bielschowsky staining, Aß(1-40) and Ngb expression by immunohistochemistry. Akt, p-Akt, CREB and p-CREB expression was evaluated by Western blot, apoptosis through cytochrome C/Apaf 1 immunocomplex formation, and TUNEL analysis. RESULTS: Bielschowsky staining and Aß(1-40) expression show few nerve connections and Aß(1-40) expression in an Aß sample, preserved neuronal cells and Aß(1-40) expression lowering in an IBU sample, mostly in IBU-LA. The Ngb level decreases in Aß samples, compared to control and IBU-LA samples. p-Akt/Akt and p-CREB/CREB ratios reveal a reduction in Aß sample, going back to the basal level in control and IBU-LA samples. Cytochrome C/Apaf 1 co-immunoprecipitate occurs and TUNEL-positive nuclei percentage decreases in Aß sample. Probe test performance shows an increased spatial reference memory in the IBU-LA compared to the Aß sample; no significant differences were seen between the IBU-LA and IBU samples. CONCLUSION: This evidence reveals that IBU-LA administration has the capability to maintain a high Ngb level allowing Ngb to perform a neuroprotective and antiapoptotic role, representing a valid tool in the therapeutic strategy of AD progression.