RESUMO
Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (n = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80+M1, CD206+M2 (anti-inflammatory macrophages), PDGFRα+MSC, and TNF-α+ cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10 ng/mL, 24 h, n = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01 mm3 vs 0.02 mm3, p<.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, p<.0001). Clodronate group showed significant reduction in mean number of CD80+, TNF-α+, PDGFRα+, and CD80+TNF-α+ cells on day 5 (306.5 vs 558.8, p<.0001; 280.5 vs 543.8, p<.0001; 365.0 vs 633.0, p<.0001, 29.0 vs 42.5, p<.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, p=.0004; 479.3 vs 384.5, p=.0008; 593.0 vs 473.0, p=.0010, 41.0 vs 32.5, p=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC's immunomodulatory capacity. In vivo, Clec4e and Gbp6 are involved in inflammation and bone formation. Clec4e, Gbp6, and Cxcl10 knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.
RESUMO
Doping offenses involve the use or attempted use of any prohibited method or substance as well as substituting samples. Consequently, it has been recommended that short tandem repeat (STR) analysis be used to determine if the doping control samples are from the same athlete. However, it has been recognized that it may be difficult to obtain full STR analysis using negligible amounts of DNA samples. Mitochondrial DNA (mtDNA) is characterized by its stability and high cellular copy number. Therefore, mtDNA testing in urine is expected to be used to analyze samples that cannot be analyzed using STR analysis. The objective of this study was to compare mtDNA testing with STR analysis by conducting sensitivity, concordance (whole blood, dried blood spot, and urine), and case-type studies. In sensitivity studies, mtDNA testing exhibited greater sensitivity compared with STR analysis. Concordance studies indicated that all samples were consistent with the mtDNA sequences and STR profiles. Allelic dropout occurred in some urine samples that were examined for STR analysis. Case-type sample studies demonstrated that mtDNA testing could be used to obtain DNA profiles of all the samples tested, including blood, dried blood spots, urine, blood residues on needles, and blood stains. In conclusion, mtDNA testing is valuable for analyzing highly degraded DNA samples, such as urine samples, compared with STR analysis. Urine testing should be performed for the initial testing procedure, because mtDNA is inherited maternally. In situations where the DNA match is detrimental to the athlete, additional blood STR analysis may be required.
RESUMO
Mesenchymal stem cells (MSCs) are known to promote tissue regeneration and suppress excessive inflammation caused by infection or trauma. Reported evidence indicates that various factors influence the expression of MSCs' endogenous immunomodulatory properties. However, the detailed interactions of MSCs with macrophages, which are key cells involved in tissue repair, and their regulatory mechanisms are not completely understood. We herein investigated how age-related immunomodulatory impairment of MSCs alters the interaction of MSCs with macrophages during bone healing using young (5-week old) and aged (50-week old) mice. To clarify the relationship between inflammatory macrophages (M1) and MSCs, their spatiotemporal localization at the bone healing site was investigated by immunostaining, and possible regulatory mechanisms were analyzed in vitro co-cultures. Histomorphometric analysis revealed an accumulation of M1 and a decrease in MSC number at the healing site in aged mice, which showed a delayed bone healing. In in vitro co-cultures, MSCs induced M1 apoptosis through cell-to-cell contact but suppressed the gene expression of pro-inflammatory cytokines by soluble factors secreted in the culture supernatant. Interestingly, interleukin 38 (Il-38) expression was up-regulated in M1 after co-culture with MSCs. IL-38 suppressed the gene expression of inflammatory cytokines in M1 and promoted the expression of genes associated with M1 polarization to anti-inflammatory macrophages (M2). IL-38 also had an inhibitory effect on M1 apoptosis. These results suggest that MSCs may induce M1 apoptosis, suppress inflammatory cytokine production by M1, and induce their polarization toward M2. Nevertheless, in aged conditions, the decreased number and immunomodulatory function of MSCs could be associated with a delayed M1 clearance (i.e., apoptosis and/or polarization) and consequent delayed resolution of the inflammatory phase. Furthermore, M1-derived IL-38 may be associated with immunoregulation in the tissue regeneration site.
Assuntos
Citocinas , Macrófagos , Camundongos , Animais , Citocinas/metabolismo , Macrófagos/metabolismo , Regeneração Óssea , Imunomodulação , ApoptoseRESUMO
The effects of an extended photoperiod (EP) on body composition of Thoroughbreds colts and fillies from December at one year old to April at two years old were investigated. Seventy-three Thoroughbreds reared and trained in Hidaka Training and Research Center, Japan Racing Association, Hokkaido were used. Forty-one horses were under the EP conditions from December 20 to April 15, and the 32 horses were under natural light alone as the control group. Body weight (BW), rump fat thickness (RFT), fat free mass (FFM) and percentage of fat (%F) were used as parameters of body composition. The present study revealed that BW and FFM increased with age in the EP group in colts. In fillies, BW increased with age in both the EP and the control group, however FFM increased with age only in the EP group. From December to April, only colts had a higher rate of increase in both BW and FFM in the EP group than in the control group. However, according to the mean rates of increase in FFM from January to March, the EP group was significantly higher than the control group in both sexes. Furthermore, monthly increase rate of FFM in March was significantly higher in the EP group than in the control group in both sexes. These results suggests that EP treatment to young Thoroughbreds in training at Hokkaido, which is shorter daylength in winter, accelerate the increase of FFM, representing muscle mass.
Assuntos
Composição Corporal , Fotoperíodo , Masculino , Cavalos , Animais , Feminino , Composição Corporal/fisiologia , Peso Corporal , JapãoRESUMO
Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the ß-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the ß-hairpin and the redox loop domains specific to CFoCF1.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons , Cloroplastos , ATPases de Cloroplastos Translocadoras de Prótons/genética , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Cloroplastos/metabolismo , Fotossíntese/genética , Oxirredução , Trifosfato de Adenosina/metabolismoRESUMO
The doping control analyses at the XXXII Olympic Games (July 23 to August 8, 2021) and the XVI Paralympic Games (August 24 to September 5, 2021) held in Tokyo, Japan, after a year of delay due to the COVID-19 pandemic are summarized in this paper. A new satellite facility at the existing World Anti-Doping Agency (WADA)-accredited Tokyo laboratory was established and fully operated by 278 staff, including 33 Tokyo laboratory staff, 49 international experts, and 196 Japanese temporary staff. The numbers of urine samples were 5081 (Olympics) and 1519 (Paralympics), and the numbers of blood samples were 1103 (Olympics) and 500 (Paralympics). The laboratory could prepare for analysis in advance using a paperless chain-of-custody system, allowing for faster turnaround time reporting. For the first time, a new polymerase chain reaction method for detecting erythropoietin (EPO) gene doping was used. The laboratory also analyzed blood samples for detecting steroid esters following the spotting of collected venous EDTA blood onto dried blood spot cards. Moreover, full-scan data acquisition using high-resolution mass spectrometers was performed for all urine samples, allowing for detecting traces of doping substances, which are not currently analyzed in the subsequent data processing. The presence of some prohibited substances was confirmed, resulting in 8 atypical findings (ATFs) and 11 adverse analytical findings (AAFs), including homologous blood transfusion (2 cases) and recombinant EPO in the blood (1 case), at the Olympics, whereas 2 ATFs and 10 AAFs were reported at the Paralympics.
Assuntos
COVID-19 , Dopagem Esportivo , Esportes , Humanos , Tóquio , Pandemias , COVID-19/diagnóstico , COVID-19/epidemiologia , Espectrometria de MassasRESUMO
Changes in the body composition of 50 Thoroughbreds colts and fillies, born between 2004 and 2010, were compared between those reared at the Hidaka Training and Research Center (Hidaka), Hokkaido, which is extremely cold in winter, and those reared at the Miyazaki Yearling Training Farm (Miyazaki), Kyushu, which is mildly cold in winter. The horses were divided into two sex groups and reared and trained in Hidaka or Miyazaki for 7 months from October of one year of age to April of two years of age. Body weight (BW), rump fat thickness (RFT), fat-free mass (FFM), and percentage of fat (%F) were used as parameters of body composition. This study revealed that BW and FFM were higher, and %F was lower in colts than in fillies at both training sites. Among colts, Miyazaki colts tended to have higher FFM values than Hidaka colts, and %F was significantly lower in Miyazaki colts than in Hidaka colts. Furthermore, from October to April, Miyazaki horses had a higher rate of increase in BW than Hidaka horses in both sexes and a higher rate of increase in FFM in colts. The higher rate of increase in FFM in Miyazaki colts suggests that training young Thoroughbreds in winter under mildly cold climate is more effective, than severely cold climate, particularly in colts.
Assuntos
Composição Corporal , Cavalos , Animais , Feminino , Masculino , Japão , Estações do Ano , Peso CorporalRESUMO
In our search for innovative drugs that could improve periodontal treatment outcomes, autophagy and its anomalies represent a potential target for therapeutic intervention. We sought to identify autophagy defects in murine experimental periodontitis and study the effectiveness of P140, a phosphopeptide known to bind HSPA8 and inhibit its chaperone properties, and that corrects autophagy dysfunctions in several autoimmune and inflammatory diseases. Experimental periodontitis was induced by placing silk ligature around mandibular first molars. Sick mice were treated intraperitoneally with either P140 or a control, scrambled peptide. After 10 days, mandibles were harvested and bone loss was measured by micro-CT. Immune cells infiltration was studied by histological analyses. Cytokines levels and autophagy-related markers expression were evaluated by qRT-PCR and western blotting. A comparison with non-affected mice revealed significant alterations in the autophagy processes in mandibles of diseased mice, especially in the expression of sequestosome 1/p62, Maplc3b, Atg5, Ulk1, and Lamp2. In vivo, we showed that P140 normalized the dysregulated expression of several autophagy-related genes. In addition, it diminished the infiltration of activated lymphocytes and pro-inflammatory cytokines. Unexpectedly P140 decreased the extent of bone loss affecting the furcation and alveolar areas. Our results indicate that P140, which was safe in clinical trials including hundreds of autoimmune patients with systemic lupus erythematosus, not only decreases the inflammatory effects observed in mandibular tissues of ligation-induced mice but strikingly also contributes to bone preservation. Therefore, the therapeutic peptide P140 could be repositioned as a decisive breakthrough for the future therapeutic management of periodontitis.
Assuntos
Fragmentos de Peptídeos , Periodontite , Animais , Citocinas/genética , Modelos Animais de Doenças , Camundongos , Fragmentos de Peptídeos/farmacologia , Periodontite/tratamento farmacológico , FosfopeptídeosRESUMO
Aging tissues present a progressive decline in homeostasis and regenerative capacities, which has been associated with degenerative changes in tissue-specific stem cells and stem cell niches. We hypothesized that amino acids could regulate the stem cell phenotype and differentiation ability of human bone marrow-derived mesenchymal stromal cells (hBMSCs). Thus, we performed a screening of 22 standard amino acids and found that D-tryptophan (10 µM) increased the number of cells positive for the early stem cell marker SSEA-4, and the gene expression levels of OCT-4, NANOG, and SOX-2 in hBMSCs. Comparison between D- and L-tryptophan isomers showed that the latter presents a stronger effect in inducing the mRNA levels of Oct-4 and Nanog, and in increasing the osteogenic differentiation of hBMSCs. On the other hand, L-tryptophan suppressed adipogenesis. The migration and colony-forming ability of hBMSCs were also enhanced by L-tryptophan treatment. In vivo experiments delivering L-tryptophan (50 mg/kg/day) by intraperitoneal injections for three weeks confirmed that L-tryptophan significantly increased the percentage of cells positive for SSEA-4, mRNA levels of Nanog and Oct-4, and the migration and colony-forming ability of mouse BMSCs. L-kynurenine, a major metabolite of L-tryptophan, also induced similar effects of L-tryptophan in enhancing stemness and osteogenic differentiation of BMSCs in vitro and in vivo, possibly indicating the involvement of the kynurenine pathway as the downstream signaling of L-tryptophan. Finally, since BMSCs migrate to the wound healing site to promote bone healing, surgical defects of 1 mm in diameter were created in mouse femur to evaluate bone formation after two weeks of L-tryptophan or L-kynurenine injection. Both L-tryptophan and L-kynurenine accelerated bone healing compared to the PBS-injected control group. In summary, L-tryptophan enhanced the stemness and osteoblastic differentiation of BMSCs and may be used as an essential factor to maintain the stem cell properties and accelerate bone healing and/or prevent bone loss.
RESUMO
Mesenchymal stem cells (MSCs) are known to play important roles in the repair of lost or damaged tissues and immunotolerance. On the other hand, aging is known to impair MSC function. However, little is currently known about how aged MSCs affect the host response to the local inflammatory condition and tissue deterioration in periodontitis, which is a progressive destructive disease of the periodontal tissue potentially leading to multiple tooth loss. In this study, we examined the relationship between aging-induced impairment of MSC function and the severity of periodontal tissue destruction associated with the decrease in host immunomodulatory response using a ligature-induced periodontitis model in young and aged mice. The results of micro computerized tomography (micro-CT) and histological analysis revealed a more severe bone loss associated with increased osteoclast activity in aged (50-week-old) mice compared to young (5-week-old) mice. Immunostaining analysis revealed that, in aged mice, the accumulation of inflammatory T and B cells was higher, whereas the percentage of platelet-derived growth factor receptor α (PDGFRα)+ MSCs, which are known to modulate the apoptosis of T cells, was significantly lower than in young mice. In vitro analysis of MSC function showed that the expression of surface antigen markers for MSCs (Sca-1, CD90, CD146), colony formation, migration, and osteogenic differentiation of aged MSCs were significantly declined compared to those of young MSCs. Moreover, a significantly higher proportion of aged MSCs were positive for the senescence-associated ß galactosidase activity. Importantly, aged MSCs presented a decreased expression of FAS-L, which was associated with a lower immunomodulatory property of aged MSCs to induce T cell apoptosis in co-cultures compared with young MSCs. In summary, this is the first study showing that aging-induced impairment of MSC function, including immunomodulatory response, is potentially correlated with progressive periodontal tissue deterioration.
Assuntos
Envelhecimento/patologia , Reabsorção Óssea/patologia , Modelos Animais de Doenças , Imunomodulação , Células-Tronco Mesenquimais/patologia , Osteogênese , Periodontite/patologia , Animais , Apoptose , Reabsorção Óssea/etiologia , Diferenciação Celular , Proliferação de Células , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/complicações , Periodontite/imunologiaRESUMO
Escherichia coli MazF is a toxin protein that cleaves RNA at ACA sequences. Its activation has been thought to cause growth inhibition, primarily through indiscriminate cleavage of RNA. To investigate responses following MazF activation, transcriptomic profiles of mazF-overexpressing and non-overexpressing E. coli K12 cells were compared. Analyses of differentially expressed genes demonstrated that the presence and the number of ACA trimers in RNA was unrelated to cellular RNA levels. Mapping differentially expressed genes onto the chromosome identified two chromosomal segments in which upregulated genes formed clusters, and these segments were absent in the chromosomes of E. coli strains other than K12. These results suggest that MazF regulates selective, rather than indiscriminate, categories of genes, and is involved in the regulation of horizontally acquired genes. We conclude that the primary role of MazF is not only cleaving RNA indiscriminately but also generating a specific cellular state.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , RNA/genética , Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , RNA/químicaRESUMO
Platinumbased antitumor agents have been widely used to treat head and neck squamous cell carcinoma (HNSCC) and numerous other malignancies. Cisplatin is the most frequently used platinumbased antitumor agent, however drug resistance and numerous undesirable side effects limit its clinical efficacy for cancer patients. Cancer cells discharge cisplatin into the extracellular space via copper transporters such as ATPase copper transporting beta (ATP7B) in order to escape from cisplatininduced cell death. In the present study, it was demonstrated for the first time that the copper chelator ammonium tetrathiomolybdate (TM) has several promising effects on cisplatin and HNSCC. First, TM suppressed the ATP7B expression in HNSCC cell lines in vitro, thereby enhancing the accumulation and apoptotic effect of cisplatin in the cancer cells. Next, it was revealed that TM enhanced the antitumor effect of cisplatin in HNSCC cell tumor progression in a mouse model of bone invasion, which is important since HNSCC cells frequently invade to facial bone. Finally, it was demonstrated that TM was able to overcome the cisplatin resistance of a human cancer cell line, A431, via ATP7B depression in vitro.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/farmacologia , ATPases Transportadoras de Cobre/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Molibdênio/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Camundongos , Molibdênio/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The γ-subunit of cyanobacterial and chloroplast ATP synthase, the rotary shaft of F1-ATPase, equips a specific insertion region that is only observed in photosynthetic organisms. This region plays a physiologically pivotal role in enzyme regulation, such as in ADP inhibition and redox response. Recently solved crystal structures of the γ-subunit of F1-ATPase from photosynthetic organisms revealed that the insertion region forms a ß-hairpin structure, which is positioned along the central stalk. The structure-function relationship of this specific region was studied by constraining the expected conformational change in this region caused by the formation of a disulfide bond between Cys residues introduced on the central stalk and this ß-hairpin structure. This fixation of the ß-hairpin region in the α3ß3γ complex affects both ADP inhibition and the binding of the ε-subunit to the complex, indicating the critical role that the ß-hairpin region plays as a regulator of the enzyme. This role must be important for the maintenance of the intracellular ATP levels in photosynthetic organisms.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Cianobactérias/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/genética , Proteínas de Bactérias/genética , Cianobactérias/genética , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/genéticaRESUMO
A deeper understanding of the detailed mechanism of in vivo tissue healing is necessary for the development of novel regenerative therapies. Among several external factors, environmental pH is one of the crucial parameters that greatly affects enzyme activity and cellular biochemical reactions involving tissue repair and homeostasis. In this study, in order to analyze the microenvironmental conditions during bone healing, we first measured the pH in vivo at the bone healing site using a high-resolution fiber optic pH microsensor directly in femur defects and tooth extraction sockets. The pH was shown to decrease from physiological 7.4 to 6.8 during the initial two days of healing (inflammatory phase). In the same initial stages of the inflammatory phase of the bone healing process, mesenchymal stem cells (MSCs) are known to migrate to the healing site to contribute to tissue repair. Therefore, we investigated the effect of a short-term acidic (pH 6.8) pre-treatment on the stemness of bone marrow-derived MSCs (BMSCs). Interestingly, the results showed that pre-treatment of BMSCs with acidic pH enhances the expression of stem cell markers (OCT-4, NANOG, SSEA-4), as well as cell viability and proliferation. On the other hand, acidic pH decreased BMSC migration ability. These results indicate that acidic pH during the initial stages of bone healing is important to enhance the stem cell properties of BMSCs. These findings may enable the development of novel methods for optimization of stem cell function towards tissue engineering or regenerative medicine.
Assuntos
Ácidos/farmacologia , Regeneração Óssea/genética , Osteogênese/efeitos dos fármacos , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Medicina Regenerativa , Antígenos Embrionários Estágio-Específicos/genética , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Cicatrização/genéticaRESUMO
Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.
Assuntos
Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2 , Microambiente Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Titânio , Animais , Células da Medula Óssea/patologia , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/farmacologia , Microambiente Celular/genética , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Cães , Feminino , Fêmur/metabolismo , Fêmur/patologia , Humanos , Camundongos , Camundongos Transgênicos , Osteoblastos/patologia , Osteogênese/genética , Titânio/química , Titânio/farmacologiaRESUMO
BACKGROUND: Commensal flora are involved in the appropriate development of the mature immune system. However, it is unclear how commensal flora contribute to immune responses against periodontal pathogens, including the response to lipopolysaccharide (LPS). The purpose of this study was to evaluate the expression of immune responses after topical application of LPS in germ-free (GF) and specific-pathogen-free (SPF) mice. METHODS: GF and SPF mice at 8 weeks of age were randomly divided into four groups each: a baseline group (n = 4/group) and three experimental groups (n = 6/group). Experimental groups received topical application of Porphyromonas gingivalis LPS (10 µg/µL) into the palatal gingival sulcus. Sampling was performed before LPS application (baseline) and at 3, 24, or 72 hours after LPS application. The numbers of neutrophils, CD4+ , and CD8+ T cells in periodontal tissue were evaluated by immunohistochemistry. Expression of genes encoding cytokines, chemokines, and a transcription factor was determined by real-time PCR. RESULTS: SPF mice, but not GF mice, showed an increased number of CD4+ T cells in the periodontal tissue at 3 hours after LPS application, compared with the number at baseline (p < 0.05). Gene expressions of tumor necrosis factor-α (Tnf-α) and forkhead box protein p3 (Foxp3) was also significantly higher in the SPF mice than in the GF mice at 3 hours after LPS application (p < 0.05). The number of neutrophils peaked at 24 hours in both GF and SPF mice. CONCLUSIONS: LPS-exposed SPF mice exhibited increases in the number of CD4+ T cells and in Tnf-α and Foxp3 gene expression in periodontal tissue compared with LPS-exposed GF mice.
Assuntos
Linfócitos T CD8-Positivos , Lipopolissacarídeos , Animais , Camundongos , Periodonto , Porphyromonas gingivalis , Organismos Livres de Patógenos Específicos , SimbioseRESUMO
BACKGROUND: Not only obesity but also sarcopenia is associated with NAFLD. The influence of altered body composition on the pathophysiology of NAFLD has not been fully elucidated. The aim of this study is to determine whether skeletal muscle mass to visceral fat area ratio (SV ratio) affects NAFLD pathophysiology. METHODS: A total of 472 subjects were enrolled. The association between SV ratio and NAFLD pathophysiological factors was assessed in a cross-sectional nature by stratification analysis. RESULTS: When the SV ratio was stratified by quartiles (Q 1-Q 4), the SV ratio showed a negative relationship with the degree of body mass index, HOMA-IR, and liver stiffness (Q 1, 8.9 ± 7.5 kPa, mean ± standard deviation; Q 2, 7.5 ± 6.2; Q 3, 5.8 ± 3.7; Q 4, 5.0 ± 1.9) and steatosis (Q 1, 282 ± 57 dB/m; Q 2, 278 ± 58; Q 3, 253 ± 57; Q 4, 200 ± 42) measured by transient elastography. Levels of leptin and biochemical markers of liver cell damage, liver fibrosis, inflammation and oxidative stress, and hepatocyte apoptosis were significantly higher in subjects in Q 1 than in those in Q 2, Q 3, or Q 4. Moreover, fat contents in femoral muscles were significantly higher in subjects in Q 1 and the change was associated with weakened muscle strength. In logistic regression analysis, NAFLD subjects with the decreased SV ratio were likely to have an increased risk of moderate-to-severe steatosis and that of advanced fibrosis. CONCLUSIONS: Decreased muscle mass coupled with increased visceral fat mass is closely associated with an increased risk for exacerbating NAFLD pathophysiology.
Assuntos
Gordura Intra-Abdominal/patologia , Músculo Esquelético/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , Adulto , Idoso , Antropometria/métodos , Apoptose/fisiologia , Composição Corporal/fisiologia , Estudos Transversais , Feminino , Hepatócitos/patologia , Humanos , Resistência à Insulina/fisiologia , Estilo de Vida , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-Idade , Força Muscular/fisiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Tamanho do Órgão/fisiologia , Estresse Oxidativo/fisiologia , Medição de Risco/métodos , Adulto JovemRESUMO
Nonalcoholic steatohepatitis (NASH) is one of the leading causes of chronic liver disease worldwide. However, details of pathogenetic mechanisms remain unknown. Deletion of both p62/Sqstm1 and Nrf2 genes spontaneously led to the development of NASH in mice fed a normal chow and was associated with liver tumorigenesis. The pathogenetic mechanism (s) underlying the NASH development was investigated in p62:Nrf2 double-knockout (DKO) mice. DKO mice showed massive hepatomegaly and steatohepatitis with fat accumulation and had hyperphagia-induced obesity coupled with insulin resistance and adipokine imbalance. They also showed dysbiosis associated with an increased proportion of gram-negative bacteria species and an increased lipopolysaccharide (LPS) level in feces. Intestinal permeability was elevated in association with both epithelial damage and decreased expression levels of tight junction protein zona occludens-1, and thereby LPS levels were increased in serum. For Kupffer cells, the foreign body phagocytic capacity was decreased in magnetic resonance imaging, and the proportion of M1 cells was increased in DKO mice. In vitro experiments showed that the inflammatory response was accelerated in the p62:Nrf2 double-deficient Kupffer cells when challenged with a low dose of LPS. Diet restriction improved the hepatic conditions of NASH in association with improved dysbiosis and decreased LPS levels. The results suggest that in DKO mice, activation of innate immunity by excessive LPS flux from the intestines, occurring both within and outside the liver, is central to the development of hepatic damage in the form of NASH.
Assuntos
Deleção de Genes , Fator 2 Relacionado a NF-E2/genética , Hepatopatia Gordurosa não Alcoólica/genética , Proteína Sequestossoma-1/genética , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Disbiose , Hiperfagia , Imunidade Inata , Resistência à Insulina , Células de Kupffer , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , ObesidadeRESUMO
Sulforaphane (SFN) plays an important role in preventing oxidative stress by activating the nuclear factor (erythroid derived 2)-like 2 (Nrf2) signalling pathway. SFN may improve exercise endurance capacity by counteracting oxidative stress-induced damage during exercise. We assessed running ability based on an exhaustive treadmill test (progressive-continuous all-out) and examined the expression of markers for oxidative stress and muscle damage. Twelve- to 13-week-old Male wild-type mice (Nrf2 +/+) and Nrf2-null mice (Nrf2 -/-) on C57BL/6J background were intraperitoneally injected with SFN or vehicle prior to the test. The running distance of SFN-injected Nrf2 +/+ mice was significantly greater compared with that of uninjected mice. Enhanced running capacity was accompanied by upregulation of Nrf2 signalling and downstream genes. Marker of oxidative stress in SFN-injected Nrf2 +/+ mice were lower than those in uninjected mice following the test. SFN produced greater protection against muscle damage during exhaustive exercise conditions in Nrf2 +/+ mice than in Nrf2 -/- mice. SFN-induced Nrf2 upregulation, and its antioxidative effects, might play critical roles in attenuating muscle fatigue via reduction of oxidative stress caused by exhaustive exercise. This in turn leads to enhanced exercise endurance capacity. These results provide new insights into SFN-induced upregulation of Nrf2 and its role in improving exercise performance.
Assuntos
Músculo Esquelético/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Resistência Física , Adenosina Trifosfatases/metabolismo , Animais , Metabolismo Energético/efeitos dos fármacos , Glutationa/metabolismo , Isotiocianatos/farmacologia , Luciferases/metabolismo , Luminescência , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Músculo Esquelético/efeitos dos fármacos , Biogênese de Organelas , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Condicionamento Físico Animal , Sulfóxidos , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The failure of Kupffer cells (KCs) to remove endotoxin is an important factor in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In this study, the effects of exercise training on KC function were studied in terms of in vivo endotoxin clearance and inflammatory responses. Mice were allocated into rest and exercise groups. KC bead phagocytic capacity and plasma steroid hormone levels were determined following exercise training. Endotoxin and inflammatory cytokine levels in plasma were determined over time following endotoxin injection. KC bead phagocytic capacity was potentiated and clearance of exogenously-injected endotoxin was increased in the exercise group. Inflammatory cytokine (TNF-α and IL-6) levels were lower in the exercise group. We found that only DHEA was increased in the plasma of the exercise group. In an in vitro experiment, the addition of DHEA to RAW264.7 cells increased bead phagocytic capacity and attenuated endotoxin-induced inflammatory responses. These results suggest that exercise training modulates in vivo endotoxin clearance and inflammatory responses in association with increased DHEA production. These exercise-induced changes in KC capacity may contribute to a slowing of disease progression in NAFLD patients.