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1.
Autoimmunity ; 57(1): 2201412, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38425093

RESUMO

OBJECTIVE: To explore the effect of CD5-like molecule (CD5L) on rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLS) and the relative molecular mechanism of CD5L in it. METHODS: Recombinant protein CD5L was used to stimulate the cultured RA-FLS cells. The inflammation-related cytokines were determined by real time-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The signal molecules and apoptosis-related molecules were detected by western blot assay (WB), and cell counting kit-8 (CCK-8) was used to detect the proliferation. RESULTS: CD5L can increase the production of IL-6, IL-8, and TNF-α and this effect can be inhibited by signal pathway inhibitor. At the same time, CD5L activated ERK1/2 MAPK signal, inhibitor treatment can weaken the intensity of phosphorylation. In addition, CD5L can enhance the proliferation ability of RA-FLS. CONCLUSION: CD5L induces the production of inflammatory cytokines in RA-FLS through the ERK1/2 MAPK pathway and increases cell survival.


Assuntos
Artrite Reumatoide , Membrana Sinovial , Humanos , Membrana Sinovial/metabolismo , Sistema de Sinalização das MAP Quinases , Artrite Reumatoide/metabolismo , Inflamação/metabolismo , Citocinas/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Proliferação de Células , Proteínas Reguladoras de Apoptose , Receptores Depuradores/metabolismo
2.
Int J Med Sci ; 21(4): 664-673, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38464837

RESUMO

N6-Methyladenosine (m6A) has been reported to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) remains unclear. Here, we found that methyltransferase-like 3 (METTL3) was up-regulated synchronously with m6A during the osteogenic differentiation of hPDLSCs. Functionally, lentivirus-mediated knockdown of METTL3 in hPDLSCs impaired osteogenic potential. Mechanistic analysis further showed that METTL3 knockdown decreased m6A methylation and reduced IGF2BP1-mediated stability of runt-related transcription factor 2 (Runx2) mRNA, which in turn inhibited osteogenic differentiation. Therefore, METTL3-based m6A modification favored osteogenic differentiation of hPDLSCs through IGF2BP1-mediated Runx2 mRNA stability. Our study shed light on the critical roles of m6A on regulation of osteogenic differentiation in hPDLSCs and served novel therapeutic approaches in vital periodontitis therapy.


Assuntos
Osteogênese , Ligamento Periodontal , Humanos , Diferenciação Celular/genética , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Osteogênese/genética , Células-Tronco
3.
Physiol Rep ; 12(5): e15971, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38467556

RESUMO

Microgravity is one of the most common causes counting for the bone loss. Mesenchymal stem cells (MSCs) contribute greatly to the differentiation and function of bone related cells. The development of novel MSCs biomarkers is critical for implementing effective therapies for microgravity induced bone loss. We aimed to find the new molecules involved in the differentiation and function of MSCs in mouse simulated microgravity model. We found CD226 was preferentially expressed on a subset of MSCs. Simulation of microgravity treatment significantly increased the proportion of CD226+ Lin- CD117- Sca1+ MSCs. The CD226+ MSCs produced higher IL-6, M-CSF, RANKL and lower CD200 expression, and promoted osteoclast differentiation. This study provides pivotal information to understand the role of CD226 in MSCs, and inspires new ideas for prevention of bone loss related diseases.


Assuntos
Células-Tronco Mesenquimais , Ausência de Peso , Animais , Camundongos , Ausência de Peso/efeitos adversos , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Simulação de Ausência de Peso
4.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 42(1): 37-45, 2024 Feb 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-38475949

RESUMO

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.


Assuntos
Benzilaminas , Ciclamos , Lipopolissacarídeos , Ligamento Periodontal , Humanos , Ligamento Periodontal/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Receptores CXCR4/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Osteogênese , Transdução de Sinais , Inflamação/metabolismo , Células-Tronco , RNA Mensageiro/metabolismo , Apoptose , Proliferação de Células , Células Estromais/metabolismo , Diferenciação Celular , Células Cultivadas
5.
Chem Biol Drug Des ; 103(3): e14454, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38477392

RESUMO

Asiatic acid (AA) is generally recognized in the treatment of various diseases and has significant advantages in the treatment of various inflammatory diseases. The treatment of rheumatoid arthritis (RA) with AA is a completely new entry point. RA is a complex autoimmune inflammatory disease, and despite the involvement of different immune and nonimmune cells in the pathogenesis of RA, fibroblast-like synoviocytes (FLS) play a crucial role in the progression of the disease. si-Nrf2 was transfected in RA-FLS and the cells were treated with AA. MTT assay and colony formation assay were used to detect the effect of AA on the viability and formation of clones of RA-FLS, respectively. Moreover, the apoptosis of RA-FLS was observed by Hoechst 33342 staining and flow cytometry. Western blot was applied to measure the expression of the Nrf2/HO-1/NF-κB signaling pathway-related proteins. Compared with the control group, RA-FLS proliferation, and clone formation were significantly inhibited by the increase of AA concentration, and further experiments showed that AA-induced apoptosis of RA-FLS. In addition, AA activated the Nrf2/HO-1 pathway to inhibit NF-κB protein expression. However, the knockdown of Nrf2 significantly offsets the effects of AA on the proliferation, apoptosis, and Nrf2/HO-1/NF-κB signaling pathway of RA-FLS cells. AA can treat RA by inhibiting the proliferation and inducing the apoptosis of RA-FLS. The mechanism may be related to the activation of the Nrf2/HO-1/NF-κB pathway.


Assuntos
Artrite Reumatoide , Triterpenos Pentacíclicos , Sinoviócitos , Humanos , NF-kappa B/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Proliferação de Células , Transdução de Sinais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Fibroblastos/metabolismo , Células Cultivadas , Apoptose
6.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473731

RESUMO

Endothelial cells in steady laminar flow assume a healthy, quiescent phenotype, while endothelial cells in oscillating disturbed flow become dysfunctional. Since endothelial dysfunction leads to atherosclerosis and cardiovascular disease, it is important to understand the mechanisms by which endothelial cells change their function in varied flow environments. Endothelial metabolism has recently been proven a powerful tool to regulate vascular function. Endothelial cells generate most of their energy from glycolysis, and steady laminar flow may reduce endothelial glycolytic flux. We hypothesized that steady laminar but not oscillating disturbed flow would reduce glycolytic flux and alter glycolytic side branch pathways. In this study, we exposed human umbilical vein endothelial cells to static culture, steady laminar flow (20 dynes/cm2 shear stress), or oscillating disturbed flow (4 ± 6 dynes/cm2 shear stress) for 24 h using a cone-and-plate device. We then measured glucose and lactate uptake and secretion, respectively, and glycolytic metabolites. Finally, we explored changes in the expression and protein levels of endothelial glycolytic enzymes. Our data show that endothelial cells in steady laminar flow had decreased glucose uptake and 13C labeling of glycolytic metabolites while cells in oscillating disturbed flow did not. Steady laminar flow did not significantly change glycolytic enzyme gene or protein expression, suggesting that glycolysis may be altered through enzyme activity. Flow also modulated glycolytic side branch pathways involved in proteoglycan and glycosaminoglycan synthesis, as well as oxidative stress. These flow-induced changes in endothelial glucose metabolism may impact the atheroprone endothelial phenotype in oscillating disturbed flow.


Assuntos
Antioxidantes , Aterosclerose , Humanos , Antioxidantes/metabolismo , Células Cultivadas , Aterosclerose/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Glicólise
7.
BMC Musculoskelet Disord ; 25(1): 213, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38481217

RESUMO

Osteoporosis is caused by the imbalance of osteoblasts and osteoclasts. The regulatory mechanisms of differentially expressed genes (DEGs) in pathogenesis of osteoporosis are of significant and needed to be further investigated. GSE100609 dataset downloaded from Gene Expression Omnibus (GEO) database was used to identified DEGs in osteoporosis patients. KEGG analysis was conducted to demonstrate signaling pathways related to enriched genes. Osteoporosis patients and the human mesenchymal stem cells (hMSCs) were obtained for in vivo and in vitro resaerch. Lentivirus construction and viral infection was used to knockdown genes. mRNA expression and protein expression were detected via qRT-PCR and western blot assay separately. Alkaline phosphatase (ALP) activity detection, alizarin Red S (ARS) staining, and expression of bone morphogenetic protein 2 (BMP2), osteocalcin (OCN) and Osterix were evaluated to determine osteoblast differentiation capacity. UL-16 binding protein 1 (ULBP1) gene was upregulated in osteoporosis and downregulated in differentiated hMSCs. Knockdown of ULBP1 increased ALP activity, mineralization ability evaluated by ARS staining, expression of BMP2, OCN and Osterix in differentiated hMSCs. Furthermore, rescue experiment demonstrated that suppressed ULBP1 boosted osteoblast differentiation by activating TNF-ß signaling pathway. Knockdown of ULBP1 gene could promoted osteoblast differentiation by activating TNF-ß signaling pathway in differentiated hMSCs. ULBP1 may be a the Achilles' heel of osteoporosis, and suppression of ULBP1 could be a promising treatment for osteoporosis.


Assuntos
Células-Tronco Mesenquimais , Osteoporose , Humanos , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Linfotoxina-alfa/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/genética , Osteoporose/genética , Proteína Smad2/metabolismo
8.
Methods Mol Biol ; 2783: 13-24, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478223

RESUMO

Adipose tissue is an abundant and accessible source of stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Adipose-derived stromal/stem cells (ASCs) have been widely used in tissue engineering and cell therapy. In addition, the clinical application of ASCs in the treatment of inflammation and injury has been proven a success. Here, we describe methods from our own laboratory and the literature for the isolation and expansion of Adipose-derived stromal/stem cells (ASCs). We present a large-scale procedure suitable for processing >100 mL volumes of lipoaspirate tissue specimens by collagenase digestion, a related procedure suitable for processing adipose tissue aspirates without digestion, and a procedure suitable for intact human adipose tissue, such as buccal fat pads in the maxillofacial region.


Assuntos
Adipócitos , Tecido Adiposo , Humanos , Células Estromais , Células-Tronco , Engenharia Tecidual/métodos , Diferenciação Celular , Células Cultivadas
9.
Methods Mol Biol ; 2783: 263-268, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478238

RESUMO

Compared to two-dimensional monolayer culture, cells cultured in three-dimensional (3D) platforms provide a more biochemically and physiologically relevant environment to study cell-cell and cell-extracellular matrix interactions in vitro. Using the liquid overlay technique, a scaffold-free method to generate 3D spheroids from human adipose-derived stem cells is described.


Assuntos
Esferoides Celulares , Células-Tronco , Humanos , Tecido Adiposo , Adipócitos , Matriz Extracelular , Células Cultivadas
10.
Methods Mol Biol ; 2783: 287-300, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478241

RESUMO

Adipose tissue is a complex and multifaceted endocrine organ located throughout the body. The dysfunction of adipose tissue is known to induce a wide variety of comorbidities that can negatively impact one's health and quality of life. In addition to behavioral changes, drugs that target dysfunctional adipose tissue to treat associated diseases are clinically needed. Regarding drug-testing platforms, animal models are the most popular models, limited by known differences from humans in genetics and physiology. Two-dimensional and static three-dimensional (3D) cell cultures are also used. Still, these in vitro models with static culture fail to recapitulate the phenotype and function of adipocytes seen in vivo. To combat this, our lab has developed an adipose tissue microphysiological system. A perfusion bioreactor with dual-flow chambers is 3D printed, which enables individualized top and bottom medium flows after adipose tissues are inserted as a barrier. Human progenitor cells, such as human mesenchymal stem cells, are embedded within a gelatin scaffold and in situ adipogenic differentiation within the bioreactor. Medium flow is established via a syringe pump system, allowing in vivo-like conditions to be maintained. The novel bioreactor-cultured adipose tissues represent a versatile disease modeling and drug-testing system. Here, we present the step-by-step methods to generate the bioreactors and adipose tissues. We also show the process of collecting and analyzing samples. In addition, we highlight the critical steps that require particular attention in notes.


Assuntos
Células-Tronco Mesenquimais , Qualidade de Vida , Animais , Humanos , Tecido Adiposo , Técnicas de Cultura de Células/métodos , Tecidos Suporte , Diferenciação Celular , Reatores Biológicos , Engenharia Tecidual , Células Cultivadas
11.
Methods Mol Biol ; 2783: 349-365, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478246

RESUMO

It is critical that human adipose-derived stromal/stem cell (hASC) tissue engineering therapies possess appropriate mechanical properties in order to restore the function of the load-bearing tissues of the musculoskeletal system. In an effort to elucidate hASC response to mechanical stimulation and develop mechanically robust tissue-engineered constructs, recent research has utilized a variety of mechanical loading paradigms, including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter will describe the methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.


Assuntos
Sistema Musculoesquelético , Ausência de Peso , Humanos , Engenharia Tecidual/métodos , Pressão Hidrostática , Diferenciação Celular , Células-Tronco , Células Cultivadas
12.
Int J Biol Sci ; 20(5): 1617-1633, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481810

RESUMO

In rheumatoid arthritis (RA), a debilitating autoimmune disorder marked by chronic synovial inflammation and progressive cartilage degradation, fibroblast-like synoviocytes (FLS) are key pathogenic players. Current treatments targeting these cells are limited. Our study focused on the Fat Mass and Obesity-associated protein (FTO), known for its roles in cell proliferation and inflammatory response modulation, and its involvement in RA. We specifically examined the inflammatory regulatory roles of FTO and CMPK2, a mitochondrial DNA synthesis protein, in FLS. Utilizing a combination of in vitro and in vivo methods, including FTO inhibition and gene knockdown, we aimed to understand FTO's influence on RA progression and chondrocyte functionality. Our findings showed that increased FTO expression in RA synovial cells enhanced their proliferation and migration and decreased senescence and apoptosis. Inhibiting FTO significantly slowed the disease progression in our models. Our research also highlighted that the FTO-CMPK2 pathway plays a crucial role in regulating synovial inflammation through the mtDNA-mediated cGAS/STING pathway, affecting chondrocyte homeostasis. This study indicates that targeting the FTO-CMPK2 axis could be a promising new therapeutic strategy for managing RA.


Assuntos
Artrite Reumatoide , Sinoviócitos , Humanos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Proliferação de Células/genética , Homeostase/genética , Fibroblastos/metabolismo , Cartilagem/metabolismo , Células Cultivadas , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo
13.
Int J Mol Sci ; 25(5)2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38473760

RESUMO

Bone differentiation is crucial for skeletal development and maintenance. Its dysfunction can cause various pathological conditions such as rickets, osteoporosis, osteogenesis imperfecta, or Paget's disease. Although traditional two-dimensional cell culture systems have contributed significantly to our understanding of bone biology, they fail to replicate the intricate biotic environment of bone tissue. Three-dimensional (3D) spheroid cell cultures have gained widespread popularity for addressing bone defects. This review highlights the advantages of employing 3D culture systems to investigate bone differentiation. It highlights their capacity to mimic the complex in vivo environment and crucial cellular interactions pivotal to bone homeostasis. The exploration of 3D culture models in bone research offers enhanced physiological relevance, improved predictive capabilities, and reduced reliance on animal models, which have contributed to the advancement of safer and more effective strategies for drug development. Studies have highlighted the transformative potential of 3D culture systems for expanding our understanding of bone biology and developing targeted therapeutic interventions for bone-related disorders. This review explores how 3D culture systems have demonstrated promise in unraveling the intricate mechanisms governing bone homeostasis and responses to pharmacological agents.


Assuntos
Técnicas de Cultura de Células , Osteogênese , Animais , Células Cultivadas , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Osso e Ossos
14.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474040

RESUMO

Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease's pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management.


Assuntos
Escleroderma Sistêmico , Doenças Vasculares , Animais , Humanos , Células Endoteliais/patologia , Pele/patologia , Escleroderma Sistêmico/patologia , Fibrose , Doenças Vasculares/patologia , Fibroblastos/patologia , Células Cultivadas
15.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474043

RESUMO

Chronic rhinosinusitis (CRS) is a disease characterised by the inflammation of the nasal and paranasal cavities. It is a widespread condition with considerable morbidity for patients. Current treatment for chronic rhinosinusitis consists of appropriate medical therapy followed by surgery in medically resistant patients. Although oral steroids are effective, they are associated with significant morbidity, and disease recurrence is common when discontinued. The development of additional steroid sparing therapies is therefore needed. Mesalazine is a commonly used therapeutic in inflammatory bowel disease, which shares a similar disease profile with chronic rhinosinusitis. This exploratory in vitro study aims to investigate whether mesalazine could be repurposed to a nasal wash, which is safe on human nasoepithelial cells, and retains its anti-inflammatory effects. CRS patients' human nasal epithelial cells (HNECs) were collected. HNECs were grown at an air-liquid interface (ALIs) and in a monolayer and challenged with mesalazine or a non-medicated control. Transepithelial electrical resistance, paracellular permeability, and toxicity were measured to assess epithelial integrity and safety. The anti-inflammatory effects of mesalazine on the release of interleukin (IL)-6 and tumour necrosis factor alpha (TNF-α) were analysed using human leukemia monocytic cell line (THP-1). mesalazine did not impact the barrier function of HNEC-ALIs and was not toxic when applied to HNECs or THP-1 cells at concentrations up to 20 mM. mesalazine at 0.5 and 1 mM concentrations significantly inhibited TNF-α release by THP-1 cells. mesalazine effectively decreases TNF-α secretion from THP-1 cells, indicating the possibility of its anti-inflammatory properties. The safety profile of mesalazine at doses up to 20 mM suggests that it is safe when applied topically on HNECs.


Assuntos
Mesalamina , Sinusite , Humanos , Mesalamina/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Sinusite/metabolismo , Mucosa Nasal/metabolismo , Interleucina-6/metabolismo , Anti-Inflamatórios/farmacologia , Doença Crônica , Células Epiteliais/metabolismo
16.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474055

RESUMO

Angiotensin-converting enzyme (ACE) plays a crucial role in the pathogenesis of hypertension. Piper sarmentosum Roxb., an herb known for its antihypertensive effect, lacks a comprehensive understanding of the mechanism underlying its antihypertensive action. This study aimed to elucidate the antihypertensive mechanism of aqueous extract of P. sarmentosum leaves (AEPS) via its modulation of the ACE pathway in phorbol 12-myristate-13-acetate (PMA)-induced human umbilical vein endothelial cells (HUVECs). HUVECs were divided into five groups: control, treatment with 200 µg/mL AEPS, induction 200 nM PMA, concomitant treatment with 200 nM PMA and 200 µg/mL AEPS, and treatment with 200 nM PMA and 0.06 µM captopril. Subsequently, ACE mRNA expression, protein level and activity, angiotensin II (Ang II) levels, and angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) mRNA expression in HUVECs were determined. AEPS successfully inhibited ACE mRNA expression, protein and activity, and angiotensin II levels in PMA-induced HUVECs. Additionally, AT1R expression was downregulated, whereas AT2R expression was upregulated. In conclusion, AEPS reduces the levels of ACE mRNA, protein and activity, Ang II, and AT1R expression in PMA-induced HUVECs. Thus, AEPS has the potential to be developed as an ACE inhibitor in the future.


Assuntos
Forbóis , Piper , Humanos , Anti-Hipertensivos/farmacologia , Miristatos/metabolismo , Miristatos/farmacologia , Angiotensina II/metabolismo , Células Endoteliais/metabolismo , Células Cultivadas , Peptidil Dipeptidase A/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , RNA Mensageiro/metabolismo , Acetatos/farmacologia , Forbóis/metabolismo , Forbóis/farmacologia
17.
Int J Mol Sci ; 25(5)2024 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-38474198

RESUMO

Periodontitis is a bacteria-induced inflammatory disease characterized by the progressive destruction of periodontal supporting tissues. Periodontal ligament stem cells (PDLSCs) are capable of differentiating into osteoblasts, which is an important stem cell source for endogenous periodontal tissue regeneration. Lysine lactylation (Kla) is a novel post-translational modification of proteins that is recently thought to be associated with osteogenic differentiation. Here, we found that lactylation levels are reduced both in the periodontal tissue of rats with periodontitis and lipopolysaccharide (LPS)-stimulated human PDLSCs. Proanthocyanidins were able to promote the osteogenesis of inflamed PDLSCs by restoring lactylation levels. Mechanistically, proanthocyanidins increased lactate production and restored the lactylation levels of PDLSCs, which recovered osteogenesis of inflamed PDLSCs via the Wnt/ß-catenin pathway. These results provide evidence on how epigenetic regulation by pharmacological agents influence the osteogenic phenotype of stem cells and the process of periodontal tissue repair. Our current study highlights the valuable potential of natural product proanthocyanidins in the regenerative engineering of periodontal tissues.


Assuntos
Periodontite , Proantocianidinas , Humanos , Ratos , Animais , Osteogênese/fisiologia , Ligamento Periodontal , Lipopolissacarídeos/metabolismo , Lisina/metabolismo , Proantocianidinas/metabolismo , Epigênese Genética , Células-Tronco/metabolismo , Periodontite/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas
18.
Int J Mol Sci ; 25(5)2024 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-38474293

RESUMO

Aortic valve disease (AVD) represents a global public health challenge. Research indicates a higher prevalence of diabetes in AVD patients, accelerating disease advancement. Although the specific mechanisms linking diabetes to valve dysfunction remain unclear, alterations of valvular endothelial cells (VECs) homeostasis due to high glucose (HG) or their crosstalk with monocytes play pivotal roles. The aim of this study was to determine the molecular signatures of VECs in HG and upon their interaction with monocytes in normal (NG) or high glucose conditions and to propose novel mechanisms underlying valvular dysfunction in diabetes. VECs and THP-1 monocytes cultured in NG/HG conditions were used. The RNAseq analysis revealed transcriptomic changes in VECs, in processes related to cytoskeleton regulation, focal adhesions, cellular junctions, and cell adhesion. Key molecules were validated by qPCR, Western blot, and immunofluorescence assays. The alterations in cytoskeleton and intercellular junctions impacted VEC function, leading to changes in VECs adherence to extracellular matrix, endothelial permeability, monocyte adhesion, and transmigration. The findings uncover new molecular mechanisms of VEC dysfunction in HG conditions and upon their interaction with monocytes in NG/HG conditions and may help to understand mechanisms of valvular dysfunction in diabetes and to develop novel therapeutic strategies in AVD.


Assuntos
Diabetes Mellitus , Células Endoteliais , Humanos , Células Endoteliais/metabolismo , Monócitos/metabolismo , Adesão Celular , Diabetes Mellitus/metabolismo , Glucose/metabolismo , Células Cultivadas
19.
Curr Protoc ; 4(3): e1005, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38465381

RESUMO

Embryonic limb bud-derived micromass cultures are valuable tools for investigating cartilage development, tissue engineering, and therapeutic strategies for cartilage-related disorders. This collection of fine-tuned protocols used in our laboratories outlines step-by-step procedures for the isolation, expansion, and differentiation of primary mouse limb bud cells into chondrogenic micromass cultures. Key aspects covered in these protocols include synchronized fertilization of mice (Basic Protocol 1), tissue dissection, cell isolation, micromass formation, and culture optimization parameters, such as cell density and medium composition (Basic Protocol 2). We describe techniques for characterizing the chondrogenic differentiation process by histological analysis (Basic Protocol 3). The protocols also address common challenges encountered during the process and provide troubleshooting strategies. This fine-tuned comprehensive protocol serves as a valuable resource for scientists working in the fields of developmental biology, cartilage tissue engineering, and regenerative medicine, offering an updated methodology for the study of efficient chondrogenic differentiation and cartilage tissue regeneration. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synchronized fertilization of mice Basic Protocol 2: Micromass culture of murine embryonic limb bud-derived cells Basic Protocol 3: Qualitative assessment of cartilage matrix production using Alcian blue staining.


Assuntos
Cartilagem , Condrogênese , Animais , Camundongos , Células Cultivadas , Diferenciação Celular , Mamíferos
20.
J Vis Exp ; (204)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38465930

RESUMO

Plant-derived cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as its ability to effectively support osteogenic differentiation. The present study further examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and energy-dispersive spectroscopy (EDS) confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young's modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.


Assuntos
Malus , Engenharia Tecidual , Ratos , Animais , Engenharia Tecidual/métodos , Osteogênese , Tecidos Suporte/química , Células Cultivadas , Osso e Ossos/cirurgia , Diferenciação Celular , Celulose , Proliferação de Células , Mamíferos
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