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1.
Stem Cell Res Ther ; 12(1): 514, 2021 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-34563249

RESUMO

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) seriously affects patient health. Despite the elusiveness of innate therapeutic effects, mesenchymal stromal cells (MSCs) hold great promise for inflammation-related diseases. Recent evidence indicates that disease-specific inflammatory cytokines could enhance the therapeutic effects of MSCs. METHODS: By establishing a CP/CPPS mouse model and pretreating MSCs with the cytokine interleukin-1ß (IL-1ß), we studied the IL-1ß-primed MSC immunoregulatory ability and targeted migration ability in vitro and in CP/CPPS mice. RESULTS: IL-1ß levels significantly increased in the prostate tissue and serum of experimental autoimmune prostatitis (EAP) mice. Pretreatment with IL-1ß enhanced the immunomodulatory potential and targeted migration of MSCs in vitro. Furthermore, intravenous infusion of IL-1ß-primed MSCs dampened inflammation in prostate tissues and alleviated hyperalgesia in EAP mice. The infused MSCs inhibited monocyte infiltration and promoted regulatory T lymphocyte formation in prostate tissue, thus remodeling the local environment. Surprisingly, IL-1ß-primed MSCs exhibited improved accumulation in the spleen but not in prostate tissue. Accordingly, infused MSCs reshaped systemic immunity by reducing the proportion of Ly6ChighCD11b+ monocytes and boosting the proportion of CD4+Foxp3+ regulatory T lymphocytes in the spleen and lung. Inflammatory chemokine (C-C motif) ligand 2 (CCL2) decreased through the downregulation of the NF-κB and JNK/MAPK pathways by inflammatory resolution via MSCs infusion to alleviate pain. CONCLUSION: In summary, IL-1ß-primed MSCs restored systemic immunologic homeostasis to alleviate CP/CPPS by modulating systemic immunity. These findings provide a novel strategy to boost the therapeutic effects of MSC-based therapy for CP/CPPS and reveal the essential role of systematic immunity in the treatment of CP/CPPS with MSC infusion.


Assuntos
Transplante de Células-Tronco Mesenquimais , Dor Pélvica , Prostatite , Animais , Interleucina-1beta/farmacologia , Masculino , Células-Tronco Mesenquimais , Camundongos , Dor Pélvica/terapia , Prostatite/terapia
2.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360888

RESUMO

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.


Assuntos
Condrócitos/metabolismo , Condrócitos/patologia , Osteoartrite/metabolismo , Transdução de Sinais/genética , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad3/química , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Hipertrofia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/genética , Osteoartrite/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Domínios Proteicos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Membrana Sinovial/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologia
3.
PLoS One ; 16(7): e0253915, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270579

RESUMO

Matrix metalloproteinases (MMPs) are involved in the pathology of numerous inflammatory retinal degenerations, including retinitis pigmentosa (RP). Our previous work revealed that intravitreal injections with tissue inhibitor of metalloproteinases 1 (TIMP-1) reduce the progression of rod cell death and inhibit cone cell remodeling that involves reactive gliosis in retinal Müller glial cells (MGCs) in rodent models. The underlying cellular and molecular mechanisms of how TIMP-1 functions in the retina remain to be resolved; however, MGCs are involved in structural homeostasis, neuronal cell survival and death. In the present study, MMP-9 and TIMP-1 expression patterns were investigated in a human MGC line (MIO-M1) under inflammatory cytokine (IL-1ß and TNF-α) and oxidative stress (H2O2) conditions. First, both IL-1ß and TNF-α, but not H2O2, have a mild in vitro pro-survival effect on MIO-M1 cells. Treatment with either cytokine results in the imbalanced secretion of MMP-9 and TIMP-1. H2O2 treatment has little effect on their secretion. The investigation of their intracellular expression led to interesting observations. MMP-9 and TIMP-1 are both expressed, not only in the cytoplasm, but also inside the nucleus. None of the treatments alters the MMP-9 intracellular distribution pattern. In contrast to MMP-9, TIMP-1 is detected as speckles. Intracellular TIMP-1 aggregation forms in the cytoplasmic area with IL-1ß treatment. With H2O2 treatments, the cell morphology changes from cobbles to spindle shapes and the nuclei become larger with increases in TIMP-1 speckles in an H2O2 dose-dependent manner. Two TIMP-1 cell surface receptors, low density lipoprotein receptor-related protein-1 (LRP-1) and cluster of differentiation 82 (CD82), are expressed within the nucleus of MIO-M1 cells. Overall, these observations suggest that intracellular TIMP-1 is a target of proinflammatory and oxidative insults in the MGCs. Given the importance of the roles for MGCs in the retina, the functional implication of nuclear TIMP-1 and MMP-9 in MGCs is discussed.


Assuntos
Núcleo Celular/metabolismo , Células Ependimogliais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Estresse Oxidativo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Linhagem Celular , Células Ependimogliais/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Interleucina-1beta/farmacologia , Proteína Kangai-1/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
4.
Biomed Res Int ; 2021: 9978651, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307684

RESUMO

Temporomandibular joint osteoarthritis (TMJOA) is characterized by chronic inflammatory degradation of mandibular condylar cartilage (MCC). Studies have found a positive correlation between inflammation and cyclooxygenase- (COX-) 2 in OA pathology. NF-κB is a crucial transcription factor of inflammatory and immune responses in the cause of TMJOA pathology. Resveratrol (RES) plays a critical role in antioxidation and anti-inflammation. But, studies on the effects of RES on TMJOA are very limited. So, the purpose of this study is to investigate the antioxidant and protective effects of RES against MCC degradation through downregulating COX-2/NF-κB expression. In vitro studies, the MCC cells were divided into three groups: the NC group, OA group, and RES group. The optimum dose of RES (10 µM) was determined. The TMJOA model of mice was created by injection of collagenase. And mice were injected with RES (100 µg/10 µl) 3 times one week for 4 weeks in the RES group. The expressions of COX-2, P65, MMP1, MMP13, COL2, and ACAN were measured by RT-PCR. Morphological changes of MCC were studied with HE staining. The results showed that inflammation could induce MCC degradation in vitro and vivo, while RES could reverse the degradation. Meanwhile, RES could downregulate COX-2/NF-κB/MMP expression and increase cartilage markers in vitro and vivo studies. The results indicated that RES treatment had antioxidant effects against chondrocyte apoptosis by downregulating the COX-2/NF-κB pathway in created TMJOA.


Assuntos
Antioxidantes/farmacologia , Apoptose , Condrócitos/patologia , Ciclo-Oxigenase 2/metabolismo , NF-kappa B/metabolismo , Osteoartrite/patologia , Substâncias Protetoras/farmacologia , Resveratrol/envenenamento , Articulação Temporomandibular/patologia , Animais , Apoptose/efeitos dos fármacos , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Modelos Animais de Doenças , Feminino , Interleucina-1beta/farmacologia , Mandíbula/patologia , Camundongos Endogâmicos C57BL , Resveratrol/farmacologia
5.
Sci Rep ; 11(1): 15508, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330980

RESUMO

Transient receptor potential vanilloid 4 (TRPV4) plays an important role in chondrocytes via Ca2+ signaling. However, its role in the progression of osteoarthritis is unclear. This study aimed to evaluate the effects of TRPV4 activation on articular cartilage and chondrocytes stimulated with interleukin (IL)-1ß. Bovine and human articular chondrocytes were stimulated with various agents, including IL-1ß, GSK1016790A (GSK101; a TRPV4 agonist), Compound C (an AMP-activated protein kinase (AMPK) inhibitor), and STO-609 (a calmodulin-dependent protein kinase kinase (CaMKK) inhibitor), and were processed for Western blot analysis and real-time PCR. The dimethylmethylene blue (DMMB) assay and Safranin O staining were also performed. GSK101 reversed the IL-1ß-induced increase in expression of matrix metalloproteinase (MMP)-13 and decrease in expression of aggrecan. GSK101 also decreased proteoglycan release in the DMMB assay and retained Safranin O staining of articular cartilage tissue. Furthermore, GSK101 increased AMPK phosphorylation and decreased IL-1ß-induced nuclear factor kappa B (NF-κB) phosphorylation. Compound C and STO-609 reversed the suppressive effects of GSK101 on NF-κB activation and MMP-13 expression. In conclusion, TRPV4 activation had chondroprotective effects on articular cartilage stimulated with IL-1ß by activating CaMKK/AMPK and suppressing the NF-κB pathway. TRPV4 activators may offer a promising therapeutic option for preventing the progression of osteoarthritis.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , NF-kappa B/metabolismo , Animais , Western Blotting , Bovinos , Feminino , Interleucina-1beta/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
6.
Sci Rep ; 11(1): 11561, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078931

RESUMO

Bone mesenchymal stem cells (BMSCs) are the most commonly investigated progenitor cells in bone defect repair and osteoarthritis subchondral bone regeneration; however, these studies are limited by complex inflammatory conditions. In this study, we investigated whether pro-autophagic γ-aminobutyric acid receptor-associated protein (GABARAP) promotes BMSCs proliferation and osteogenic differentiation by modulating autophagy in the presence or absence of interleukin-1 beta (IL-1ß) in vitro. The expression levels of all relevant factors were evaluated by qRT-PCR or western blotting where appropriate. BMSCs differentiation were assessed by Alizarin Red, alkaline phosphatase, safranin O, and Oil Red O staining. Furthermore, the interactions between autophagy and osteogenic differentiation were investigated by co-treatment with the autophagy inhibitor 3-methyladenine (3-MA). As the results, we found that treatment with recombinant human His6-GABARAP protein promoted cell proliferation, inhibited apoptosis, and reduced ROS generation by increasing autophagic activity, particularly when co-cultured with IL-1ß. Moreover, His6-GABARAP could effectively increase the osteogenic differentiation of BMSCs. The expression levels of inflammatory factors were significantly decreased by His6-GABARAP treatment, whereas its protective effects were attenuated by 3-MA. This study demonstrates that GABARAP maintains BMSCs survival and strengthens their osteogenic differentiation in an inflammatory environment by upregulating mediators of the autophagy pathway.


Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Autofagia/efeitos dos fármacos , Inflamação/prevenção & controle , Interleucina-1beta/farmacologia , Proteínas Associadas aos Microtúbulos/farmacologia , Osteogênese/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Inflamação/etiologia , Células-Tronco Mesenquimais , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Regulação para Cima/efeitos dos fármacos
7.
Invest Ophthalmol Vis Sci ; 62(7): 25, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34160563

RESUMO

Purpose: The ocular surface is considered an important route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. The expression level of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is vital for viral infection. However, the regulation of ACE2 expression on the ocular surface is still unknown. We aimed to determine the change in ACE2 expression in inflamed corneal epithelium and explore potential drugs to reduce the expression of ACE2 on the ocular surface. Methods: The expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in human corneal epithelial cells (HCECs) was examined by qPCR and Western blotting. The altered expression of ACE2 in inflammatory corneal epithelium was evaluated in TNFα- and IL-1ß-stimulated HCECs and inflamed mouse corneal epithelium, and the effect of resveratrol on ACE2 expression in HCECs was detected by immunofluorescence and Western blot analysis. Results: ACE2 and TMPRSS2 are expressed on the human corneal epithelial cells. ACE2 expression is upregulated in HCECs by stimulation with TNFα and IL-1ß and inflamed mouse corneas, including dry eye and alkali-burned corneas. In addition, resveratrol attenuates the increased expression of ACE2 induced by TNFα in HCECs. Conclusions: This study demonstrates that ACE2 is highly expressed in HCECs and can be upregulated by stimulation with inflammatory cytokines and inflamed mouse corneal epithelium. Resveratrol may be able to reduce the increased expression of ACE2 on the inflammatory ocular surface. Our work suggests that patients with an inflammatory ocular surface may display higher ACE2 expression, which increases the risk of SARS-CoV-2 infection.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Inibidores Enzimáticos/farmacologia , Epitélio Corneano/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Ceratite/enzimologia , Resveratrol/farmacologia , SARS-CoV-2/fisiologia , Adulto , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Western Blotting , Células Cultivadas , Epitélio Corneano/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Interleucina-1beta/farmacologia , Ceratite/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Virais/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima
8.
Sci Rep ; 11(1): 12516, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131243

RESUMO

We recently reported that cyclin-dependent kinase inhibitor 1 (p21) deficiency induces osteoarthritis susceptibility. Here, we determined the mechanism underlying the effect of p21 in synovial and cartilage tissues in RA. The knee joints of p21-knockout (p21-/-) (n = 16) and wild type C57BL/6 (p21+/+) mice (n = 16) served as in vivo models of collagen antibody-induced arthritis (CAIA). Arthritis severity was evaluated by immunological and histological analyses. The response of p21 small-interfering RNA (siRNA)-treated human RA FLSs (n = 5 per group) to interleukin (IL)-1ß stimulation was determined in vitro. Arthritis scores were higher in p21-/- mice than in p21+/+ mice. More severe synovitis, earlier loss of Safranin-O staining, and cartilage destruction were observed in p21-/- mice compared to p21+/+ mice. p21-/- mice expressed higher levels of IL-1ß, TNF-α, F4/80, CD86, p-IKKα/ß, and matrix metalloproteinases (MMPs) in cartilage and synovial tissues via IL-1ß-induced NF-kB signaling. IL-1ß stimulation significantly increased IL-6, IL-8, and MMP expression, and enhanced IKKα/ß and IκBα phosphorylation in human FLSs. p21-deficient CAIA mice are susceptible to RA phenotype alterations, including joint cartilage destruction and severe synovitis. Therefore, p21 may have a regulatory role in inflammatory cytokine production including IL-1ß, IL-6, and TNF-α.


Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inflamação/genética , Interleucina-1beta/genética , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Antígeno B7-2/genética , Proteínas de Ligação ao Cálcio/genética , Cartilagem/metabolismo , Cartilagem/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1beta/efeitos adversos , Interleucina-1beta/farmacologia , Interleucina-6/genética , Articulação do Joelho , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/genética , Líquido Sinovial/metabolismo , Fator de Necrose Tumoral alfa/genética
9.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064989

RESUMO

SGLT2 inhibitor-related nephroprotection is-at least partially-mediated by anti-inflammatory drug effects, as previously demonstrated in diabetic animal and human studies, as well as hyperglycemic cell culture models. We recently presented first evidence for anti-inflammatory potential of empagliflozin (Empa) under normoglycemic conditions in human proximal tubular cells (HPTC) by demonstrating Empa-mediated inhibition of IL-1ß-induced MCP-1/CCL2 and ET-1 expression on the mRNA and protein level. We now add corroborating evidence on a genome-wide level by demonstrating that Empa attenuates the expression of several inflammatory response genes in IL-1ß-induced (10 ng/mL) normoglycemic HPTCs. Using microarray-hybridization analysis, 19 inflammatory response genes out of >30.000 human genes presented a consistent expression pattern, that is, inhibition of IL-1ß (10 ng/mL)-stimulated gene expression by Empa (500 nM), in both HK-2 and RPTEC/TERT1 cells. Pathway enrichment analysis demonstrated statistically significant clustering of annotated pathways (enrichment score 3.64). Our transcriptomic approach reveals novel genes such as CXCL8/IL8, LOX, NOV, PTX3, and SGK1 that might be causally involved in glycemia-independent nephroprotection by SGLT2i.


Assuntos
Compostos Benzidrílicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Inflamação/imunologia , Interleucina-1beta/farmacologia , Túbulos Renais Proximais/imunologia , Perfilação da Expressão Gênica , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
10.
Eur J Pharmacol ; 905: 174216, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34058204

RESUMO

Glioblastoma (GBM) is the most common and lethal brain tumor with high inflammation. GBM cells infiltrate microglia and macrophages and are surrounded by pro-inflammatory cytokines. Interleukin (IL)-1ß, which is abundantly expressed in the tumor microenvironment, is involved in tumor progression. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mediate cell-cell interactions, and these cell adhesion molecules (CAMs) can be regulated by cytokines in immune cells or cancer cells in the inflammatory tumor microenvironment. In this study, we found that ICAM-1 and VCAM-1 expression was induced when GBM cells were treated with IL-1ß, and that adhesive interaction between monocytes and GBM cells increased accordingly. The levels of soluble CAMs (sICAM-1 and sVCAM-1) were also increased in the supernatants induced by IL-1ß. Furthermore, the conditioned media contained sICAM-1 and sVCAM-1, which further promoted IL-6 and CCL2 expression in differentiated macrophages. IL-1ß downregulated Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1) in GBM. The expression of ICAM-1 and VCAM-1 was regulated by p38, AKT, and NF-κB signaling pathways, which were modulated by SHP-1 signaling. The present study suggests that IL-1ß-induced protein expression of ICAM-1 and VCAM-1 in GBM may modulate the adhesive interaction between GBM and monocytes. In addition, IL-1ß also induced the soluble form of ICAM-1 and VCAM-1 in GBM, which plays a key role in the regulation of tumor-associated monocyte/macrophage polarization.


Assuntos
Glioblastoma/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/farmacologia , Monócitos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Molécula 1 de Adesão Intercelular/genética , Macrófagos/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/genética , eIF-2 Quinase/metabolismo
11.
PLoS One ; 16(5): e0252163, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019587

RESUMO

Human umbilical cord Wharton's jelly derived mesenchymal stem cells (hUCMSCs), a source of cell therapy, have received a great deal of attention due to their homing or migrating ability in response to signals emanating from damaged sites. It has been found that IL-1ß possesses the ability to induce the expression of matrix metalloproteinase-3 (MMP-3) in bone marrow MSCs. MMP-3 is involved in cell migration in various types of cells, including glioblastoma, vascular smooth muscle, and adult neural progenitor cells. In this study, we proposed that IL-1ß influences hUCMSCs migration involving MMP-3. The expression level of MMP-3 in IL-1ß-induced hUCMSCs was verified using cDNA microarray analysis, quantitative real-time PCR, ELISA and Western blot. Wound-healing and trans-well assay were used to investigate the cell migration and invasion ability of IL-1ß-treated hUCMSCs. In addition, we pre-treated hUCMSCs with interleukin-1 receptor antagonist, MMP-3 inhibitors (ALX-260-165, UK 356618), or transfected with MMP-3 siRNA to confirm the role of MMP3 in IL-1ß-induced cell migration. Our results showed that IL-1ß induced MMP-3 expression is related to the migration of hUCMSCs. Moreover, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) inhibitor U0126, p38 inhibitor SB205380, JNK inhibitor SP600125 and Akt inhibitor GSK 690693 decreased IL-1ß-induced MMP-3 mRNA and protein expression. The migration and invasion ability analyses showed that these inhibitors attenuated the IL-1ß-induced migration and invasion ability of hUCMSCs. In conclusion, we have found that IL-1ß induces the expression of MMP-3 through ERK1/2, JNK, p38 MAPK and Akt signaling pathways to enhance the migration of hUCMSCs. These results provide further understanding of the mechanisms in IL-1ß-induced hUCMSCs migration to injury sites.


Assuntos
Interleucina-1beta/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinase 3 da Matriz/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Transdução de Sinais/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
12.
J Neurochem ; 158(4): 898-911, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34050952

RESUMO

Both spinal tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to the development of "mechanical" spinal hyperexcitability in inflammatory pain states. Recently, we found that spinal sensitization by TNF was significantly reduced by blockade of spinal IL-6 signaling suggesting that IL-6 signaling is involved in spinal TNF effects. Here, we explored whether spinal interleukin-1ß (IL-1ß), also implicated in inflammatory pain, induces "mechanical" spinal hyperexcitability, and whether spinal IL-1ß effects are related to TNF and IL-6 effects. We recorded the responses of spinal cord neurons to mechanical stimulation of the knee joint in vivo and used cellular approaches on microglial and astroglial cell lines to identify interactions of IL-1ß, TNF, and IL-6. Spinal application of IL-1ß in anesthetized rats modestly enhanced responses of spinal cord neurons to innocuous and noxious mechanical joint stimulation. This effect was blocked by minocycline indicating microglia involvement, and significantly attenuated by interfering with IL-6 signaling. In the BV2 microglial cell line, IL-1ß, like TNF, enhanced the release of soluble IL-6 receptor, necessary for spinal IL-6 actions. Different to TNF, IL-1ß caused SNB-19 astrocytes to release interleukin-11. The generation of "mechanical" spinal hyperexcitability by IL-1ß was more pronounced upon spinal TNF neutralization with etanercept, suggesting that concomitant TNF limits IL-1ß effects. In BV2 cells, TNF stimulated the release of IL-1Ra, an endogenous IL-1ß antagonist. Thus, spinal IL-1ß has the potential to induce spinal hyperexcitability sharing with TNF dependency on IL-6 signaling, but TNF also limited IL-1ß effects explaining the modest effect of IL-1ß.


Assuntos
Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Neurônios/efeitos dos fármacos , Medula Espinal/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Interleucina-11/metabolismo , Articulações/inervação , Microglia/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Estimulação Física , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
13.
Front Immunol ; 12: 668483, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33968073

RESUMO

To fully perform their functions, T lymphocytes migrate within organs' parenchyma and interact with local cells. Infiltration of T lymphocytes within the central nervous system (CNS) is associated with numerous neurodegenerative disorders. Nevertheless, how these immune cells communicate and respond to neural cells remains unresolved. To investigate the behavior of T lymphocytes that reach the CNS, we have established an in vitro co-culture model and analyzed the spatiotemporal interactions between human activated CD8+ T lymphocytes and primary human astrocytes and neurons using time-lapse microscopy. By combining multiple variables extracted from individual CD8+ T cell tracking, we show that CD8+ T lymphocytes adopt a more motile and exploratory behavior upon interacting with astrocytes than with neurons. Pretreatment of astrocytes or neurons with IL-1ß to mimic in vivo inflammation significantly increases CD8+ T lymphocyte motility. Using visual interpretation and analysis of numerical variables extracted from CD8+ T cell tracking, we identified four distinct CD8+ T lymphocyte behaviors: scanning, dancing, poking and round. IL-1ß-pretreatment significantly increases the proportion of scanning CD8+ T lymphocytes, which are characterized by active exploration, and reduces the proportion of round CD8+ T lymphocytes, which are less active. Blocking MHC class I on astrocytes significantly diminishes the proportion of poking CD8+ T lymphocytes, which exhibit synapse-like interactions. Lastly, our co-culture time-lapse model is easily adaptable and sufficiently sensitive and powerful to characterize and quantify spatiotemporal interactions between human T lymphocytes and primary human cells in different conditions while preserving viability of fragile cells such as neurons and astrocytes.


Assuntos
Astrócitos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Comunicação Celular , Microscopia de Vídeo , Neurônios/metabolismo , Imagem com Lapso de Tempo , Adulto , Astrócitos/efeitos dos fármacos , Astrócitos/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/efeitos dos fármacos , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Inflamação/imunologia , Interleucina-1beta/farmacologia , Masculino , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Fenótipo , Fatores de Tempo , Adulto Jovem
14.
Mol Cell Biochem ; 476(10): 3623-3633, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34046827

RESUMO

Osteoarthritis (OA) is a chronic disease characterized by articular cartilage degeneration and uncontrolled chondrocyte apoptosis. At present, accumulating evidence introduces that circular RNA (circRNA) is involved in the development of OA. The aim of our study was to explore the role and the functional mechanism of circ_0020093 in OA cell model. Human chondrocytes were treated with interleukin-1 beta (IL-1ß) to construct OA model. The expression of circ_0020093, miR-23b, and Sprouty 1 (SPRY1) mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis was assessed by flow cytometry assay. The expression of extracellular matrix (ECM)-associated markers and SPRY1 protein level was detected by qRT-PCR and Western blot. Bioinformatics analysis-predicted relationship between miR-23b and circ_0020093 or SPRY1 was further verified by dual-luciferase reporter assay and RNA pull-down assay. In this study, we found that the expression of circ_0020093 and SPRY1 was declined, while miR-23b expression was elevated in IL-1ß-treated chondrocytes. IL-1ß induced chondrocyte apoptosis and ECM degradation, while these negative effects were alleviated by circ_0020093 overexpression or miR-23b inhibition. MiR-23b was a target of circ_0020093, and SPRY1 was a downstream target of miR-23b. Rescue experiments showed that miR-23b enrichment reversed the role of circ_0020093 overexpression, and SPRY1 knockdown also reversed the effects of miR-23b inhibition. Importantly, circ_0020093 positively regulated SPRY1 expression by targeting miR-23b. In conclusion, circ_0020093 ameliorates IL-1ß-induced apoptosis and ECM degradation of human chondrocytes by regulating the miR-23b/SPRY1 axis.


Assuntos
Apoptose/efeitos dos fármacos , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Interleucina-1beta/farmacologia , Proteínas de Membrana/biossíntese , Fosfoproteínas/biossíntese , RNA Circular/metabolismo , Regulação para Cima/efeitos dos fármacos , Apoptose/genética , Matriz Extracelular/genética , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fosfoproteínas/genética , RNA Circular/genética
15.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946948

RESUMO

Neurodegenerative diseases are characterized by increased permeability of the blood-brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1ß-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope-receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit ß-1. Among them, only laminin subunit ß-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit ß-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.


Assuntos
Barreira Hematoencefálica , Células Endoteliais/metabolismo , Inflamação/metabolismo , Laminina/metabolismo , Animais , Bacteriófago M13 , Biomarcadores , Células Cultivadas , Reagentes para Ligações Cruzadas , Fibronectinas/metabolismo , Ontologia Genética , Humanos , Interleucina-1beta/farmacologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos
16.
Methods Mol Biol ; 2271: 317-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33908017

RESUMO

Glycan "node" analysis is the process by which pooled glycans within complex biological samples are chemically deconstructed in a way that facilitates the analytical quantification of uniquely linked monosaccharide units (glycan "nodes"). It is based on glycan methylation analysis (a.k.a. linkage analysis) that has historically been applied to pre-isolated glycans. Thus, when using glycan node analysis, unique glycan features within whole biospecimens such as "core fucosylation," "α2-6 sialylation," "ß1-6 branching," "ß1-4 branching," and "bisecting GlcNAc," are captured as single analytical signals by GC-MS. Here we describe the use of this methodology in cell culture supernatant and in the analysis of IgG (alpha-1 antitrypsin) glycans. The effect of IL-6 and IL-1ß cytokines on secreted hepatocyte protein glycan features is demonstrated; likewise, the impact of neuraminidase treatment of IgG is illustrated. For the majority of glycan nodes, the assay is consistent and reproducible on a day-to-day basis; because of this, relatively subtle shifts in the relative abundance of glycan features can be captured using this approach.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Glicômica , Glicoproteínas/análise , Hepatócitos/metabolismo , Imunoglobulina G/análise , Polissacarídeos/análise , Processamento de Proteína Pós-Traducional , Meios de Cultivo Condicionados/metabolismo , Glicosilação , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Interleucina-6/farmacologia , Metilação , Neuraminidase/metabolismo , Projetos de Pesquisa , Via Secretória , Fluxo de Trabalho
17.
Cells ; 10(4)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918659

RESUMO

Chronic brain hypoperfusion is the primary cause of vascular dementia and has been implicated in the development of white matter disease and lacunar infarcts. Cerebral hypoperfusion leads to a chronic state of brain inflammation with immune cell activation and production of pro-inflammatory cytokines, including IL-1ß. In the present study, we induced chronic, progressive brain hypoperfusion in mice using ameroid constrictor, arterial stenosis (ACAS) surgery and tested the efficacy of an IL-1ß antibody on the resulting brain damage. We observed that ACAS surgery causes a reduction in cerebral blood flow (CBF) of about 30% and grey and white matter damage in and around the hippocampus. The IL-1ß antibody treatment did not significantly affect CBF but largely eliminated grey matter damage and reduced white matter damage caused by ACAS surgery. Over the course of hypoperfusion/injury, grip strength, coordination, and memory-related behavior were not significantly affected by ACAS surgery or antibody treatment. We conclude that antibody neutralization of IL-1ß is protective from the brain damage caused by chronic, progressive brain hypoperfusion.


Assuntos
Isquemia Encefálica/prevenção & controle , Encéfalo/patologia , Interleucina-1beta/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Isquemia Encefálica/patologia , Circulação Cerebrovascular/efeitos dos fármacos , Substância Cinzenta/efeitos dos fármacos , Substância Cinzenta/patologia , Masculino , Camundongos Endogâmicos C57BL , Substância Branca/efeitos dos fármacos , Substância Branca/patologia
18.
FASEB J ; 35(5): e21525, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33817836

RESUMO

Glycolysis is a well-known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin-1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis. In addition, numerous glycolytic enzymes are known to be redox regulated. However, whether and how redox chemistry regulates metabolic reprogramming more generally remains unclear. In this study, we employed a multi-omics approach in primary mouse airway basal cells to evaluate the role of protein redox biochemistry, specifically protein glutathionylation, in mediating metabolic reprogramming. Our findings demonstrate that IL1B induces glutathionylation of multiple proteins involved in metabolic regulation, notably in the glycolysis pathway. Cells lacking Glutaredoxin-1 (Glrx), the enzyme responsible for reversing glutathionylation, show modulation of multiple metabolic pathways including an enhanced IL1B-induced glycolytic response. This was accompanied by increased secretion of thymic stromal lymphopoietin (TSLP), a cytokine important in asthma pathogenesis. Targeted inhibition of glycolysis prevented TSLP release, confirming the functional relevance of enhanced glycolysis in cells stimulated with IL1B. Collectively, data herein point to an intriguing link between glutathionylation chemistry and glycolytic reprogramming in epithelial cells and suggest that glutathionylation chemistry may represent a therapeutic target in pulmonary pathologies with perturbations in the glycolysis pathway.


Assuntos
Reprogramação Celular , Glutarredoxinas/fisiologia , Glutationa/metabolismo , Glicólise , Inflamação/imunologia , Interleucina-1beta/farmacologia , Pulmão/imunologia , Animais , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução
19.
Neurochem Res ; 46(7): 1781-1793, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33864170

RESUMO

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.


Assuntos
Anti-Inflamatórios/farmacologia , Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Mercaptopurina/farmacologia , Neurônios/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Animais , Células Cultivadas , Sistema Nervoso Entérico/citologia , Inflamação/induzido quimicamente , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos , Camundongos , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
20.
Clin Immunol ; 227: 108718, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33819576

RESUMO

BACKGROUND: Osteoarthritis (OA) is a common inflammatory disease characterized by articular cartilage degeneration and injury. Circular RNAs (circRNAs) are widely involved in the development of human diseases, including OA. The objective of this study was to investigate the function and functional mechanism of circ_0001103 in OA. METHODS: Cell model of OA was established by treating chondrocytes with interleukin-1ß (IL-1ß). The expression of circ_0001103, miR-375 and sirtuin 1 (SIRT1) mRNA was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was assessed using cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined using flow cytometry assay. The expression levels of inflammatory factors were quantified by qRT-PCR. The expression of extracellular matrix (ECM) metabolism-related markers, including Collagen Type II Alpha 1 Chain (COL2A1) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), was detected by western blot. Predicted target relationship between miR-375 and circ_0001103 or SIRT1 by the bioinformatics tools was validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. RESULTS: Circ_0001103 was downregulated in OA tissues and IL-1ß-induced chondrocytes. Overexpression of circ_0001103 attenuated IL-1ß-induced chondrocyte apoptosis, inflammatory responses and ECM degradation. MiR-375 was targeted by circ_0001103, and miR-375 could bind to SIRT1. Circ_0001103 overexpression increased the expression of SIRT1 by suppressing miR-375. Rescue experiments suggested that miR-375 restoration reversed the effects of circ_0001103 overexpression, and SIRT1 knockdown overturned the effects of miR-375 inhibition. CONCLUSION: Circ_0001103 governed the miR-375/SIRT1 axis to ameliorate IL-1ß-induced chondrocyte injuries, implying that circ_0001103 was a promising biomarker in OA pathogenesis.


Assuntos
Apoptose/genética , Condrócitos/metabolismo , Inflamação/genética , MicroRNAs/genética , Osteoartrite do Joelho/genética , RNA Circular/genética , Sirtuína 1/genética , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Estudos de Casos e Controles , Condrócitos/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Interleucina-1beta/farmacologia , MicroRNAs/metabolismo , Osteoartrite do Joelho/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo
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