RESUMEN
Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.
Asunto(s)
Dependovirus , Factor VIII , Vectores Genéticos , Hemofilia A , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 3 de la Matriz , ARN Largo no Codificante , Animales , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia A/complicaciones , Dependovirus/genética , ARN Largo no Codificante/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz/genética , Ratones , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/administración & dosificación , Factor VIII/genética , Factor VIII/metabolismo , Artropatías/terapia , Artropatías/genética , Artropatías/etiología , Humanos , Terapia Genética/métodos , Ratones Endogámicos C57BL , Cartílago Articular/metabolismo , Cartílago Articular/patología , Modelos Animales de Enfermedad , MasculinoRESUMEN
OBJECT: To clarify the involvement of clock genes in the production of inflammatory mediators from RA-FLS, we examined the role of Bmal1, one of the master clock genes. METHODS: RA-FLSs were stimulated with IL-1ß (0, 20 ng/mL), IL-6 (0, 20 ng/mL), IL-17 (0, 20 ng/mL), TNF-α (0, 20 ng/mL) or IFN-γ (0, 20 ng/mL) to examine the expression of Bmal1, MMP-3, CCL2, IL-6, IL-7 and IL-15 by qPCR and immunofluorescence staining. After silencing Bmal1, RA-FLSs were stimulated with IL-1ß (0, 20 ng/mL), TNF-α (0, 20 ng/mL) or IFN-γ (0, 20 ng/mL) to examine the expressions of inflammatory mediators; MMP-3, CCL2, IL-6 and IL-15 by qPCR, ELISA and immunofluorescence staining. RESULTS: Bmal1 expressions were increased by IL-1ß, TNF-α and IFN-γ stimulations. Under stimulations with TNF-α, IL-1ß, and IFN-γ, mRNA and protein expressions of MMP-3, CCL2 and IL-6 were suppressed by siBmal1. CONCLUSION: Results indicate that Bmal1 contributes the production of MMP-3, CCL2, and IL-6 from RA-FLS, implying Bmal1 is involved in the pathogenesis of RA by regulating the inflammation.
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Artritis Reumatoide , Sinoviocitos , Humanos , Sinoviocitos/metabolismo , Membrana Sinovial/metabolismo , Interleucina-15/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Mediadores de Inflamación/metabolismo , Artritis Reumatoide/patología , Fibroblastos/metabolismo , Células CultivadasRESUMEN
Oncolytic virotherapy is a promising therapeutic approach for glioblastoma (GBM) treatment, although the outcomes are partially satisfactory. Hence, more effective strategies are needed urgently to modify therapeutic viruses to enhance their efficiency and safety in killing tumor cells and improve the survival rate of GBM patients. This study generated a new-generation oncolytic adenovirus Ad5 KT-E1A-IL-15 (TS-2021) and evaluated its antitumor efficacy. Ex vivo analyses revealed Ki67 and TGF-ß2 co-localized in GBM cells. In addition, TS-2021 selectively replicated in GBM cells, which was dependent on the expression of Ki67 and TGF-ß2. The immunocompetent mice model of GBM demonstrated the in vivo efficacy of TS-2021 by inhibiting tumor growth and improving survival proficiently. Notably, TS-2021 effectively reduced MMP3 expression by inactivating the MKK4/JNK pathway, thereby reducing tumor invasiveness. Altogether, the findings of the present study highlight that TS-2021 can effectively target GBM cells expressing high levels of Ki67 and TGF-ß2, exerting potent antitumor effects. Additionally, it can improve efficacy and suppress tumor invasiveness by inhibiting the MKK4/JNK/MMP3 pathway. Thus our study demonstrates the efficiency of the novel TS-2021 in the mouse model and provides a potential therapeutic option for patients with GBM.
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Infecciones por Adenoviridae , Glioblastoma , Animales , Ratones , Humanos , Adenoviridae/genética , Glioblastoma/terapia , Glioblastoma/genética , Glioblastoma/patología , Regiones no Traducidas 5' , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Interleucina-15/metabolismo , Línea Celular TumoralRESUMEN
The proinflammatory cytokines and arachidonic acid (AA)-derived eicosanoids play a key role in cartilage degeneration in osteoarthritis (OA). The lysophosphatidylcholine acyltransferase 3 (LPCAT3) preferentially incorporates AA into the membranes. Our recent studies showed that MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that LPCAT3 expression was up-regulated in both human and mice articular cartilage of OA, and correlated with severity of OA. The IL-1ß-induces cell death via upregulation of LPCAT3, MMP3, ADAMTS5, and eicosanoids via MALT1. Gene silencing or pharmacological inhibition of LPCAT3 or MALT1 in chondrocytes and human cartilage explants notably suppressed the IL-1ß-induced cartilage catabolism through inhibition of expression of MMP3, ADAMTS5, and also secretion of cytokines and eicosanoids. Mechanistically, overexpression of MALT1 in chondrocytes significantly upregulated the expression of LPCAT3 along with MMP3 and ADAMTS5 via c-Myc. Inhibition of c-Myc suppressed the IL-1ß-MALT1-dependent upregulation of LPCAT3, MMP3 and ADAMTS5. Consistent with the in vitro data, pharmacological inhibition of MALT1 or gene silencing of LPCAT3 using siRNA-lipid nanoparticles suppressed the synovial articular cartilage erosion, pro-inflammatory cytokines, and eicosanoids such as PGE2, LTB4, and attenuated osteoarthritis induced by the destabilization of the medial meniscus in mice. Overall, our data reveal a previously unrecognized role of the MALT1-LPCAT3 axis in osteoarthritis. Targeting the MALT1-LPCAT3 pathway with MALT1 inhibitors or siRNA-liposomes of LPCAT3 may become an effective strategy to treat OA by suppressing eicosanoids, matrix-degrading enzymes, and proinflammatory cytokines.
Asunto(s)
Cartílago Articular , Osteoartritis , Animales , Humanos , Ratones , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/farmacología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocitos/metabolismo , Citocinas/metabolismo , Eicosanoides/metabolismo , Eicosanoides/farmacología , Eicosanoides/uso terapéutico , Células Endoteliales/metabolismo , Interleucina-1beta/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/farmacología , Metaloproteinasa 3 de la Matriz/uso terapéutico , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Osteoartritis/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
OBJECTIVES: The aberrant expression of omentin-1 had been reported in type 2 diabetes and cardiovascular disease. Here, we investigated the expression and role of omentin-1 in rheumatoid arthritis (RA). METHODS: The expression of omentin-1 in RA and in the normal population was detected by ELISA and immunohistochemistry, and collagen-induced arthritis (CIA) mice were used to detect the role of omentin-1 in RA. RESULTS: We found that the expression of omentin-1 was elevated in serum of RA patients compared with healthy controls (p=0.004), and in the RA disease activity group compared with the disease remission group (p<0.001). In addition, the level of omentin-1 in RA patients was positively correlated with CRP (r=0.427, p=0.002), ESR (r=0.454, p<0.001) and DAS28 (r=0.496, p<0.001; r=0.661, p<0.001, respectively). Multivariable analysis showed that omentin-1 alone was associated with disease activity state (OR=1.018, p=0.004). Immunohistochemical results showed that omentin-1 was increased in the synovium of RA and CIA mice. Omentin-1 injection resulted in an earlier onset of arthritis, an aggravated arthritic progression, more severe synovial hyperplasia and bone erosion in CIA mice. Moreover, omentin-1 treatment markedly enhanced IL-6, TNF-α, MMP-3, MMP-13 and RANKL in the joint tissue of CIA mice. CONCLUSIONS: Our results suggested that omentin-1 was up-regulated in RA and can exacerbate synovitis and joint destruction which may provide new insight into the pathogenesis of RA.
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Artritis Experimental , Artritis Reumatoide , Citocinas , Proteínas Ligadas a GPI , Lectinas , Ratones Endogámicos DBA , Sinovitis , Artritis Reumatoide/metabolismo , Animales , Proteínas Ligadas a GPI/metabolismo , Lectinas/metabolismo , Citocinas/metabolismo , Persona de Mediana Edad , Sinovitis/patología , Sinovitis/metabolismo , Masculino , Artritis Experimental/metabolismo , Artritis Experimental/patología , Humanos , Femenino , Estudios de Casos y Controles , Articulaciones/patología , Articulaciones/metabolismo , Adulto , Metaloproteinasa 3 de la Matriz/metabolismo , Regulación hacia Arriba , Ratones , Biomarcadores/sangre , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , AncianoRESUMEN
INTRODUCTION AND HYPOTHESIS: The objective was to investigate the correlation between endogenous vaginal microecological alterations and female pelvic organ prolapse (POP). METHODS: Patients who underwent vaginal hysterectomy were retrospectively analyzed as the POP group (n = 30) and the non-POP group (n = 30). The vaginal microbial metabolites and enzyme levels were tested using the dry chemoenzymatic method. The mRNA and protein expression were tested using real-time quantitative PCR and immunohistochemistry. SPSS version 25.0 and GraphPad Prism 8.0 were performed for statistical analysis. RESULTS: Compared with the non-POP group, the vaginal pH, H2O2 positivity and leukocyte esterase positivity were higher in patients with POP (all p < 0.05). Further analysis showed that patients with pelvic organ prolapse quantification (POP-Q) stage IV had higher rates of vaginal pH, H2O2 positivity and leukocyte esterase positivity than those with POP-Q stage III. Additionally, the mRNA expression of decorin (DCN), transforming growth factor beta 1 (TGF-ß1), and matrix metalloproteinase-3 (MMP-3) in uterosacral ligament tissues were higher, whereas collagen I and III were lower. Similarly, the positive expression of MMP-3 in uterosacral ligament tissue was significantly upregulated in the POP group compared with the non-POP group (p = 0.035), whereas collagen I (p = 0.004) and collagen III (p = 0.019) in uterosacral ligament tissue were significantly downregulated in the POP group. Correlation analysis revealed that there was a significant correlation between vaginal microecology and collagen metabolism. In addition, MMP-3 correlated negatively with collagen I and collagen III (p = 0.002, r = -0.533; p = 0.002, r = -0.534 respectively), whereas collagen I correlated positively with collagen III (p = 0.001, r = 0.578). CONCLUSIONS: Vaginal microecological dysbiosis affects the occurrence of female POP, which could be considered a novel therapeutic option.
Asunto(s)
Prolapso de Órgano Pélvico , Vagina , Femenino , Humanos , Prolapso de Órgano Pélvico/metabolismo , Persona de Mediana Edad , Estudios Retrospectivos , Metaloproteinasa 3 de la Matriz/metabolismo , Decorina/metabolismo , Decorina/genética , Anciano , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Histerectomía Vaginal , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , ARN Mensajero/metabolismo , Ligamentos/metabolismo , Microbiota , AdultoRESUMEN
The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide-specific phospholipase C (PI-PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI-PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI-PLC γ1 was produced. The decrease of PI-PLC γ1 was associated with the decrease of metalloprotease-3 (MMP-3), and MMP-13, and ADAMTS-5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI-PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI-PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI-PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.
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Fibroblastos , Intrones , Fosfolipasa C gamma , ARN Mensajero , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Fosfolipasa C gamma/metabolismo , Fosfolipasa C gamma/genética , Células Cultivadas , Osteoartritis/metabolismo , Osteoartritis/patología , Membrana Sinovial/metabolismo , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacos , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Sinoviocitos/metabolismo , Sinoviocitos/efectos de los fármacos , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz/genéticaRESUMEN
BACKGROUND: The increasing production and usage of copper oxide nanoparticles (Nano-CuO) raise human health concerns. Previous studies have demonstrated that exposure to Nano-CuO could induce lung inflammation, injury, and fibrosis. However, the potential underlying mechanisms are still unclear. Here, we proposed that matrix metalloproteinase-3 (MMP-3) might play an important role in Nano-CuO-induced lung inflammation, injury, and fibrosis. RESULTS: Exposure of mice to Nano-CuO caused acute lung inflammation and injury in a dose-dependent manner, which was reflected by increased total cell number, neutrophil count, macrophage count, lactate dehydrogenase (LDH) activity, and CXCL1/KC level in bronchoalveolar lavage fluid (BALF) obtained on day 3 post-exposure. The time-response study showed that Nano-CuO-induced acute lung inflammation and injury appeared as early as day 1 after exposure, peaked on day 3, and ameliorated over time. However, even on day 42 post-exposure, the LDH activity and macrophage count were still higher than those in the control group, suggesting that Nano-CuO caused chronic lung inflammation. The Nano-CuO-induced pulmonary inflammation was further confirmed by H&E staining of lung sections. Trichrome staining showed that Nano-CuO exposure caused pulmonary fibrosis from day 14 to day 42 post-exposure with an increasing tendency over time. Increased hydroxyproline content and expression levels of fibrosis-associated proteins in mouse lungs were also observed. In addition, Nano-CuO exposure induced MMP-3 overexpression and increased MMP-3 secretion in mouse lungs. Knocking down MMP-3 in mouse lungs significantly attenuated Nano-CuO-induced acute and chronic lung inflammation and fibrosis. Moreover, Nano-CuO exposure caused sustained production of cleaved osteopontin (OPN) in mouse lungs, which was also significantly decreased by knocking down MMP-3. CONCLUSIONS: Our results demonstrated that short-term Nano-CuO exposure caused acute lung inflammation and injury, while long-term exposure induced chronic pulmonary inflammation and fibrosis. Knocking down MMP-3 significantly ameliorated Nano-CuO-induced pulmonary inflammation, injury, and fibrosis, and also attenuated Nano-CuO-induced cleaved OPN level. Our study suggests that MMP-3 may play important roles in Nano-CuO-induced pulmonary inflammation and fibrosis via cleavage of OPN and may provide a further understanding of the mechanisms underlying Nano-CuO-induced pulmonary toxicity.
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Líquido del Lavado Bronquioalveolar , Cobre , Metaloproteinasa 3 de la Matriz , Neumonía , Fibrosis Pulmonar , Animales , Cobre/toxicidad , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Neumonía/inducido químicamente , Neumonía/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , Líquido del Lavado Bronquioalveolar/química , Ratones Endogámicos C57BL , Pulmón/patología , Pulmón/efectos de los fármacos , Masculino , Nanopartículas del Metal/toxicidad , Nanopartículas del Metal/químicaRESUMEN
Ischemic stroke followed by reperfusion (IR) leads to extensive cerebrovascular injury characterized by neuroinflammation and brain cell death. Inhibition of matrix metalloproteinase-3 (MMP-3) emerges as a promising therapeutic approach to mitigate IR-induced stroke injury. We employed middle cerebral artery occlusion with subsequent reperfusion (MCAO/R) to model ischemic stroke in adult mice. Specifically, we investigated the impact of MMP-3 knockout (KO) on stroke pathophysiology using RNA sequencing (RNA-seq) of stroke brains harvested 48 h post-MCAO. MMP-3 KO significantly reduced brain infarct size following stroke. Notably, RNA-seq analysis showed that MMP-3 KO altered expression of 333 genes (252 downregulated) in male stroke brains and 3768 genes (889 downregulated) in female stroke brains. Functional pathway analysis revealed that inflammation, integrin cell surface signaling, endothelial- and epithelial-mesenchymal transition (EndMT/EMT), and apoptosis gene signatures were decreased in MMP-3 KO stroke brains. Intriguingly, MMP-3 KO downregulated gene signatures more profoundly in females than in males, as indicated by greater negative enrichment scores. Our study underscores MMP-3 inhibition as a promising therapeutic strategy, impacting multiple cellular pathways following stroke.
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Infarto Cerebral , Modelos Animales de Enfermedad , Accidente Cerebrovascular Isquémico , Metaloproteinasa 3 de la Matriz , Ratones Noqueados , Animales , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Masculino , Femenino , Ratones , Accidente Cerebrovascular Isquémico/genética , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/patología , Infarto Cerebral/genética , Infarto Cerebral/patología , Infarto Cerebral/metabolismo , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/patología , Ratones Endogámicos C57BL , Transcriptoma , Regulación de la Expresión Génica , Encéfalo/metabolismo , Encéfalo/patologíaRESUMEN
Keloids, marked by abnormal cellular proliferation and excessive extracellular matrix (ECM) accumulation, pose significant therapeutic challenges. Ethyl pyruvate (EP), an inhibitor of the high-mobility group box 1 (HMGB1) and TGF-ß1 pathways, has emerged as a potential anti-fibrotic agent. Our research evaluated EP's effects on keloid fibroblast (KF) proliferation and ECM production, employing both in vitro cell cultures and ex vivo patient-derived keloid spheroids. We also analyzed the expression levels of ECM components in keloid tissue spheroids treated with EP through immunohistochemistry. Findings revealed that EP treatment impedes the nuclear translocation of HMGB1 and diminishes KF proliferation. Additionally, EP significantly lowered mRNA and protein levels of collagen I and III by attenuating TGF-ß1 and pSmad2/3 complex expression in both human dermal fibroblasts and KFs. Moreover, metalloproteinase I (MMP-1) and MMP-3 mRNA levels saw a notable increase following EP administration. In keloid spheroids, EP induced a dose-dependent reduction in ECM component expression. Immunohistochemical and western blot analyses confirmed significant declines in collagen I, collagen III, fibronectin, elastin, TGF-ß, AKT, and ERK 1/2 expression levels. These outcomes underscore EP's antifibrotic potential, suggesting its viability as a therapeutic approach for keloids.
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Fibroblastos , Queloide , Piruvatos , Esferoides Celulares , Humanos , Queloide/metabolismo , Queloide/patología , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Piruvatos/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Factor de Crecimiento Transformador beta1/metabolismo , Proteína HMGB1/metabolismo , Proteína HMGB1/genética , Colágeno/metabolismo , Colágeno/biosíntesis , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Proteína Smad2/metabolismo , Proteína Smad2/genética , Proteína smad3/metabolismo , Regulación hacia Arriba/efectos de los fármacos , MasculinoRESUMEN
Fibrosis is a ubiquitous pathology, and prior studies have indicated that various artemisinin (ART) derivatives (including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA)) can reduce fibrosis in vitro and in vivo. The medicinal plant Artemisia annua L. is the natural source of ART and is widely used, especially in underdeveloped countries, to treat a variety of diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared the effects of A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART), on fibroblast viability and expression of key fibrotic marker genes after 1 and 4 days of treatment. AS, DHA, and Artemisia teas reduced fibroblast viability 4 d post-treatment in up to 80% of their respective controls. After 4 d of treatment, AS DHA and Artemisia teas downregulated ACTA2 up to 10 fold while ART had no significant effect, and AM increased viability by 10%. MMP1 and MMP3 were upregulated by AS, 17.5 and 32.6 fold, respectively, and by DHA, 8 and 51.8 fold, respectively. ART had no effect, but A. annua and A. afra teas increased MMP3 5 and 16-fold, respectively. Although A. afra tea increased COL3A1 5 fold, MMP1 decreased >7 fold with no change in either transcript by A. annua tea. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART alone but was less effective than A. afra. Immunofluorescent staining for smooth-muscle α-actin (α-SMA) correlated well with the transcriptional responses of drug-treated fibroblasts. Together, proliferation, qPCR, and immunofluorescence results show that treatment with ART, AS, DHA, and the two Artemisia teas yield differing responses, including those related to fibrosis, in human dermal fibroblasts, with evidence also of remodeling of fibrotic ECM.
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Artemisia , Artemisininas , Fibroblastos , Fibrosis , Humanos , Artemisininas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Artemisia/química , Extractos Vegetales/farmacología , Extractos Vegetales/química , Supervivencia Celular/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Actinas/metabolismo , Actinas/genética , Artesunato/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Arteméter/farmacología , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patologíaRESUMEN
BACKGROUND: Endoplasmic reticulum (ER) stress has been shown to play an important role in osteoarthritis (OA). OBJECTIVE: This study was aimed at assessing the relationship of endoplasmic reticulum (ER) stress-related glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) concentrations in the serum/synovial fluid (SF) with disease severity of primary knee osteoarthritis (pkOA). METHODS: Patients with pkOA together with healthy individuals were consecutively recruited from our hospital. The levels of GRP78 and CHOP in serum / SF were detected using enzyme-linked immunosorbent assay. The levels of IL-6 and MMP-3 were also examined. Radiographic progression of pkOA was evaluated based on Kellgren-Lawrence (K-L) grades. Receiver Operating Characteristic (ROC) curves were used to assess the diagnostic value of GRP78/CHOP levels with regard to K-L grades. The assessment of clinical severity was conducted using the visual analogue scale (VAS), Oxford knee score (OKS), and Lequesne algofunctional index (LAI). RESULTS: A total of 140 pkOA patients and 140 healthy individuals were included. Serum GRP78 and CHOP levels in pkOA patients were not significantly different from those in healthy individuals. The SF GRP78 and CHOP levels in healthy controls were not detected due to ethical reasons. Compared to those with K-L grade 2 and 3, the pkOA patients with K-L grade 4 had higher GRP78 and CHOP levels in the SF with statistical significance. In addition, the pkOA patients with K-L grade 3 exhibited drastically upregulated GRP78 and CHOP concentrations in the SF compared to those with K-L grade 2. Positive correlations of GRP78 and CHOP levels with K-L grades, IL-6, and MMP-3 levels in the SF were observed. ROC curve analysis indicated that both GRP78 and CHOP levels may act as decent indicators with regard to OA. GRP78 and CHOP concentrations in the SF were positively correlated with VAS/LAI score and negatively associated with OKS score. CONCLUSION: The study indicated that GRP78 and CHOP levels in the SF but not the serum were positively correlated with disease severity of pkOA.
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Osteoartritis de la Rodilla , Humanos , Osteoartritis de la Rodilla/diagnóstico por imagen , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Estudios Transversales , Chaperón BiP del Retículo Endoplásmico , Interleucina-6/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , Progresión de la EnfermedadRESUMEN
Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.
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Metaloproteinasa 3 de la Matriz , Inhibidor Tisular de Metaloproteinasa-1 , Humanos , Metaloproteinasa 10 de la Matriz/química , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/química , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Ingeniería de Proteínas , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
BACKGROUND: Geese exhibit relatively low reproductive performance, and follicular atresia is an important factor that restricts the egg production of geese. Systematic analysis of the regulation of follicle atresia in geese through transcriptome and proteome levels could provide meaningful information on clarifying the mechanism of follicle atresia in poultry. RESULT: The granulosa cell layer was loose, disintegrated and showed apoptosis in atretic follicles and remained intact in normal follicles. The hormone levels of FSH and LH were significantly decreased in the atresia follicles compared to the normal follicles (P < 0.05). A total of 954 differentially expressed genes (DEGs, 315 increased and 639 decreased) and 161 differentially expressed proteins (DEPs, 61 increased and 100 decreased) were obtained in atresia follicles compared to normal follicles, of which, 15 genes were differentially expressed in both transcriptome and proteome. The DEGs were mainly enriched in sodium transmembrane transport, plasma membrane, and transmembrane transporter activity based on the GO enrichment analysis and in the cell cycle pathway based on the KEGG enrichment analysis. The DEPs were mainly enriched in localization, lysosome, and phospholipid-binding based on the GO enrichment analysis. Candidate genes Smad2/3, Smad4, Annexin A1 (ANXA1), Stromelysin-1 (MMP3), Serine/threonine-protein kinase (CHK1), DNA replication licensing factor (MCM3), Cyclin-A2 (CCNA2), mitotic spindle assembly checkpoint protein (MAD2), Cyclin-dependent kinase 1 (CDK1), fibroblast growth factor 12 (FGF12), and G1/S-specific cyclin-D1 (CCND1) were possibly responsible for the regulation of atresia. CONCLUSION: The cell cycle is an important pathway for the regulation of follicular atresia. Sodium outflow and high expression of MMP3 and MMP9 could be responsible for structural destruction and apoptosis of follicular cells.
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Atresia Folicular , Gansos , Metaloproteinasa 3 de la Matriz , Animales , Femenino , Apoptosis/genética , Ciclo Celular/genética , Ciclinas/genética , Ciclinas/metabolismo , Atresia Folicular/genética , Gansos/genética , Células de la Granulosa/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Proteoma/metabolismo , TranscriptomaRESUMEN
Akt is a central node of many signaling pathways, which plays important roles in cell survival, proliferation, migration, metabolism and collagen synthesis. Conjunctivochalasis (CCH) is one of the most common age-related ocular superficial diseases related to abnormalities in conjunctival extracellular matrix. Here, we studied the role of Akt regulating collagens and MMPs in the pathogenesis of CCH. Primary conjunctival fibroblasts were obtained from CCH patients (n = 13) and age-matched normal controls (n = 10). The levels of Akt, collagen type I, collagen type III, MMP1, and MMP3 were determined by Western blot, qRT-PCR, immunohistochemistry, and immunofluorescence staining. Normal control conjunctival fibroblasts were treated with Akt inhibitor A6730, and CCH fibroblasts were transfected with Akt overexpression vector. The expression of Akt in CCH was significantly lower than that in normal control of conjunctival tissues and cultured fibroblasts. Blocking Akt signaling with Akt inhibitor could inhibit the expression of collagen type I and collagen type III and upregulate the expression of MMP1 and MMP3. Meanwhile, compared with CCH fibroblasts transfected with control mimics, the protein and mRNA expression of collagen type I and collagen type III were increased significantly in Akt overexpression group, while the results of MMP1 and MMP3 in transfected fibroblasts were opposite. Taken together, Akt upregulated the expression of collagen type I and collagen type III and downregulated the expression of MMP1 and MMP3. Akt signaling pathway could provide a direct negative contribution to CCH and might be an attractive target for CCH therapy.
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Colágeno , Enfermedades de la Conjuntiva , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 3 de la Matriz , Proteínas Proto-Oncogénicas c-akt , Humanos , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III , Enfermedades de la Conjuntiva/metabolismo , Fibroblastos/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Post-traumatic osteoarthritis in the temporomandibular joint (TMJ OA) is associated dysfunctional cellmatrix mediated signalling resulting from changes in the pericellular microenvironment after injury. Matrix metalloproteinase (MMP)-13 is a critical enzyme in biomineralisation and the progression of OA that can both degrade the extracellular matrix and modify extracellular receptors. This study focused on MMP-13 mediated changes in a transmembrane proteoglycan, Neuron Glial antigen 2 (NG2/CSPG4). NG2/CSPG4 is a receptor for type VI collagen and a known substrate for MMP-13. In healthy articular layer chondrocytes, NG2/CSPG4 is membrane bound but becomes internalised during TMJ OA. The objective of this study was to determine if MMP-13 contributed to the cleavage and internalisation of NG2/CSPG4 during mechanical loading and OA progression. Using preclinical and clinical samples, it was shown that MMP-13 was present in a spatiotemporally consistent pattern with NG2/CSPG4 internalisation during TMJ OA. In vitro, it was illustrated that inhibiting MMP-13 prevented retention of the NG2/CSPG4 ectodomain in the extracellular matrix. Inhibiting MMP-13 promoted the accumulation of membrane-associated NG2/CSPG4 but did not affect the formation of mechanical-loading dependent variant specific fragments of the ectodomain. MMP- 13 mediated cleavage of NG2/CSPG4 is necessary to initiate clathrin-mediated internalisation of the NG2/ CSPG4 intracellular domain following mechanical loading. This mechanically sensitive MMP-13-NG2/CSPG4 axis affected the expression of key mineralisation and OA genes including bone morphogenetic protein 2, and parathyroid hormone-related protein. Together, these findings implicated MMP-13 mediated cleavage of NG2/CSPG4 in the mechanical homeostasis of mandibular condylar cartilage during the progression of degenerative arthropathies such as OA.
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Cartílago Articular , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Homeostasis , Cóndilo Mandibular , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , NeuronasRESUMEN
OBJECTIVE: The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity of BBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. METHODS: The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. RESULTS: Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5 µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. CONCLUSION: MMP-9 and MMP-3 disrupt the BBB by degrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic protein against MMP-related diseases where BBB disruption plays a role.
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Células Endoteliales , Inhibidor Tisular de Metaloproteinasa-1 , Ratas , Animales , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Células Endoteliales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/farmacología , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/farmacología , Uniones Estrechas/metabolismo , Encéfalo/metabolismo , Barrera Hematoencefálica/metabolismoRESUMEN
BACKGROUND AND AIMS: Helicobacter pylori (H. pylori)-induced gastric pathology involves remodeling of extracellular matrix mediated by aberrant activity of matrix metalloproteinases (MMPs). We have previously shown that in vitro H. pylori infection leads to MMP-3 and MMP-9 overexpression, associated with phosphorylation of bacterial oncoprotein CagA. We extended these findings in an in vivo model of H. pylori infection and further assessed the involvement of MAPK pathways in MMP expression. MATERIALS AND METHODS: C57BL/6 mice were infected with H. pylori strains HPARE, HPARE ΔCagA, and SS1, for 6 and 9 months. Transcriptional expression of Mmp-3 and Mmp-9 was evaluated via qPCR while respective protein levels in the gastric mucosa were determined immunohistochemically. Epithelial cell lines AGS and GES-1 were infected with H. pylori strain P12 in the presence of chemical inhibitors of JNK, ERK1/2, and p38 pathways, for 24 h. mRNA and protein expression of MMP-3 and MMP-9 were determined via qPCR and Western blot, respectively. RESULTS: We observed transcriptional activation of Mmp-3 and Mmp-9 as well as aberrant MMP-3 and MMP-9 protein expression in murine gastric tissue following H. pylori infection. CagA expression was associated with MMP upregulation, particularly during the early time points of infection. We found that inhibition of ERK1/2 resulted in reduced mRNA and protein expression of MMP-3 and MMP-9 during H. pylori infection, in both cell lines. Expressed protein levels of both MMPs were also found reduced in the presence of JNK pathway inhibitors in both cell lines. However, p38 inhibition resulted in a more complex effect, probably attributed to the accumulation of phospho-p38 and increased phospho-ERK1/2 activity due to crosstalk between MAPK pathways. CONCLUSIONS: H. pylori colonization leads to the upregulation of MMP-3 and MMP-9 in vivo, which primarily involves ERK1/2 and JNK pathways. Therefore, their inhibition may potentially offer a protective effect against gastric carcinogenesis and metastasis.
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Infecciones por Helicobacter , Helicobacter pylori , Metaloproteinasa 3 de la Matriz , Metaloproteinasa 9 de la Matriz , Animales , Ratones , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Células Epiteliales/metabolismo , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos C57BL , ARN MensajeroRESUMEN
The exposure to UVA (320-400 nm) irradiation is a major threat to human skin concerning photoaging and carcinogenesis. It has been shown that UVA irradiation can induce reactive oxygen species (ROS) and DNA mutations, such as 8-hydroxydeoxyguanosine. Furthermore, UVA induces the expression of photoaging-associated matrix metalloproteases (MMPs), especially of matrix metalloprotease 1 (MMP 1) and matrix metalloprotease 3 (MMP 3). In addition to this, it was recently shown that UVA-induced ROS also increase glucose metabolism of melanoma cells, however, the influence of UVA on the glucose metabolism of non-malignant cells of the human skin has, so far, not been investigated in detail. Here, we investigated the UVA-induced changes in glucose metabolism and the functional relevance of these changes in primary fibroblasts-normal non-malignant cells of the skin. These cells showed an UVA-induced enhanced glucose consumption and lactate production and changes in pyruvate production. As it has been proposed that pyruvate could have antioxidant properties we tested the functional relevance of pyruvate as protective agent against UVA-induced ROS. Our initial experiments support earlier publications, demonstrating that pyruvate treated with H2O2 is non-enzymatically transformed to acetate. Furthermore, we show that this decarboxylation of pyruvate to acetate also occurs upon UVA irradiation. In addition to this, we could show that in fibroblasts pyruvate has antioxidant properties as enhanced levels of pyruvate protect cells from UVA-induced ROS and partially from a DNA mutation by the modified base 8-hydroxydeoxyguanosine. Furthermore, we describe for the first time, that the interaction of UVA with pyruvate is relevant for the regulation of photoaging-associated MMP 1 and MMP 3 expression.
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Antioxidantes , Envejecimiento de la Piel , Humanos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Especies Reactivas de Oxígeno/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Peróxido de Hidrógeno/metabolismo , Piel/efectos de la radiación , Glucosa , Piruvatos/farmacología , Piruvatos/metabolismo , Rayos Ultravioleta , Fibroblastos/metabolismo , Células CultivadasRESUMEN
Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1ß (IL-1ß)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1ß was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1ß-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.