RESUMEN
The senescence of aortic valve interstitial cells (VICs) plays a critical role in the progression of calcific aortic valve disease (CAVD). However, the precise mechanisms underlying the senescence of VICs remain unclear, demanding the identification of a novel target to mitigate this process. Previous studies have highlighted the anti-aging potential of morusin. Thus, this study aimed to explore the therapeutic potential of morusin in CAVD. Cellular experiments reveal that morusin effectively suppresses cellular senescence and cause a shift toward osteogenic differentiation of VICs in vitro. Mechanistically, morusin activate the Nrf2-mediated antiaging signaling pathway by downregulating CCND1 expression and aiding Keap1 degradation through Trim 25. This activation lead to the upregulated expression of antioxidant genes, thus reducing reactive oxygen species production and thereby preventing VIC osteogenic differentiation. In vivo experiments in ApoE-/- mice on a high-fat Western diet demonstrate the positive effect of morusin in mitigating aortic valve calcification. These findings emphasize the antiaging properties of morusin and its potential as a therapeutic agent for CAVD.
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Estenosis de la Válvula Aórtica , Calcinosis , Senescencia Celular , Flavonoides , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/patología , Calcinosis/metabolismo , Calcinosis/genética , Senescencia Celular/efectos de los fármacos , Ciclina D1/metabolismo , Ciclina D1/genética , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Flavonoides/administración & dosificaciónRESUMEN
BACKGROUND AND AIMS: Although calcific aortic valve disease (CAVD) is a common valvular disease among elderly populations and its incidence has markedly increased in recent decades, the pathogenesis of CAVD remains unclear. In this study, we explored the potential role of interleukin (IL)-22 and the underlying molecular mechanism in CAVD. METHODS AND RESULTS: Our results showed that IL-22 was upregulated in calcific aortic valves from CAVD patients, and its main sources were CD3+ T cells and CD68+ macrophages. Human aortic valve interstitial cells (VICs) expressed the IL-22-specific receptor IL-22R1, and IL-22R1 expression also was elevated in calcified valves. Treatment of cultured human VICs with recombinant human IL-22 resulted in markedly increased expression of osteogenic proteins Runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), as well as increased matrix calcium deposition. Moreover, siRNA silencing of IL-22R1 blocked the pro-osteogenic effect of IL-22 in VICs. In IL-22-treated VICs, we also observed increased phosphorylation of JAK3 and STAT3 and nuclear translocation of STAT3. Pretreatment with a specific JAK3 inhibitor, WHIP-154, or siRNA knockout of STAT3 effectively mitigated the IL-22-induced osteoblastic trans-differentiation of human VICs. CONCLUSIONS: Together, these data indicate that IL-22 promotes osteogenic differentiation of VICs by activating JAK3/STAT3 signaling. Based on our results demonstrating a pro-osteogenic role of IL-22 in human aortic valves, pharmacological inhibition of IL-22 signaling may represent a potential strategy for alleviating CAVD.
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Estenosis de la Válvula Aórtica , Válvula Aórtica , Calcinosis , Interleucina-22 , Anciano , Humanos , Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/metabolismo , Células Cultivadas , Osteogénesis , ARN Interferente Pequeño/metabolismoRESUMEN
Background: Type A aortic dissection (TAAD) is a cardiovascular emergency condition with high mortality rate. Hybrid total aortic arch replacement using endovascular graft for the descending aorta repair results in favorable outcomes and has been recommended as an alternative procedure for the higher-risk category patients. Our institution started applying the upper ministernotomy incision technique for the hybrid procedures back in 2018. Methods: We collected patients who underwent hybrid total arch replacement (HTAR) via ministernotomy (96) and total arch replacement with frozen elephant trunk (TAR + FET) procedures (99), between 2018 and 2021. The baseline information, intraoperative and postoperative characteristics have been compared. Kaplan-Meier analysis was used for survival evaluation. Cox regression were applied to identify the independent predictor of mortality. Results: The baseline characteristics between the two patient groups were compared and found similar, except that RBC counts were higher (p = 0.038) and the ascending aorta diameter was smaller (P = 0.019) in the "HTAR" group relative to the "TAR + FET" group. The cardiopulmonary bypass time (P < 0.001), the aortic cross clamp time (P < 0.001), the operation duration (P = .029), ICU (P = 0.037) and postoperative hospital stay (P = 0.002) were shorter in the "HTAR" group. The "HTAR" group exhibited also significantly lower levels of intraoperative transfusion (all <0.001) characteristics than the "TAR + FET" group. The hospital mortality and 1-year mortality revealed similar patterns in both groups. Conclusion: HTAR via ministernotomy have similar short term prognosis, and also reduced the ICU and postoperative hospital stay. In all, The application of the ministernotomy technique in HTAR was safe and technically feasible and may benefit individual patients as well as hospitals in general.
RESUMEN
BACKGROUND: IRF4 is the pioneer factor for effector T cell maturation. Here we investigated the function of IRF4 in maintaining OX40-related T cell responses following alloantigen activation in a mouse heart transplantation model. METHODS: Irf4flox/flox mice were bred with Ox40cre/+ mice to generate Irf4flox/floxOx40cre/+ mice. Wild type C57BL/6, Irf4flox/floxOx40cre/+ mice were transplanted with BALB/c heart allografts, with or without BALB/c skin-sensitization. CD4+ TEa T cells co-transfer experiments and flow cytometric analysis were conducted to investigate the amount of CD4+ T cells and the percentage of the T effector subset. RESULTS: Irf4flox/floxOx40cre/+ and Irf4flox/floxOx40cre/+ TEa mice were constructed successfully. IRF4 ablation in activated OX40-mediated alloantigen specific CD4+ TEa T cells reduced effector T cell differentiation (CD44hiCD62Llo, Ki67, IFN-γ), which caused long-term allograft survival (> 100 d) in the chronic rejection model. In the donor skin-sensitized heart transplantation model, the formation and function of alloantigen-specific memory CD4+ TEa cells were also impaired in Irf4flox/floxOx40cre/+ mice. Additionally, deletion of IRF4 after T cell activation in Irf4flox/floxOx40cre/+ mice reduced T cell reactivation in vitro. CONCLUSIONS: IRF4 ablation after OX40-related T cell activation could reduce effector and memory T cell formation and inhibit their function in response to alloantigen stimulation. These findings could have significant implications in targeting activated T cells to induce transplant tolerance.
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Trasplante de Corazón , Células T de Memoria , Animales , Ratones , Memoria Inmunológica , Isoantígenos , Células T de Memoria/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
INTRODUCTION: Calcific aortic valve disease (CAVD) is an active and cellular-driven fibrocalcific process characterised by differentiation of valve interstitial cells (VICs) towards an osteogenic-like phenotype. A recently identified lncRNA, lncTSI, has been reported to inhibit fibrogenesis through transforming growth factor (TGF)-ß/Smad3 pathway. Here, the present study aimed to investigate the role of lncTSI in CAVD. METHODS: The effect of TGF-ß1 on lncTSI of VICs was measured. TGF-ß1, RUNX2 and collagen I expression between calcified aortic valve tissue and normal samples by immunohistochemistry and western blotting. Human VICs were cultured and treated with TGF-ß1. SiRNA and pcDNA3.1-lncTSI plasmid transfection were used to silence and overexpress lncTSI in VICs for 48 h, Smads phosphorylation, RUNX2 and collagen I expression were then verified by western blotting. In ApoE-/- mice fed with 0.25 % high-cholesterol diet, AAV2-lncTSI were injected intravenously to observe their effect on the formation of aortic valve calcification. RESULTS: lncTSI was highly expressed in VICs treated with TGF-ß1. lncTSI was transcriptionally regulated by Smad3 and reversely inhibited TGF-ß1-induced Smad3 phosphorylation and downregulated profibrotic gene expression. Silencing lncTSI increased TGF-ß1-induced Smad3 phosphorylation, and subsequently, upregulated RUNX2 and collagen I expressions in VICs. While overexpression of lncTSI reversed the production of RUNX2 and collagen I in VICs. In a mouse CAVD model of 24 week 0.25 % high-cholesterol diet feeding, overexpression of lncTSI significantly reduced calcium deposition, RUNX2, pSmad3, and collagen I expression in aortic valve leaflets, with less aortic valve stenosis. CONCLUSIONS: The novel findings of present study suggested that lncTSI alleviated aortic valve calcification through negative regulation of the TGF-ß/Smad3 pathway. The results may help elucidate new diagnostic and therapeutic targets to prevent CAVD progression.
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Estenosis de la Válvula Aórtica , Válvula Aórtica , ARN Largo no Codificante , Animales , Humanos , Masculino , Ratones , Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/metabolismo , Células Cultivadas , Colesterol/metabolismo , Colágeno/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Osteogénesis , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , ARN Largo no Codificante/genéticaRESUMEN
Interleukin 17A (IL17A) is reported to be involved in many inflammatory processes, but its role in aortic valve diseases remains unknown. We examined the role of IL17A based on an ApoE-/- mouse model with strategies as fed with high-fat diet or treated with IL17A monoclonal antibody (mAb). 12 weeks of high-fat diet feeding can elevate cytokines secretion, inflammatory cells infiltration and myofibroblastic transition of valvular interstitial cells (VICs) in aortic valve. Moreover, diet-induction accelerated interleukin 17 receptor A (IL17RA) activation in VICs. In an IL17A inhibition model, the treatment group was intra-peritoneally injected with anti-IL17A mAb while controls received irrelevant antibody. Functional blockade of IL17A markedly reduced cellular infiltration and transition in aortic valve. To investigate potential mechanisms, NF-κB was co-stained in IL17RA+ VICs and IL17RA+ macrophages, and further confirmed by Western blotting in VICs. High-fat diet could activate NF-κB nuclear translocation in IL17RA+ VICs and IL17RA+ macrophages and this process was depressed after IL17A mAb-treatment. In conclusion, high-fat diet can lead to IL17A upregulation, VICs myofibroblastic transition and inflammatory cells infiltration in the aortic value of ApoE-/- mice. Blocking IL17A with IL17A mAb can alleviate aortic valve inflammatory states.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Estenosis de la Válvula Aórtica/tratamiento farmacológico , Apolipoproteínas E/deficiencia , Dieta Alta en Grasa/efectos adversos , Interleucina-17/genética , Receptores de Interleucina-17/genética , Animales , Anticuerpos Monoclonales/farmacología , Estenosis de la Válvula Aórtica/inducido químicamente , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/metabolismo , Núcleo Celular/metabolismo , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Humanos , Inyecciones Intraperitoneales , Interleucina-17/antagonistas & inhibidores , Masculino , Ratones , FN-kappa B/metabolismo , Transporte de Proteínas , Receptores de Interleucina-17/metabolismo , Regulación hacia ArribaRESUMEN
Atherosclerosis is a leading cause of mortality worldwide. Artery tertiary lymphoid organ (ATLO) neogenesis is affected by abdominal aorta atherosclerosis, which may lead to an immune response. The present study obtained microarray data to investigate the gene expression differences underlying the potential pathogenesis of atherosclerosis and to elucidate the mechanisms underlying ATLO development. Microarray studies of the aorta, plaques, adventitia, blood, spleen, renal lymph nodes and ATLO were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in aorta clusters and ATLO clusters. Kyoto Encyclopedia of Genes and Genomes enrichment and Gene Ontology (GO) analyses were conducted to predict the biological functions of DEGs. The results demonstrated that interleukin 7 receptor (Il7r), CXC motif chemokine ligand (Cxcl)16, Cxcl13, Cxcl12, CC motif chemokine receptor 2, CC motif chemokine ligand (Ccl)8, Ccl5 and Ccl12 may function through pathways associated with 'cytokinecytokine receptor interaction' and 'chemokine signaling pathway' in ATLO. Gene expression alterations were validated by reverse transcriptionquantitative polymerase chain reaction. Il7r appeared to be the central gene involved in these events, and chemokines and/or chemokine receptors were visualized by GO enrichment. A proteinprotein interaction network was constructed, which suggested that Il7r had a core function in all clusters. Taken together, the results indicated that Il7r upregulation may serve an important role in ATLO development via 'cytokinecytokine receptor interaction' and 'chemokine signaling pathway'. This may provide novel perspectives for understanding ATLO development and the regulation of the immune response in atherosclerosis.
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Aterosclerosis/genética , Aterosclerosis/metabolismo , Redes Reguladoras de Genes , Tejido Linfoide/irrigación sanguínea , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Transducción de Señal , Factores de Edad , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/patología , Biomarcadores , Biología Computacional/métodos , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Ontología de Genes , Tejido Linfoide/patología , Masculino , Ratones , Ratones Noqueados , TranscriptomaRESUMEN
Osteoblastic differentiation of aortic valve interstitial cells (AVICs) is the central process in the development of calcific aortic valve disease (CAVD). Metformin is a widely used first-line antidiabetic drug, and recently, pleiotropic benefits of metformin beyond hypoglycemia have been reported in the cardiovascular system. Here, we examined the effect of metformin on the osteoblastic differentiation of human AVICs. Our results showed that metformin ameliorated TGF-ß1-induced production of osteogenic proteins Runx2 and osteopontin as well as calcium deposition in the cultured human AVICs. Experiments using AICAR, Compound C and AMPKα siRNA showed that the beneficial effect of metformin on TGF-ß1-induced osteoblastic differentiation of human AVICs was mediated by AMPKα. Moreover, metformin inhibited the TGF-ß1-induced activation of ß-catenin, and ß-catenin siRNA blocked TGF-ß1-induced osteoblastic differentiation of AVICs. Smad2/3 and JNK were phosphorylated to promote the TGF-ß1-induced activation of ß-catenin and osteoblastic differentiation of AVICs, and metformin also alleviated TGF-ß1-induced activation of Smad2/3 and JNK. In conclusion, our results suggest a beneficial effect of metformin based on the prevention of osteoblastic differentiation of human AVICs via inhibition of ß-catenin, which indicates the therapeutic potential of metformin for CAVD.
Asunto(s)
Válvula Aórtica/citología , Diferenciación Celular/efectos de los fármacos , Metformina/farmacología , Osteoblastos/citología , Transducción de Señal , Factor de Crecimiento Transformador beta1/farmacología , beta Catenina/metabolismo , Adenilato Quinasa/metabolismo , Adulto , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Persona de Mediana Edad , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Aims: Emerging evidence indicates that long non-coding RNAs (lncRNAs) play a vital role in cardiovascular physiology and pathology. Although the lncRNA TUG1 is implicated in atherosclerosis, its function in calcific aortic valve disease (CAVD) remains unknown. Methods and results: In this study, we found that TUG1 was highly expressed in human aortic valves and primary valve interstitial cells (VICs). Moreover, TUG1 knockdown induced inhibition of osteoblast differentiation in CAVD both in vitro and in vivo. Mechanistically, silencing of TUG1 increased the expression of miR-204-5p and subsequently inhibited Runx2 expression at the post-transcriptional level. Importantly, TUG1 directly interacted with miR-204-5p and downregulation of miR-204-5p efficiently reversed the suppression of Runx2 induced by TUG1 short hairpin RNA (shRNA). Thus, TUG1 positively regulated the expression of Runx2, through sponging miR-204-5p, and promoted osteogenic differentiation in CAVD. Conclusion: All together, the evidence generated by our study elucidates the role of lncRNA TUG1 as a miRNA sponge in CAVD, and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD.
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Válvula Aórtica/metabolismo , Calcinosis/metabolismo , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Enfermedades de las Válvulas Cardíacas/metabolismo , MicroARNs/metabolismo , Osteoblastos/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Válvula Aórtica/patología , Calcinosis/genética , Calcinosis/patología , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Enfermedades de las Válvulas Cardíacas/genética , Enfermedades de las Válvulas Cardíacas/patología , Humanos , Ratones Noqueados para ApoE , MicroARNs/genética , Osteoblastos/patología , ARN Largo no Codificante/genética , Transducción de SeñalRESUMEN
Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type â ¡ collagen. The ER stress-mediated apoptosis of tunicamycin (TM)stimulated chondrocytes was detected using 4-phenylbutyric acid (4PBA). We found that 4PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4',6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TMinduced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), Xbox binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBPhomologous protein (Chop), caspase9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3 and Bax were significantly decreased, whereas the mRNA and protein expression levels of Xbp1 and Bcl-2 were significantly increased compared with the TMstimulated chondrocytes not treated with BZD. Additionally, all our findings demonstrated that there was no significant difference between the TMstimulated chondrocytes treated with BZD and those treated with 4PBA. Taken together, our results indicate that BZD inhibits TMinduced chondrocyte apoptosis mediated by ER stress. Thus, BZD may be a potential therapeutic agent for use in the treatment of OA.
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Apoptosis/efectos de los fármacos , Condrocitos/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Tunicamicina/farmacología , Animales , Western Blotting , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fenilbutiratos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-Dawley , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Our previous study showed that Duhuo Jisheng decoction (DHJSD) inhibited chondrocyte apoptosis by the mitochondria-dependent signaling pathway. Endoplasmic reticulum (ER) stress is upstream of the mitochondria-dependent signaling pathway and has been shown to promote chondrocyte apoptosis that occurs in osteoarthritis (OA). The present study aimed to evaluate whether DHJSD inhibits the chondrocyte apoptosis by regulating ER stress. DHJSD enhanced the viability of tunicamycin (TM)exposed chondrocytes, a model of ER stress-induced apoptosis, in a dose and timedependent manner, as shown by MTT assay. The present results showed that DHJSD and sodium 4-phenylbutyrate (PBA), an ER stress inhibitor, reduced TMinduced chondrocyte apoptosis by 4',6-diamidino2-phenylindole staining. To gain insight into the mechanisms of DHJSD that are responsible for enhancing the viability and inhibiting TMinduced chondrocyte apoptosis, the associated mRNA expressions and protein levels were detected by reverse transcriptionpolymerase chain reaction (RTPCR) and western blot analysis, respectively. The results showed that the expression levels of Xbp1, Xbp1s and Bcl2 were increased, and the expression levels of Bip, Atf4, Chop, Bax, caspase9 and 3 were decreased in the TMexposed chondrocytes treated with DHJSD or PBA compared with that in the TMexposed chondrocytes. To identify the possible mechanisms, the expression of miR34a was examined by the TaqMan microRNA assay, and was downregulated in the TMexposed chondrocytes treated with DHJSD or PBA compared with that in the TM-exposed chondrocytes. DHJSD inhibits ER stress in chondrocytes induced by exposure to TM by downregulating miR34a, suggesting that DHJSD may be a potential therapeutic agent for OA.
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Condrocitos/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , MicroARNs/genética , Tunicamicina/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Regulación hacia Abajo/genética , Estrés del Retículo Endoplásmico/genética , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Fenilbutiratos/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wildtype mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpressionassociated congenital dysplasia of the TMJ in mice.
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Enfermedades del Desarrollo Óseo/patología , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Trastornos de la Articulación Temporomandibular/patología , Animales , Apoptosis , Enfermedades del Desarrollo Óseo/congénito , Enfermedades del Desarrollo Óseo/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Cavidad Glenoidea/metabolismo , Proteínas Hedgehog/genética , Proteínas de Homeodominio/genética , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Factor de Transcripción SOX9/metabolismo , Trastornos de la Articulación Temporomandibular/congénito , Trastornos de la Articulación Temporomandibular/metabolismo , Regulación hacia ArribaRESUMEN
Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation. The present study, therefore, focused on the effect of BZD on chondrocyte proliferation, exploring the mechanism of BZD on the inhibition of cartilage degradation. Chondrocytes isolated from the knee articular cartilage of Sprague Dawley rats were cultured and identified by type II collagen immunohistochemistry. It was found that BZD promoted chondrocyte viability in a dose- and time-dependent manner. To investigate if BZD promoted the chondrocyte viability by stimulating the cell cycle progression a flow cytometer was used, and the results showed that the percentage proportion of G0/G1 cells was significantly lower, and the percentage proportion of S cells was significantly higher, in treated cells compared with that in untreated cells. To gain insight into the mechanism underlying the effect of BZD on the cell cycle progression, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and p21 was measured by reverse transcription-polymerase chain reaction and western blotting, respectively. The mRNA and protein expression of cyclin D1, CDK4 and CDK6 in the BZD-treated chondrocytes was significantly upregulated, while the mRNA and protein expression of p21 was significantly downregulated, compared with that in the untreated chondrocytes. These results suggested that BZD promoted chondrocyte proliferation by accelerating G1/S transition, indicating that BZD is a potential therapeutic agent for the treatment of OA.
RESUMEN
Indian hedgehog (Ihh), one of the hedgehog gene families, is indicated in the regulation of chondrocyte differentiation. Tougu Xiaotong formula (TXF), a traditional Chinese medicinal compound, has been used for the treatment of bone and joint disease. However, the underlying molecular mechanisms of TXF on the function of bone marrow stromal cells (BMSCs) remain unclear. In the present study, the affect of TXF on proliferation and chondrogenic differentiation was investigated in primary BMSCs from fourweekold Sprague Dawley rats. The cell viability in BMSCs treated with TXF was higher compared to the untreated cells. Additionally, the percentage of G(0)/G(1) phase cells was significantly decreased, whereas that of the S phase cells was significantly increased. Furthermore, following TXF treatment, cyclin D1, cyclindependent kinase 4 (CDK4) and CDK6 expression in BMSCs was significantly enhanced. The results showed that TXF had no cytotoxicity to BMSCs. To explore the effect of TXF on the differentiation in BMSCs, whether TXF induced chondrogenic differentiation of BMSCs by the regulation of Ihh signaling pathway was investigated. The protein expression of Ihh, Patched and Smoothened in the induction group were significantly increased when compared to those in the control group, and the highest protein level of Ihh was in the induction group that was treated with the combination of TXF and transforming growth factorß1 (TGFß1). In addition, TXF combined with TGFß1 significantly induced the protein expression of cartilage oligomeric matrix protein and collagen II compared to the TGFß1 group. Taken together, these results indicate that TXF promotes the proliferation via accelerating the G(1)/S transition, and induces chondrogenic differentiation in BMSCs by activation of the Ihh signaling pathway in association with TGFß1.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Animales , Ciclo Celular/efectos de los fármacos , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Medicamentos Herbarios Chinos/química , Expresión Génica , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratas , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Chondrocyte apoptosis activated by the mitochondrial-dependent signaling pathway plays a crucial role in the cartilage degeneration of osteoarthritis. Duhuo Jisheng decoction (DHJSD), a herbal formula from traditional Chinese medicine, has been widely used for treating osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effect of DHJSD remain to be elucidated. In the present study, the effects of DHJSD on the mitochondrial-dependent signaling pathway in sodium nitroprussiate (SNP)-induced chondrocyte apoptosis were investigated. Chondrocytes, from the knee articular cartilage of Sprague Dawley rats, were identified by type II collagen immunohistochemistry. The chondrocytes, stimulated with or without SNP to induce apoptosis, were treated by DHJSD for various concentrations and times. The viability of SNP-induced chondrocytes treated with DHJSD was enhanced compared to SNP-induced chondrocytes in a dose- and time-dependent manner, as assessed by the MTT assay. The apoptosis of SNP-induced chondrocytes treated by DHJSD was significantly decreased compared to SNP-induced chondrocyte, as shown by 4',6-diamidino-2-phenylindole and Annexin V/propidium iodide staining. The mitochondrial membrane potential (∆Ψm) of SNP-induced chondrocytes treated by DHJSD was significantly decreased compared to SNP-induced chondrocyte, as shown by JC-1 staining. To understand the mechanism, the mRNA and protein levels of Bax, B-cell lymphoma 2 (Bcl-2), caspase-9 and caspase-3 were detected by reverse transcriptionpolymerase chain reaction and western blot analysis, respectively. In SNP-induced chondrocyte treated by DHJSD, the Bcl-2 expression was increased, whereas the expression of Bax, caspase-9 and caspase-3 was decreased compared to SNP-induced chondrocyte. Taken together, these results indicated that DHJSD inhibits the apoptosis of SNP-induced chondrocyte by the mitochondrial-dependent apoptotic pathway, and this may partly explain its therapeutic efficacy for OA.
Asunto(s)
Apoptosis/efectos de los fármacos , Condrocitos/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Mitocondrias/efectos de los fármacos , Nitroprusiato/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Cartílago Articular/citología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 9/genética , Caspasa 9/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/fisiología , Donantes de Óxido Nítrico/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Achyranthes bidentata polysaccharides (ABPS) are the active components of Radix Achyranthis Bidentatae (AB), which has been extensively used in Traditional Chinese medicine (TCM) in the treatment of osteoarthritis (OA). Our previous study provided evidence that ABPS regulated the G1/S transition to promote chondrocyte proliferation. However, the precise mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of ABPS on the Wnt/ßcatenin signaling pathway in chondrocytes. Chondrocytes, obtained from the knee cartilage of Sprague-Dawley rats, were identified by type II collagen immunohistochemistry. ABPS upregulated the expression of Wnt-4, Frizzled-2, ß-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3ß (GSK-3ß), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced ß-catenin nuclear translocation. Importantly, the expression of ß-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/ß-catenin signaling pathway. In addition, we found that ABPS increased the expression of type II collagen in chondrocytes. These results suggest that ABPS promote chondrocyte proliferation by activating the Wnt/ß-catenin signaling pathway.
Asunto(s)
Achyranthes/química , Condrocitos/citología , Condrocitos/metabolismo , Polisacáridos/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/enzimología , Colágeno Tipo II/metabolismo , Ciclina D1/metabolismo , Receptores Frizzled/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , beta Catenina/metabolismoRESUMEN
Our previous study reported that inactivation of Shox2 led to dysplasia and ankylosis of the temporomandibular joint (TMJ), and that replacing Shox2 with human Shox partially rescued the phenotype with a prematurely worn out articular disc. However, the mechanisms of Shox2 activity in TMJ development remain to be elucidated. In this study, we investigated the molecular and cellular basis for the congenital dysplasia of TMJ in Wnt1-Cre; pMes-stop Shox2 mice. We found that condyle and glenoid fossa dysplasia occurs primarily in the second week after the birth. The dysplastic TMJ of Wnt1-Cre; pMes-stop Shox2 mice exhibits a loss of Collagen type I, Collagen type II, Ihh and Gli2. In situ zymography and immunohistochemistry further demonstrate an up-regulation of matrix metalloproteinases (MMPs), MMP9 and MMP13, accompanied by a significantly increased cell apoptosis. In addition, the cell proliferation and expressions of Sox9, Runx2 and Ihh are no different in the embryonic TMJ between the wild type and mutant mice. Our results show that overexpression of Shox2 leads to the loss of extracellular matrix and the increase of cell apoptosis in TMJ dysplasia by up-regulating MMPs and down-regulating the Ihh signaling pathway.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Articulación Temporomandibular/fisiopatología , Animales , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación hacia Abajo , Embrión de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Expresión Génica , Técnicas de Sustitución del Gen , Proteínas Hedgehog/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Ratones , Fenotipo , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Transducción de Señal , Articulación Temporomandibular/patologíaRESUMEN
We have previously reported that Tougu Xiaotong capsule (TXC) inhibits tidemark replication and cartilage degradation by regulating chondrocyte autophagy in vivo. Autophagy, a cell protective mechanism for maintaining cellular homeostasis, has been shown to be a constitutively active and protective process for chondrocyte survival. However, it remains unclear whether TXC promotes chondrocyte autophagy by regulating the autophagy-related (Atg)12/microtubule-associated protein 1 light chain 3 (LC3) conjugation systems. Thus, in the present study, we investigated the effects of TXC on primary chondrocytes treated with cobalt chloride (CoCl2). We found that CoCl2 induced a decrease in chondrocyte viability and the autophagosome formation of chondrocytes, indicating that CoCl2 induced autophagic death in a dose- and time-dependent manner. To determine the effects of TXC on CoCl2-exposed chondrocytes, we assessed cell viability by MTT assay. Our results revealed that TXC enhanced the viability of CoCl2-exposed chondrocytes. To gain insight into the mechanisms responsible for the enhancing effects of TXC on CoCl2-exposed chondrocytes, the expression of Atg genes was assessed in chondrocytes exposed to CoCl2 and treated with or without TXC. The results revealed that the expression of beclin 1, Atg3, Atg5, Atg7, Atg10, Atg12 and LC3 II/LC3 I in the chondrocytes treated with TXC increased, compared to that in the untreated chondrocytes. In addition, ultrastructural analysis indicated that treated chondrocytes contained more autophagosomes than the untreated cells, suggesting that TXC increased the formation of autophagosomes in the chondrocytes to clear the CoCl2-induced autophagic death. Therefore, these data suggest that TXC is a potential therapeutic agent for the reduction of cartilage degradation that occurs in osteoarthritis.
Asunto(s)
Autofagia/efectos de los fármacos , Medicamentos Herbarios Chinos/administración & dosificación , Proteínas Asociadas a Microtúbulos/biosíntesis , Osteoartritis/tratamiento farmacológico , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/biosíntesis , Apoptosis/efectos de los fármacos , Proteína 12 Relacionada con la Autofagia , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Línea Celular , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Cobalto/toxicidad , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Fagosomas/efectos de los fármacos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genéticaRESUMEN
Bauhinia championi (Benth.) Benth. polysaccharides (BCBPs), extracted from Bauhinia championi (Benth.) Benth., which has been used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA), are the bioactive constituents of Bauhinia championi (Benth.) rattan. However, the molecular mechanisms responsible for their effects on OA are poorly understood. The Wnt/ß-catenin signaling pathway plays an important role in the proliferation of chondrocytes. In the present study, the effects of BCBPs on Wnt/ß-catenin signaling in chondrocytes were investigated. BCBPs were obtained by hot-water extraction and identified by the modified high performance liquid chromatography (HPLC) method. Chondrocytes were isolated from the knees of SpragueDawley rats and identified by type II collagen immunohistochemistry. The chondrocytes were treated with or without BCBPs for 48 h. Cell viability was evaluated by MTT assay. The mRNA and protein levels of Wnt-4, ß-catenin, Frizzled-2, glycogen synthase kinase (GSK)-3ß, cyclin D1 and collagen II were detected by western blot analysis and reverse transcription PCR (RT-PCR), respectively. We found that the BCBPs contained at least seven monosaccharides, including D-mannose, rhamnose, D-(+) glucuronic acid, D-(+) galacturonic acid, D-glucose, galactose and arabinose. The cell viability of the chondrocytes treated with 50, 100 and 200 µg/ml BCBPs was significantly higher than that of the chondroctyes in the control group (treated with 0 µg/ml BCBPs). Furthermore, compared with the control group, the mRNA and protein expression of Wnt-4, ß-catenin, Frizzled-2 and cyclin D1 in the BCBP-treated groups markedly increased, whereas the mRNA and protein expression of GSK-3ß significantly decreased. Of note, the dose of 100 µg/ml BCBPs was more effective than the dose of 50 µg/ml BCBPs and 200 µg/ml BCBPs. In addition, we found that treatment with BCBPs upregulated the protein levels of collagen II in the chondrocytes. These results indicate that BCBPs upregulate Wnt/ß-catenin signaling, thus promoting chondrocyte proliferation.