RESUMEN
Fungal keratitis (FK) is a refractory keratitis caused by excessive inflammation and fungal damage. Excessive inflammation can lead to tissue damage and corneal opacity, resulting in a poor prognosis for FK. Oxymatrine (OMT) is a natural alkaloid, which has rich pharmacological effects, such as antioxidant and anti-inflammation. However, its antifungal activity and the mechanism of action in FK have not been elucidated. This study confirmed that OMT suppressed Aspergillus fumigatus growth, biofilm formation, the integrity of fungal cell and conidial adherence. OMT not only effectively reduced corneal fungal load but also inflammation responses. OMT lessened the recruitment of neutrophils and macrophages in FK. In addition, OMT up-regulated the expression of Nrf2 and down-regulated the expression of IL-18, IL-1ß, caspase-1, NLRP3 and GSDMD. Pre-treatment with Nrf2 inhibitor up-regulated the expression of IL-1ß, IL-18, caspase-1, NLRP3 and GSDMD supressed by OMT. In conclusion, OMT has efficient anti-inflammatory and antifungal effects by suppressing fungal activity and restricting pyroptosis via Nrf2 pathway. OMT is considered as a potential option for the treatment of FK.
Asunto(s)
Aspergilosis , Úlcera de la Córnea , Infecciones Fúngicas del Ojo , Queratitis , Matrinas , Animales , Ratones , Aspergillus fumigatus/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR , Interleucina-18 , Aspergilosis/tratamiento farmacológico , Aspergilosis/metabolismo , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Piroptosis , Factor 2 Relacionado con NF-E2 , Queratitis/microbiología , Inflamación , Infecciones Fúngicas del Ojo/tratamiento farmacológico , Infecciones Fúngicas del Ojo/metabolismo , Caspasa 1/metabolismo , Ratones Endogámicos C57BLRESUMEN
Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5ß1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5ß1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5ß1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/ß-catenin signal. The interaction of FN with the integrin receptor α5ß1 in the constructed model, which may act through the Wnt/ß-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV.
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Modelos Animales de Enfermedad , Fibronectinas , Integrina alfa5beta1 , Ratones Endogámicos C57BL , Vía de Señalización Wnt , Animales , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfa5beta1/genética , Ratones , Vía de Señalización Wnt/fisiología , Movimiento Celular/fisiología , Western Blotting , Macaca mulatta , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , beta Catenina/metabolismo , Inmunohistoquímica , Reacción en Cadena en Tiempo Real de la Polimerasa , Masculino , Células CultivadasRESUMEN
Adipose tissue-derived mesenchymal stem cells (ADSCs) have promising effects on nerve repair due to the differentiation ability to neural cells. Ghrelin has been shown to promote the neural differentiation of ADSCs. This work was designed to explore its underlying mechanism. Herein, we found high expression of LNX2 in ADSCs after neuronal differentiation. Knockdown of LNX2 might block neuronal differentiation of ADSCs, as evidenced by the decreased number of neural-like cells and dendrites per cell, and the reduced expressions of neural markers (including ß-Tubulin III, Nestin, and MAP2). We also demonstrated that LNX2 silencing suppressed the nuclear translocation of ß-catenin in differentiated ADSCs. Luciferase reporter assay indicated that LNX2 inhibited wnt/ß-catenin pathway by reducing its transcriptional activity. In addition, results showed that LNX2 expression was increased by ghrelin, and its inhibition diminished the effects of ghrelin on neuronal differentiation. Altogether, the results suggest that LNX2 is involved in the role of ghrelin to facilitate neuronal differentiation of ADSCs.
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Ghrelina , Células Madre Mesenquimatosas , beta Catenina , beta Catenina/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Ghrelina/farmacología , Ghrelina/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neuronas/metabolismo , HumanosRESUMEN
Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.
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Neovascularización Coroidal , Degeneración Macular , Ratones , Animales , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales/metabolismo , Neovascularización Coroidal/metabolismo , Transducción de Señal/fisiología , ARN Interferente Pequeño/genética , Degeneración Macular/metabolismo , Proliferación Celular , Rayos LáserRESUMEN
The protein GSDMD is an important performer of pyroptosis and a universal substrate for the inflammatory caspase. However, the role and regulatory mechanism of GSDMD in Aspergillus fumigatus keratitis is remains unknown. Here we detected GSDMD protein in the cornea of normal and fungal-infected C57BL/6 mice. Human corneal epithelial cell (HCECs) were preincubated with a hydrochloride solution (IFNR inhibitor), ruxolitinib (JAK/STAT inhibitor), belnacasan (caspase-1 inhibitor) before infection with A. fumigatus conidia. Mice corneas were infected with Aspergillus fumigatus after pretreatment of GSDMD siRNA via subconjunctival injection. After, samples were harvested at specific time points and the expression of GSDMD and IL-1ß was assessed by PCR, Western blot and immunofluorescence staining. Compared with the control group, we observed that the expression of GSDMD in fungal-infected mice cornea was significantly increased. After pretreatment with IFNR, JAK/STAT and caspase-1 inhibitors before fungal infection, the expression of GSDMD was significantly inhibited compared to the DMSO control in HCECs. Moreover, the GSDMD siRNA treatment have significantly weaken corneal inflammatory response, decreasing the proinflammatory factor IL-1ß secretion and reducing neutrophils and macrophages recruitment in mice infected corneas. In summary, the data here provided evidences that GSDMD, an executor of pyroptosis, is involved in the early immune response of A. fumigatus keratitis. Additionally, the inhibition of GSDMD expression can affect the secretion of IL-1ß and the recruitment of neutrophil and macrophages by blocking IFNR, JAK/STAT and caspase-1 signaling pathway. The protein GSDMD may emerge as a potential therapeutic target for A. fumigatus keratitis.
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Aspergilosis/metabolismo , Epitelio Corneal/metabolismo , Infecciones Fúngicas del Ojo/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Queratitis/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Piroptosis , Animales , Aspergilosis/microbiología , Aspergilosis/patología , Aspergillus fumigatus/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Epitelio Corneal/microbiología , Epitelio Corneal/patología , Infecciones Fúngicas del Ojo/microbiología , Infecciones Fúngicas del Ojo/patología , Femenino , Humanos , Queratitis/microbiología , Queratitis/patología , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Angiogenin (ANG) is known to alter multiple cell behaviors by directly targeting downstream targets, but its role in hepatocellular carcinoma (HCC) remains to be elucidated. The expression of ANG in HCC cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The effects of ANG expression on cell proliferation, cell migration, and hallmarks of epithelial-mesenchymal transition (EMT) process were also investigated. The relationship between ANG and high mobility group AT-hook 2 (HMGA2) was evaluated. ANG expression was increased in HCC cell lines. Downregulating of ANG inhibits proliferation, migration, and EMT of HCC cells. The direct regulation of ANG on HMGA2 was verified by luciferase activity reporter assay and western blot assay. Furthermore, overexpression of HMGA2 reversed the inhibitory effects of ANG downregulation on HCC cell behaviors. Our results illustrated the mechanism that ANG promote the EMT of HCC through targeting HMGA2.
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Carcinoma Hepatocelular/metabolismo , Proteína HMGA2/metabolismo , Neoplasias Hepáticas/metabolismo , Ribonucleasa Pancreática/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Regulación hacia Abajo , Transición Epitelial-Mesenquimal , Humanos , Neoplasias Hepáticas/patología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Ribonucleasa Pancreática/biosíntesis , Ribonucleasa Pancreática/genética , Transfección , Regulación hacia ArribaRESUMEN
Diabetic cardiomyopathy (DCM) is defined as ventricular dysfunction occurring independently of a recognized cause such as hypertension or coronary artery disease. Liver X receptor α (LXRα), a subtype of ligand-activated transcription factors LXRs, has been considered as a potential pharmacological target in the pathogenesis of cardiovascular and metabolic diseases. However, the potential mechanism of how LXRα is regulated in cardiomyocytes is still unclear. This study investigated the effect of activating LXRα with GW3965 on cardiomyocyte apoptosis and its upstream regulator in glucose-induced H9C2 cells. Our data indicated that GW3965 up-regulated the expression of LXRα, inhibited cardiomyocyte apoptosis, and altered the apoptosis-related proteins in glucose-induced H9C2 cells. In addition, GW3965 restored the mitochondrial membrane potential level and decreased the ROS production induced by glucose. Moreover, LXRα was confirmed as a direct target of microRNA-1 (miR-1) that was involved in cardiomyocyte apoptosis of DCM, and overexpression of miR-1 abrogated the inhibiting effect of GW3965 on glucose-induced apoptosis in H9C2 cells. This study highlights an important role of LXRα in the development of DCM and brings new insights into the complex mechanisms involved in the pathogenesis of DCM.
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Apoptosis/efectos de los fármacos , Receptores X del Hígado/antagonistas & inhibidores , MicroARNs/farmacología , Mitocondrias/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Receptores X del Hígado/metabolismo , MicroARNs/genética , Mitocondrias/metabolismo , RatasRESUMEN
Understanding the relationship between acid detergent lignin (ADL) and lodging resistance index (LRI) is essential for breeding new varieties of brown midrib (bmr) sudangrass (Sorghum sudanense (Piper) Stapf.). In this study, bmr-12 near isogenic lines and their wild-types obtained by back cross breeding were used to compare relevant forage yield and quality traits, and to analyze expression of the caffeic acid O-methyltransferase (COMT) gene using quantitative real time-PCR. The research showed that the mean ADL content of bmr-12 mutants (20.94 g kg(-1)) was significantly (P < 0.05) lower than measured in N-12 lines (43.45 g kg(-1)), whereas the LRI of bmr-12 mutants (0.29) was significantly (P < 0.05) higher than in N-12 lines (0.22). There was no significant correlation between the two indexes in bmr-12 materials (r = -0.44, P > 0.05). Sequence comparison of the COMT gene revealed two point mutations present in bmr-12 but not in the wild-type, the second mutation changed amino acid 129 of the protein from Gln (CAG) to a stop codon (UAG). The relative expression level of COMT gene was significantly reduced, which likely led to the decreased ADL content observed in the bmr-12 mutant.
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To investigate the anti-inflammatory and antifungal effects of plumbagin (PL) in Aspergillus fumigatus (A. fumigatus) keratitis, the minimum inhibitory concentration (MIC), time-killing curve, spore adhesion, crystal violet staining, calcium fluoride white staining, and Propidium Iodide (PI) staining were employed to assess the antifungal activity of PL in vitro against A. fumigatus. The cytotoxicity of PL was assessed using the Cell Counting Kit-8 (CCK8). The impact of PL on the expression of HMGB1, LOX-1, TNF-α, IL-1ß, IL-6, IL-10 and ROS in A. fumigatus keratitis was investigated using RT-PCR, ELISA, Western blot, and Reactive oxygen species (ROS) assay. The therapeutic efficacy of PL against A. fumigatus keratitis was assessed through clinical scoring, plate counting, Immunofluorescence and Hematoxylin-Eosin (HE) staining. Finally, we found that PL inhibited the growth, spore adhesion, and biofilm formation of A. fumigatus and disrupted the integrity of its cell membrane and cell wall. PL decreased IL-6, TNF-α, and IL-1ß levels while increasing IL-10 expression in fungi-infected mice corneas and peritoneal macrophages. Additionally, PL significantly attenuated the HMGB1/LOX-1 pathway while reversing the promoting effect of Boxb (an HMGB1 agonist) on HMGB1/LOX-1. Moreover, PL decreased the level of ROS. In vivo, clinical scores, neutrophil recruitment, and fungal burden were all significantly reduced in infected corneas treated with PL. In summary, the inflammatory process can be inhibited by PL through the regulation of the HMGB-1/LOX-1 pathway. Simultaneously, PL can exert antifungal effects by limiting fungal spore adhesion and biofilm formation, as well as causing destruction of cell membranes and walls.
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AIM: To compare the macular ganglion cell-inner plexiform layer (GCIPL) thickness, retinal nerve fiber layer (RNFL) thickness, optic nerve head (ONH) parameters, and retinal vessel density (VD) measured by spectral-domain optical coherence tomography (SD-OCT) and analyze the correlations between them in the early, moderate, severe primary angle-closure glaucoma (PACG) and normal eyes. METHODS: Totally 70 PACG eyes and 20 normal eyes were recruited for this retrospective analysis. PACG eyes were further separated into early, moderate, or severe PACG eyes using the Enhanced Glaucoma Staging System (GSS2). The GCIPL thickness, RNFL thickness, ONH parameters, and retinal VD were measured by SD-OCT, differences among the groups and correlations within the same group were calculated. RESULTS: The inferior and superotemporal sectors of the GCIPL thickness, rim area of ONH, average and inferior sector of the retinal VD were significantly reduced (all P<0.05) in the early PACG eyes compared to the normal and the optic disc area, cup to disc ratio (C/D), and cup volume were significantly higher (all P<0.05); but the RNFL was not significant changes in early and moderate PACG. In severe group, the GCIPL and RNFL thickness were obvious thinning with retinal VD were decreasing as well as C/D and cup volume increasing than other three groups (all P<0.01). In the early PACG subgroup, there were significant positive correlations between retinal VD and GCIPL thickness (except superonasal and inferonasal sectors, r=0.573 to 0.641, all P<0.05), superior sectors of RNFL thickness (r=0.055, P=0.049). More obvious significant positive correlations were existed in moderate PACG eyes between retinal VD and superior sectors of RNFL thickness (r=0.650, P=0.022), and temporal sectors of RNFL thickness (r=0.740, P=0.006). In the severe PACG eyes, neither GCIPL nor RNFL thickness was associated with retinal VD. CONCLUSION: The ONH damage and retinal VD loss appears earlier than RNFL thickness loss in PACG eyes. As the PACG disease progressed from the early to the moderate stage, the correlations between the retinal VD and RNFL thickness increases.
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Fungal keratitis is a refractory kind of keratopathy. We attempted to investigate the anti-inflammatory role of thymol on Aspergillus fumigatus (A. fumigatus) keratitis. Wound healing and fluorescein staining of the cornea were applied to verify thymol's safety. Mice models of A. fumigatus keratitis underwent subconjunctival injection of thymol. The anti-inflammatory roles of thymol were verified by hematoxylin-eosin (HE) staining, slit lamp observation, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. In contrast with the DMSO group, more transparent corneas and less inflammatory cells infiltration were detected in mice treated with 50 µg/ml thymol. Thymol downregulated the synthesis of TLR4, MyD88, NF-kB, IL-1ß, NLRP3, caspase 1, caspase 8, GSDMD, RIPK3 and MLKL. In summary, we proved that thymol played a protective part in A. fumigatus keratitis by cutting down inflammatory cells aggregation, downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß signal expression and reducing necroptosis and pyroptosis.
Asunto(s)
Aspergilosis , Queratitis , Animales , Ratones , Antiinflamatorios/uso terapéutico , Aspergilosis/tratamiento farmacológico , Aspergilosis/metabolismo , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Modelos Animales de Enfermedad , Queratitis/tratamiento farmacológico , Queratitis/metabolismo , Queratitis/microbiología , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Necroptosis , FN-kappa B/genética , FN-kappa B/metabolismo , Piroptosis , Timol/farmacología , Timol/uso terapéutico , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Adipose tissuederived mesenchymal stem cells (ADMSCs) differentiate into cardiomyocytes and may be an ideal cell source for myocardial regenerative medicine. Ghrelin is a gastricsecreted peptide hormone involved in the multilineage differentiation of MSCs. To the best of our knowledge, however, the role and potential downstream regulatory mechanism of ghrelin in cardiomyocyte differentiation of ADMSCs is still unknown. The mRNA and protein levels were measured by reverse transcriptionquantitative PCR and western blotting. Immunofluorescence staining was used to show the expression and cellular localization of cardiomyocyte markers and ßcatenin. RNA sequencing was used to explore the differentially expressed genes (DEGs) that regulated by ghrelin. The present study found that ghrelin promoted cardiomyocyte differentiation of ADMSCs in a concentrationdependent manner, as shown by increased levels of cardiomyocyte markers GATA binding protein 4, αmyosin heavy chain (αMHC), ISL LIM homeobox 1, NK2 homeobox 5 and troponin T2, cardiac type. Ghrelin increased ßcatenin accumulation in nucleus and decreased the protein expression of secreted frizzledrelated protein 4 (SFRP4), an inhibitor of Wnt signaling. RNA sequencing was used to determine the DEGs regulated by ghrelin. Functional enrichment showed that DEGs were more enriched in cardiomyocyte differentiationassociated terms and Wnt pathways. Deadbox helicase 17 (DDX17), an upregulated DEG, showed enhanced mRNA and protein expression levels following ghrelin addition. Overexpression of DDX17 promoted protein expression of cardiacspecific markers and ßcatenin and enhanced the fluorescence intensity of αMHC and ßcatenin. DDX17 upregulation inhibited protein expression of SFRP4. Rescue assay confirmed that the addition of SFRP4 partially reversed ghrelinenhanced protein levels of cardiacspecific markers and the fluorescence intensity of αMHC. In conclusion, ghrelin promoted cardiomyocyte differentiation of ADMSCs by DDX17mediated regulation of the SFRP4/Wnt/ßcatenin axis.
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Células Madre Mesenquimatosas , Miocitos Cardíacos , Miocitos Cardíacos/metabolismo , Ghrelina/farmacología , Ghrelina/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Diferenciación Celular/genética , Células Madre Mesenquimatosas/metabolismo , Vía de Señalización Wnt , ARN Mensajero/metabolismoRESUMEN
This study aimed to evaluate whether combination therapy of bone marrow stromal cells (BMSCs) transplantation and chondroitinase ABC (ChABC) treatment further enhances axonal regeneration and functional recovery after acellular nerve allograft repair of the sciatic nerve gap in rats. Eight Sprague-Dawley rats were used as nerve donors, and 32 Wistar rats were randomly divided into four groups: Group I: acellular rat sciatic nerve (ARSN) group; Group II: ChABC treatment; Group III: BMSCs transplantation; and Group IV: ChABC treatment and BMSCs transplantation. The results showed that compared with ARSN control group, BMSC transplantation promoted axonal regeneration, the secretion of neural trophic factors NGF, BDNF and axon angiogenesis in nerve graft. ChABC treatment degraded chondroitin sulfate proteoglycans in ARSN in vitro and in vivo and improved BMSCs survival in ARSN. The combination therapy caused much better beneficial effects evidenced by increasing sciatic function index, nerve conduction velocity, restoration rate of tibialis anterior wet muscle weight, and myelinated nerve number, but did not further boost the therapeutic effects on neurotrophic factor production, axon angiogenesis, and sensory functional recovery by BMSC transplantation. Taken together, for the first time, we demonstrate the synergistic effects of BMSC transplantation and BMSCs treatment on peripheral nerve regeneration, and our findings may help establish novel strategies for cell transplantation therapy for peripheral nerve injury.
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Trasplante de Médula Ósea/métodos , Condroitina ABC Liasa/administración & dosificación , Trasplante de Células Madre Mesenquimatosas/métodos , Regeneración Nerviosa/fisiología , Neuropatía Ciática/tratamiento farmacológico , Neuropatía Ciática/cirugía , Animales , Células Cultivadas , Femenino , Masculino , Regeneración Nerviosa/efectos de los fármacos , Tejido Nervioso/enzimología , Tejido Nervioso/trasplante , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Neuropatía Ciática/metabolismo , Trasplante Homólogo/métodosRESUMEN
Acellular nerves possess the structural and biochemical features similar to those of naive endoneurial tubes, and have been proved bioactive for allogeneil graft in nerve tissue engineering. However, the source of allogenic donators is restricted in clinical treatment. To explore sufficient substitutes for acellular nerve allografts (ANA), we investigated the effectiveness of acellular nerve xenografts (ANX) combined with bone marrow stromal cells (BMSCs) on repairing peripheral nerve injuries. The acellular nerves derived from Sprague-Dawley rats and New Zealand rabbits were prepared, respectively, and BMSCs were implanted into the nerve scaffolds and cultured in vitro. All the grafts were employed to bridge 1 cm rat sciatic nerve gaps. Fifty Wistar rats were randomly divided into five groups (n = 10 per group): ANA group, ANX group, BMSCs-laden ANA group, BMSCs-laden ANX group, and autologous nerve graft group. At 8 weeks post-transplantation, electrophysiological study was performed and the regenerated nerves were assayed morphologically. Besides, growth-promoting factors in the regenerated tissues following the BMSCs integration were detected. The results indicated that compared with the acellular nerve control groups, nerve regeneration and functional rehabilitation for the xenogenic nerve transplantation integrated with BMSCs were advanced significantly, and the rehabilitation efficacy was comparable with that of the autografting. The expression of neurotrophic factors in the regenerated nerves, together with that of brain-derived neurotrophic factor (BDNF) in the spinal cord and muscles were elevated largely. In conclusion, ANX implanted with BMSCs could replace allografts to promote nerve regeneration effectively, which offers a reliable approach for repairing peripheral nerve defects.
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Trasplante de Médula Ósea/fisiología , Regeneración Nerviosa , Nervios Periféricos/trasplante , Nervio Ciático/fisiología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Nervio Ciático/cirugía , Trasplante Heterólogo , Trasplante HomólogoRESUMEN
Recently it was demonstrated that the exposure of the developing brain during the period of synaptogenesis to drugs that block NMDA glutamate receptors can trigger widespread apoptotic neurodegeneration. Sevoflurane is a new inhalation anesthetic agent commonly used in the clinic. Here we address whether sevoflurane could induce neurotoxicity in the developing brain. Sevoflurane was administered to rats before pregnancy and pregnant rats on embryonic days E6, E10, E14, and E18 1MAC for 6 h, and we employed histopathological, immunochemistry, semiquantitative RT-PCR, and Western blot to investigate the effect of the exposure of pregestation and gestation rats to sevoflurane on the offspring brain development. The results showed that the exposure of gestation but not pregestation rats to sevoflurane-induced extensive apoptotic neurodegeneration in the hippocampus of offspring at P0, P7, and P14, accompanied by altered expression of casepase-3, GAP-43, nNOS, NMDAR1, NMDAR2A, and NMDAR2B. Furthermore, upregulation of PKCα and p-JNK and downregulation of p-ERK and FOS protein levels were observed in the hippocampus of offspring at P0, P7, and P14 from rats exposed to sevoflurane at gestation, but not pregestation. In summary, our data suggest that sevoflurane induces developmental neurotoxicity in rats and this may be attributed to the upregulation of PKCα and p-JNK and downregulation of p-ERK and FOS protein in the hippocampus.
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Anestésicos por Inhalación/toxicidad , Encéfalo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Éteres Metílicos/toxicidad , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/patología , Factores de Edad , Animales , Apoptosis/efectos de los fármacos , Análisis de los Gases de la Sangre , Encéfalo/efectos de los fármacos , Encéfalo/patología , Femenino , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microscopía Electrónica de Transmisión , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/ultraestructura , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Proteínas Oncogénicas v-fos/genética , Proteínas Oncogénicas v-fos/metabolismo , Embarazo , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , SevofluranoRESUMEN
Silages processed with gramineous grass and wood feed mixtures may have positive associative effects on silage quality and animal performance when ruminants are fed those silages. The present study was conducted to evaluate the fermentation quality, chemical composition, and in vitro rumen digestion characteristics of silages produced with mixtures of sweet sorghum (SS) and korshinsk pea shrub (KP) in six different ratios of fresh weights: 100:0 (0%KP), 80:20 (20%KP), 60:40 (40%KP), 40:60 (60%KP), 20:80 (80%KP), and 0:100 (100%KP). As the proportion of KP increased in the silages, the contents of lactic acid, acetic acid, starch, total phenolics, hydrolysable tannins, and condensed tannins decreased (p < 0.05), while the pH value and the contents of neutral detergent fiber, acid detergent fiber, ether extract, crude protein, and ash increased (p < 0.05). The silages were evaluated in 48 h incubations with rumen liquor. The in vitro dry matter digestibility, in vitro neutral detergent fiber digestibility, and in vitro gas production decreased as the proportion of KP increased (p < 0.05). This study suggests that ensiling SS-KP at a ratio of 80:20 is a practical method for preserving such low-DM forages (SS) and that these silage mixtures offer an opportunity to optimize the nutrient supply for ruminant production.
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Rumen , Sorghum , Animales , Digestión , Fermentación , Pisum sativum , Rumen/metabolismoRESUMEN
PURPOSE: To explore the role of caspase-8 in mediating the transition between different death modes in fungal keratitis. METHODS: The expression of caspase-8 in Aspergillus fumigatus (A. fumigatus) keratitis was detected using western blotting and immunofluorescence. After subconjunctival injection of Z-IETD-FMK (caspase-8 inhibitor) or VX765 (caspase-1 inhibitor), the mice corneas of A. fumigatus keratitis were observed and scored under a slit lamp. Colony plate count, immunofluorescence staining, western blotting and qRT-PCR experiments were used to detect fungal load, inflammatory cells, and the production of related mRNAs and proteins. In vitro experiments, the LDH release test, Cell Count Kit-8(CCK-8) assay, ELISA, qRT-PCR and western blotting were used to detect cell viability, related mRNAs and proteins. RESULTS: The caspase-8 protein was upregulated following fungal infection. Compared with the A. fumigatus keratitis group, the mice treated with Z-IETD-FMK had heavier corneal turbidity, higher clinical scores, more fungal load and fewer inflammatory cells. The expression of NLRP3, cleaved-caspase-1, N-GSDMD, and IL-1ß in the fungal infection group after Z-IETD-FMK pretreatment were downregulated, while RIPK3 and p-MLKL were upregulated. In the fungal infection group after VX765 pretreatment, the expression of cleaved-caspase-8 was up-regulated, while N-GSDMD was downregulated. CONCLUSIONS: Caspase-8 is involved in the early immune defense response of A. fumigatus keratitis. It is essential for the recruitment of inflammatory cells and the clearance of the fungus. In A. fumigatus keratitis, activated caspase-8 promoted the caspase-1/GSDMD signaling pathway to participate in pyroptosis, inhibited RIPK3/MLKL signaling pathway-mediated necroptosis, and promoted IL-1ß maturation and release by activating the NLRP3 inflammasomes.
Asunto(s)
Aspergilosis , Caspasa 8 , Queratitis , Animales , Ratones , Aspergillus fumigatus , Caspasa 1/metabolismo , Caspasa 8/metabolismo , Inhibidores de Caspasas/farmacología , Queratitis/microbiología , Ratones Endogámicos C57BL , Necroptosis , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , PiroptosisRESUMEN
Our previous study has shown that the transcription factor Krüppel-like factor 7 (KLF7) promotes peripheral nerve regeneration and motor function recovery after spinal cord injury. KLF7 also participates in traumatic brain injury, but its regulatory mechanisms remain poorly understood. In the present study, an HT22 cell model of traumatic brain injury was established by stretch injury and oxygen-glucose deprivation. These cells were then transfected with an adeno-associated virus carrying KLF7 (AAV-KLF7). The results revealed that, after stretch injury and oxygen-glucose deprivation, KLF7 greatly reduced apoptosis, activated caspase-3 and lactate dehydrogenase, downregulated the expression of the apoptotic markers B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and cleaved caspase-3, and increased the expression of ßIII-tubulin and the antiapoptotic marker Bcl-2. Furthermore, KLF7 overexpression upregulated Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in HT22 cells treated by stretch injury and oxygen-glucose deprivation. Immunoprecipitation assays revealed that KLF7 directly participated in the phosphorylation of STAT3. In addition, treatment with AG490, a selective inhibitor of JAK2/STAT3, weakened the protective effects of KLF7. A mouse controlled cortical impact model of traumatic brain injury was then established. At 30 minutes before modeling, AAV-KLF7 was injected into the ipsilateral lateral ventricle. The protein and mRNA levels of KLF7 in the hippocampus were increased at 1 day after injury and recovered to normal levels at 3 days after injury. KLF7 reduced ipsilateral hippocampal atrophy, decreased the injured cortex volume, downregulated Bax and cleaved caspase-3 expression, and increased the number of 5-bromo-2'-deoxyuridine-positive neurons and Bcl-2 protein expression. Moreover, KLF7 transfection greatly enhanced the phosphorylation of JAK2 and STAT3 in the ipsilateral hippocampus. These results suggest that KLF7 may protect hippocampal neurons after traumatic brain injury through activation of the JAK2/STAT3 signaling pathway. The study was approved by the Institutional Review Board of Mudanjiang Medical University, China (approval No. mdjyxy-2018-0012) on March 6, 2018.
RESUMEN
To explore the biocompatibility of acellular nerves of different mammalian species, for the acellular nerves derived from rats and rabbits, the morphology, immunocompatibility, and cytocompatibility with bone marrow stromal cells (BMSCs) were evaluated. The results indicated that the tridimensional architecture and main proteins of endoneurial tubes in both biomaterials were well retained. The nerve scaffolds did not show immunogenicity or cytotoxicity, but facilitated growth of BMSCs and secretion of neurotrophic factors in vitro. In conclusion, acellular nerves of different species possess favorable biocompatibility, and xenogenic acellular nerves combined with BMSCs have potential to replace allografts for peripheral nerve reconstruction.