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1.
Gene ; 766: 145149, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-32971185

RESUMEN

BACKGROUND: Crosstalk between posterior cruciate ligament fibroblasts (PCLfs) and synoviocytes (SCs) significantly modifies the homeostatic balance of the extracellular matrix (ECM) and appears to post a prominent affection for wound healing of PCL. Interleukin-1ß (IL-1ß) is regarded as a critical factor in acute inflammatory events during ligament injury. METHODS: In order to confirm the capability of SCs the response of lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs) to IL-1ß, the complex cues of the joint cavity following PCL injury were simulated and the effect of IL-1ß on the expression of LOXs and MMPs in PCLfs were investigated. PCLfs in both the mono- and co-culture conditions were treated with IL-1ß. Cell lysates were collected from the PCLfs and LOXs and MMP-1, 2, 3 expression quantified using quantitative real-time PCR and western bolting. RESULTS: The results indicated that injury alone elevated the expression of LOXs and MMP-1, 2 and 3. But IL-1ß significantly decreased the LOX, LOXL1, and LOXL3 expression, and simultaneously increased MMP-1, 2 and 3 expressions in injured PCLfs. Furthermore, co-culture further suppressed LOXs, but stimulated MMP-1, 2 and 3 expressions when subjected to both mechanical injury and IL-1ß treatment. This possibly suggests that a number of soluble factors are secreted that act as mediators that amplify the response of SCs. CONCLUSION: The results indicated that the SCs could affect the IL-1ß-induction of LOXs inhibition and MMPs accumulation, which may be the underlying mechanism of the the poor healing response following PCL injury.


Asunto(s)
Fibroblastos/metabolismo , Interleucina-1beta/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Sinoviocitos/metabolismo , Células Cultivadas , Técnicas de Cocultivo/métodos , Matriz Extracelular/metabolismo , Humanos , Membrana Sinovial/metabolismo , Cicatrización de Heridas/fisiología
2.
Gene ; 766: 145022, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-32758579

RESUMEN

BACKGROUND: A better understanding of the mechanism(s) underlying cardioembolic stroke can promote recovery and reduce the risk of recurrent embolisms. METHODS: Peripheral blood mononuclear cell (PBMC) gene expression datasets from cardioembolic patients and healthy controls were obtained from the Gene Expression Omnibus (GEO) database (GSE58294). The Limma software package was utilized to identify differentially-expressed genes (DEGs). Protein-protein interaction (PPI) analysis of the DEGs was performed using STRING. A weighted gene co-expression network analysis (WGCNA) was used to build a gene co-expression network. In vitro experiments assessed the effects on neutrophils exposed to oxygen and glucose-deprived (OGD) cortical neurons. An in vivo murine model of thromboembolic stroke was constructed through thrombin injection to examine effects on circulating neutrophils. Mechanistic in vitro studies were conducted using the proteasome inhibitor MG132, the p53-Mdm2 binding inhibitor Nutlin-3a, Mdm2 small-interfering RNA (siRNA), and Ctnnb1 siRNA. RESULTS: DEG analysis identified 44 upregulated and 66 downregulated genes in cardioembolic stroke PBMCs. PPI analysis of these DEGs yielded one eight-node protein module with ß-catenin (CTNNB1) as the central hub protein. Integration of the DEGs with WGCNA-derived hub genes revealed the key hub DEGs CTNNB1 and mouse double minute 2 (MDM2). Follow-up experiments revealed Mdm2, p53, and phospho-ß-catenin upregulation in neutrophils exposed to OGD neurons in vitro and following thromboembolic stroke in vivo. Mechanistic studies revealed that neutrophils transcriptionally upregulate Ctnnb1 expression to compensate for Mdm2/p53-mediated ß-catenin degradation induced by exposure to OGD neurons, thereby promoting neutrophil survival. CONCLUSION: Compensatory Ctnnb1 transcriptional upregulation in neutrophils induced by ischemic neuron exposure may be involved in promoting neutrophil survival following cardioembolic stroke.


Asunto(s)
Neutrófilos/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/genética , beta Catenina/genética , Animales , Células Cultivadas , Regulación hacia Abajo/genética , Expresión Génica/genética , Redes Reguladoras de Genes/genética , Corazón/fisiopatología , Humanos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética
3.
Gene ; 764: 145094, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-32860898

RESUMEN

Long chain acyl-CoA synthetases (ACSLs), which drive the conversion of long chain fatty acid into acyl-CoA, an ingredient of lipid synthesis, have been well-acknowledged to exert an indispensable role in many metabolic processes in mammals, especially lipid metabolism. However, in chicken, the evolutionary characteristics, expression profiles and regulatory mechanisms of ACSL gene family are rarely understood. Here, we analyzed the genomic synteny, gene structure, evolutionary event and functional domains of the ACSL gene family members using bioinformatics methods. The spatiotemporal expression profiles of ACSL gene family, and their regulatory mechanism were investigated via bioinformatics analysis incorporated with in vivo and in vitro estrogen-treated experiments. Our results indicated that ACSL2 gene was indeed evolutionarily lost in the genome of chicken. Chicken ACSLs shared an AMP-binding functional domain, as well as highly conversed ATP/AMP and FACS signature motifs, and were clustered into two clades, ACSL1/5/6 and ACSL3/4, based on high sequence similarity, similar gene features and conversed motifs. Chicken ACSLs showed differential tissue expression distributions, wherein the significantly decreased expression level of ACSL1 and the significantly increased expression level of ACSL5 were found, respectively, the expression levels of the other ACSL members remained unchanged in the liver of peak-laying hens versus pre-laying hens. Moreover, the transcription activity of ACSL1, ACSL3 and ACSL4 was silenced and ACSL6 was activated by estrogen, but no response to ACSL5. In conclusion, though having highly conversed functional domains, chicken ACSL gene family is organized into two separate groups, ACSL1/5/6 and ACSL3/4, and exhibits varying expression profiles and estrogen effects. These results not only pave the way for better understanding the specific functions of ACSL genes in avian lipid metabolism, but also provide a valuable evidence for gene family characteristics.


Asunto(s)
Pollos/genética , Coenzima A Ligasas/genética , Evolución Molecular , Metabolismo de los Lípidos/genética , Familia de Multigenes/genética , Acilcoenzima A/metabolismo , Animales , Células Cultivadas , Embrión de Pollo , Pollos/crecimiento & desarrollo , Pollos/metabolismo , Coenzima A Ligasas/metabolismo , Biología Computacional , Estrógenos/metabolismo , Ácidos Grasos/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hepatocitos , Cultivo Primario de Células , Dominios Proteicos/genética , Análisis Espacio-Temporal , Sintenía
4.
Gene ; 766: 145153, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-32950633

RESUMEN

AIM: Acute lung injury (ALI) is the mild form of acute respiratory distress syndrome (ARDS) which is a common lung disease with a high incidence and mortality rate. Recent studies manifested that some circular RNAs were associated with ALI. In this study, we aimed to uncover the effect of circular RNA circ_0054633 on ALI initiation and progression and proposed a new mechanism related to ALI. METHODS: The lipopolysaccharides (LPS)-induced acute lung injury model were build both in vivo of rat and in vitro of primary murine pulmonary microvascular endothelial cells (MPVECs). Hematoxylin and eosin (H&E) was employed to observe the tissue morphology and estimate the degree of lung damage. We used real-time quantitative polymerase chain reaction (RT-qPCR) to measure the expression level of circ_0054633. The expression levels of inflammatory cytokines IL-17A and tumor necrosis factor-α (TNF-α) were detected by ELISA. The effects of circ_0054633 on MPVECs proliferation and apoptosis were detected with the help of CCK-8 and apoptosis assay, separately. The expression level of NF-κB p65 protein was measured by Western blot. RESULTS: circ_0054633, IL-17A, TNF-α and NF-κB p65 were all overexpressed in LPS-treated rat and MPVECs, and LPS enhanced the proliferation and apoptosis of MPVECs. While circ_0054633 silencing reversed the above promotion effects of LPS on IL-17A, TNF-α expression and MPVECs proliferation and apoptosis. CONCLUSIONS: Quietness of circ_0054633 alleviated LPS-induced ALI via NF-κB signaling pathway, implicating circ_0054633 may be a potential biomarker for diagnose and therapy of ALI.


Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Proliferación Celular/fisiología , Células Endoteliales/metabolismo , Inflamación/metabolismo , FN-kappa B/metabolismo , ARN Circular/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Inflamación/inducido químicamente , Interleucina-17/metabolismo , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
5.
Head Face Med ; 16(1): 26, 2020 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-33190638

RESUMEN

BACKGROUND: Periodontal ligament (PDL) cells initiate local immune responses, similar to microglia regulating primary host defense mechanisms in neuroinflammatory events of the central nervous system. As these two cell types manifest similarities in their immunomodulatory behavior, this study investigated the thesis that the immunological features of PDL cells might be modulated by the endocannabinoid system, as seen for microglia. METHODS: A human PDL cell line and an Embryonic stem cell-derived microglia (ESdM) cell line were grown in n = 6 experimental groups each, incubated with cannabinoid receptor agonists arachidonoylethanolamine (AEA) (50 µM) or Palmitoylethanolamide (PEA) (50 µM) and challenged with centrifugation-induced inflammation (CII) for 6 and 10 h. Untreated samples served as controls. Quantitative real-time polymerase chain reaction was applied for gene expression analyses of inflammatory cytokines, cannabinoid receptors and ionized calcium binding adaptor molecule 1 (IBA-1). Microglia marker gene IBA-1 was additionally verified on protein level in PDL cells via immunocytochemistry. Proliferation was determined with a colorimetric assay (WST-1 based). Statistical significance was set at p < 0.05. RESULTS: IBA-1 was inherently expressed in PDL cells both at the transcriptional and protein level. AEA counteracted pathological changes in cell morphology of PDL cells and microglia caused by CII, and PEA contrarily enhanced them. On transcriptional level, AEA significantly downregulated inflammation in CII specimens more than 100-fold, while PEA accessorily upregulated them. CII reduced cell proliferation in a time-dependent manner, synergistically reinforced by PEA decreasing cell numbers to 0.05-fold in PDL cells and 0.025-fold in microglia compared to controls. CONCLUSION: PDL cells and microglia exhibit similar features in CII with host-protective effects for AEA through dampening inflammation and preserving cellular integrity. In both cell types, PEA exacerbated proinflammatory effects. Thus, the endocannabinoid system might be a promising target in the regulation of periodontal host response.


Asunto(s)
Endocannabinoides , Ligamento Periodontal , Proliferación Celular , Células Cultivadas , Humanos , Microglía
6.
J Biomed Nanotechnol ; 16(6): 965-974, 2020 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33187591

RESUMEN

As an important recycling and degradation system, autophagy is considered to be critical in regulating stem cell differentiation. It has been shown that graphene oxide quantum dots (GOQDs) are a robust biological labelling tool for stem cells with little cytotoxicity. In this study, we explored the role of autophagy in regulating the impact of GOQDs on the odontoblastic differentiation of DPSCs during autophagy. Western blotting and immunofluorescence staining were used to evaluate the autophagic activity of DPSCs. Quantitative PCR, alizarin red S staining, and alkaline phosphatase staining were used to examine DPSC odontoblastic differentiation. The impacts of ROS scavengers on autophagy induction and reactive oxygen species (ROS) levels were also measured. Lentiviral vectors carrying Beclin1 siRNA sequences, as well as autophagy inhibitors (3-MA and bafilomycin A1), were used to inhibit autophagy. Initial exposure to GOQDs increased autophagic activity and enhanced DPSC mineralization. Autophagy inhibition suppressed GOQD-induced odontoblastic differentiation. Moreover, GOQD treatment induced autophagy in a ROS-dependent manner. GOQDs promoted differentiation, which could be modulated via ROS-induced autophagy.


Asunto(s)
Puntos Cuánticos , Autofagia , Diferenciación Celular , Células Cultivadas , Pulpa Dental , Grafito , Odontoblastos , Puntos Cuánticos/toxicidad , Especies Reactivas de Oxígeno , Células Madre
7.
Zhongguo Zhong Yao Za Zhi ; 45(19): 4712-4718, 2020 Oct.
Artículo en Chino | MEDLINE | ID: mdl-33164437

RESUMEN

To observe the effect of shikonin on the proliferation, migration, adhesion and invasion of rheumatoid arthritis(RA) fibroblast like synoviocytes induced by tumor necrosis factor-α(TNF-α), and to explore its mechanism of action from aspects of protein kinase B(Akt) and mitogen activated protein kinase(MAPK) signaling pathways. TNF-α(20 ng·mL~(-1)) was used in this experiment to induce human RA fibroblast like synovial cell line(MH7 A). After addition of different concentrations of shikonin(0.025, 0.05, 0.1 pmol·L~(-1)), the proliferation, migration, adhesion and invasion ability of MH7 A cells were detected by MTT test, scratch test, adhesion test, Transwell invasion test, respectively. Protein expression of Akt and MAPK signaling pathway molecules in MH7 A cells was detected by Western blot. The results showed that as compared with the control group, TNF-α could significantly induce the proliferation, migration, adhesion and invasion of MH7 A cells, and increase the phosphorylation level of Akt, JNK, p38 and extracellular regulatory protein kinase(ERK). As compared with the TNF-α group, shikonin had no significant effect on TNF-α-induced proliferation of MH7 A cells after 24 h treatment, and it could reduce the TNF-α-induced proliferation of MH7 A cells in a concentration dependent manner after 48 h treatment. Shikonin also significantly reduced the TNF-α-induced migration, adhesion, invasion and phosphorylation levels of Akt, JNK, p38, ERK in MH7 A cells within 24 h. These results suggested that shikonin could reduce the proliferation, migration, adhesion and invasion ability of MH7 A cells induced by TNF-α, and its mechanism may be related to the down-regulation of Akt and MAPK signaling pathway activation.


Asunto(s)
Artritis Reumatoide , Sinoviocitos , Artritis Reumatoide/tratamiento farmacológico , Movimiento Celular , Proliferación Celular , Células Cultivadas , Fibroblastos , Humanos , Naftoquinonas , Membrana Sinovial , Factor de Necrosis Tumoral alfa/genética
8.
Polim Med ; 50(1): 41-51, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33150750

RESUMEN

BACKGROUND: Skin, the first barrier to pathogens, loses its integrity and function after an injury. The presence of an antibacterial dressing at the wound site may prevent bacterial invasion and also improve the healing process. OBJECTIVES: The current study aimed to fabricate a biomimetic membrane with antibacterial properties for healing chronic wounds. MATERIAL AND METHODS: The membranes, fabricated through electrospinning, are comprised of poly(ethylene oxide) (PEO) and zinc oxide nanoparticles (ZnO-NPs) as the main biomaterial and antibacterial agent, respectively. Antibacterial activity, cell attachment and viability were tested to evaluate the biological properties of the membranes. The optimal cell compatible concentration of ZnO-NPs was determined for further studies. In vitro characterization of the membranes was performed to confirm their suitable properties for wound healing. RESULTS: The antibacterial PEO/ZnO-NP membrane containing 2% of nanoparticles showed no cell toxicity, and human fibroblast cells were able to adhere and proliferate on the scaffold. The in vitro results from the tensile test, wettability, porosity, and protein adsorption revealed appropriate properties of the membrane as a scaffold for skin tissue engineering. CONCLUSIONS: Synthetic polymers have been widely used for tissue engineering applications. The proper characteristics of PEO nanofibers, including a high ratio of surface/volume, moderate hydrophilicity and good mechanical properties, make this polymer interesting for skin regeneration. The results demonstrate the potential of the antibacterial PEO/ZnO-NP membrane to be used as an engineered scaffold to improve the wound healing process.


Asunto(s)
Quitosano , Nanofibras , Polietilenglicoles , Andamios del Tejido , Óxido de Zinc , Antibacterianos/uso terapéutico , Células Cultivadas , Etilenos , Fibroblastos/citología , Humanos , Cicatrización de Heridas
9.
Int J Clin Pharmacol Ther ; 58(12): 678-686, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33141018

RESUMEN

Although medication treatment in COVID-19 patients would have no direct effect on the spread of the disease, a shortening of the period of hospitalization by only a few days would release 25 - 30% of critical-care resources. However, there appears to be no well-established medication treatment available that can do this reliably at the present time. Anti-malarials currently being evaluated, i.e., chloroquine and hydroxychloroquine, are not yet established as effective medications, and antiviral agents, including remdesivir, are only weakly active. This position paper report is focused on the modulation of the cytokine storm since it appears to be a major cause of the multi-organ failure in COVID-19. Whereas corticosteroids are not recommended in patients not on mechanical ventilation, immunotherapy with convalescent plasma and intravenous immunoglobulin (IVIG) have been used with some success in COVID-19. There is emerging new evidence that polyvalent immunoglobulins (PVIG) from bovine colostrum given orally can also modulate the immune response. Research using lipopolysaccharide-stimulated peripheral blood mononuclear cells from colorectal cancer patients (a so called micro-cytokine storm) has shown that PVIG block the expression of pro-inflammatory cytokines and stimulate the expression of anti-inflammatory cytokines. We have been able to confirm these results in a similar model using mononuclear cells from healthy subjects and could demonstrate that the modulations produced by PVIG are quantitatively and qualitatively similar to those obtained using human immunoglobulin (IVIG). Both immunoglobulins reduce the lipopolysaccharide-induced increase in inflammatory cytokines, interleukin (IL-) 12/23p40 (-90%), IL-6 (-75%) and TNF-α (-60%) and increased the levels of the anti-inflammatory cytokine, IL-10 (+75%). Evidence is presented that PVIG can produce anti-inflammatory effects similar to these after oral application in patients. Its use is contraindicated in patients with lactose intolerance but is otherwise safe and free of complications in clinical studies including the treatment of infants with gastrointestinal disorders. Conclusion: PVIG appears to be a potential and safe anti-inflammatory agent and can be recommended as a candidate medication for studies in COVID-19 patients.


Asunto(s)
Infecciones por Coronavirus/terapia , Síndrome de Liberación de Citoquinas/terapia , Neumonía Viral/terapia , Animales , Betacoronavirus , Bovinos , Células Cultivadas , Síndrome de Liberación de Citoquinas/virología , Citocinas , Humanos , Inmunización Pasiva , Inmunoglobulinas Intravenosas/uso terapéutico , Leucocitos Mononucleares , Pandemias
10.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(11): 1438-1445, 2020 Nov 15.
Artículo en Chino | MEDLINE | ID: mdl-33191703

RESUMEN

Objective: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. Methods: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. Results: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). Conclusion: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.


Asunto(s)
Células Madre Mesenquimatosas , Proteínas del Tejido Nervioso , Receptores de Factores de Crecimiento , Animales , Células de la Médula Ósea , Matriz Ósea , Diferenciación Celular , Células Cultivadas , Silenciador del Gen , Lentivirus , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Osteogénesis , Ratas , Ratas Sprague-Dawley , Receptor de Factor de Crecimiento Nervioso , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Transfección
11.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(11): 1446-1453, 2020 Nov 15.
Artículo en Chino | MEDLINE | ID: mdl-33191704

RESUMEN

Objective: To investigate the effect of microencapsulated transgenic bone marrow mesenchymal stem cells (BMSCs) transplantation on early steroid induced osteonecrosis of femoral head (SONFH) in rabbits. Methods: Alginate poly- L-lysine-sodium alginate (APA) microencapsulated transgenic BMSCs with high expression of Foxc2 were prepared by high-voltage electrostatic method. Part of the cells were cultured in osteoblasts and observed by alizarin red staining at 2 and 3 weeks. Forty New Zealand white rabbits were used to prepare SONFH models by using hormone and endotoxin. Thirty two rabbits who were successful modeling were screened out by MRI and randomly divided into 4 groups (groups A, B, C and D, n=8); another 6 normal rabbits were taken as normal control (group E). The rabbits in group A did not receive any treatment; and in groups B, C, and D were injected with normal saline, allogeneic BMSCs, and APA microencapsulated transgenic BMSCs respectively after core decompression. At 6 and 12 weeks after operation, specimens of femoral head were taken for HE staining to observe bone ingrowth; the expressions of osteocalcin (OCN), peroxisome proliferative activated receptor γ 2 (PPARγ-2), and vascular endothelial growth factor (VEGF) proteins were observed by immunohistochemistry staining. At 12 weeks after operation, the bone microstructure was observed by transmission electron microscope, and the maximum compressive strength and average elastic modulus of cancellous bone and subchondral bone were measured by biomechanics. Results: After 2 and 3 weeks of induction culture, alizarin red staining showed the formation of calcium nodules, and the number of calcium nodules increased at 3 weeks when compared with 2 weeks. The rabbits in each group survived until the experiment was completed. Compared with groups A, B, and C, the trabeculae of group D were more orderly, the empty bone lacunae were less, there were abundant functional organelles, and obvious osteogenesis was observed, and the necrotic area was completely repaired at 12 weeks. Immunohistochemical staining showed that, at 6 and 12 weeks after operation, the expressions of OCN and VEGF in groups A, B, and C were significantly lower than those in groups D and E, while those in groups B and C were significantly higher than those in group A, and in group E than in group D ( P<0.05). The expression of PPARγ-2 was significantly higher in groups A, B, and C than in groups D and E, and in group A than in groups B and C, and in group D than in group E ( P<0.05). At 12 weeks after operation, biomechanical test showed that the average elastic modulus and maximum compressive strength of cancellous bone and subchondral bone in groups D and E were significantly higher than those in groups A, B, and C ( P<0.05); there was no significant difference between groups A, B, and C and between groups D and E ( P>0.05). Conclusion: In vivo transplantation of microencapsulated transgenic BMSCs can repair early SONFH in rabbits.


Asunto(s)
Células Madre Mesenquimatosas , Osteonecrosis , Animales , Células de la Médula Ósea , Células Cultivadas , Cabeza Femoral , Osteogénesis , Conejos , Factor A de Crecimiento Endotelial Vascular
12.
Nat Commun ; 11(1): 5171, 2020 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-33057002

RESUMEN

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function.


Asunto(s)
Región CA3 Hipocampal/fisiología , Proteínas Portadoras/metabolismo , Potenciales Postsinápticos Excitadores/fisiología , Proteínas de la Membrana/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Células Piramidales/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Cultivo Primario de Células , Proteómica , Ratas , Sinaptosomas/metabolismo
13.
J Pharmacol Sci ; 144(4): 197-203, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33070838

RESUMEN

The role of cytoskeleton dynamics in the oxidative stress toward human vasculature has been unclear. The current study examined whether the cytoskeleton-disrupting agent cytochalasin B reduces oxidative stress caused by high glucose in the human arterial smooth muscle. All experiments in the human omental arteries without endothelium or the cultured human coronary artery smooth muscle cells were performed in d-glucose (5.5 mmol/L). The exposure toward d-glucose (20 mmol/L) for 60 min reduced the relaxation or hyperpolarization to an ATP sensitive K+ channel (KATP) opener levcromakalim (10-8 to 3 × 10-6 mol/L and 3 × 10-6 mol/L, respectively). Cytochalasin B and a superoxide inhibitor Tiron, restored them similarly. Cytochalasin B reduced the NADPH oxidase activity, leading to a decrease in superoxide levels of the arteries treated with high d-glucose. Also, cytochalasin B impaired the F-actin constitution and the membrane translocation of an NADPH oxidase subunit p47phox in artery smooth muscle cells treated with high d-glucose. A clinical concentration of cytochalasin B prevented human vascular smooth muscle malfunction via the oxidative stress caused by high glucose. Regulation of the cytoskeleton may be essential to keep the normal vascular function in patients with hyperglycemia.


Asunto(s)
Citocalasina B/farmacología , Citoesqueleto/metabolismo , Glucosa/efectos adversos , Hiperglucemia/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Estrés Oxidativo/efectos de los fármacos , Adulto , Anciano , Células Cultivadas , Cromakalim/farmacología , Femenino , Humanos , Hiperglucemia/fisiopatología , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Relajación Muscular/efectos de los fármacos , NADPH Oxidasas/metabolismo , Superóxidos/metabolismo
14.
Int J Nanomedicine ; 15: 6975-6991, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33061363

RESUMEN

Purpose: Small extracellular vesicles (sEV) are a heterogeneous group of vesicles that consist of proteins, lipids and miRNA molecules derived from the cell of origin. Although xenogeneic sEV have been applied for soft tissue regeneration successfully, the regeneration effect of allogeneic and xenogeneic sEV has not been compared systematically. Methods: Our previous study has shown that sEV derived from rat adipose tissue successfully induced neoadipose regeneration. In this study, sEV were isolated from rat adipose tissue (r-sEV-AT) and porcine adipose tissue (p-sEV-AT), the morphology, size distribution and marker proteins expression of r-sEV-AT and p-sEV-AT were characterized. Besides, the sEV/AT ratio was evaluated and compared between r-sEV-AT and p-sEV-AT. Rat adipose-derived stromal/stem cells (rASCs) and rat aorta endothelial cells (rECs) were adopted to test the cellular response to allogeneic and xenogeneic sEV-AT. The effects of allogeneic and xenogeneic sEV-AT on host cells migration and neoadipose formation were evaluated in a subcutaneous custom-designed model. A full-thickness skin wound healing model was used to further compare the ability of allogeneic and xenogeneic sEV-AT in inducing complex soft tissue regeneration. Results: p-sEV-AT showed similar morphology and size distribution to r-sEV-AT. Marker proteins of sEV were detected in both r-sEV-AT and p-sEV-AT. The sEV/AT ratio of porcine was slightly higher than that of rat. The effects of r-sEV-AT and p-sEV-AT on the differentiation of rASCs and rECs showed no significant difference. When allogeneic and xenogeneic sEV-AT were subcutaneously implanted into the back of SD rats, the host cells chemotactic infiltration was observed in 1 week and neoadipose tissue formation was induced in 8 weeks; no significant difference was observed between allogeneic and xenogeneic sEV-AT. For complex soft tissue regeneration, both allogeneic and xenogeneic sEV-AT significantly promoted wound re-epithelialization, granulation tissue formation and hair follicle regeneration and then accelerated skin wound healing. Conclusion: Our results demonstrated that sEV derived from the same tissues of different species might be loaded with similar therapeutic substance benefitting tissue repair and regeneration, and paved the way for future research aimed at xenogeneic sEV application.


Asunto(s)
Tejido Adiposo/fisiología , Vesículas Extracelulares/trasplante , Trasplante Heterólogo , Trasplante Homólogo , Adipocitos , Tejido Adiposo/citología , Animales , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Células Endoteliales/citología , Espacio Extracelular , MicroARNs , Ratas Sprague-Dawley , Regeneración , Porcinos , Trasplante Heterólogo/métodos , Trasplante Homólogo/métodos , Cicatrización de Heridas
15.
Nat Commun ; 11(1): 5319, 2020 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-33087700

RESUMEN

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1.


Asunto(s)
Células Endoteliales/citología , Células Endoteliales/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Remodelación Vascular/fisiología , Animales , Animales Modificados Genéticamente , Tamaño de la Célula , Células Cultivadas , Factores de Intercambio de Guanina Nucleótido/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Modelos Cardiovasculares , Factor de Crecimiento Placentario/genética , Factor de Crecimiento Placentario/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Remodelación Vascular/genética , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/fisiología
16.
Nat Commun ; 11(1): 5318, 2020 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-33087709

RESUMEN

Synaptic vesicles (SVs) can be pooled across multiple synapses, prompting questions about their dynamic allocation for neurotransmission and plasticity. We find that the axonal traffic of recycling vesicles is not supported by ubiquitous microtubule-based motility but relies on actin instead. Vesicles freed from synaptic clusters undergo ~1 µm bouts of active transport, initiated by nearby elongation of actin filaments. Long distance translocation arises when successive bouts of active transport were linked by periods of free diffusion. The availability of SVs for active transport can be promptly increased by protein kinase A, a key player in neuromodulation. Vesicle motion is in turn impeded by shutting off axonal actin polymerization, mediated by nitric oxide-cyclic GMP signaling leading to inhibition of RhoA. These findings provide a potential framework for coordinating post-and pre-synaptic strength, using retrograde regulation of axonal actin dynamics to mobilize and recruit presynaptic SV resources.


Asunto(s)
Citoesqueleto de Actina/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Óxido Nítrico/fisiología , Vesículas Sinápticas/fisiología , Animales , Transporte Axonal/fisiología , Transporte Biológico Activo , Células Cultivadas , GMP Cíclico/fisiología , Femenino , Hipocampo/citología , Hipocampo/fisiología , Proteínas Luminiscentes/metabolismo , Masculino , Neuronas/fisiología , Nocodazol/farmacología , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología , Vesículas Sinápticas/efectos de los fármacos
17.
Nat Commun ; 11(1): 5292, 2020 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-33087715

RESUMEN

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.


Asunto(s)
Técnicas de Reprogramación Celular/métodos , Células Endoteliales/citología , Hepatocitos/citología , Células Madre/citología , Animales , Conductos Biliares/citología , Conductos Biliares/fisiología , Agregación Celular , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Células Endoteliales/fisiología , Femenino , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/fisiología , Factor Nuclear 3-gamma del Hepatocito/genética , Factor Nuclear 3-gamma del Hepatocito/fisiología , Factor Nuclear 6 del Hepatocito/genética , Factor Nuclear 6 del Hepatocito/fisiología , Hepatocitos/fisiología , Hepatocitos/trasplante , Xenoinjertos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Esferoides Celulares/citología , Esferoides Celulares/fisiología , Células Madre/fisiología
18.
Toxicol Lett ; 334: 87-93, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-33002526

RESUMEN

The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 µM) and IAD (IC50 10 µM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.


Asunto(s)
Microbioma Gastrointestinal , Ácidos Indolacéticos/metabolismo , Indoles/metabolismo , Mucosa Intestinal , Receptor X de Pregnano/agonistas , Triptófano/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Genes Reporteros , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ligandos , Masculino , Receptor X de Pregnano/genética , Unión Proteica , Transfección
19.
Toxicol Lett ; 334: 94-101, 2020 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-33010382

RESUMEN

Silica dust mainly attacks alveolar macrophages (AMs). The apoptosis of AMs is correlated with the progress of silicosis. Our previous study showed that autophagic degradation was blocked in AMs from silicosis patients. However, the effects of nicotine on AM autophagy and apoptosis in silicosis are unknown. In this study, we collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups, according to the tuberous pathological changes observed by X-ray. The AMs from both groups were exposed to nicotine. We found increased levels of LC3, p62, and cleaved caspase-3, decreased levels of LAMP2, and damaged lysosomes after nicotine stimulation of the AMs from both groups. We also found that the autophagy inhibitor 3-methyladenine (3MA) inhibited nicotine-induced apoptosis in the AMs. Furthermore, 3MA reversed both the nicotine-induced decrease in Bcl-2 and the increase in Bax in both groups. These results suggest that nicotine may induce apoptosis by blocking AM autophagic degradation in human silicosis.


Asunto(s)
Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos , Nicotina/toxicidad , Silicosis/patología , Adenina/análogos & derivados , Adenina/farmacología , Caspasa 3/metabolismo , Células Cultivadas , Humanos , Etiquetado Corte-Fin in Situ , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/patología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Dióxido de Silicio/toxicidad , Silicosis/metabolismo
20.
Medicine (Baltimore) ; 99(42): e22438, 2020 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-33080678

RESUMEN

BACKGROUND: Myeloid cell leukemia-1 (Mcl-1) plays an important role in the clearance of Mycobacterium tuberculosis (MTB) infection. It has the effect of anti-apoptosis, protecting macrophages that have engulfed pathogens and preventing pathogen clearance. Meanwhile, the MAPK signaling pathway plays a significant role in regulating Mcl-1 expression during tuberculosis infection. In the case of latent infection and active infection, the apoptosis and polarization of macrophages have a great influence during MTB infection, so we discussed the effect of Mcl-1 on apoptosis and polarization. Then, further discussed its mechanism. METHODS: An infected RAW264.7 macrophage model was established to investigate the regulatory role and mechanism of the Mcl-1 pathway inhibition during apoptosis and polarization of H37Rv infection. First, Mcl-1 protein and mRNA was identified by western blotting and Real-Time Polymerase Chain Reaction (RT-PCR). RAW264.7 macrophage apoptosis was detected by flow cytometry. RT-PCR was utilized to detect Bax, Caspase-3, Cyt-c and Bcl-2 mRNA expression. Next, Then the expression levels of inflammation factors CD86, CD206, iNOS, Fizz1, IL-6, IL-10, TNF-α, and TGF-ß was detected by ELISA. SEM was used to observe macrophages phenotype. Finally, Bax, Bcl-2 and Bcl-xl the expression was detected by western blotting. Confocal microscopy was used to analyze mitochondrial membrane potential using the JC-10 kit. RESULTS: In this study, we found that inhibiting the Mcl-1 expression signaling pathway led to infection by different virulence Mycobacterium tuberculosis, as well as changes in Mcl-1 protein and mRNA expression. Concomitantly macrophage apoptosis rate also changed, While, two phenotypic states of M1 and M2 appeared in the infected cells. We also found that the mitochondrial pathway was activated, the expression of its related genes Bax, casepase3, and Cyt-c, increased, whereas that of Bcl-2 decreased, and the mitochondrial membrane depolarization function was changed. CONCLUSIONS: We found that Mcl-1 affected the apoptosis and polarization of macrophages infected by Mycobacterium tuberculosis, mainly M1 in the early stage and M2 in the later stage. In addition, mitochondria played a crucial role in this process.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/inmunología , Hidrolasas/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Activación de Macrófagos/inmunología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/inmunología , Células Cultivadas , Regulación hacia Abajo , Activación Enzimática , Humanos , Macrófagos/inmunología , Potenciales de la Membrana , Fenotipo , Virulencia
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