ABSTRACT
Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB. In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain.
Subject(s)
Drug Carriers/pharmacology , I-kappa B Kinase/antagonists & inhibitors , Nanoparticles/therapeutic use , Neuralgia/drug therapy , Polylactic Acid-Polyglycolic Acid Copolymer/pharmacology , RNA, Small Interfering/pharmacology , Animals , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Male , Microglia/pathology , Neuralgia/genetics , Neuralgia/metabolism , Neuralgia/pathology , RNA, Small Interfering/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Sphingolipids (SPLs) have been proposed as potential therapeutic targets for strokes, but no reports have ever profiled the changes of the entire range of SPLs after a stroke. This study applied sphingolipidomic methods to investigate the temporal and individual changes in the sphingolipidome including the effect of atorvastatin after ischemic brain injury. We conducted sphingolipidomic profiling of mouse brain tissue by liquid chromatography-electrospray ionization tandem mass spectrometry at 3 h and 24 h after 1 h of middle cerebral artery occlusion (MCAO), and SPL levels were compared with those of the Sham control group. At 3 h post-MCAO, ceramides (Cers) exhibited an increase in levels of long-chain Cers but a decrease in very-long-chain Cers. Moreover, sphingosine, the precursor of sphingosine-1-phosphate (S1P), decreased and S1P increased at 3 h after MCAO. In contrast to 3 h, both long-chain and very-long-chain Cers showed an increased trend at 24 h post-MCAO. Most important, the administration of atorvastatin improved the neurological function of the mice and significantly reversed the SPL changes resulting from the ischemic injury. Furthermore, we used plasma samples from nonstroke control and stroke patients at time points of 72 h after a stroke, and found a similar trend of Cers as in the MCAO model. This study successfully elucidated the overall effect of ischemic injury on SPL metabolism with and without atorvastatin treatment. The network of SPL components that change upon ischemic damage may provide novel therapeutic targets for ischemic stroke.
Subject(s)
Brain Injuries/genetics , Brain Ischemia/genetics , Sphingolipids/genetics , Stroke/genetics , Animals , Atorvastatin/pharmacology , Brain/metabolism , Brain/pathology , Brain Injuries/drug therapy , Brain Injuries/metabolism , Brain Injuries/pathology , Brain Ischemia/drug therapy , Brain Ischemia/metabolism , Brain Ischemia/pathology , Ceramides/genetics , Ceramides/metabolism , Chromatography, High Pressure Liquid/methods , Disease Models, Animal , Humans , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Lipidomics/methods , Lysophospholipids/metabolism , Mice , Sphingolipids/isolation & purification , Sphingolipids/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Stroke/drug therapy , Stroke/metabolism , Stroke/pathology , Tandem Mass Spectrometry/methodsABSTRACT
Transplantation of encapsulated porcine islets is proposed to treat type 1 diabetes. However, the envelopment of fibrous tissue and the infiltration of immune cells impair islet function and eventually cause implant failure. It is known that hemodialysis using an ethylene vinyl alcohol (EVOH) membrane results in minor tissue responses. Therefore, we hypothesized that using a low-adhesive EVOH membrane for encapsulation may prevent host cell accumulation and fibrous capsule formation. In this study, rat islets suspended in chitosan gel were encapsulated in bags made from highly porous EVOH membranes, and their in vitro insulin secretion function as well as in vivo performance was evaluated. The results showed that the EVOH bag did not affect islet survival or glucose-stimulated insulin secretion. Whereas naked islets were dysfunctional after 7 days of culture in vitro, islets within the EVOH bag produced insulin continuously for 30 days. Streptozotocin-induced diabetic mice were given islets-chitosan gel-EVOH implants intraperitoneally (650-800 islets equivalent) and exhibited lower blood glucose levels and regained body weight during a 4-week observation period. The transplanted mice had higher levels of serum insulin and C-peptide, with an improved blood glucose disappearance rate. Retrieved implants had minor tissue adhesion, and histology showed a limited number of mononuclear cells and fibroblasts surrounding the implants. No invasion of host cells into the EVOH bags was noticed, and the encapsulated islets were intact and positive for insulin-glucagon immunostaining. In conclusion, an EVOH bag can protect encapsulated islets, limit fibrous capsule formation, and extend graft function.
Subject(s)
Cells, Immobilized/metabolism , Diabetes Mellitus, Experimental/therapy , Diabetes Mellitus, Type 1/therapy , Insulin/metabolism , Islets of Langerhans Transplantation/methods , Islets of Langerhans/metabolism , Animals , Blood Glucose , Body Weight , Cell Survival , Graft Survival , Mice , Polyvinyls , Rats , Treatment OutcomeABSTRACT
BACKGROUND: Acetaminophen (APAP) overdose causes acute liver failure (ALF) in animals and humans via the rapid depletion of intracellular glutathione (GSH) and the generation of excess reactive oxygen species (ROS) that damage hepatocytes. Stem cell therapy is a potential treatment strategy for ALF. METHODS: We isolated mesenchymal stem cells (MSCs) from mice omentum adipose tissue-derived stem cells (ASCs) and transplanted them into a mouse model of APAP-induced ALF to explore their therapeutic potential. In addition, we performed in vitro co-culture studies with omentum-derived ASCs and primary isolated hepatocytes to demonstrate the hepatoprotective effect of omentum-derived ASCs on hepatocytes that were subjected to APAP-induced damage. RESULT: ASC transplantation significantly improved the survival rate of mice with ALF and attenuated the severity of APAP-induced liver damage by suppressing cytochrome P450 activity to reduce the accumulation of toxic nitrotyrosine and the upregulation of NF-E2-related factor 2 (Nrf2) expression, resulting in an increase in the subsequent antioxidant activity. These effects protected the hepatocytes from APAP-induced damage through the suppression of downstream MAPK signal activation and inflammatory cytokine production. CONCLUSIONS: our results demonstrate that omentum-derived ASCs are an alternative source of ASCs that regulate the antioxidant response and may represent a beneficial therapeutic strategy for ALF.
Subject(s)
Acetaminophen/adverse effects , Adipose Tissue/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Gene Expression Regulation, Enzymologic/drug effects , Liver Failure, Acute , NF-E2-Related Factor 2/metabolism , Omentum/metabolism , Stem Cell Transplantation , Stem Cells/metabolism , Acetaminophen/pharmacology , Animals , Liver Failure, Acute/chemically induced , Liver Failure, Acute/metabolism , Liver Failure, Acute/prevention & control , Male , MiceABSTRACT
The maintenance of human telomeres requires the ribonucleoprotein enzyme telomerase, which is composed of telomerase reverse transcriptase (TERT), telomerase RNA component, and several additional proteins for assembly and activity. Telomere elongation by telomerase in human cancer cells involves multiple steps including telomerase RNA biogenesis, holoenzyme assembly, intranuclear trafficking, and telomerase recruitment to telomeres. Although telomerase has been shown to accumulate in Cajal bodies for association with telomeric chromatin, it is unclear where and how the assembly and trafficking of catalytically active telomerase is regulated in the context of nuclear architecture. Here, we show that the catalytically active holoenzyme is initially assembled in the dense fibrillar component of the nucleolus during S phase. The telomerase RNP is retained in nucleoli through the interaction of hTERT with nucleolin, a major nucleolar phosphoprotein. Upon association with TCAB1 in S phase, the telomerase RNP is transported from nucleoli to Cajal bodies, suggesting that TCAB1 acts as an S-phase-specific holoenzyme component. Furthermore, depletion of TCAB1 caused an increase in the amount of telomerase RNP associated with nucleolin. These results suggest that the TCAB1-dependent trafficking of telomerase to Cajal bodies occurs in a step separate from the holoenzyme assembly in nucleoli. Thus, we propose that the dense fibrillar component is the provider of active telomerase RNP for supporting the continued proliferation of cancer and stem cells.
Subject(s)
Cell Nucleolus/enzymology , S Phase , Telomerase/metabolism , Cell Line, Tumor , Flow Cytometry , Holoenzymes/metabolism , HumansABSTRACT
Methods for detecting circulating microRNAs (miRNAs), small RNAs that control gene expression, at high sensitivity and specificity in the blood have been reported in recent studies. The goal of this study was to determine if detectable levels of specific miRNAs are released into the circulation for bevacizumab-induced cardiotoxicity. A miRNA array analysis was performed using RNA isolated from 10 control patients in bevacizumab treatment, and n=10 patients have been confirmed to have bevacizumab-induced cardiotoxicity. From the array, we selected 19 candidate miRNA for a second validation study in 90 controls and 88 patients with bevacizumab-induced cardiotoxicity. Consistent with the data obtained from the microRNA array, circulating levels of five miRNAs were significantly increased in patients with bevacizumab-induced cardiotoxicity compared with controls. To confirm these data, we compared selected miRNAs in the plasma of patients with bevacizumab-induced cardiotoxicity with those of 66 patients with acute myocardial infarction (AMI). Moreover, we went on to analyze what factors may influence the levels of potential biomarker miRNAs. Consistent with the data obtained from the microRNA array, circulating levels of five miRNAs were significantly increased in patients with bevacizumab-induced cardiotoxicity compared with those of healthy bevacizumab treatment controls. However, only miRNA1254 and miRNA579 showed high specificity in the validation experiments. Moreover, we went on to analyze what factors may influence the levels of potential biomarker miRNAs. We identify two miRNAs that are specifically elevated in patients with bevacizumab-induced cardiotoxicity, miR1254 and miRNA579, and miRNA1254 shows the strongest correlation to the clinical diagnosis of bevacizumab-induced cardiotoxicity.
Subject(s)
Angiogenesis Inhibitors/adverse effects , Antibodies, Monoclonal, Humanized/adverse effects , Colorectal Neoplasms/drug therapy , Heart/drug effects , MicroRNAs/blood , Aged , Bevacizumab , Colorectal Neoplasms/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle AgedABSTRACT
Continuous-wave (CW) room-temperature (RT) laser operation with low energy consumption is an ultimate goal for electrically driven lasers. A monolithically integrated perovskite laser in a chip-level fiber scheme is ideal. However, because of the well-recognized air and thermal instabilities of perovskites, laser action in a perovskite has mostly been limited to either pulsed or cryogenic-temperature operations. Most CW laser operations at RT have had poor durability. Here, crystal fibers that have robust and high-heat-load nature are shown to be the key to enabling the first demonstration of ultralow-threshold CW RT laser action in a compact, monolithic, and inexpensive crystal fiber/nanoperovskite hybrid architecture that is directly pumped with a 405 nm diode laser. Purcell-enhanced light-matter coupling between the atomically smooth fiber microcavity and the perovskite nanocrystallites gain medium enables a high Q (≈1500) and a high ß (0.31). This 762 nm laser outperforms previously reported structures with a record-low threshold of 132 nW and an optical-to-optical slope conversion efficiency of 2.93%, and it delivers a stable output for CW and RT operation. These results represent a significant advancement toward monolithic all-optical integration.
ABSTRACT
Subcutaneous pouch is a potential site for islet transplantation. However, insufficient oxygen supply remains challenging. Pretreatment of neovascularization using basic fibroblast growth factor can solve this, but it needs 2× operations. We developed a device that contains rat islets in chitosan gel packed in a bag made of highly biocompatible ethylene vinyl alcohol copolymer porous membrane. This study investigated whether coencapsulation of hepatocyte growth factor (HGF) with islets in the device enables novel method of prevascularization-free primary subcutaneous transplantation. METHODS: In vitro experiments examined slow release of HGF from the chitosan gel and islet-protection effect of HGF against hypoxia. In the latter, rat islets with/without HGF (200 ng/mL) was cultured in 1% oxygen. In in vivo experiment, fabricated device with/without HGF (10 µg/device) containing rat islets was primarily transplanted to streptozotocin-induced diabetic mice subcutaneously. RESULTS: In vitro experiments showed sustained release of HGF for 28 d and alleviating effect of HGF on cell death and glucose-responsive insulin release after hypoxic culture. Islet + HGF mice, but not islet-alone mice, showed decreased nonfasting blood glucose and regained body weight after transplantation. In intraperitoneal glucose tolerance test, islet + HGF mice exhibited decreased fasting blood glucose (200 ± 55 mg/dL) and good blood glucose disappearance rate (K value) (0.817 ± 0.101) comparing to normal mice (123 ± 28 mg/dL and 1.074 ± 0.374, respectively). However, in islet-alone mice, fasting blood glucose was high (365 ± 172 mg/dL) and K value was indeterminable. Serum insulin in islet + HGF mice (1.58 ± 0.94 µg/L) was close to normal mice (1.66 ± 0.55 µg/L), whereas those in islet-alone mice (0.279 ± 0.076 µg/L) and diabetic mice (0.165 ± 0.079 µg/L) were low. Immunohistochemical examination showed intact insulin- and glucagon-positive islets in retrieved devices with HGF, but no intact islet was found in the device without HGF. CONCLUSIONS: HGF could enhance islet survival in hypoxia and enhance in vivo function of encapsulated islets after primary subcutaneous transplantation.
ABSTRACT
BACKGROUND: Subcutaneous pockets provide an extrahepatic transplant site for islet grafting to treat type 1 diabetes. However, a hypoxic environment may cause central necrosis to islets and lead to graft failure. Our previous studies focused on a pre-treated subcutaneous site with basic fibroblast growth factor (bFGF) for the formation of vascular bed. In addition to neovascularization, bFGF was also shown to protect islets against oxidative stress and chemical-induced damage in vitro. Accordingly, we propose that subcutaneous islet transplantation with a bFGF-slow releasing device simultaneously can improve islet survival in vivo. METHODS: A bFGF-impregnated collagen sheet was implanted in the right back of a streptozotocin-induced diabetic mouse for neovascularization. After 10 days, the sheet was removed and the rat islet-embedding gel within the immune-isolation device was transplanted (2-time operation [OP]). In another group, the diabetic mice received bFGF-impregnated gel with rat islets within the immune-isolation device simultaneously (1-time OP). RESULTS: Diabetic mice in 2-time OP group experienced a decrease in their non-fasting blood glucose level for a period of 10 days, and the glucose levels were lower than those of untreated diabetic mice post-implantation. However, the mice in the 1-time OP group remained hyperglycemic post-operation and showed no improvements in body weight or the area under curve in intraperitoneal glucose tolerance test. Furthermore, mice in the 2-time OP had relatively higher serum insulin levels with improved renal and metabolic biomarkers. CONCLUSION: Our findings suggest that bFGF had no beneficial effect on a 1-time operation in subcutaneous islet transplantation.
Subject(s)
Diabetes Mellitus, Experimental/surgery , Fibroblast Growth Factor 2/administration & dosage , Islets of Langerhans Transplantation/instrumentation , Islets of Langerhans Transplantation/methods , Subcutaneous Tissue , Animals , Blood Glucose , Diabetes Mellitus, Experimental/metabolism , Disease Models, Animal , Heterografts , Male , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic/drug effects , Rats , Rats, Inbred Lew , Subcutaneous Tissue/surgeryABSTRACT
Periodontitis is an inflammatory disease caused by bacteria. In periodontitis, reactive oxygen species (ROS) are released from inflammatory cells in response to bacteria. Interleukin (IL)-8 is one of pro-inflammatory cytokines. To investigate the role of ROS in pathogenesis of periodontitis, we estimated the effect of H(2)O(2), one of ROS, on the expression of IL-8 in human periodontal ligament (PDL) cells. PDL cells were treated with H(2)O(2). IL-8 expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38) and c-jun NH(2)-terminal kinase (JNK) was estimated by Western blotting. Treatment with H(2)O(2) at concentration of up to 250 microM increased IL-8 mRNA expression and production in a concentration-dependent manner. However, treatment with 500 microM H(2)O(2) did not increase IL-8 production. Catalase, an inhibitor of H(2)O(2), down-regulated the production of IL-8 induced by H(2)O(2). H(2)O(2) increased the phosphorylation of ERK, p38, and JNK. Pretreatment with PD98059 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (JNK inhibitor) decreased the IL-8 production induced by H(2)O(2). These results indicate that H(2)O(2) acts as an inducer of IL-8 secretion via activation of ERK, p38, and JNK in PDL cells. H(2)O(2) deposited in periodontal tissue during inflammation against bacteria may accelerate tissue destruction via induction of IL-8 in PDL cells.
Subject(s)
Hydrogen Peroxide/pharmacology , Interleukin-8/immunology , Periodontal Ligament/drug effects , Periodontal Ligament/immunology , Periodontitis/immunology , Up-Regulation/drug effects , Cell Survival/drug effects , Cells, Cultured , Gene Expression/drug effects , Humans , Interleukin-8/genetics , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Periodontitis/microbiology , Phosphorylation , Signal Transduction/drug effectsABSTRACT
Cellular lipidomic studies have been favored approaches in many biomedical research areas. To provide fair comparisons of the studied cells, it is essential to perform normalization of the determined concentration before lipidomic analysis. This study proposed a cellular lipidomic normalization method by measuring the phosphatidylcholine (PC) and sphingomyelin (SM) contents in cell extracts. To provide efficient analysis of PC and SM in cell extracts, flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) with a precursor ion scan (PIS) of m/z 184 was used, and the parameters affecting the performance of the method were optimized. Good linearity could be observed between the cell extract dilution factor and the reciprocal of the total ion chromatogram (TIC) area in the PIS of m/z 184 within the dilution range of 1- to 16-fold (R2 = 0.998). The calibration curve could be used for concentration adjustment of the unknown concentration of a cell extract. The intraday and intermediate precisions were below 10%. The accuracy ranged from 93.0% to 105.6%. The performance of the new normalization method was evaluated using different numbers of HCT-116 cells. Sphingosine, ceramide (d18:1/18:0), SM (d18:1/18:0) and PC (16:1/18:0) were selected as the representative test lipid species, and the results showed that the peak areas of each lipid species obtained from different cell numbers were within a 20% variation after normalization. Finally, the PIS of 184 normalization method was applied to study ischemia-induced neuron injury using oxygen and glucose deprivation (OGD) on primary neuronal cultured cells. Our results showed that the PIS of 184 normalization method is an efficient and effective approach for concentration normalization in cellular lipidomic studies.
Subject(s)
Chromatography, Liquid , Phosphatidylcholines/analysis , Spectrometry, Mass, Electrospray Ionization , Sphingomyelins/analysis , Tandem Mass Spectrometry , Animals , Cell Line, Tumor , Flow Injection Analysis , Humans , Neurons , Rats, Sprague-DawleyABSTRACT
PURPOSE: Globular adiponectin (gAd) is a type of adipocytokine, which is mainly produced by adipose tissue. It has been reported that gAd acts as a pro- as well as an anti-inflammatory factor. Interleukin (IL)-6 and IL-8 are pro-inflammatory cytokines. To investigate the role of gAd on periodontal tissues, the expression of adiponectin receptor 1 (AdipoR1) and the effect of gAd on the expression of IL-6 and IL-8 were investigated in periodontal ligament (PDL) and gingival fibroblasts. METHODS: PDL and gingival fibroblasts were cultured from human periodontal tissues. gAd derived from Escherichia coli and murine myeloma cells were used. The expression of AdipoR1 was estimated by reverse transcription-polymerase chain reaction and western blot. The expression of cytokines was measured by enzyme-linked immunosorbent assay. RESULTS: PDL and gingival fibroblasts expressed both mRNA and protein of AdipoR1. gAd derived from E. coli increased the production of IL-6 and IL-8, but polymyxin B, an inhibitor of lipopolysaccharide (LPS), inhibited IL-6 and IL-8 production induced by gAd in both types of cells. gAd derived from murine myeloma cells did not induce IL-6 and IL-8 production in those cells. gAd derived from E. coli contained higher levels of LPS than gAd derived from murine myeloma cells. LPS increased production of IL-6 and IL-8 in PDL and gingival fibroblasts, but pretreatment of cells with gAd derived from murine myeloma cells did not inhibit LPS-induced IL-6 and IL-8 expression. CONCLUSIONS: Our results suggest that PDL and gingival fibroblasts express AdipoR1 and that gAd does not act as a modulator of IL-6 and IL-8 expression in PDL and gingival fibroblasts.
ABSTRACT
The establishment of a tracheal airway with direct laryngoscopy can either be difficult or impossible in children with airway pathology. Multiple direct laryngoscopic attempts cause oedema and/or bleeding with subsequent difficult ventilation. The techniques utilizing the laryngeal mask airway (LMATM) and the fibreoptic bronchoscope have been reported. The case of a child with lingual thyroglossal duct cyst in which the LMA was useful to secure the airway and as a conduit for fibreoptic tracheal intubation is reported.