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@#BACKGROUND: Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury. Mesenchymal stem cell (MSC) transplantation is used to reduce tissue damage, but exosomes are more stable and highly conserved than MSCs. This study was conducted to investigate the therapeutic effects of MSC-derived exosomes (MSC-Exo) on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R), and to explore the underlying mechanisms. METHODS: Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment, with or without MSC-Exo treatment. Exosomal integration, cell viability, mitochondrial membrane potential, and generation of reactive oxygen species (ROS) were examined. Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick-end labeling (TUNEL) staining was performed to detect neuronal apoptosis. Moreover, mitochondrial function-associated gene expression, Nrf2 translocation, and expression of downstream antioxidant proteins were determined. RESULTS: MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation (P<0.05). The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus (2.14±0.65 vs. 5.48±1.09, P<0.01) and increased the intracellular expression of antioxidative proteins, including superoxide dismutase and glutathione peroxidase (17.18±0.97 vs. 14.40±0.62, and 20.65±2.23 vs. 16.44±2.05, respectively; P<0.05 for both). OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial function-associated genes, such as PINK, DJ1, LRRK2, Mfn-1, Mfn-2, and OPA1. The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons. CONCLUSIONS: MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons. Therefore, MSC-Exo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.
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ObjectiveHyperlipidemic severe acute pancreatitis, which has a poor prognosis, is a common acute and critical disease. To discuss the reactive factors and prognosis analysis of serum lipid level to plasma exchange in patients with hyperlipidemic severe acute pancreatitis (HL-SAP).MethodsA retrospective study was conducted on the clinical data of 70 HL-SAP patients admitted to the department of critical care medicine in Nanjing drum tower hospital from January 2010 to May 2018. All patients received plasma exchange therapy, and were divided into the high-response group (>60%) and the low-response group (< 60%) according to the decrease of serum triglyceride (TG) level. Single factor analysis was conducted with χ2 or Mann-Whitney U test in the patients' gender, age, body mass index (BMI), Ranson score, clinical acute physiology assessment and chronic health evaluation (APA-CHE), sequential organ failure assessment(SOFA), start time from incidence to plasmapheresis, plasma exchange dosage, blood flow velocity, serum amylase, albumin, red blood cells hematocrit (Hct), blood TG before plasma exchange, total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL). The factors, which include gender, start time from incidence to plasmapheresis, TG, and Hct, have statistical differences in single factor analysis and were incorporated into the Logstic regression analysis for a multifactor analysis.ResultsTG levels ranged from 10.10 to 53.60 mmol/L in 70 patients with an average of (21.45±13.56) mmol/L before plasma exchange while it ranged from 1.97 to 20.00 mmol/L with an average of (6.10±3.58) mmol/L after plasma exchange, and the difference was statistically significant (P<0.05). In this group, the TG decreased by 12.75%~89.43%, which included 46 patients in the high-response group and 24 patients in the low-response group. Compared with the low-response group, the high-response group has statistically significant (P<0.05) in the differences of gender, start time from incidence to plasmapheresis, serum amylase, plasma exchange dosage, red blood cells hematocrit (Hct), blood lipid before exchange, TC, and HDL. Multivariate Logistic regression analysis showed that changes in blood lipid level after plasmapheresis in HL-SAP patients were significantly correlated with gender, start time from incidence to plasmapheresis, red blood cells hematocrit (Hct), blood lipid before plasmapheresis (P<0.05). Compared with the low-response group, it has statistically significant in difference between acute kidney injury and mortality in the high-response group (P<0.05).ConclusionThe reactive factors for the efficacy of plasma exchange in patients with HL-SAP are gender, tart time from incidence to plasmapheresis, red blood cells hematocrit (Hct), and blood lipid before plasmapheresis. The low response group had a higher incidence of acute kidney injury and a poor prognosis.
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OBJECTIVE: To construct a novel fusion protein contained insulin-like growth factor 1 (IGF-1) and lidamycin (LDM) and evaluate its antitumor activity on non-small cell lung cancer (NSCLC). METHODS: DNA fragment coding for fusion protein (ldp-igf) was synthesized by linking apoprotein of lidamycin (ldp) with igf-1, and then was cloned into the plasmid pET30a. Fusion protein LDP-IGF was expressed in E.coli as inclusion bodies and was purified by Ni2+ affinity chromatography. Binding affinity of LDP-IGF to NSCLC cells was evaluated by immunofluorescence assay and flow cytometry-based binding assay. MTT assay was used to measure the in vitrocytotoxicity of LDP-IGF and its enediyne-energized analogue LDP-IGF-AE. PI staining assay and Annexin V-FITC/PI staining assay were used to analyze the cell cycle arrest and cell apoptosis after treatment with LDP-IGF-AE, respectively. RESULTS: Active soluble LDP-IGF protein was prepared by isolation, purification, denaturation and refolding, and the production of LDP-IGF was 12 mg per liter fermentation broth. Both of immunofluorescence assay and flow cytometry-based binding assay showed that LDP-IGF has strong binding activity to NSCLC cells. Enediyne-energized fusion protein LDP-IGF-AE exhibited potent cytotoxicity to NSCLC cells in vitro, and it is more potent than that of LDM. Furthermore, fusion protein LDP-IGF without active enediyne was also cytotoxic to A549 cells at high concentrations (50 and 100 μg·mL-1). LDP-IGF-AE could cause significant G2-M arrest in A549 and H460 cells, and it also induced the apoptosis in NSCLC cells in a concentration-dependent manner. CONCLUSION: Fusion protein LDP-IGF-AE shows potent antitumor efficacy in vitro on NSCLC, suggesting it could be a promising candidate for targeted therapy.
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<p><b>OBJECTIVE</b>To investigate the safety and efficacy of low-concentration inhaled nitric oxide (NO) in the treatment of hypoxic respiratory failure (HRF) among premature infants.</p><p><b>METHODS</b>Sixty premature infants (gestational age ≤ 34 weeks) with HRF were randomized into NO and control groups between 2012 and 2013, with 30 cases in each group. Both groups received nasal continuous positive airway pressure (nCPAP) or mechanical ventilation. NO inhalation was continued for at least 7 days or until weaning in the NO group. The general conditions, blood gas results, complications, and clinical outcomes of the two groups were analyzed.</p><p><b>RESULTS</b>The NO group showed significantly more improvement in blood gas results than the control group after 12 hours of treatment (P<0.05). After that, the change in oxygenation status over time showed no significant difference between the two groups (P>0.05). There were no significant differences in total time of assisted ventilation and duration of oxygen therapy between the two groups (P>0.05). The incidence of bronchopulmonary dysplasia (BPD), patent ductus arteriosus, necrotizing enterocolitis, retinopathy of prematurity, and pneumothorax in infants showed no significant differences between the NO and control groups (P>0.05), but the incidence of IVH and mortality were significantly lower in the NO group than in the control group (7% vs 17%, P<0.05; 3% vs 13%, P<0.05).</p><p><b>CONCLUSIONS</b>NO inhalation may improve oxygenation status and reduce the mortality in premature infants with HRF, but it cannot reduce the incidence of BPD and the total time of mechanical ventilation or nCPAP and duration of oxygen therapy. NO therapy may have a brain-protective effect for premature infants with HRF and does not increase clinical complications.</p>
Subject(s)
Humans , Infant, Newborn , Administration, Inhalation , Blood Gas Analysis , Bronchopulmonary Dysplasia , Epidemiology , Hypoxia , Incidence , Infant, Premature , Nitric Oxide , Respiratory Insufficiency , Blood , Drug TherapyABSTRACT
The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.
Subject(s)
Humans , Cell Differentiation , Cells, Cultured , Dendritic Cells , Cell Biology , Metabolism , Ligands , Lipopolysaccharides , Mannose-Binding Lectin , Pharmacology , Monocytes , Cell Biology , Metabolism , Toll-Like Receptor 4 , MetabolismABSTRACT
OBJECTIVE: To construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth factor receptor 2(HER2) and lidamycin(LDM), and investigate its antitumor activity. METHODS: Coding sequences of oligopeptides from complementarity determining region 3(CDR3) of anti-HER2 antibody C6.5 heavy chain was fused to apoprotein of lidamycin to obtain the fusion gene ldp-Hr. Fusion protein LDP-Hr was expressed in E. coli and purified by affinity chromatography. The purity of LDP-Hr was analyzed by high HPLC. Immunofluorescence assay and flow cytometry-based binding assay were used to investigate the binding activity of LDP-Hr to HER2 overexpressed cancer cells. The energized fusion protein LDP-Hr-AE was prepared by integrating the active enediyne chromophore (AE) of lidamycin into the LDP-Hr protein. MTT assay was used to measure the in vitro cytotoxicity of LDP-Hr-AE and Annexin V-FITC/PI staining assay was used to analyze its apoptosis-inducing efficacy. RESULTS: Fusion protein LDP-Hr was constructed correctly and expressed in E. coli in a secretory manner. The production of LDP-Hr was 40 mg per liter fermentation broth, and the purity of fusion protein was 97.4% as analyzed by HPLC. LDP-Hr showed strong binding activity to cancer cells highly expressing HER2, such as SK-BR-3 and SK-OV-3 cells. The energized fusion protein LDP-Hr-AE exhibited more potent cytotoxicity to SK-BR-3 and SK-OV-3 cells than LDM as measured by MTT assay. The results from Annexin V-FITC/PI staining assay also revealed that LDP-Hr-AE significantly induced cell apoptosis even at very low concentrations. CONCLUSION: The novel energized fusion protein LDP-Hr-AE bounds to HER2 specifically, and shows potent cytotoxicity and apoptosis-inducing activity to cancer cells, which suggests that it would be a promising candidate for targeted cancer therapy.
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<p><b>OBJECTIVE</b>To assess the association of haptoglobin (HP)1/2 polymorphism with coronary heart disease (CHD) in Chinese Hans.</p><p><b>METHODS</b>One hundred and eighty-nine CHD patients and 242 healthy controls confirmed with angiography were recruited. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was utilized to genotype the HP1 and HP2 alleles and genotype frequencies in cases and controls were compared.</p><p><b>RESULTS</b>The frequency of HP2-2 genotype was significantly higher in CHDs than in controls (0.54 vs.0.35, P = 0.000). The HP2-2 genotype significantly increased the risk for CHD in univariable analysis (OR = 2.166, 95%CI: 1.467-3.196). Multifactor Logistic regression analysis indicated that HP2-2 genotype is an independent risk factor to CHD (P = 0.002; OR = 2.101, 95%CI: 1.311-3.367). Similarly, the HP2 allele frequency in the CHD group was significantly higher than that in the control subjects (0.74 vs.0.61, P = 0.000).</p><p><b>CONCLUSION</b>The HP2-2 genotype is associated with CHD in Chinese. HP2-2 genotype may be an independent risk factor to CHD, and HP2 allele may be a genetic susceptibility factor to CHD in Chinese.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Asian People , Genetics , Coronary Disease , Genetics , Gene Frequency , Genotype , Haptoglobins , Genetics , Logistic Models , Polymorphism, GeneticABSTRACT
<p><b>OBJECTIVE</b>Lidamycin (LDM) can be dissociated to an apoprotein (LDP) and an active enediyne chromophore (AE). The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy.</p><p><b>METHODS</b>A method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L(9) (3(4)) was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated.</p><p><b>RESULTS</b>A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature>time>pH>molar ratio of AE to protein and all were statistically significant (P<0.01). The optimal assembly conditions were temperature at 10°C, time of 12 h, pH 7.0, and the molar ratio of AE: protein of 5:1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization.</p><p><b>CONCLUSION</b>The assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.</p>
Subject(s)
Humans , Aminoglycosides , Chemistry , Pharmacology , Antibiotics, Antineoplastic , Chemistry , Pharmacology , Apoproteins , Chemistry , Cell Line, Tumor , Cell Survival , Chromatography, High Pressure Liquid , Drug Design , Enediynes , Chemistry , Pharmacology , Recombinant Fusion Proteins , Chemistry , Single-Chain Antibodies , ChemistryABSTRACT
<p><b>BACKGROUND</b>von Willebrand factor (vWF) mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arterial stenosis. On release from endothelial cells, vWF is rapidly cleaved by ADAMTS13/vWF-cleaving protease (vWF-CP). We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease.</p><p><b>METHODS</b>Plasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay (ELISA) and residual collagen binding assay respectively in patients with lupus nephritis (n = 31), primary nephritic syndrome (n = 25), diabetic nephropathy (n = 45), chronic glomerulonephritis (n = 38) and 40 normal controls. The relation of their levels with pathological and renal status was analyzed.</p><p><b>RESULTS</b>In all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls (both P < 0.01). vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity. vWF-CP activity was not changed two weeks after renal transplantation. Renal biopsy demonstrated that the vWF level in stage IV was higher than in stages II and III while vWF-CP activity was lower in patients with lupus nephritis. After eight-week treatment, the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema, disease activity index < 9, but not with index = 9. Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease, mesangial proliferative glomerulonephritis or IgA glomerulonephritis, the vWF level was not significantly different.</p><p><b>CONCLUSIONS</b>The alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies. Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF level and lower vWF-CP activity in chronic renal disease and thus a mechanism for worsening of chronic renal disease and thrombosis.</p>