A novel electrochemical IL-6 sensor based on Au nanoparticles-modified platinum carbon electrode.

Introduction: Interleukin-6 (IL-6) is a multifunctional polypeptide cytokine composed of two glycoprotein chains, which plays an important role in many cellular reactions, pathological processes, diagnosis and treatment of diseases and so on. The detection of IL-6 plays a promising role in the cognition of clinical diseases. Methods: 4-mercaptobenzoic acid (4-MBA) was immobilized on the gold nanoparticles modified platinum carbon (PC) electrode with the linker IL-6 antibody, and finally formed an electrochemical sensor that specifically recognized IL-6. Through the highly specific antigen-antibody reaction, the IL-6 concentration of the samples to be detected. The performance of the sensor was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Results: The experimental results showed that the linear detection range of the sensor for IL-6 was 100 pg/mL-700 pg/mL and the detection limit was 3 pg/mL. In addition, the sensor had the advantages of high specificity, high sensitivity, high stability and reproducibility under the interference environment of bovine serum albumin (BSA), glutathione (GSH), glycine (Gly) and neuron specific enolase (NSE), which provided a prospect for specific antigen detection sensor. Discussion: The prepared electrochemical sensor successfully detected the content of IL-6 in standard and biological samples, showing excellent detection performance. No significant difference was found between the detection results of the sensor and that of ELISA. The sensor showed a very broad prospect in the application and detection of clinical samples.

Prevalence and correlates of sleep problems among Chinese prisoners.

PURPOSE: This study aimed to investigate the prevalence and correlates of sleep problems in a large prison in China. METHODS: A total of 1491 incarcerated male adults (35.44 ± 9.67 years, range 18-69) were assessed by a self-administered structured questionnaire. Sleep duration, insomnia, sleep quality, substance abuse history, gambling history, traumatic life events, posttraumatic stress disorder (PTSD) and depressive symptoms were measured. Type of offense, history of incarceration, sentence length, and duration in prison were recorded. Multivariate logistic regression was used to examine correlated factors of sleep problems. RESULTS: Overall, 17.4% (95% CI 15.6-19.5%) slept less than 6 h at night, 35.6% (95% CI 33.2-38.0%) slept 6-7 h, and 47.0% (95% CI 44.5-49.6%) slept 7 h or more. The prevalence rates were 26.2% (95% CI 24.0-28.5%) for insomnia and 45.9% (95% CI 43.4-48.4%) for poor sleep quality. Multiple models showed that older age, being divorced/widowed, poor physical health, long duration in prison, drug use before incarceration, PTSD and depression were associated with short sleep duration; while older age, poor physical health, PTSD, depression, and gambling before incarceration were associated with increased incidence of insomnia, and that being divorced/widowed, poor physical health, PTSD, depression, smoking before incarceration were related to poor sleep quality. CONCLUSIONS: These findings indicate that sleep loss, insomnia, and poor sleep quality are common in prisoners, and that sleep problems are associated with multiple psychosocial factors.

Grape Seed Proanthocyanidin Extract Ameliorates Cardiac Remodelling After Myocardial Infarction Through PI3K/AKT Pathway in Mice.

Myocardial infarction is one of the most serious fatal diseases in the world, which is due to acute occlusion of coronary arteries. Grape seed proanthocyanidin extract (GSPE) is an active compound extracted from grape seeds that has anti-oxidative, anti-inflammatory and anti-tumor pharmacological effects. Natural products are cheap, easy to obtain, widely used and effective. It has been used to treat numerous diseases, such as cancer, brain injury and diabetes complications. However, there are limited studies on its role and associated mechanisms in myocardial infarction in mice. This study showed that GSPE treatment in mice significantly reduced cardiac dysfunction and improved the pathological changes due to MI injury. In vitro, GSPE inhibited the apoptosis of H9C2 cells after hypoxia culture, resulting in the expression of Bax decreased and the expression of Bcl-2 increased. The high expression of p-PI3K and p-AKT was detected in MI model in vivo and in vitro. The use of the specific PI3K/AKT pathway inhibitor LY294002 regressed the cardio-protection of GSPE. Our results showed that GSPE could improve the cardiac dysfunction and remodeling induced by MI and inhibit cardiomyocytes apoptosis in hypoxic conditions through the PI3K/AKT signaling pathway.

Bioequivalence of Oral Formulations of Anastrozole in Healthy Chinese Male Volunteers: A Randomized, Single-Dose, Two-Period, Two-Sequence Crossover Study.

Anastrozole is currently used as first-line treatment in locally advanced or metastatic breast cancer. A generic anastrozole tablet was developed to offer an alternative to the marketed tablet formulation. The aim of the current study was to evaluate the bioequivalence between the test and reference formulations of anastrozole in a single-dose, 2-period, 2-sequence crossover study with a 14-day washout interval. A total of 20 healthy male Chinese volunteers were enrolled and completed the study, after oral administration of a single dose of 1.0-mg test and reference formulations of anastrozole. The blood samples were collected at different times and were determined by a fully validated high-pressure liquid chromatography-tandem mass spectrometry method. The evaluated pharmacokinetic parameters, including Cmax , AUC0-t , and AUC0-∞ , were assessed for bioequivalence based on current guidelines. The observed pharmacokinetic parameters of anastrozole of the test drug were similar to those of the reference formulation. The 90% confidence intervals of test/reference ratios for Cmax , AUC0-t , and AUC0-∞ were within the bioequivalence acceptance range of 80%-125%. The results obtained from these healthy Chinese subjects in this study suggest that the test formulation of anastrozole 1.0-mg tablet is bioequivalent to the reference formulation (Arimidex 1.0-mg tablet).

Etoposide combined with ruxolitinib for refractory hemophagocytic lymphohistiocytosis during pregnancy: a case report and literature review.

Hemophagocytic lymphohistiocytosis (HLH) is an immune-mediated disorder caused by uncontrolled inflammatory responses and the activation of T lymphocytes. This life-threatening disease, characterized by fever, cytopenia and hepatosplenomegaly, is extremely rare during pregnancy with high mortality. Despite the improvement of treatment regimen in recent years, HLH is still a great challenge for clinicians. Here, we described a 26-year-old woman who admitted to our hospital at her first pregnancy with pyrexia. Her condition continued to deteriorate after receiving broad-spectrum antimicrobials, presenting with fever, pancytopenia, hepatosplenomegaly, ferritin ≥ 500 µg/L, hemophagocytosis and low NK-cell activity. HLH was eventually diagnosed by clinical manifestation and laboratory examination results. Then the patient recovered well after treatment with etoposide combined with ruxolitinib therapy and underwent successful induced-labor operation. Additionally, we summarized similar cases from the literature to improve the management of HLH during pregnancy. In conclusion, this study highlights the challenges and difficulties in the diagnosis and management of patients with HLH during pregnancy. Moreover, this is the first case report of etoposide combined with ruxolitinib in the treatment of patients with refractory secondary HLH during pregnancy.

Osthole attenuates myocardial ischemia/reperfusion injury in rats by inhibiting apoptosis and inflammation.

This study was performed to evaluate the cardioprotective effects of osthole (OST) in a rat model of myocardial ischemia/reperfusion injury (MI/RI) and the underlying mechanism. We exposed rat hearts to left anterior descending coronary artery ligation for 30 min followed by 24 h of reperfusion. The results showed that pretreatment with OST ameliorated MI/RI as evidenced by histopathological examination. Moreover, the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay demonstrated that OST suppressed myocardial apoptosis, which may be related to an increase in the Bcl-2/Bax ratio and inhibition of caspase-3 and caspase-9 activation. Furthermore, we determined that OST ameliorated impaired mitochondrial morphology and the oxidation system; OST also attenuated levels of pro-inflammatory cytokines, including tumor necrosis factor α and interleukins 6 and 1ß. In conclusion, OST exerted a strong favorable cardioprotective effect on MI/RI, possibly by suppressing the inflammatory response and inhibiting cell apoptosis.

Effects of small interfering RNA targeting TLR4 on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome with hypertension in a rat model.

We explored the effects of RNA interference-mediated silencing of TLR4 gene on expressions of adipocytokines in obstructive sleep apnea hyponea syndrome (OSAS) with hypertension in a rat model. Systolic blood pressure of caudal artery and physiological changes were observed when establishing rat models of OSAS with hypertension. Mature rat adipocytes were induced from separated and cultured primary rat adipocytes. To transfect rat mature adipocytes, TLR4 siRNA group and negative control (NC) siRNA group were established. Expressions of TLR4 mRNA of adipocytes were examined after silenced by siRNA by quantitative real-time polymerase chain reaction (qRT-PCR). By enzyme-linked immunosorbent assay (ELISA), expressions of inflammatory cytokines, and adipocytokines of adipocytes were detected. Blood pressure in rat caudal artery was higher in the intermittent hypoxia group than that of the blank control group by 29.87 mmHg, and cardiocytes in the former group showed physiological changes, which indicated successful establishment of rat models of OSAS with hypertension. Red particles could be seen in mature rat adipocytes when stained with Oil Red O. Transfection of TLR4 mRNA was significantly suppressed in the TLR4 siRNA group, which didn't happen in the untransfected control group. Rats in the TLR4 siRNA group had significantly reduced expressions of such inflammatory cytokines as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) and such adipocytokines as visfatin, adiponectin (ADN), and leptin than those in the untransfected control group. RNA interference-mediated silencing of TLR4 gene could regulate occurrence and development of OSAS with hypertension in rats by downregulating expressions of adipocytokines.

Downregulated SOCS1 expression activates the JAK1/STAT1 pathway and promotes polarization of macrophages into M1 type.

Macrophage polarization is flexible, and involves in different signaling pathways and various transcription factors. Suppressor of cytokine signaling (SOCS) is an important inhibitor of cytokine signaling pathways and also a key physiological regulator for natural and acquired immunity systems. Following transfection of SOCS1 short hairpin (sh)RNA into mouse macrophage cells, reverse transcription­quantitative polymerase chain reaction demonstrated that the mRNA levels of Janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)1 increased significantly. In addition, western blotting indicated that JAK1, STAT1 and p­STAT1 expression was significantly enhanced. Fludarabine can inhibit phosphorylation of STAT1 and SOCS1 expression. When fludarabine was added and SOCS1 shRNA was transfected, the inhibition of fludarabine was weakened, and p­STAT1 expression was upregulated. Flow cytometry detection indicated that, following the downregulation of SOCS1 expression, M1­type cells significantly increased, but the proportion of M2­type cells did not change significantly. Fludarabine can reduce the effect of SOCS1 shRNA on promoting M1­type cell polarization, and macrophages can polarize into both M1 and M2 phenotypes. Further ELISA results presented that, when downregulating SOCS1 expression, interleukin (IL)­4 and IL­10 expression was both downregulated, and tumor necrosis factor (TNF)­α and interferon (IFN)­Î³ expression was significantly upregulated. When adding fludarabine or injecting with the traditional Chinese medicine Xuebijing, IL­4 and IL­10 expression was both significantly upregulated, and TNF­α and IFN­Î³ expression was significantly downregulated. When adding fludarabine and downregulating SOCS1, IL­4, IL­10, TNF­α and IFN­Î³ expression presented no significant changes. The above results indicated that, when SOCS1 expression is downregulated, it will activate the JAK1/STAT1 pathway, and thereby promote the polarization of macrophages into M1 type. The findings are of great importance for understanding occurrence, development and treatment of various immune­related diseases.

Rhein attenuates inflammation through inhibition of NF-κB and NALP3 inflammasome in vivo and in vitro.

Rhein is an important component in traditional Chinese herbal medicine formulations for gastrointestinal disorders, including inflammatory bowel diseases such as ulcerative colitis. In this study, we investigated the beneficial effects of rhein in inflammation models in the transgenic zebrafish line TG (corolla eGFP), in which both macrophages and neutrophils express eGFP and RAW264.7 macrophages. We found that the tail-cutting-induced migration of immune cells was significantly reduced in transgenic zebrafish treated with rhein. In addition, the production of proinflammatory cytokines, including IL-6, IL-1ß, and tumor necrosis factor-α, were significantly reduced in lipopolysaccharide (LPS)-induced RAW264.7 macrophages treated with rhein. Parallel to the inhibition of proinflammatory cytokines, rhein significantly reduced phosphorylation levels of NF-κB p65 and inducible nitric oxide synthase, as well as COX-2 protein expression levels. Furthermore, rhein significantly reduced NALP3 and cleaved IL-1ß expression in LPS + ATP-induced RAW264.7 macrophages. Thus, the present study demonstrates that rhein may exhibit its anti-inflammatory action via inhibition of NF-κB and NALP3 inflammasome pathways.

Soluble Egg Antigen Activates M2 Macrophages via the STAT6 and PI3K Pathways, and Schistosoma Japonicum Alternatively Activates Macrophage Polarization to Improve the Survival Rate of Septic Mice.

Sepsis is one of the most challenging health problems worldwide. Our previous study showed that chronic schistosoma japonica (SJ) infection might increase serum anti-inflammatory factors to play a protective role, thus improving the survival rate of septic mice. Further research revealed that SJ infection promoted J774A.1 macrophage differentiation into M2 macrophages; suppressed LPS-induced activation of M1 macrophages; up-regulated CD163, IL-10, and TGF-ß1 expression; inhibited TNF-α and iNOS expression; and blocked the effect of LPS-promoted TNF-α and iNOS expression. Furthermore, adoptive transfer of ex vivo programed M2 macrophages significantly increased the survival rate of septic mice. In vitro studies suggested that soluble egg antigen (SEA) from SJ played the same role as worm infection but had no impact on M1 macrophages. SEA reduced LPS-induced TNF-α and iNOS expression, decreased the inhibitory effect of LPS on IL-10 and TGF-ß1 expression, increased STAT6 phosphorylation, and up-regulated PI3K and Akt expression but inhibited SOCS1 expression. When PI3K inhibitors were added, SEA-induced expression of CD163, IL-10, and arg1 might be reduced. Therefore, worm infection has a protective effect in septic mice in which SEA may play a key role via the STAT6 and PI3K pathways. This finding may provide a favorable solution for the treatment of sepsis, especially early cases. J. Cell. Biochem. 118: 4230-4239, 2017. © 2017 Wiley Periodicals, Inc.

Bone marrow mesenchymal stem cells inhibit dendritic cells differentiation and maturation by microRNA-23b.

Research on regulation and its mechanism of bone marrow mesenchymal stem cells (BMSCs) on dendritic cells (DCs), which is the initiating factor in immune response has applicable clinical value. Although BMSCs have a significant regulatory effect on the maturation of DCs, its molecular mechanism is still unclear. BMSCs and DCs, were co-cultured by different concentration ratios. Flow cytometry was used to detect the expression of DC markers (CD83, CD11c). Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of related genes in RNA level. Expression of the target proteins was detected with using Western blot assay. miRNA inhibitor and miRNA mimic were used to suppress and up-regulate the expression of the target gene. In this research, our results demonstrated that BMSCs notably inhibited maturation of DCs in the co-culture system of BMSCs and DCs and confirmed that this inhibition is due to overexpression of miR-23b Furthermore, this research found that miR-23b overexpression inhibited the expression of p50/p65, thus blocked the activation of the NF-κB pathway. In conclusion, BMSCs affected the activation of NF-κB pathway through miR-23b overexpression resulting in inhibition of the maturation and differentiation of DCs.

The effects of different vascular carrier patterns on the angiogenesis and osteogenesis of BMSC-TCP-based tissue-engineered bone in beagle dogs.

Bone repair using tissue-engineered bone (TEB) in a large defect or accompanied by a poor recipient vascular bed is a long-standing challenge. Surgical vascular carrier patterns of vascular bundle (VB) and arteriovenous loop (AV loop) have been shown to improve the vascularization and repair capacity of TEB. However, the effects of these different vascular carrier patterns on angiogenesis and osteogenesis in TEB have never been evaluated. Here, TEB was constructed with bone marrow mesenchymal stem cells (BMSCs) and ß-TCP and prevascularized by the VB or AV loop technique in beagle dogs. The vascularization and bone formation in TEB were quantitatively compared using Microfil perfusion, histological examination and CT and micro-CT analyses. The distribution and constitution of the neovasculature were analysed to determine the underlying mechanism of angiogenesis. The results showed that prevascularized TEB generated bone tissue faster and more homogeneously than untreated TEB. The VB technique was found to strike a better balance between bone regeneration and ß-TCP scaffold degradation than the AV loop strategy, which resulted in more vascularization but less bone yield, due to faster degradation of the ß-TCP scaffold. This study indicates that a suitable triangular relationship, composed of bone regeneration, scaffold degradation and vasculature, is critical to TEB construction. Copyright © 2015 John Wiley & Sons, Ltd.

Investigating ego modules involved in TGFß3-induced chondrogenesis in mesenchymal stem cells based on ego network.

OBJECTIVE: This paper aimed to investigate ego modules for TGFß3-induced chondrogenesis in mesenchymal stem cells (MSCs) using ego network algorithm. METHODS: The ego network algorithm comprised three parts, extracting differential expression network (DEN) based on gene expression data and protein-protein interaction (PPI) data; exploring ego genes by reweighting DEN; and searching ego modules by ego gene expansions. Subsequently, permutation test was carried out to evaluate the statistical significance of the ego modules. Finally, pathway enrichment analysis was conducted to investigate ego pathways enriched by the ego modules. RESULTS: A total of 15 ego genes were obtained from the DEN, such as PSMA4, HNRNPM and WDR77. Starting with each ego genes, 15 candidate modules were gained. When setting the thresholds of the area under the receiver operating characteristics curve (AUC) ≥0.9 and gene size ≥4, three ego modules (Module 3, Module 8 and Module 14) were identified, and all of them had statistical significances between normal and TGFß3-induced chondrogenesis in MSCs. By mapping module genes to confirmed pathway database, their ego pathways were detected, Cdc20:Phospho-APC/C mediated degradation of Cyclin A for Module 3, Mitotic G1-G1/S phases for Module 8, and mRNA Splicing for Module 14. CONCLUSIONS: We have successfully identified three ego modules, evaluated their statistical significances and investigated their functional enriched ego pathways. The findings might provide potential biomarkers and give great insights to reveal molecular mechanism underlying this process.

Recombinant Trichinella spiralis 53-kDa protein activates M2 macrophages and attenuates the LPS-induced damage of endotoxemia.

Sepsis is a serious clinical condition of excessive systemic immune response to microbial infection. The pro-inflammatory stage of sepsis is generally launched by innate cells such as macrophages. They release inflammatory cytokines, activate other immune cells and cause severe tissue/organ damage. In this study, we have revealed that recombinant Trichinella spiralis (TS) excretory-secretory protein (rTsP53) exhibited anti-inflammatory properties and rescued mice from LPS-induced endotoxemia, which is a common model for sepsis study, potentially through the induction of M2 macrophages. rTsP53 treatment significantly decreased inflammatory cytokines (IL-6, IFN-γ and TNF-α) and increased IL-4, IL-10, IL-13 and TGF-ß secretion, both in circulation and in tissues. rTsP53 also induced the activation and infiltration of F4/80(+)CD163(+) macrophages to inflammatory tissues, increased M2 macrophage-related Arg1 and Fizz1 expression, and decreased M1 macrophage-related iNOS expression. PCR array showed that rTsP53 activated several genes that involve the survival of macrophages and also anti-inflammatory genes such as SOCS3. Together, our results show that rTsP53 activates M2 macrophages, which has strong anti-inflammatory potential to prevent LPS-induced lethal sepsis.

Influence of Immunogenicity of Allogeneic Bone Marrow Mesenchymal Stem Cells on Bone Tissue Engineering.

Allogeneic bone marrow mesenchymal stem cell (allo-BMSC)-based tissue-engineered bone (TEB) has great potential for bone defect repair. However, the immunogenicities and biological roles of allo-BMSCs are still controversial. In this study, we established an animal model of critical-sized mandibular defect in beagle dogs and compared the repairing effects of allo-BMSC-based TEB with autogenic BMSC (auto-BMSC)-based TEB without the administration of immunosuppressants. During the first 2 weeks postimplantation, a transient immune response in the allo-BMSC group was detected with an increase in proinflammation cytokines TNF-α, IFN-γ, and IL-2, a declination of anti-inflammation cytokine IL-10, and an increase in percentages of CD4(+) and CD8(+) T-cell subsets in peripheral blood. Nevertheless, there was no significant difference in bone union achievement, bone mineral density, and biomechanical properties between the two groups at 12 and 24 weeks postimplantation. Further subcutaneous implantation of allo-BMSCs/scaffold also exhibited the similar transient immune responses in the first 2 weeks postimplantation but followed by a decreased bone formation at 4 and 8 weeks postimplantation. These findings indicate that allo-BMSCs can induce a transient immunoreaction, which may temporally delay the osteogenesis of allo-BMSC/scaffold complex in early stage of in vivo implantation, whereas the long-term engineered bone formation was not affected.

[Protective effects of recombinant trichinella spiralis-53 000 protein combining with imipenem on polymicrobial septic mice].

Objective: To observe the protective effects of recombinant trichinella spiralis-53 000 protein (rTsP53 protein) combining with imipenem (IMP) on septic mice and their underlying mechanisms. . Methods: Male BALB/c mice were divided into five groups randomly. Cecal ligature and puncture (CLP) operation was used for building polymicrobial septic model (CLP group).Mice in sham group were only subjected to laparoromy and abdominal closure without cecum ligation. At 6 hours post CLP, mice in CLP+IMP,CLP+rTsP53,and CLP+IMP+rTsP53 groups were injected intraperitoneally with IMP (20 mg/kg) + 0.1 mL albumin,rTsP53 protein (6 mg/kg) + 0.1 mL normal saline (NS),IMP (20 mg/kg) + rTsP53 protein (6 mg/kg) respectively, mice in sham group and CLP group were injected intraperitoneally with 0.1 mL albumin + 0.1 mL NS, then these therapies were repeated every 12 hours until the experiment ended. Twenty mice were extracted randomly from each group for surveying 72-hour survival rate.At 0,6,12,24,36,48,72 hours post CLP,3 mice in each group were collected and cytokines in serum were tested by enzyme-linked immunosorbent assay (ELISA).Whole blood was cultured, then the numbers of bacteria colony-forming units (CFU) were counted. Mice were executed at 24 hours, then the epithelial cells ultrastructures of the mice small intestinal mucosa were observed by transmission electron microscope (TEM). Results: ① Compared with CLP,CLP+IMP or CLP+rTsP53 group,72-hour survival rate of the mice in CLP+IMP+rTsP53 group was significantly elevated (85％ vs.20％,55％,25％,all P ＜ 0.05).② No bacteria was found in cultured whole blood of mice in sham group at all time-points. At 6 hours post CLP operation, the number of bacterial clone of all experimental groups was rose significantly. The changed trend of bacterial number in CLP group was rising at the beginning, then declining, and the bacterial number reached the peak at 24 hours (× 106 cfu/L:12.74± 2.33).From 12 hours, the bacterial numbers of CLP+rTsP53 group were higher than those of CLP group, and reached the peak at 36 hours (× 106 cfu/L:22.13 ± 4.28),then declined gradually. The bacterial numbers of CLP+IMP and CLP+IMP+rTsP53 groups reached the peak at 6 hours (× 106 cfu/L:5.72 ± 0.50,5.49 ± 0.59),then declined. They were significantly less than those of CLP group from 12 hours.③ From 6 hours after CLP,the cytokines levels of mice in all experimental groups were higher than those in sham group. The tumor necrosis factor-α (TNF-α) levels in CLP group showed a trend of elevation in the beginning, and decrease thereafter. It reached the peak at 36 hours (ng/L:1 422.67 ±72.19).The TNF-α level peak time of CLP+IMP group,CLP+rTsP53 group,CLP+IMP+rTsP53 group was advanced to 12 hours post CLP (ng/L:1376.29±44.67,929.36±40.42,809.61±22.61).At 24 hours post CLP, the interleukin-6 (IL-6) level of CLP group and CLP+IMP group reached the peak (ng/L:215.39 ± 16.05,191.63 ± 8.99).The peak time of CLP+rTsP53 group and CLP+IMP+rTsP53 was advanced to 12 hours post CLP (ng/L:113.01 ± 12.11,92.43±6.11).The level of IL-4,IL-10 in CLP group raised gradually to the highest at 72 hours (ng/L:366.25 ±24.25,923.14±30.36).The IL-4 and IL-10 levels of CLP+IMP group raised to their maximum value at 12 hours and 24 hours respectively (ng/L:281.47±16.33,555.67±13.57),then declined. The IL-4 and IL-10 levels of CLP+rTsP53 group and CLP+IMP+rTsP53 group gradually ascended their peak value at 72 hours [IL-4 (ng/L) was 453.14±18.53,410.43 ± 15.75,IL-10 (ng/L) was 1 185.61 ± 16.74,1 006.77 ± 36.91,respectively].From 12 hours, the pro-inflammatory cytokines levels of CLP+IMP+rTsP53 group were significantly less than those of CLP+IMP group and CLP+rTsP53 group.④ At 24 hours post CLP, compared with mice in CLP,CLP+IMP, or CLP+rTsP53 group, mice in CLP+IMP+rTsP53 group had slighter ultra structure injuries in the microvilli, cell junction and mitochondria of small intestinal mucosa epithelial cells. Conclusions: The levels of pro-inflammatory cytokines were reduced and the levels of anti-inflammatory eytokines were escalated by intervention of rTsP53 protein combining with IMP boosted in polymierobial septic mice serum, and enhanced the survival rate of the mice. The injection of rTsP53 protein alone had no protective effects on polymicrobial septic mice,because the amount of bacteria in mice blood was augmented

Micrometam C Protects against Oxidative Stress in Inflammation Models in Zebrafish and RAW264.7 Macrophages.

Micrometam C is a core of novel marine compound isolated from the mangrove associates Micromelum falcatum. In this study, we investigated the protective effects of micrometam C in inflammation models in the transgenic zebrafish line Tg (corola: eGFP) and RAW264.7 macrophages. We found that micrometam C significantly suppressed the migration of immune cells in tail-cutting-induced inflammation in transgenic zebrafish and reduced lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) in both zebrafish and macrophages. In addition, micrometam C also restored LPS-induced reduction of endogenous antioxidants, such as catalase (CAT), glutathione (GSH) and superoxide dismutase (SOD). The protective effects of micrometam C were in parallel to its inhibition of NADPH oxidase and nuclear factor-kappa-binding (NF-κB) activity. Thus, the present results demonstrate that micrometam C protects against LPS-induced inflammation possibly through its antioxidant property.

Effect of rTsP53 on the M1/M2 activation of bone-marrow derived macrophage in vitro.

We investigated that if rTsP53 could be used to activate bone-marrow derived macrophage (BMDM) into M2 macrophage and stop M1 macrophage activation. After 72 h incubation in blank culture medium, cells with PE-CCR7 (-) and FITC-CD206 (-) was extracted and its mean proportion was 92.30 ± 0.22%. With the stimulation of 20 µg/ml IFN-γ for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 16.24 ± 0.82%. With the stimulation of IL-3/IL-14 (both 10 µg/ml) for 72 h, cells with FICT-CD206 (+) was extracted and its mean proportion was 87.32 ± 4.29%. Co-incubation with different dose of rTsP53 (0.001 µg/ml, 0.01 µg/ml, 0.1 µg/ml, 1 µg/ml, 2 µg/ml, 5 µg/ml, 10 µg/ml, respectively) for 72 h, FITC-CD206 (+) macrophage was extracted. The mean proportion in each group was 1.09 ± 0.22%, 2.13 ± 0.13%, 4.91 ± 0.07%, 5.48 ± 0.29%, 9.81 ± 0.06%, 12.83 ± 0.55%, 17.87 ± 0.02%, respectively. The dose of rTsP53 was significantly positive correlated to the proportion of FITC-CD206 (+) macrophage. Co-incubation with 20 µg/ml IFN-γ and 5 µg/ml rTsP53 for 72 h, cells with PE-CCR7 (+) was extracted and its mean proportion was 10.60 ± 0.19%. Compared to that of mere co-incubation with IFN-γ, there was significant difference between the two groups. ELISA showed that Th1 cytokines' (IFN-γ, IL-6 and TNF-α) level decreased in the culture medium supernatant of BMDM co-incubated with rTsP53. There was negative correlation between the Th1 cytokines' level and the dose of rTsP53. Both Th2 cytokines (IL-4 and IL-13) and regulatory cytokines in the culture medium increased. There was positive correlation between the Th2 cytokines' level and the dose of rTsP53. There was also positive correlation between the regulatory cytokines' level and the dose of rTsP53. Compared to that of BMDM co-incubated with IFN-γ, levels of TNF-α and IL-6 were significant lower than that of BMDM co-incubated with both IFN-γ and rTsP53 (both P < 0.05), while the levels of IL-4 and TGF-ß were significant higher (both P < 0.05). There was no significant difference in the levels of IL-13 and IL-10 between the two groups.

[Expression and effects of microRNA-155 in the livers of septic mice].

OBJECTIVE: To evaluate the effects of microRNA-155 (miR-155) on liver injury in mice with sepsis. METHODS: One hundred and twenty BALB/c mice were randomly divided into two groups of equal number according to random number table. Sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS,20 mg/kg). The mice were sacrificed at the time-points of 0, 2, 6, 12, 24, 48 hours. Blood and liver tissue were collected, and the levels of tumor necrosis factor- α (TNF- α ), interleukin (IL-6, IL-10) in serum and liver homogenate and alanine transaminase (ALT) in serum were determined by enzyme linked immunosorbent assay(ELISA). The injury of liver tissue was evaluated by histopathology. The expression of miR-155 in liver tissue was assessed by fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The levels of TNF- α , IL-6 and IL-10 in serum and liver homogenate of septic mice increased with passage of time, and then the levels of TNF-α and IL-6 lowered after reaching the peak value, but remained higher than that of control group.TNF-α (ng/L) reached the peak value at 2 hours post-LPS-injection (serum: 1538.46 ± 102.12 vs. 64.52 ± 18.44,liver homogenate: 255.26 ± 41.23 vs. 60.21 + 13.55, both P<0.05). The level of IL-6 (µg/L) reached the peak value at 6 hours post-LPS-injection (serum: 875.33 ± 102.37 vs. 153.72 ± 20.67, liver homogenate: 9.22 + 0.82 vs. 3.35 ±0.36, both P<0.05), and that of IL-10 (ng/L) reached the peak value at 48 hours post-LPS-injection (serum: 520.13 ± 88.52 vs. 23.43 3.01, liver homogenate: 260.12 + 50.38 vs. 16.37 ± 3.71, both P<0.05). There were significant differences in above indexes between septic and control group (all P<0.05). The serum level of ALT (U/L) rose with passage of time, reaching the peak value at 48 hours post-LPS-injection (603.26 + 70.21 vs. 45.84 + 5.64, P<0.05). The values showed significant differences between septic and control group (P<0.05). A large number of leucocytic infiltration was found in liver. Hepatic tissue showed architectural distortion. Hepatocyte vacuolation and nodular necrosis were obvious at 12 hours post-LPS-injection. Relative expression of miR-155 was found to be increased at 2 hours post-LPS-injection, reaching its peak value at 12 hours post-LPS-injection [(72.96 ± 9.34)-fold of control group, P<0.05]. CONCLUSION: The increase in miR-155 expression might play an important role in the mechanism of liver injury during sepsis.

Vascular and micro-environmental influences on MSC-coral hydroxyapatite construct-based bone tissue engineering.

Bone tissue engineering (BTE) has been demonstrated an effective approach to generate bone tissue and repair bone defect in ectopic and orthotopic sites. The strategy of using a prevascularized tissue-engineered bone grafts (TEBG) fabricated ectopically to repair bone defects, which is called live bone graft surgery, has not been reported. And the quantitative advantages of vascularization and osteogenic environment in promoting engineered bone formation have not been defined yet. In the current study we generated a tissue engineered bone flap with a vascular pedicle of saphenous arteriovenous in which an organized vascular network was observed after 4 weeks implantation, and followed by a successful repaire of fibular defect in beagle dogs. Besides, after a 9 months long term observation of engineered bone formation in ectopic and orthotopic sites, four CHA (coral hydroxyapatite) scaffold groups were evaluated by CT (computed tomography) analysis. By the comparison of bone formation and scaffold degradation between different groups, the influences of vascularization and micro-environment on tissue engineered bone were quantitatively analyzed. The results showed that in the first 3 months vascularization improved engineered bone formation by 2 times of non-vascular group and bone defect micro-environment improved it by 3 times of ectopic group, and the CHA-scaffold degradation was accelerated as well.