De-ubiquitination of p300 by USP12 Critically Enhances METTL3 Expression and Ang II-induced cardiac hypertrophy.

Stresses, such as neurohumoral activation, induced pathological cardiac hypertrophy is the main risk factor for heart failure. The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role and mechanism of deubiquitinating enzymes (DUBs) in cardiac hypertrophy is limited. Here, we observe that the deubiquitinating enzyme ubiquitin-specific protease 12(USP12) is upregulated in Ang II-induced hypertrophic hearts and primary neonatal rat cardiomyocytes (NRCMs). Inhibition of USP12 ameliorate Ang II-induced myocardial hypertrophy, while overexpression of USP12 have the opposite effect. USP12 deficiency also significantly attenuate the phenotype of Ang II-induced cardiac hypertrophy in vivo. Moreover, we demonstrate that USP12 aggravate Ang II-induced cardiac hypertrophy by enhancing METTL3, a methyltransferase which catalyze N6-methyladenosine (m6A) modification on messenger RNA and acts as a harmful factor in pathological cardiac hypertrophy. Upregulation of METTL3 reverse the reduction of myocardial hypertrophy induced by USP12 silencing in NRCMs. In contrast, knockdown of METTL3 attenuate the aggravation of myocardial hypertrophy in USP12-overexpressing NRCMs. Furthermore, we discover that USP12 promote the expression of METTL3 via upregulating p300. Mechanistically, USP12 binds and stabilizes p300, thereby activating the transcription of its downstream gene METTL3. Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy. Taken together, our results demonstrate that USP12 acts as a pro-hypertrophic deubiquitinating enzyme via enhancing p300/METTL3 axis, indicating that targeting USP12 could be a potential treatment strategy for pathological cardiac hypertrophy.

Pyruvate alleviates lipid peroxidation and multiple-organ dysfunction in rats with hemorrhagic shock.

OBJECTIVE: Pyruvate can reduce lipid peroxidation, which plays a critical role in organ injury, in various models. However, it is not fully understood if this inhibition occurs in resuscitation of hemorrhagic shock (HS). This study examines effects of pyruvate Ringer solution (PR) in this respect in rats. METHODS: Rats, subjected to 45% blood loss, were randomly allocated to the 3 groups (n = 18): HS with no fluid resuscitation (group NR), HS resuscitated with lactated Ringer solution (LR) (group LR), and HS resuscitated with PR (group PR). Mean arterial pressure, plasma levels of thiobarbituric acid reactive substances (TBARS), and superoxide dismutase were measured at various time points until 360 minutes after hemorrhage. Visceral organs were harvested at the end for evaluations of the TBARS, antioxidant enzyme, and tissue water content. Other 54 rats with identical procedures without sampling were documented for 24-hour survival rates (n = 18) after fluid resuscitation. RESULTS: Pyruvate Ringer solution significantly increased mean arterial pressure and decreased blood TBARS levels after lethal HS. It also reduced TBARS concentrations and glutathione peroxidase activities but significantly enhanced glutathione reductase activities in most organs and greatly improved the ratios of reduced glutathione over oxidized glutathione in various organs in group PR, compared to group LR. Furthermore, PR significantly improved various organ function and water contents relative to LR. Group PR showed a more than 2-fold higher 24-hour survival rate of group LR. CONCLUSIONS: Pyruvate Ringer solution alleviated organ edema and injury and prompted survival partially through inhibition of lipid peroxidation in various organs in severe HS rats.

Pyruvate oral rehydration solution improved visceral function and survival in shock rats.

BACKGROUND: Recent findings showed advantages of a novel pyruvate-enriched oral rehydration solution (Pyr-ORS) in resuscitation of burns. This study focused on effects of Pyr-ORS on the visceral blood perfusion (VBP), gastrointestinal function, and survival rate, compared with the bicarbonate-based World Health Organization-guided oral rehydration solution (WHO-ORS), during intragastric rehydration of lethal hemorrhagic shock in rats. METHODS: Sixty adult rats were subjected to 45% total blood volume loss and were randomly allocated to the following three groups (n = 20): group NR (no fluid resuscitation), group PORS (oral Pyr-ORS rehydration), and group BORS (oral WHO-ORS rehydration), respectively. Other 10 rats were served as group NH (the sham group). Enteral rehydration lasted for 4 h after hemorrhage. The mean arterial pressure (MAP), VBP, and plasma enzymes activities of heart, liver, and kidney, and intestinal fatty acid binding protein were measured. Liver, kidney, and ileum were harvested for the evaluation of activities of oxidative enzymes and intestinal barrier protein (ZO-1). Other 84 rats with identical procedures without sampling were observed for their 24-h survival rates. RESULTS: Pyr-ORS was more effective in enhancing the MAP and VBP, inhibiting tissue oxidative damage, and improving organ function, compared with WHO-ORS. Hypoxic lactic acidosis was fully corrected in group PORS in 4 h, whereas it worsened in group BORS, and the 24-h survival rate was twice higher in group PORS than in group BORS (45.8 versus 20.8%, P < 0.05). CONCLUSIONS: A small amount of pyruvate in Pyr-ORS was more therapeutically beneficial than equivalent bicarbonate in WHO-ORS and greatly raised survival in enteral rehydration of lethal hemorrhagic shock. The Pyr-ORS may be an ideal oral fluid in resuscitation of hypovolemic shock, especially in prehospital and resource-poor settings.

Role of mitofusin-2 in high mobility group box-1 protein-mediated apoptosis of T cells in vitro.

BACKGROUND: High mobility group box-1 protein (HMGB1), a ubiquitous nuclear protein, which is recognized as a danger-associated molecular pattern (DAMP) triggering activation of the innate immune system. Previous studies have shown that HMGB1 also plays a role in T cell-mediated immunity, but the effect of HMGB1 on apoptosis of T cells and its precise mechanism remain to be determined. METHODS: Two kinds of apoptosis assay techniques were used, i.e., Annexin V-FITC conjunction with PI to identify early apoptotic cells, Hoechst 33342 staining for double-stranded DNA to observe nuclear fragmentation or apoptotic body. The activation status of caspase-3, caspase-8, as well as caspase-9 was examined by colorimetric assay. The dynamic changes in intracellular calcium concentration ([Ca(2+)]i) was monitored by flow cytometry. Overexpression of Mfn2 was preformed by lentiviral vector transfection. The mRNA and protein levels of Mfn2 were determined by RT-PCR and Western-blotting. RESULTS: Treatment of Jurkat T cells with recombinant human HMGB1 (rhHMGB1) causes a significant dose-dependent increase in percentage of apoptotic cells. When T cells are incubated with HMGB1 they express decreased mitochondria fusion-related protein mitofusin-2 (Mfn2) and activate mitochondrial apoptotic pathway via elevation of [Ca(2+)]i, Bax insertion, and activation of caspase. Furthermore, overexpression of Mfn2 ameliorates the apoptosis of T cells induced by HMGB1. This occurs at least partly through Mfn2 keeps Ca(2+) homeostasis in T cells evidenced by monitoring [Ca(2+)]i dynamics. CONCLUSION: HMGB1 can trigger apoptosis of T lymphocytes through mitochondrial death pathway associated with [Ca(2+)]i elevation. Mfn2 plays a pivotal role in this process, and it might be a novel therapeutic target in T cell apoptosis related disorders.

Anti-RAGE antibody ameliorates severe thermal injury in rats through regulating cellular immune function.

AIM: The receptor of advanced glycation end products (RAGE) participates in a variety of pathophysiological processes and inflammatory responses. The aim of this study was to investigate the therapeutic potential of an anti-RAGE neutralizing antibody for severe thermal injury in rats, and to determine whether the treatment worked via modulating cellular immune function. METHODS: Full-thickness scald injury was induced in Wistar rats, which were treated with the anti-RAGE antibody (1 mg/kg, iv) at 6 h and 24 h after the injury. The rats were sacrificed on d 1, 3, 5, and 7. Blood and spleen samples were harvested to monitor organ function and to analyze dendritic cell (DC) and T cell cytokine profiles. The survival rate was analyzed up to d 7 after the injury. RESULTS: Administration of the antibody significantly increased the 7 d survival rate in thermally injured rats (6.67% in the model group; 33.33% in anti-RAGE group). Treatment with the antibody also attenuated the multiple organ dysfunction syndrome (MODS) following the thermal injury, as shown by significant decreases in the organ dysfunction markers, including serum ALT, AST, blood urea nitrogen, creatinine and CK-MB. Moreover, treatment with the antibody significantly promoted DC maturation and T cell activation in the spleens of thermally injured rats. CONCLUSION: Blockade of the RAGE axis by the antibody effectively ameliorated MODS and improved the survival rate in thermally injured rats, which may be due to modulation of cellular immune function.

Up-regulation of mitofusin-2 protects CD4+ T cells from HMGB1-mediated immune dysfunction partly through Ca(2+)-NFAT signaling pathway.

High mobility group box 1 protein (HMGB1) was recently discovered to be a critical late-acting cytokine and innate immune-modulating factor in sepsis, but the potential role and mechanism of HMGB1 in adaptive immunity remains elusive. The present study demonstrated that HMGB1 had a dual influence on immune function of CD4(+) T lymphocytes. Low dose of HMGB1 had no effect on the proliferation activity of CD4(+) T lymphocytes, but the Th1 cytokines production was increased. In contrast, treatment with high amount of HMGB1 suppressed the proliferative response and induced Th2 polarization of CD4(+) T lymphocytes. We found that the expression of mitofusin-2 (Mfn2; also named hyperplasia suppressor gene), a member of the mitofusin family, was decreased in CD4(+) T lymphocytes when stimulated with high dose of HMGB1. Up-regulation of Mfn2 attenuated the suppressive effect of HMGB1 on CD4(+) T lymphocytes, which was associated with profound elevation of intracellular calcium concentration ([Ca(2+)](i)) and nuclear factor of activated T cells (NFAT) activity. These results indicate that HMGB1 have a direct role on adaptive immunity, and the decrease of Mfn2 expression may be a major cause of HMGB1-mediated immune dysfunction and Ca(2+)-NFAT signaling defect of CD4(+) T lymphocytes.

Treatment with gelsolin reduces brain inflammation and apoptotic signaling in mice following thermal injury.

BACKGROUND: Burn survivors develop long-term cognitive impairment with increased inflammation and apoptosis in the brain. Gelsolin, an actin-binding protein with capping and severing activities, plays a crucial role in the septic response. We investigated if gelsolin infusion could attenuate neural damage in burned mice. METHODS: Mice with 15% total body surface area burns were injected intravenously with bovine serum albumin as placebo (2 mg/kg), or with low (2 mg/kg) or high doses (20 mg/kg) of gelsolin. Samples were harvested at 8, 24, 48 and 72 hours postburn. The immune function of splenic T cells was analyzed. Cerebral pathology was examined by hematoxylin/eosin staining, while activated glial cells and infiltrating leukocytes were detected by immunohistochemistry. Cerebral cytokine mRNAs were further assessed by quantitative real-time PCR, while apoptosis was evaluated by caspase-3. Neural damage was determined using enzyme-linked immunosorbent assay of neuron-specific enolase (NSE) and soluble protein-100 (S-100). Finally, cerebral phospho-ERK expression was measured by western blot. RESULTS: Gelsolin significantly improved the outcomes of mice following major burns in a dose-dependent manner. The survival rate was improved by high dose gelsolin treatment compared with the placebo group (56.67% vs. 30%). Although there was no significant improvement in outcome in mice receiving low dose gelsolin (30%), survival time was prolonged against the placebo control (43.1 ± 4.5 h vs. 35.5 ± 5.0 h; P < 0.05). Burn-induced T cell suppression was greatly alleviated by high dose gelsolin treatment. Concurrently, cerebral abnormalities were greatly ameliorated as shown by reduced NSE and S-100 content of brain, decreased cytokine mRNA expressions, suppressed microglial activation, and enhanced infiltration of CD11b+ and CD45+ cells into the brain. Furthermore, the elevated caspase-3 activity seen following burn injury was remarkably reduced by high dose gelsolin treatment along with down-regulation of phospho-ERK expression. CONCLUSION: Exogenous gelsolin infusion improves survival of mice following major burn injury by partially attenuating inflammation and apoptosis in brain, and by enhancing peripheral T lymphocyte function as well. These data suggest a novel and effective strategy to combat excessive neuroinflammation and to preserve cognition in the setting of major burns.

High mobility group box 1 protein suppresses T cell-mediated immunity via CD11c(low)CD45RB(high) dendritic cell differentiation.

AIM: High mobility group box 1 protein (HMGB1) has been identified as a late proinflammatory cytokine and plays a key role in immune regulation. However, it is not yet clear whether HMGB1 can induce the activation and differentiation of dendritic cell (DC) subsets and subsequently modulate immune function of T cells. This study was performed to investigate the effect of HMGB1 on the differentiation of splenic DCs and its influence on T cell-mediated immunity in terms of DC subsets CD11c(low)CD45RB(high) DCs and CD11c(high)CD45RB(low) DCs in male BALB/c mice spleens in vitro. RESULTS: MACS microbeads were used to isolate splenic DCs, CD11c(low)CD45RB(high) DCs, CD11c(high)CD45RB(low) DCs and CD4(+) T cells. The percentage of CD11c(low)CD45RB(high) DCs was significantly increased after treatment with HMGB1 compared to their counterparts (CD11c(high)CD45RB(low) DCs). It was found that unlike the gradually increasing interleukin (IL)-12 secretion of CD11c(high)CD45RB(low) DCs induced by HMGB1, CD11c(low)CD45RB(high) DCs showed a obvious dose-dependent response between IL-10 production and HMGB1 stimulation. In order to verify whether the alteration of CD4(+) T cells was mainly associated with the differentiation of splenic DCs mediated by HMGB1 to CD11c(low)CD45RB(high) DCs, anti-IL-12 receptor (IL-12R) or anti-IL-10R monoclonal antibody was used to inhibit the effect of CD11c(high)CD45RB(low) DCs or CD11c(low)CD45RB(high) DCs in CD4(+) T cells mixed lymphocyte reaction culture. After treatment with anti-IL-12R or anti-IL-10 monoclonal antibody in CD4(+) T cells+CD11c(high)CD45RB(low) DCs or CD11c(low)CD45RB(high) DCs mixed lymphocyte reaction, the induction of these DCs on T cells was inhibited dramatically. CONCLUSION: These data demonstrated that HMGB1 might induce the differentiation of splenic DCs to CD11c(low)CD45RB(high) DCs followed by shifting of Th1 to Th2 with enhancement of T lymphocyte immune function in vitro. Also, the effect of HMGB1 on T cell differentiation to Th2 was not associated with the inhibition of IL-12 production in CD11c(high)CD45RB(low) DCs.

Association of high mobility group box-1 protein levels with sepsis and outcome of severely burned patients.

BACKGROUND: The study was performed to observe the systemic release and kinetics of high mobility group box-1 protein (HMGB1) in burned patients. METHODS: 106 patients were included, and they were divided into three groups with different burn sizes: group I, group II and group III. Healthy volunteers served as normal controls (n=25). The peripheral blood samples were collected on postburn days (PBD) 1, 3, 7, 14, and 21. The blood samples were used to detect levels of HMGB1 in plasma by ELISA kits for human. Gene expression of HMGB1 in peripheral blood mononuclear cells was assessed by real-time quantitative PCR taking GAPDH as the internal standard. RESULTS: The levels of HMGB1 were significantly elevated on PBD 1-21 in patients with various burn sizes compared with normal controls, and there were obvious differences between group I and group III. The HMGB1 levels were significantly higher in septic patients than those without sepsis on PBD 7-21. Among septic patients, the HMGB1 levels in the survival group were markedly lower than those with fatal outcome on PBD 3-21. CONCLUSIONS: Extensive burn injury could result in significantly increased HMGB1 levels, which appears to be associated with the development of sepsis and fatal outcome of major burns.

High mobility group box-1 protein regulate immunosuppression of regulatory T cells through toll-like receptor 4.

INTRODUCTION: High mobility group box-1 protein (HMGB1), a recently recognized mediator of immune response might contribute to immune suppression when released extracellularly. The present study was performed to clarify effects of HMGB1 on regulatory T cells (Tregs) and the involvement of toll-like receptor (TLR) 4 signaling. METHODS: CD4(+)CD25(+)Tregs, isolated from spleens of normal mice and treated with HMGB1 in vitro, and those isolated from HMGB1-treated C3H/HeN (wild type) or C3H/HeJ (TLR4 mutant type) mice, were analyzed for expressions of cytotoxic T lymphocyte-associated antigen (CTLA)4, forkhead/winged helix transcription factor p3 (Foxp3) and interleukin (IL)-10 secretion. RESULTS: HMGB1-treatment was found to markedly decrease the expressions of CTLA4 and Foxp3, as well as IL-10 secretion. Administration of TLR4 neutralizing antibody abolished the phenotypic and functional changes in Tregs induced by HMGB1. Tregs from HMGB1-treated normal mice showed lower expression of CTLA4, Foxp3, and IL-10 secretion when compared with non-treated mice. Yet opposite results were observed in that of C3H/HeJ mice. Moreover, HMGB1 stimulation could down-regulate the expression of TLR4 on Tregs. CONCLUSION: Our data suggest that HMGB1 has the ability to directly modulate the suppressive capacity of CD4(+)CD25(+)Tregs, and TLR4 might be a potential receptor essential for the negative effect of HMGB1 on CD4(+)CD25(+)Tregs activity.

[The effects of administration of valproic acid on organ function and outcome in a canine lethal hemorrhagic shock model].

OBJECTIVE: To investigate the effects of valproic acid (histone deacetylase inhibitor) on visceral function and outcome in a canine lethal hemorrhage model. METHODS: Twenty male Beagle canines were subjected to an about 42% of total blood volume loss to reproduce a lethal hemorrhage shock model. Animals were randomly divided into shock control group (SC group) and valproic acid treatment group (VPA group), each group n=10. Canines in SC group and VPA group were intravenously injected either 20 ml saline or valproic acid (100 mg/kg) in 20 ml saline 1.5 hours after hemorrhage. Canines in each group were given delayed intravenous fluid resuscitation 24 hours after bleeding. The mean arterial pressure (MAP) was measured at 0 hour and at different time points without anesthesia, and the plasma levels of alanine aminotransferase (ALT), creatinine (Cr) and isoenzyme of creatine kinase (CK-MB) were measured before hemorrhage (0 hour), and at different time points after hemorrhage. Urinary output and survival rate 72 hours after hemorrhage were also recorded. RESULTS: The levels of MAP in both groups were significantly lowered from 2 hours after bleeding. The level of MAP (mm Hg, 1 mm Hg=0.133 kPa) in VPA group recovered rapidly and exceeded with statistically significant difference compared with those of SC group after hemorrhage (4 hours: 58.4±7.6 vs. 40.3±5.0, 8 hours: 84.4±8.0 vs. 56.4±4.4, 24 hours: 92.6±10.3 vs. 72.6±8.9, P<0.05 or P<0.01). The amount of urinary output of VPA group was significantly higher than that of SC group during the period of 0-8 hours, 8-24 hours, 24-48 hours, and 48-72 hours, but it was still lower than that before hemorrhage (0 hour). The plasma parameters for visceral function in both groups were significantly elevated compared with 0 hour. The plasma levels of ALT, Cr and CK-MB in VPA group were obviously lower than those in SC group from 4 hours after hemorrhage [at 4 hours after bleeding, ALT (U/L): 80.1±9.8 vs. 112.2±10.1, Cr (µmol/L): 74.5±8.3 vs. 88.0±7.6, CK-MB (kU/L): 10.39± 1.10 vs. 13.67±1.46, P<0.05 or P<0.01], but the visceral functional parameters at 72 hours after hemorrhage in VPA group were obviously higher than those at 0 hour [ALT (U/L):79.5±7.1 vs. 40.5±4.4; Cr (µmol/L): 85.6±7.1 vs. 46.6±4.8; CK-MB (kU/L): 7.63±0.86 vs. 1.66±0.21, all P<0.01]. The survival rate of VPA group 72 hours after bleeding was significantly higher than that of SC group [70% (7/10) vs. 20% (2/10), P<0.05]. CONCLUSION: The results indicate that intravenous injection of VPA promote MAP, increase urinary output, alleviate visceral injury and improve the survival rate at 72 hours in canines suffering from 42% blood volume loss, it might be an effective drug for hypovolemic shock, especially in war or other site of mass casualties in an austere environment.

Stimulation of α7 nicotinic acetylcholine receptor by nicotine increases suppressive capacity of naturally occurring CD4+CD25+ regulatory T cells in mice in vitro.

α7 Nicotinic acetylcholine receptor (α7 nAChR) has been found in several non-neuronal cells and is described as an important regulator of cellular function. Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. The present study investigated whether naturally occurring Tregs expressed α7 nAChR and investigated the functionary role of this receptor in controlling suppressive activity of these cells. We found that CD4(+)CD25(+) Tregs from naive C57BL/6J mice positively expressed α7 nAChR, and its activation by nicotine enhanced the suppressive capacity of Tregs. Nicotine stimulation up-regulated the expression of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on Tregs but had no effect on the production of interleukin (IL)-10 and transforming growth factor-ß1 by Tregs. In the supernatants of CD4(+)CD25(+) Tregs/CD4(+)CD25(-) T-cell cocultures, we observed a decrease in the concentration of IL-2 in nicotine-stimulated groups, but nicotine stimulation had no effect on the ratio of IL-4/interferon (IFN)-γ, which partially represented T-cell polarization. The above-mentioned effects of nicotine were reversed by a selective α7 nAChR antagonist, α-bungarotoxin. In addition, the ratio of IL-4/IFN-γ was increased by treatment with α-bungarotoxin. We conclude that nicotine might increase Treg-mediated immune suppression of lymphocytes via α7 nAChR. The effect is related to the up-regulation of CTLA-4 as well as Foxp3 expression and decreased IL-2 secretion in CD4(+)CD25(+) Tregs/CD4(+)CD25(-) T-cell coculture supernatants. α7 nAChR seems to be a critical regulator for immunosuppressive function of CD4(+)CD25(+) Tregs.

Association between regulatory T cell activity and sepsis and outcome of severely burned patients: a prospective, observational study.

INTRODUCTION: To investigate the significance of changes in regulatory T cells (Tregs) activity and its relationship with sepsis, as well as outcome of patients with major burns. METHODS: The periphery blood samples of 106 patients were collected on post-burn days 1, 3, 7, 14, and 21. Tregs were isolated and their phenotypes (cytotoxic T-lymphocyte-associated antigen 4 and forkhead/winged helix transcription factor p3) were analyzed by flow cytometry, and the contents of cytokines (interleukin-10 and transforming growth factor-beta1) released into supernatants by Tregs were also determined by enzyme-linked immunosorbent assay kits. Gene expressions of cytokines were assessed by real-time quantitative polymerase chain reaction. RESULTS: Expressions of Tregs phenotypes and gene/protein expression of cytokines were all elevated after burn, and there were obvious differences among patients with various burn sizes. They were also higher in septic patients than those without sepsis. Among septic patients, the expressions of Tregs phenotypes and the levels of cytokines were markedly lower in the survival group than those in patients with fatal outcome. CONCLUSIONS: Severe burn injury per se could lead to the changes in Tregs activities. Elevated levels of cytokines produced by Tregs and activation markers on Tregs surface might play an important role in the pathogenesis of sepsis and mortality in burned patients.

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors.

AIM: To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. METHODS: Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. RESULTS: Carbachol (2 µmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 µg/mL. CONCLUSION: These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor.

[Cellular phenotype transdifferentiation of adipose-derived stem cells into endothelial cells].

OBJECTIVE: To investigate the possibility of adipose derived stem cells (ADSCs) for wound healing by detecting cellular phenotype conversion of ADSCs into endothelial cells (ECs). METHODS: ADSCs were isolated and cultured from adipose tissue derived from SD rats (n = 8), and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) in vitro. The marker antigen of P3 ADSCs was detected by analysis CD49d and CD106 antigens expression using flow cytometry, and the multipotential differentiation of P3 ADSCs were identified by specific medium inducing to differentiate into osteoblasts and adipocytes. And then, the ADSCs were cultured and induced for 3 days by condition culture medium (containing 30% superior of homogenating rat blood vessels in 10%FBS DMEM) as experimental group, and were cultured by 10% FBS DMEM as control group, and the expression of CD34 and von Willebrand factor (vWF) in ADSCs were analyzed by flow cytometry. RESULTS: Flow cytometry analysis showed that the expression of CD49d and CD106 in ADSCs were positive (98.32 ± 0.37)% and negative (1.67 ± 0.61)%, respectively. The multipotential differentiation experiment demonstrated that the cultured P3 ADSCs can be induced to differentiate into osteoblasts and adipocytes in vitro. The positive rate of CD34 and vWF were (77.14 ± 0.76)% and (75.46 ± 0.37)% in condition medium group, higher than (1.38 ± 0.31)% and (1.70 ± 0.23)% in 10% FBS DMEM control group, respectively (P < 0.01). CONCLUSION: The ADSCs can be induced to differentiated into ECs, suggesting that ADSCs have potential to take part in wound repair and angiogenesis.

Sevoflurane-induced cognitive decline in aged mice: Involvement of toll-like receptors 4.

Toll-like receptors 4 (TLR4) contributes to the pathogenesis of some neurodegenerative diseases. However, little is known about whether TLR4 is associated with sevoflurane-induced cognitive decline. This investigation aims to address the effect of global TLR4 gene knockout on cognitive decline following sevoflurane exposure to mice. Wild-type and TLR4-/- mice were exposed to 3% sevoflurane. Novel object recognition test and Y-maze test were used to analyze cognitive function. Tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in plasma and hippocampus were measured by ELISA. Peripheral administration of recombinant TNF-α to TLR4-/- mice was used to observed the role of TNF-α in cognitive function following sevoflurane. Our results showed that, in contrast to wild-type mice, TLR4 deficiency protected against the cognitive function impairment following sevoflurane exposure, and abrogated IL-1ß, IL-6 and TNF-α response to sevoflurane in the system and the hippocampus. Subcutaneous administration of recombinant TNF-α elevated these cytokine levels in the hippocampus, and resulted in cognitive decline in TLR4-/- mice exposed to sevoflurane. Taken together, our results identify the crucial role of TLR4 in sevoflurane-induced cognitive decline, and showed that TLR4 mediated pro-inflammatory cytokine response to sevoflurane, and consequent cognitive decline in aged mice exposed to sevoflurane, and imply a novel target for improvement and therapy of sevoflurane-associated cognitive decline.

Angiotensin II induces type I collagen gene expression in human dermal fibroblasts through an AP-1/TGF-beta1-dependent pathway.

Angiotensin II is critically involved in skin wound healing, but the underlying mechanism remains unclear. This study investigated the effect of angiotensin II on type I collagen gene activation in human dermal fibroblasts and the possible mechanism involved. Angiotensin II stimulated the mRNA and protein expression of type I collagen and TGF-beta1. Effects were abolished by the angiotensin AT1 receptor antagonist ZD7155 but not by the AT2 blocker PD123319. Blockade of TGF-beta1 markedly inhibited angiotensin II-induced type I collagen gene expression. Activator protein-1 (AP-1) decoy ODNs transfection suppressed angiotensin II-induced TGF-beta1 expression, and also, diminished type I collagen expression. These data indicated that angiotensin II induces collagen gene activation in human dermal fibroblasts through an AT1-mediated AP-1/TGF-beta1 signaling pathway.

Effect of high mobility group box-1 protein on apoptosis of peritoneal macrophages.

The present study was performed to clarify the effects of high mobility group box-1 protein (HMGB1), a recently described late-acting pro-inflammatory cytokine, on apoptosis of macrophages. Treatment with HMGB1 (0.01, 0.1, 1, 10 microg/ml for 24 h, or 10 microg/ml for 6, 12, 24, 48 h) resulted in a dose- and time-dependent induction of apoptosis in mice macrophages, peaked at 24 h after 10 microg/ml HMGB1 stimulation. HMGB1 treatment enhanced the receptor for advanced glycation end products (RAGE) expression and caspase-3 activation in macrophages. Blockage of RAGE and caspase-3 activation could significantly reduce apoptosis of macrophages induced by HMGB1. The activity of NF-kappaB/p65 in macrophages was decreased after HMGB1 treatment. These results suggested that HMGB1-induced apoptosis of macrophages in a dose- and time-dependent manner. RAGE, together with caspase-3 activation and inhibition of NF-kappaB signaling pathways, might be involved in the pathogenesis of macrophage apoptosis induced by HMGB1.

Regulation of signaling pathways downstream of IGF-I/insulin by androgen in skeletal muscle of glucocorticoid-treated rats.

BACKGROUND: The mechanisms by which androgens ameliorate glucocorticoid-induced muscle wasting are still under investigation. In the present study, we tested the hypothesis that androgen's effects in reversing muscle wasting are related to activating the signaling pathways downstream of insulin-like growth factor-1 (IGF-I)/insulin. METHODS: Forty female Sprague-Dawley rats were randomly divided into four groups: control group, dexamethasone (DEX) group, testosterone (TES) group, and TES + DEX group. Each group was injected with saline or DEX (0.1 mg/100 g/d) for 10 days and sesame oil or TES (0.5 mg/100 g/d) for 13 days. Several downstream targets of IGF-I/insulin in skeletal muscle including protein kinase B (Akt), p70 ribosomal protein S6 kinase (p70S6K), and glycogen synthase kinase-3beta (GSK-3beta) that are associated with protein synthesis were examined. Two proteolysis-related ubiquitin E3-ligases, muscle atrophy F-box, and muscle RING finger-1 that are also regulated by IGF-I/insulin were also assessed. RESULTS: TES attenuated gastrocnemius muscle atrophy induced by DEX. TES prevented the DEX-induced decrease of IGF-I expression in gastrocnemius muscle, but not in serum. TES ameliorated DEX-induced dephosphorylation of Akt and p70S6K and promoted the phosphorylation of GSK-3beta in gastrocnemius muscle. The total amount of Akt, p70S6K, or GSK-3beta proteins was not changed among these groups. TES did not show any effects on the DEX-induced upregulation of muscle atrophy F-box, and muscle RING finger-1 mRNA in gastrocnemius muscle. CONCLUSION: This findings suggest that the effects of TES in reversing DEX-induced muscle atrophy are related to signaling pathways downstream of IGF-I/insulin that are associated with protein synthesis.