Adjudin Attenuates Cerebral Edema and Improves Neurological Function in Mice with Experimental Traumatic Brain Injury.

Adjudin, a small molecular compound that is used as a male contraceptive, has been reported to play a neuroprotective role in an ischemic stroke injury model. However, its effect on traumatic brain injury (TBI) has not been assessed. Hence, we investigated the effects of adjudin on cerebral edema using a mouse model of TBI and explored the underlying mechanisms. Adult male C57BL/6 mice received controlled cortical impact (CCI) injury, then an injection of adjudin (50 mg/kg). The mice were euthanized 3 days post-CCI injury, and samples were collected for further analysis. Cultured primary mouse astrocytes were used for in vitro experiments. Adjudin treatment significantly attenuated cerebral edema on Day 3 and improved neurobehavioral outcomes on Days 3, 7, and 14 after CCI injury, compared with the vehicle group. Additionally, the evaluation of Evans blue extravasation and expression of tight junction proteins demonstrated remarkable effects of adjudin on blood-brain barrier protection. Further, adjudin treatment significantly decreased the gene and protein expression of aquaporin 4 in post-injury mice and inhibited progression of neuroinflammation in both mice and cultured astrocytes. The Western blot results of the peritraumatic protein samples demonstrated that adjudin significantly blocked the phosphorylation of IKKα, IκBα/ß, and NF-κB p65, which resulted in a reduction of NF-κB p65 nuclear translocation. In conclusion, adjudin attenuated the development of TBI-induced cerebral edema at least partly via anti-inflammatory effects and inhibition of the NF-κB pathway. These findings suggest that adjudin is a potential therapeutic intervention preventing the development of cerebral edema after TBI.

Sirt3 deficiency impairs neurovascular recovery in ischemic stroke.

AIMS: Sirt3 is one member of the NAD+ -dependent protein deacetylase family and plays crucial roles in diverse aspects of mammalian biological function. Then the role of Sirt3 on ischemia stroke is unknown. METHODS: To examine the effect of Sirt3 on ischemic stroke, we performed transient middle cerebral artery occlusion (tMCAO) in adult male Sirt3 knockout (KO) and wild-type (WT) mice. RESULTS: The level of Sirt3 in infarct region is decreased after ischemic stroke. In addition, we found that Sirt3 KO mice showed worse neurobehavioral outcome compared with WT mice, accompanied by decreased neurogenesis and angiogenesis as shown by the reduction in number of DCX+ /BrdU+ cells, NeuN+ /BrdU+ cells, and CD31+ /BrdU+ cells in the perifocal region during recovery phase after ischemic stroke. Furthermore, Sirt3 deficiency reduced the activation of vascular endothelial growth factor (VEGF), AKT, and extracellular signal-regulated kinases (ERK) signaling pathways. CONCLUSION: Our results indicated that Sirt3 is beneficial to neurovascular and functional recovery following chronic ischemic stroke.

Novel double-layer Silastic testicular prosthesis with controlled release of testosterone in vitro, and its effects on castrated rats.

Testicular prostheses have been used to deal with anorchia for nearly 80 years. Here, we evaluated a novel testicular prosthesis that can controllably release hormones to maintain physiological levels of testosterone in vivo for a long time. Silastic testicular prostheses with controlled release of testosterone (STPT) with different dosages of testosterone undecanoate (TU) were prepared and implanted into castrated Sprague-Dawley rats. TU oil was applied by oral administration to a separate group of castrated rats. Castrated untreated and sham-operated groups were used as controls. Serum samples from every group were collected to measure the levels of testosterone (T), follicle-stimulating hormone and luteinizing hormone (LH). Maximum intracavernous penile pressure (ICPmax) was recorded. The prostates and seminal vesicles were weighed and subjected to histology, and a terminal dexynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) assay was used to evaluate apoptosis. Our results revealed that the weights of these tissues and the levels of T and LH showed significant statistical differences in the oral administration and TU replacement groups compared with the castrated group (P < 0.05). Compared with the sham-operated group, the ICPmax, histology and TUNEL staining for apoptosis, showed no significant differences in the hormone replacement groups implanted with medium and high doses of STPT. Our results suggested that this new STPT could release TU stably through its double semi-permeable membranes with excellent biocompatibility. The study provides a new approach for testosterone replacement therapy.

Postreperfusion hyperkalemia in liver transplantation using donation after cardiac death grafts with pathological changes.

BACKGROUND: With the increasing use of donation after cardiac death (DCD), especially of the graft liver with steatosis or other pathological changes, the frequency of postreperfusion hyperkalemia in liver transplantation has increased significantly. The present study aimed to determine the factors associated with developing postreperfusion hyperkalemia in liver transplantation from DCD. METHODS: One hundred thirty-one consecutive adult patients who underwent orthotopic liver transplantation from DCD were retrospectively studied. Based on serum potassium within 5 minutes after reperfusion, recipients were divided into two groups: hyperkalemia and normokalemia. According to preoperative biopsy results, the DCD graft livers were classified into five categories. Univariate analysis was performed using Chi-square test to identify variables that were significantly different between two groups. Multivariate logistic regression was used to confirm the risk factors of developing hyperkalemia and postreperfusion syndrome. Correlation analysis was used to identify the relationship between the serum concentration of potassium within 5 minutes after reperfusion and the difference in mean arterial pressure values before and within 5 minutes after reperfusion. RESULTS: Twenty-two of 131 liver recipients had hyperkalemia episodes within 5 minutes after reperfusion. The rate of hyperkalemia was significantly higher in recipients of macrosteatotic DCD graft liver (78.6%, P<0.001) than that in recipients of non-macrosteatotic DCD graft liver. The odds ratio of developing postreperfusion hyperkalemia in recipients of macrosteatotic DCD graft liver was 51.3 (P<0.001). Macrosteatosis in the DCD graft liver was an independent risk factor of developing hyperkalemia within 5 minutes after reperfusion. The highest rate of postreperfusion syndrome also occurred in the recipients with macrosteatotic DCD graft liver (71.4%, P<0.001). A strong relationship existed between the serum potassium within 5 minutes after reperfusion and the difference in mean arterial pressure values before and within 5 minutes after reperfusion in macrosteatotic DCD graft liver recipients. CONCLUSION: Macrosteatosis in the DCD graft liver was an independent risk factor of developing hyperkalemia and postreperfusion syndrome in the recipients.

GIT1 enhances neurite outgrowth by stimulating microtubule assembly.

GIT1, a G-protein-coupled receptor kinase interacting protein, has been reported to be involved in neurite outgrowth. However, the neurobiological functions of the protein remain unclear. In this study, we found that GIT1 was highly expressed in the nervous system, and its expression was maintained throughout all stages of neuritogenesis in the brain. In primary cultured mouse hippocampal neurons from GIT1 knockout mice, there was a significant reduction in total neurite length per neuron, as well as in the average length of axon-like structures, which could not be prevented by nerve growth factor treatment. Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice. The GIT1 N terminal region, including the ADP ribosylation factor-GTPase activating protein domain, the ankyrin domains and the Spa2 homology domain, were sufficient to enhance axonal extension. Importantly, GIT1 bound to many tubulin proteins and microtubule-associated proteins, and it accelerated microtubule assembly in vitro. Collectively, our findings suggest that GIT1 promotes neurite outgrowth, at least partially by stimulating microtubule assembly. This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

[Risk factors of biliary complications after liver transplantation from donation after cardiac death].

OBJECTIVE: To analyze the risk factors for biliary complications of liver transplantation from donation after cardiac death (DCD). METHODS: Clinical data of 109 patients undergoing liver transplantation from DCD in First Affiliated Hospital of Zhejiang University School of Medicine from October 2010 to October 2013 were studied retrospectively. The risk factors of biliary complications following DCD liver transplantation were analyzed. RESULTS: Twenty-four (22%) patients developed biliary complications after DCD liver transplantation. Univariate analysis showed that biliary complications were associated with warm ischemia time (P<0.001) and length of ICU stay (P=0.013), but not associated with ABO blood types match (P>0.05). Administration of inotropic agents and fatty liver increased the trend of biliary complications. Multivariate analysis demonstrated that warm ischemia time and length of ICU stay were independent risk factors for predicting biliary complications. CONCLUSION: Warm ischemia time and days of ICU stay are independent risk factors for predicting biliary complications after DCD liver transplantation.

Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells.

Spermatogonial stem cells (SSCs) divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells (hdFs). Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen (SSEA)-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction (RT-PCR) analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the 'niche' that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Evaluation of hepatitis B virus replication and proteomic analysis of HepG2.2.15 cell line after cyclosporine A treatment.

AIM: The effect of cyclosporine A (CsA) on hepatitis B virus (HBV) replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication. METHODS: Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens (HBsAg) and Hepatitis B e antigens (HBeAg) in culture supernatant, while the intracellular HBV DNA replication level was analyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search. RESULTS: CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed significantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors (eIF) 3k, otubain 1, 14.3.3 protein, eIF2-1 alpha, eIF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The downregulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa (ECP-51), stathmin 1/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally controlled 1, was shifted, suggesting it had undergone posttranslational modifications. CONCLUSION: Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.

Pretreatment of hypertonic saline can increase endogenous interleukin 10 release to attenuate hepatic ischemia reperfusion injury.

Ischemia-reperfusion (I/R) injury of the liver occurs in many clinical cases. Many steps are associated with hepatic I/R injury, including the release of many inflammatory molecules and infiltration of neutrophils into the liver. Recent studies revealed that hypertonic saline (HTS) has a strong anti-inflammatory effect and can inhibit a varity of neutrophil functions. So pretreatment with HTS may attenuate the liver injury associated with I/R. In this study, rats were divided into three groups: the sham group (S group), hepatic I/R group (I/R group), and HTS pretreatment group (HTS group). Serum ALT and myeloperoxidase (MPO) activity were determined. Serum tumor necrosis factor alpha (TNF-alpha) and interleukin 10 (IL-10) were determined by enzyme-linked immunosorbent assay (ELISA). Reverse transcription polymerase chain reaction analysis was used to assess the mRNA expressions of TNF-alpha and IL-10. Protein expressions of TNF-alpha, IL-10, STAT3, and phosphorylated STAT3 were analyzed by Western blot. Results showed that HTS pretreatment can augment the release of endogenous IL-10 by activating STAT3 in the process of hepatic I/R injury. Serum ALT levels, MPO activity in liver, generation of TNF-alpha, and infiltration of neutrophils in liver were inhibited in the HTS group. So we concluded that HTS pretreatment attenuates hepatic I/R injury by increasing the release of endogenous IL-10.

[Effects of ciclosporin and tacrolimus on replication of hepatitis B virus in vitro: a comparative study].

OBJECTIVE: To investigate the effects of ciclosporin (CsA) and tacrolimus (FK506) on replication of hepatitis B virus (HBV) in vitro. METHODS: HBV genome permanently transfected human liver cancer cells of the line HepG2.2.15 were cultured. CsA and FK506 at different concentrations were added into the culture fluid so as to identify the nontoxic concentrations by MTT method. Then the HepG2.2.15 cells were treated by CsA and FK506 at different nontoxic concentrations respectively for 4 days. ELISA was used to detect the HB surface antigen (HBsAg) and HB e antigen (HBeAg) in the supernatant. The relative replication level of HBV DNA was detected by slot blot analysis. RESULTS: MTT method confirmed that the nontoxic concentrations of CsA and FK506 were 0-40.0 microg/ml and 0-400 ng/ml respectively. After the treatment of CsA at the concentration of 1.3, 2.5, and 5.0 microg/ml, in comparison to the control group, the suppression rates of HBsAg expression in the HepG2.2.15 cells were 16.5% +/- 9.4%, 21.5% +/- 8.9%, and 33.1% +/- 5.3% respectively (all P < 0.05); the suppression rates of HBeAg expression in the HepG2.2.15 cells were 7.8% +/- 2.2%, 11.0% +/- 2.3%, and 20.8% +/- 1.5% respectively (all P < 0.05); and the HBV DNA replication levels were 56 +/- 16, 42 +/- 11, and 40 +/- 10 respectively (P > 0.05, P < 0.05, and P > 0.05). However, FK506 at different nontoxic concentrations showed no significant inhibitory effect on the levels of HBsAg, HBeAg, and HBV DNA. CONCLUSION: CsA dose-dependently inhibits the HBV replication in vitro, and FK506 does not exercise similar effects.

[Effects of hypertonic saline on expression of heme oxygenase enzyme-1 in hepatic ischemia/reperfusion injury rats].

OBJECTIVE: To explore the effect of the pretreatment with hypertonic saline (HTS) in the hepatic ischemia/reperfusion (I/R) injury. METHODS: Twenty-five SD rats were randomly divided into sham operation group, heme oxygenase-1 (HO-1) blocker ZnPP group, I/R group, HTS pretreatment group and ZnPP intervention group (n=5). The rat model of partial hepatic I/R injury was reproduced by isolating the portal venous and hepatic arterial branches to the left and median hepatic lobes, and they were occluded with a microvascular clamp for 30 minutes, followed by reperfusion. In HTS pretreatment group, the rats received 4 ml/kg volume of HTS (7.5%) intravenous 1 hour before the occlusion of the vessels. The rats were sacrificed 6 hours after reperfusion. The levels of alanine aminotransferase (ALT) and tumor necrosis factor-alpha (TNF-alpha) in serum, liver myeloperoxidase (MPO) activity and liver endothelin-1 (ET-1) were determined. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to examine the expression of HO-1 in the liver. The pathological changes in the liver, including hepatic sinusoid, were evaluated in hematoxylin and eosin (HE)-stained sections and transmission electron microscopy (TEM) of liver specimens. The effect of HTS pretreatment was also assessed in the rats pretreated with ZnPP. RESULTS: The levels of serum ALT and TNF-alpha, ET-1 and MPO activity in hepatic tissues increased after hepatic I/R injury (all P<0.01), and expression of HO-1 mRNA and protein were also increased. RT-PCR and Western-blot revealed that the expression of HO-1 in the liver was upgraded significantly, and the ALT level, serum TNF-alpha, liver MPO activity and liver ET-1 were suppressed significantly after I/R injury in the HTS pretreatment group. In this group, there were moderate swelling of hepatocytes and mild neutrophils infiltration in the liver. The hepatic microcirculatory dysfunction was ameliorated. All these findings showed that HTS pretreatment produced the effect of prevention on hepatic I/R injury. However, the adjunctive infusion of ZnPP abrogated the beneficial effects. CONCLUSION: Pretreatment of HTS has the effect of the prevention of hepatic I/R injury by promotion of the expression of HO-1.

Inhibitory effect of cyclosporine A on hepatitis B virus replication in vitro and its possible mechanisms.

BACKGROUND: Hepatitis B related end-stage liver disease is recently acknowledged as one of the main indications for orthotopic liver transplantation (OLT). However, the high recurrence rate of hepatitis B virus infection following transplantation is regarded as a major factor affecting the long-term survival of transplant recipients especially in China. Cyclosporine A (CsA), which is routinely used to prevent the allograft rejection, is reported to have the inhibitory activity on hepatitis B virus (HBV) replication in vitro. In this paper, we review the inhibitory effect and its possible mechanisms of CsA on HBV replication in vitro. DATA RESOURCES: An English-language literature search was conducted using MEDLINE (1990-2004) on cyclosporine A, hepatitis B virus, mitochondria, calcium and other related reports and review articles. RESULTS: Hepatitis B x protein (HBx) is essential to HBV replication. The cytosolic calcium signaling mediated by mitochondria and the Src kinase pathway were involved during HBx activation of HBV replication. CsA inhibits the HBV replication in vitro by its binding to mitochondrial cyclophilin D, then blocking the mitochondria-mediated cytosolic calcium signaling. The derivates of CsA also have the HBV replication inhibitory effect in vitro. CONCLUSIONS: By interacting with mitochondria, preventing the release of intramitochondrial calcium, and then blocking the cytosolic calcium signaling, CsA inhibits the HBV replication in vitro. The derivates of CsA also have this activity.