Protective effect of zinc on liver injury induced byd-galactosamine in rats

The protective effects of zinc on liver injury induced byd-galactosamine (GalN) were investigated in rats in vivo and in vitro. Zinc supplementation (50 mg/kg/d) for 5 d of rats treated with GalN (1.5 g/kg, ip) could reduce their mortality rate, restore liver pathomorphological changes, maintain zinc content, inhibit the lipid peroxidation, hasten the protein synthesis, and improve liver function. In vitro, zinc supplement could abate the death of GalN-intoxicated hepatocytes, decrease malonaldehyde (MDA) content, and maintain reduced glutathione (GSH). It is concluded that zinc has protective effects on GalN-induced liver damage. Its effects may be owing to inhibition of lipid peroxidation and hastening of protein syntheses.     

Regenerative capacity of the dystrophic (mdx) diaphragm after induced injury

Duchenne muscular dystrophy is characterized by myofiber necrosis, muscle replacement by connective tissue, and crippling weakness. Although the mdx mouse also lacks dystrophin, most muscles show little myofiber loss or functional impairment. An exception is the mdx diaphragm, which is phenotypically similar to the human disease. Here we tested the hypothesis that the mdx diaphragm has a defective regenerative response to necrotic injury, which could account for its severe phenotype. Massive necrosis was induced in mdx and wild-type (C57BL10) mouse diaphragms in vivo by topical application of notexin, which destroys mature myofibers while leaving myogenic precursor satellite cells intact. At 4 h after acute exposure to notexin, >90% of diaphragm myofibers in both wild-type and mdx mice demonstrated pathological sarcolemmal leakiness, and there was a complete loss of isometric force-generating capacity. Both groups of mice showed strong expression of embryonic myosin within the diaphragm at 5 days, which was largely extinguished by 20 days after injury. At 60 days postinjury, wild-type diaphragms exhibited a persistent loss ( approximately 25%) of isometric force-generating capacity, associated with a trend toward increased connective tissue infiltration. In contrast, mdx diaphragms achieved complete functional recovery of force generation to noninjured values, and there was no increase in muscle connective tissue over baseline. These data argue against any loss of intrinsic regenerative capacity within the mdx diaphragm, despite characteristic features of major dystrophic pathology being present. Our findings support the concept that significant latent regenerative capacity resides within dystrophic muscles, which could potentially be exploited for therapeutic purposes.     

The expression changes of Numblike in rat brain cortex after traumatic brain injury

Numblike (Numbl) plays an important role in ependymal wall integrity and subventricular zone neuroblast survival. And Numbl is specifically expressed in the brain. However, its expression and function in the central nervous system lesion are still unclear. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of Numbl expression in the brain cortex. Western blot and immunohistochemistry analysis revealed that Numbl was present in normal brain. It gradually decreased, reached the lowest point at day 3 after TBI, and then increased during the following days. Double immunofluorescence staining showed that Numbl immunoreactivity was found in neurons, but not astrocytes and microglia. Moreover, the 3rd day post injury was the apoptotic peak implied by the alteration of caspase-3. All these results suggested that Numbl may be involved in the pathophysiology of TBI and further research is needed to have a good understanding of its function and mechanism.     

Increased expression of BAG-1 in rat brain cortex after traumatic brain injury

BAG-1 protein was initially identified as a Bcl-2-binding protein. It was reported to enhance Bcl-2 protection from cell death, suggesting that BAG-1 represents a new type of anti-cell death gene. Moreover, recent study has shown that BAG-1 can enhance the proliferation of neuronal precursor cells, attenuate the growth inhibition induced by siah1. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of BAG-1 expression in the brain cortex. Double immunofluorescence staining revealed that BAG-1 was co-expressed with NEURON and glial fibrillary acidic protein (GFAP). In addition, we detected that proliferating cell nuclear antigen had the co-localization with GFAP, and BAG-1. All our findings suggested that BAG-1 might involve in the pathophysiology of brain after TBI.     

Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury

Background

Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury.

Methods and Findings

Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH⁺ and Iba-1⁺ cells acutely died, and as the penumbra the area surrounding the core where Iba-1⁺ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d.

Conclusions

Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic.     

Metallothionein expression and tissue metal kinetics after partial hepatectomy in mice

To better elucidate previous results showing that partial hepatectomy noticeably changes the tissue content of zinc, calcium, magnesium, and iron(II) ions in regenerating the liver, thymus, and spleen, we report on the correlation of these metal tissue kinetics in these organs with the expression of metallothionein-I+II (MT-I+II) proteins and MT-I mRNA in early postoperative period (1, 2, 6, 12, and 24 h) after one-third hepatectomy (pHx). The results showed that 2 h after pHx the regenerating liver accumulated Zn²⁺, Ca²⁺, Mg²⁺, and Fe²⁺ ions while decreasing the concentration of all these metals in the spleen and of Zn²⁺ in the thymus. On the 24th h, a new high accumulation of Zn²⁺ and Ca²⁺ was seen in the regenerating liver and of Zn²⁺, Ca²⁺, and Fe²⁺ in the spleen. Simultaneously, MT-I mRNA increased in the liver and spleen. In hepatocytes and on several spleen and thymus mononuclear lymphatic cells, the increased expression of MT proteins was found mainly in the cytoplasm and nuclei. The areas expressing MTs in regenerating liver inversely correlated with those containing apoptotic cells, suggesting that these proteins participate in tissue restoration through reduction or increase of metal ions after injury to the liver.     

Zinc-induced upregulation of metallothionein (MT)-2A is predicted by gene expression of zinc transporters in healthy adults

The usefulness of zinc transporter and metallothionein (MT) gene expressions to detect changes in zinc intake remains unclear. This pilot study aimed to determine the effects of zinc supplementation on zinc transporter and MT gene expressions in humans. Healthy adults (n = 39) were randomised to zinc treatment (ZT), receiving 22 mg Zn/day (n = 19), or no treatment (NT) (n = 20). Blood samples were collected on Days 0, 2, 7, 14, and 21. Plasma zinc and serum C-reactive protein concentrations were analysed. Gene expression of zinc transporters and MT in peripheral blood mononuclear cells was analysed using real-time PCR. Using repeated-measures ANOVA, MT-2A gene expression and fold change were found to be higher in the ZT group (P = 0.025 and P = 0.016, respectively) compared to the NT group, specifically at Day 2 (40 ± 18 % increase from baseline, P = 0.011), despite no significant increase in plasma zinc concentration. In a multiple regression model exploring the changes in gene expressions between Days 0 and 21, the change in MT-2A gene expression was correlated with changes in all zinc transporter expressions (r² = 0.54, P = 0.029); the change in ZIP1 expression emerged as a univariate predictor (P = 0.003). Dietary zinc intake was predictive of zinc transporter and MT expressions (P = 0.030). Physical activity level was positively correlated with baseline ZIP7 expression (r = 0.36, P = 0.029). The present study shows that MT-2A expression is related to changing expression of zinc transporter genes, specifically ZIP1, in response to zinc supplementation. The current report adds to our understanding of MT in the coordinated nature of cellular zinc homeostasis.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0494-y) contains supplementary material, which is available to authorized users.     

Caspase 7: increased expression and activation after traumatic brain injury in rats

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.     

Cu/Zn- and Mn-superoxide dismutases are specifically up-regulated in neurons after focal brain injury

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury.     

柽柳金属硫蛋白基因(MT2)的过量表达对烟草耐Cd2+性的促进效应

将在柽柳(Tamarix androssowii)中克隆的金属硫蛋白基因MT2(GenBank登录号:AY620987)构建到载体pROKII上。用农杆菌介导的叶盘法转化烟草‘龙江911’,获得了抗卡那霉素的转基因植株。PCR-Southern和Northern blot检测证明外源基因已整合进烟草基因组并可正常表达。Cd2 抗性实验证明柽柳金属硫蛋白基因(MT2)的表达可提高转基因烟草的抗Cd2 性。     

Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic

The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.     

Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic

The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.     

(Copper, zinc)--thionein in pig liver.

The concentrations of zinc and/or copper in the hepatic metallothionein of young (1–11 days, and 8 week-old) pigs correlate with the concentration of whole liver zinc. In the livers of adult animals only thionein-bound zinc correlates with total zinc. Although thionein-bound copper correlates with total copper in the livers of developing pigs, it shows a higher degree of correlation with whole liver zinc.Hepatic (copper, zinc) -thioneins, from either the young or the adult pig, in which the Cu : Zn ratio is high, are denatured on columns of DE cellulose, or during preparative electrophoresis. Preparations in which the Cu : Zn ratio is lower are resolved by ion-exchange chromatography into three forms, all of which contain both cations, but in differing ratios. Electrophoresis of a (copper, zinc) thionein that contains the two cations in 1 : atomic ratio yields zinc-thionein as a single distinct molecular species.     

Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice

Stem cell factor (SCF) and its receptor c-kit are important in hematopoiesis and cellular proliferation. c-kit has also been identified as a cell surface marker for progenitor cells. We have previously shown that there is a large reservoir of hepatic SCF, and this molecule plays a significant role in liver regeneration after 70% hepatectomy. In the current study, we further examined the expression of SCF and c-kit in acetaminophen (APAP)-induced liver injury in C57BL/6J mice or SCF-deficient sl-sld mice and their appropriate wild-type controls. Following APAP-induced liver injury, c-kit mRNA expression increased, with peak levels detected 48 h postinjury. Hepatic SCF mRNA levels after APAP injury were also increased, with peak levels seen 16 h post-APAP. The mortality rate in SCF-deficient mice treated with APAP was significantly higher than that of wild-type mice; furthermore, administration of exogenous SCF significantly reduced the mortality of APAP-treated wild-type mice. Bromodeoxyuridine incorporation experiments showed that SCF significantly increased hepatocyte proliferation at 48 and 72 h in APAP-treated mice. SCF inhibited APAP-induced hepatocyte apoptosis and increased Bcl-2 and Bcl-xL expression, suggesting that this decrease in hepatocyte apoptosis is mediated through Bcl-2 and Bcl-xL. In summary, SCF and c-kit expression was increased after APAP-induced liver injury. Administration of exogenous SCF reduces mortality in APAP-treated mice, increases hepatocyte proliferation, and prevents hepatocyte apoptosis induced by APAP, suggesting that these molecules are important in the liver's recovery from these injuries.