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Brain-derived neurotrophic factor (BDNF) plays an important role in promoting the growth, differentiation, survival and synaptic stability of neurons. Presently, the transplantation of neural stem cells (NSCs) is known to induce neural repair to some extent after injury or disease. In this study, to investigate whether NSCs genetically modified to encode the BDNF gene (BDNF/NSCs) would further enhance synaptogenesis, BDNF/NSCs or naive NSCs were directly engrafted into lesions in a rat model of traumatic brain injury (TBI). Immunohistochemistry, western blotting and RT-PCR were performed to detect synaptic proteins, BDNF-TrkB and its downstream signaling pathways, at 1, 2, 3 or 4 weeks after transplantation. Our results showed that BDNF significantly increased the expression levels of the TrkB receptor gene and the phosphorylation of the TrkB protein in the lesions. The expression levels of Ras, phosphorylated Erk1/2 and postsynaptic density protein-95 were elevated in the BDNF/NSCs-transplanted groups compared with those in the NSCs-transplanted groups throughout the experimental period. Moreover, the nuclear factor (erythroid-derived 2)-like 2/Thioredoxin (Nrf2/Trx) axis, which is a specific therapeutic target for the treatment of injury or cell death, was upregulated by BDNF overexpression. Therefore, we determined that the increased synaptic proteins level implicated in synaptogenesis might be associated with the activation of the MAPK/Erk1/2 signaling pathway and the upregulation of the antioxidant agent Trx modified by BDNF-TrkB following the BDNF/NSCs transplantation after TBI.

Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

Cytoskeletal proteins are involved in neuronal survival. Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury. However, the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear. To examine this, we established a rat model of traumatic brain injury by controlled cortical impact. At 72 hours after injury, 2 × 107 cells/mL neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells (3 mL) were injected into the injured cortex. At 1–3 weeks after transplantation, expression of neurofilament 200, microtubule-associated protein 2, actin, calmodulin, and beta-catenin were remarkably increased in the injury sites. These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival, growth, and differentiation in the injury sites. The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.

Long-term abuse of alcohol results in chronic alcoholic myopathy which is associated with increased oxidative stress. Ginkgo biloba extract (EGB) is widely used as a therapeutic agent to treat certain cardiovascular and neurological disorders. Although EGB is known to possess antioxidant functions and potent cytoprotective effects, its protective mechanism on alcohol-induced oxidative damage in C2C12 myoblasts remains unclear. In this study, we investigated the cytoprotective mechanisms of EGB against alcohol-derived oxidative stress in mouse C2C12 myoblasts. Challenge with alcohol (100mM) caused an increase in intracellular reactive oxygen species in mouse C2C12 myoblasts, which was not alleviated by treatment with EGB. These results indicate that EGB does not seem to act as an ROS scavenger in this experimental model. Additionally, EGB produced activation of ERK and JNK [two major mitogen-activated protein kinases (MAPKs)], an increase in the nuclear level of nuclear factor erythroid-2-related factor 2 (Nrf2) and upregulation of heme oxygenase-1 (HO-1, a stress-responsive protein with antioxidant function). Pretreatment with inhibitors of MAPKs PD98059 (a specific inhibitor of ERK), SP600125 (a specific inhibitor of JNK) abolished both EGB-induced Nrf2 nuclear translocation and HO-1 up-regulation. We conclude that EGB confers cytoprotective effects from oxidative stress induced by alcohol in mouse C2C12 myoblasts depend on transcriptional upregulation of HO-1 by EGB via the MAPKs/Nrf2 pathway.

This study evaluated the effects of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) on injured gastrocnemius, soleus and plantaris muscles, induced by alcohol in rats. A total of 60 male Sprague-Dawley rats (2.5 months old, 200 ± 20 g) were divided into 6 groups: i) untreated skeletal muscle and analyzed 2 weeks later (A1 group, 5 rats); ii) untreated skeletal muscle and analyzed 6 weeks later (A2 group, 5 rats); iii) untreated skeletal muscle and analyzed 12 weeks later (A3 group, 5 rats); iv) injured skeletal muscle and analyzed 2 weeks later (B1 group, 15 rats); v) injured skeletal muscle and analyzed 6 weeks later (B2 group, 15 rats); and vi) injured skeletal muscle and analyzed 12 weeks later (B3 group, 15 rats). The injured and uninjured muscles were observed by light microscopy and polarization microscopy. The MMP activity was evaluated through zymography, and messenger RNA (mRNA) of MMP-9 and of MMP-2 were assessed by RT-PCR. The expression of MMP-9 was assessed by Western blot analysis. The plantaris and gastrocnemius muscles in the rats subjected to alcohol ingestion were found to have a high expression of MMP-9, but not of MMP-2. Picrosirius red staining was used to assess whether increased fibrosis in the skeletal muscle was associated with alcohol exposure in rats. The study indicated that alcohol may be involved in the skeletal muscle interstitial fibrosis in our model of alcohol-exposed rats through increased MMP-9 expression, and that this increased expression may aggravate the development of prolonged alcohol muscle injury.

RIMs (Rab3-interacting molecules) are synaptic proteins essential for neural transmission and plasticity. RIM1alpha has been implicated in membrane trafficking and regulation of secretory vesicle exocytosis in eukaryotic cells. Little information is as yet available on RIM3gamma. In the present study, we investigated the cellular expression, subcellular distribution, and possible functions of RIM3gamma in the rat CNS. Rim3gamma cDNA was subcloned and the protein expressed in vitro for the generation and purification of a rabbit anti-RIM3gamma polyclonal antibody. In situ hybridization histochemistry, immunohistochemistry, and immunoelectron microscopy were performed to map expression of the mRNA and protein in the rat CNS. Our results indicated widespread distribution of RIM3gamma in diverse CNS neuronal cell types. The mRNA was found mainly in the cell bodies, whereas the protein immunoreactivity was localized chiefly to neuronal dendrites and to the postsynaptic densities as visualized under the light and electron microscope. This postsynaptic placement of RIM3gamma is distinct from the presynaptic localization of RIM1alpha but may contribute to regulating synaptic transmission and plasticity. The identification of RIM3gamma as a postsynaptic protein has functional implications for CNS synapse functions.

Previous studies showed that nitricoxide synthase (NOS) and oxidative stress can induce skeletal muscle atrophy in the muscular dystrophy and inclusion-body myopathy. There is a correlation between NOS and oxidative stress. However, it is not clear, whether there are some changes of the NOS activity in prolonged alcoholic myopathy (PAM), and whether NOS activity has relation to amyotrophy of PAM. We established experimental alcoholic myopathy model of rats by prolonged alcohol intake. We found that there is a reduction in GSH-px (P0.05) and an increase of SOD (P0.05), MDA (P0.05) and iNOS (P0.05) in the plantaris of the experimental group by spectrophotometer. In the soleus of the experimental group, except for MDA showed an increase (P0.05), the other enzymes showed no obvious difference (P0.05). The immunohistochemistry results showed that there was obvious expression of iNOS in the cytoplasm of plantaris in the experimental group and there was no expression of iNOS in the control group. There was a decrease of nNOS expression on the membranes of the plantaris cells in the experimental group by immunofluorescence. Meanwhile, we found the expression of nNOS in some cytoplasm. Our results suggested that NOS might be an important factor during the development of PAM. We could infer that there are some disturbances with regard to output and scavenging of free radical in PAM. Alcohol can induce the oxidative stress reaction and further result in imbalance of the oxidant-antioxidant status in the organism.

CXC chemokine recepter-4 (CXCR4) and its ligand, stromal cell-derived factor-1alpha (SDF-1alpha) have been implicated in the organ-specific metastasis of several malignancies. Hca-F and its syngeneic cell line Hca-P are mouse hepatocarcinoma cell lines with high and low potential of lymphatic metastasis, respectively. Previous studies showed that the secretion of matrix metalloproteinases (MMPs) associated with the metastatic ability of Hca-F and Hca-P cell line depending on the lymph node environment. However, the mechanism of this process has remained unclear. This study investigated the roles of CXCR4 on Hca-F cell and SDF-1alpha of lymph node in lymphatic metastasis. The RT-PCR and Flow cytometry analysis results show that Hca-F cells express higher level CXCR4 mRNA and cell-surface CXCR4 protein, as compared with Hca-P cells. Treatment of recombinant SDF-1alpha proteins induced greater amount of calcium-flux in Hca-F cells than that in Hca-P cells, demonstrating higher functional CXCR4 expression on Hca-F cells than that on Hca-P cells. Furthermore, both the cell-free extratcs of lymph node and recombinant SDF-1alpha proteins induced secretions of active MMP-9 and MMP-2 from Hca-F cells in vitro. But those secretions were significantly reduced by blockade of cell surface CXCR4 with rabbit anti-mouse CXCR4 polyclonal antibody (pAb) and neutralization of SDF-1alpha in lymph node extracts with rabbit anti-mouse SDF-1alpha pAb as well. These results suggest that the CXCR4/SDF-1alpha system mediates active MMP-9 and MMP-2 secretion from Hca-F and Hca-P cells, which facilitates lymphogenous metastasis of those cells consequently.

In the process of screening cell-type-specific genes, we identified juxtanodin (JN), an oligodendroglial protein featuring a putative C-terminal actin-binding domain. At the cellular level, JN in the rat CNS colocalized with 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a cytoskeleton-related oligodendroglial protein. In the myelin sheath, JN was found mainly in the abaxon and the lateral few terminal loops. Its apposition to the myelinated axon, through the latter, defined an axonal subregion, herewith termed juxtanode, at the Ranvier node-paranode junction. During forebrain ontogenesis, JN expression paralleled that of MBPs but lagged behind CNPase. Juxtanodin transfection promoted arborization of cultured OLN-93 cells and augmented endogenous CNPase expression and transport to the process arbors of cultured primary oligodendrocyte precursors. These results reveal JN as a cytoskeleton-related oligodendroglial protein that delineates the juxtanode and might serve oligodendrocyte motility, differentiation, or myelin-axon signaling. Functionally, JN may be involved in CNS myelination and/or specialization of the node of Ranvier.