Simvastatin induces neuroprotection in 6-OHDA-lesioned PC12 via the PI3K/AKT/caspase 3 pathway and anti-inflammatory responses.

BACKGROUND: In addition to their original applications for lowering cholesterol, statins display multiple neuroprotective effects. Inflammatory reactions and the PI3K/AKT/caspase 3 pathway are strongly implicated in dopaminergic neuronal death in Parkinson's disease (PD). This study aims to investigate how simvastatin affects 6-hydroxydopamine-lesioned PC12 via regulating PI3K/AKT/caspase 3 and modulating inflammatory mediators. METHODS: 6-hydroxydopamine-treated PC12 cells were used to investigate the neuroprotection of simvastatin, its association with the PI3K/AKT/caspase 3 pathway, and antiinflammatory responses. Dopamine transporters (DAT) and tyrosine hydroxylase (TH) were examined in 6-hydroxydopamine-treated PC12 after simvastatin treatment. RESULTS: Simvastatin-mediated neuroprotection was associated with a robust reduction in the upregulation induced by 6-OHDA of inflammatory mediators including IL-6, COX2, and TNF-α. The downregulated DAT and TH levels in 6-OHDA-lesioned PC12 were restored after simvastatin treatment. Simvastatin reversed 6-OHDA-induced downregulation of PI3K/Akt phosphorylation and attenuated 6-OHDA-induced upregulation of caspase 3 in PC12. Furthermore, the PI3K inhibitor LY294002 pronouncedly abolished the simvastatin-mediated attenuation in caspase 3. CONCLUSIONS: Our results demonstrate that simvastatin provides robust neuroprotection against dopaminergic neurodegeneration, partially via antiinflammatory mechanisms and the PI3K/Akt/caspase 3 pathway. These findings contribute to a better understanding of the critical roles of simvastatin in treating PD and might elucidate the molecular mechanisms of simvastatin effects in PD.

[Bone marrow stromal cells transfected with ciliary neurotrophic factor gene ameliorates the symptoms and inflammation in C57BL/6 mice with experimental allergic encephalomyelitis].

OBJECTIVE: To investigate the anti-inflammatory effect of bone marrow stromal cells (MSCs) transfected with recombinant adenovirus-mediated ciliary neurotrophic factor (CNTF) gene in C57BL/6 mice with experimental allergic encephalomyelitis (EAE). METHODS: An adenovirus vector containing CNTF gene Ad-CNTF-IRES-GFP was constructed and transfected in the MSCs (MSC-CNTF). After examination of CNTF expression, the transfected cells were transplanted in C57BL/6 mice with MOG 35-55-induced EAE, which were monitored for the changes in the symptoms scores. The levels of tumor necrosis factor-alpha (TNF-alpha), inteferon-gamma (IFN-gamma), interleukin-12P35 (IL-12P35), and IL-10 in the peripheral blood of the mice were detected, and the number of MSC-CNTF cells in the spleen and spinal cord was counted. CD3+ T cell infiltration and TNF-alpha and IFN-gamma expressions in the lesions were also observed after the cell transplantation. RESULTS: CNTF gene transfection resulted in significantly increased CNTF expression in the MSCs. The mice receiving MSC-CNTF transplantation exhibited significantly improved symptoms with shortened disease course and lessened disease severity. The cell transplantation also resulted in significantly decreased peripheral blood TNF-alpha levels, ameliorated CD3+T cell infiltrations and lowered TNF-alpha expression in the lesions, while the levels of IFN-gamma underwent no significant changes. CONCLUSION: Transplantation of CNTF gene-transfected MSCs results in decreased peripheral blood TNF-alpha and IFN-gamma levels and reduced inflammatory cells, CD3-positive cells and TNF-alpha expression in the lesion of EAE, therefore providing better effect than MSCs in relieving the symptoms of EAE in mice.

Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) inhibits experimental autoimmune encephalomyelitis in dark Agouti (DA) rats.

Previous studies have shown that vascular endothelial growth factor (VEGF) expression is up-regulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a model for MS, and may exacerbate the disease. However, it remains unknown whether anti-VEGF modalities could serve as a potential treatment for such central nervous system (CNS) autoimmune diseases. We constructed a recombinant adenoviral vector carrying FLAG-tagged sFlt-1(1-3) (the first three extracellular domains of Flt-1, the hVEGF receptor-1). Intramuscular transfection of the recombinant adenoviral vector suppressed VEGF-induced inflammatory cell infiltration in matrigel plugs. When given intracerebrally to EAE rats, recombinant sFlt-1(1-3) adenoviral vector significantly reduced disease severity compared to untreated rats. sFlt-1(1-3) gene transfer blocked VEGF and greatly reduced the number of cells that express VEGF and ED1-positive cells in CNS in EAE rats. This study demonstrates that sFlt-1(1-3) gene transfer into the brain ameliorates the severity of EAE by inhibiting monocyte recruitment in the CNS of dark Agouti rats.

Translocation and neuroprotective properties of transactivator-of-transcription protein-transduction domain-neuroglobin fusion protein in primary cultured cortical neurons.

Ngb (neuroglobin) is a newly discovered hexaco-ordinate globin that is expressed in vertebrate brain and peripheral nervous systems. Expression of Ngb increases in response to oxygen deprivation and protects neurons from hypoxia in vitro and in vivo. However, the lack of its transduction ability into cells resulted in limited neuroprotection. To educe its neuroprotection under hypoxia, a cell-permeable Ngb fusion protein was generated. A rat brain Ngb gene was cloned and fused with a gene fragment encoding the nine-amino-acid TAT PTD (transactivator-of-transcription protein-transduction domain; RKKRRQRRR) of HIV-1 in a prokaryotic expression vector to generate a genetic in-frame N-terminal hexahistidine-tagged) TAT PTD-Ngb fusion protein. It was expressed in soluble form in Escherichia coli BL21(DE3)plysS and purified with Ni(2+)-affinity chromatography. The results showed that the purified fusion protein TAT PTD-Ngb can enter into the primary cultured cortical neurons in a dose-dependent manner when added exogenously to the culture media and can be detected in cells within 48 h. The cell viability under hypoxia was increased and apoptosis induced by hypoxia was decreased after TAT PTD-Ngb was transduced into cortical neurons. The results provide a clue for the research of Ngb and suggest that transduction of TAT PTD-Ngb may be one of the ways for the therapy of CNS (central nervous system) diseases, especially cerebrovascular diseases and neurodegenerative diseases.

Identification and characterization of the CD226 gene promoter.

CD226 is one of the main activating receptors on natural killer cells, and it can induce cytotoxicity to target cells through interaction with its ligands CD155 or CD112. CD226 is also involved in T cell differentiation, activation, and cytotoxicity. The expression of CD226 on natural killer cells and T cells can be regulated by cytokines and chemical stimuli; however, the mechanism of the regulation of the CD226 gene is still unknown. In this study, we have identified two promoters in the human CD226 gene named P1 and P2, which are located at -810 to -287 bp and +33 to +213 bp, respectively, and a negative regulation element between P1 and P2. Both P1 and P2 can be regulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and calcium ionophore (A23187). Bioinformatics analysis shows that, within this CD226 gene region, there are putative binding sites for transcription factors AP-1, Sp1, PEA3, and Ets-1. We have found that transcription factor activating protein-1 (AP-1) can up-regulate CD226 promoters P1 and P2 in human hepatocarcinoma cells, a hepatocarcinoma cell line with low expression of endogenous AP-1 and Ets-1. Interestingly, the transcription factor Ets-1 promotes AP-1-induced P2 activity but inhibits AP-1-induced P1 activity for which a 10-bp AP-1/Ets-1 composite site (CCTTCCTTCC) in P1 may be responsible.

[Detection of proinflammatory cytokine levels in sera from patients with Kaschin-Beck disease].

AIM: To explore the role of proinflammatory cytokines (TNF, IL-1beta and IL-6) in the pathogenesis of Kaschin-Beck disease (KBD). METHODS: The levels of serum TNF, IL-1beta and IL-6 from 62 KBD patients and 60 healthy persons were detected by double antibody sandwich ELISA. RESULTS: The levels of serum IL-1beta and IL-6 in KBD patients were (238.4+/-698.5) ng/L and (164.4+/-661.4) ng/L, respectively, but they were higher than those in healthy persons, being (74.5+/-130.0) ng/L and (52.2+/-154.6) ng/L, respectively. There were no significant differences between them (P>0.05). However, the level of serum TNF in KBD patients [(109.2+/-145.3) ng/L] was significantly higher than that in healthy persons [(40.9+/-89.7) ng/L] (P<0.01). Moreover, there was no correlation between serum TNF and IL-1beta levels (r=0.0387, P>0.05) and TNF and IL-6 in KBD patients (r=0.2135, P>0.05), but there was positive correlation between serum IL-1beta and IL-6 levels (r=0.346, P<0.01). CONCLUSION: The elevated proinflammatory cytokine levels in sera may relate to the pathogenesis of Kaschin-Beck disease.

[Preparation and characterization of monoclonal antibodies against human RANTES molecule].

AIM: To prepare monoclonal antibodies against human RANTES molecule and identify the expression of RANTES in rat small intestine after small bowel transplantation. METHODS: Murine mAbs were prepared by B lymphocyte hybridoma technique. The expression of RANTES in rat small intestine after small bowel transplantation was detected by immunohistochemistry. RESULTS: Four hybridoma cell lines secreting monoclonal antibodies to human RANTES, FMU-RANTES 1, FMU-RANTES 2, FMU-RANTES 3 and FMU-RANTES 4, were established. The titers of a scetic mAbs reached to 1 x 10(-6) and the Ig subclass of FMU-RANTES 1, FMU-RANTES 3 and FMU-RANTES 4 was IgG1(kappa) and that of FMU-RANTES 2 was IgG2b(kappa). Among these mAbs, FMU-RANTES 1, FMU-RANTES 2 and FMU-RANTES 3 could bind human RANTES protein in Western bolt. FMU-RANTES 1, FMU-RANTES 2 and FMU-RANTES 4 could be used in immunohistochemistry staining. Rat RANTES molecule could be detected in the cyto plasm of epithelial cells in rat small intestine after small bowel transplantation. CONCLUSION: Four mAbs against RANTES molecule were prepared, which can provide a useful tool in research on the structure and function of RANTES molecule. High expression of RANTES may be involved in the rejection of allogeneic graft.

[The inhibition of TNF-induced NF-kappaB nuclear translocation in ECV304 cells by mAbs against human TNF].

AIM: To identify the inhibition of TNF-induced NF-kappaB nuclear translocation by three anti-human TNF mAbs, D2, E6 and F6. METHODS: TNF solutions were pretreated with mAbs D2, E6 and F6 as well as control mAb at 37 degrees Celsius for 1 h, respectively, and then they were added to ECV304 cell cultures. After 1 hour, the cells were harvested and nuclear proteins were extracted. The NF-kappaB activity in nuclear extract was detected by electrophoretic mobility shift assay (EMSA). RESULTS: All of the three anti-TNF mAbs could inhibit TNF-induced NF-kappaB nuclear translocation in a dose-dependent manner. At the concentrations of 10 mg/L and 0.1 mg/L, the inhibition rates of mAb D2, E6 and F6 were 94.2% and 75.1%, 64.9% and 28.6%, 70.3% and 49.5% respectively, while the inhibition rate of control mAb was only 20.0% and 11.1%. CONCLUSION: mAbs D2, E6 and F6 can specifically inhibit TNF-induced NF-kappaB nuclear translocation, which lays the foundation for preparation of therapeutic chimeric anti-human TNF antibody for treatment of infectious and autoimmune diseases.

[Characterization of neutralizing activity of murine monoclonal antibodies for construction of anti-human TNF chimeric antibody].

AIM: To screen high quality murine monoclonal antibodies (mAb) for construction of anti-human TNF chimeric antibody. METHODS: 3 hybridoma cells D2, E6 and F6 were selected from one group of hybridoma cells secreting anti-human TNF mAbs and then ascitic fluids were prepared according to routine protocol. After salted out with saturated ammonium sulphate, the mAb (IgG2b) secreted by D2 cells was purified through protein A affinity chromatography column. The mAbs (IgG1) secreted by E6 and F6 cells were purified through QFF anion exchange chromatography column. The titers of 3 mAbs were detected by indirect ELISA before and after purification. Inhibition of TNF induced cell death of L929 cells and ICAM-1 upregulation on ECV304 cells were assayed as indexes of neutralizing activity of the mAbs. RESULTS: ELISA results showed that the titers of 3 mAbs D2, E6, and F6 before purification were 1x10(-6 ), 1x10(-7 ), and 1x10(-7 ), respectively, and 0.01, 0.002 and 0.002 mg/L after purification, respectively. 0.16 mg/L D2, 0.40 mg/L E6, or 0.50 mg/L F6 could protect 50% L929 cells from cell death induced by 2x10(4) U/L TNF, and the inhibition rates of ICAM-1 upregulation by 2x10(5) U/L TNF of 1.0 mg/L D2, E6 or F6 were 70.1%, 60.1%, and 60.1%, respectively. The inhibition rates still reached 60.1%,53.7% and 59.0%, respectively, when the concentrations of 3 mAbs D2, E6 and F6 decreased to 0.1 mg/L. CONCLUSION: 3 mAbs with high titers and neutralizing activities were obtained, which paves the way for construction of anti-human TNF chimeric antibody.