[Effect of acupuncture combined with repetitive transcranial magnetic stimulation on motor function and cerebral hemodynamics in children with spastic cerebral palsy with spleen-kidney deficiency].

OBJECTIVE: To investigate the clinical effect of acupuncture combined with repetitive transcranial magnetic stimulation in the treatment of children with spastic cerebral palsy with spleen-kidney deficiency, as well as its effect in improving cerebral hemodynamics. METHODS: A total of 220 children with spastic cerebral palsy were divided into observation group and control group using a random number table, with 110 children in each group. The children in the control group were given rehabilitation training and repetitive transcranial magnetic stimulation, and those in the observation group were given acupuncture in addition to the treatment in the control group. Acupuncture was performed at Zusanli (ST36), Xuanzhong (GB39), Sanyinjiao (SP6), Pishu (BL20), Shenshu (BL23), Qihai (CV6), Quchi (LI11), Neiguan (PC6), Hegu (LI4) and Tianshu (ST25) once every other day, three times a week for 3 consecutive months. The two groups were compared in terms of Gross Motor Function Measure (GMFM), Fine Motor Function Measure (FMFM), comprehensive function score for children with cerebral palsy, clinical outcome, and related cerebral hemodynamic parameters (mean blood flow velocity [Vm], systolic peak velocity [Vs], and resistance index [RI] of the cerebral artery). RESULTS: After treatment, both groups had significant increases in the scores of GMFM, FMFM and comprehensive function (cognitive function, speech function, motor ability, self-care, and social adaptability,P<0.01), and the observation group had significantly better improvements in the scores of GMFM (domains A, B and C), FMFM (domains B, C, D and E), and comprehensive function than those of the control group (P<0.01). The therapeutic effect of the observation group (93/110, 84.55%)was superior to that of the control group (80/110, 72.73%, P<0.05). The observation group had significantly higher Vs and Vm and a significantly lower RI than the control group (P<0.01). CONCLUSION: In the treatment of children with spasmodic cerebral palsy with spleen-kidney deficiency, acupuncture combined with repeated transcranial magnetic stimulation can significantly improve their motor function, comprehensive function, and clinical outcome, which may be associated with the regulation of cerebral hemodynamics.

Modification of TAK1 by O-linked N-acetylglucosamine facilitates TAK1 activation and promotes M1 macrophage polarization.

Macrophages play many different roles in tissue inflammation and immunity, and the plasticity of macrophage polarization is closely associated with acute inflammatory responses. O-GlcNAcylation is an important type of post-translational modification, which subtly modulates inflammation responses. Transforming growth factor (TGF)-ß-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as TGF-ß, tumor necrosis factor (TNF), and interleukin-1 (IL-1). It is here reported that TGFß-activated kinase (TAK1) is modified with N-acetylglucosamine (O-GlcNAc) on S427. Both IL-1 and osmotic stress, which are known as the TAK1-signaling inducers, significantly trigger the O-GlcNAcylation of TAK1 in macrophages. By overexpressing wild-type (WT) or S427A TAK1 mutant into macrophages, it was determined that O-GlcNAcylation of TAK1 on S427 is required for T187/S192 phosphorylation and full activation of TAK1 upon stimulation with IL-1α and NaCl. Aborting O-GlcNAcylation of TAK1 on S427 was found to inhibit the downstream JNK and nuclear factor-κB activation and reduce the final amount of cytokines produced in activated macrophages to a great extent. Results also showed that overexpression of the O-GlcNAcylation-deficient mutant of TAK1 promotes LPS-mediated apoptosis in macrophages. Importantly, TAK1 O-GlcNAcylation was found to promote M1 macrophage polarization in activated macrophages. Taken together, these data demonstrate that O-GlcNAcylation of TAK1 on S427 critically regulates the pro-inflammatory activation and M1 polarization of macrophages via modulation of the TAK1/JNK/NF-κB signaling pathway.

Upregulated Expression of Karyopherin α2 is Involved in Neuronal Apoptosis Following Intracerebral Hemorrhage in Adult Rats.

Karyopherin α2 (KPNA2) plays a central role in nucleocytoplasmic transport. It is involved in controlling the flow of genetic information and the modulation of diverse cellular activities. Here we explored the KPNA2's roles during the pathophysiological processes of intracerebral hemorrhage (ICH). An ICH rat model was built and evaluated according to behavioral testing. Using Western blot, immunohistochemistry, and immunofluorescence, significant upregulation of KPNA2 was found in neurons in brain areas surrounding the hematoma following ICH. Increasing KPNA2 level was found to be accompanied by the upregulation of active caspase-3, Bax, and decreased expression of Bcl-2. Besides, KPNA2 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. What's more, knocking down KPNA2 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, KPNA2 may play a role in promoting the brain secondary damage following ICH.

USP11, Deubiquitinating Enzyme, Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage.

Protein ubiquitination is a dynamic two-way process that can be reversed or regulated by deubiquitinating enzymes (DUB). USP11, located on the X chromosome, 6 is a member of USP subclass of the DUB family. Here, we demonstrate that USP11 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). From the results of Western blot, immunohistochemistry, and immunofluorescence, we obtained a significant up-regulation of USP11 in neurons adjacent to the hematoma following ICH. Increasing USP11 level was found to be accompanied by the up-regulation of active caspase-3, Fas receptor (Fas), Fas ligand (FasL), and active caspase-8. Besides, USP11 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. What is more, knocking down USP11 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, USP11 may play a role in promoting the brain secondary damage following ICH.

CRMP1 Interacted with Spy1 During the Collapse of Growth Cones Induced by Sema3A and Acted on Regeneration After Sciatic Nerve Crush.

CRMP1, a member of the collapsin response mediator protein family (CRMPs), was reported to regulate axon outgrowth in Sema3A signaling pathways via interactions with its co-receptor protein neuropilin-1 and plexin-As through the Fyn-cyclin-dependent kinase 5 (CDK5) cascade and the sequential phosphorylation of CRMP1 by lycogen synthase kinase-3ß (GSK-3ß). Using yeast two-hybrid, we identified a new molecule, Speedy A1 (Spy1), a member of the Speedy/RINGO family, with an interaction with CRMP1. Besides, for the first time, we observed the association of CRMP1 with actin. Based on this, we wondered the association of them and their function in Sema3A-induced growth cones collapse and regeneration process after SNC. During our study, we constructed overexpression plasmid and short hairpin RNA (shRNA) to question the relationship of CRMP1/Spy1 and CRMP1/actin. We observed the interactions of CRMP1/Spy1 and CRMP1/actin. Besides, we found that Spy1 could affect CRMP1 phosphorylation actived by CDK5 and that enhanced CRMP1 phosphorylation might disturb the combination of CRMP1 and actin, which would contribute to abnormal of Sema3A-induced growth cones collapse and finally lead to influent regeneration process after rat sciatic nerve crush. Through rat walk footprint test, we also observed the variance during regeneration progress, respectively. We speculated that CRMP1 interacted with Spy1 which would disturb the association of CRMP1 with actin and was involved in the collapse of growth cones induced by Sema3A and regeneration after sciatic nerve crush.

Up-regulation of VCAM1 Relates to Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats.

Vascular cell adhesion molecule 1 (VCAM1) is a member of the Immunoglobulin superfamily and encodes a cell surface sialoglycoprotein expressed in cytokine-activated endothelium. This type I membrane protein mediates leukocyte-endothelial cell adhesion, facilitates the downstream signaling, and may play a role in the development of artherosclerosis and rheumatoid arthritis. Accumulating evidence has demonstrated that VCAM1 exerts an anti-apoptotic effect in several tumor tissues such as ovarian cancer and breast cancer. Intracerebral hemorrhage (ICH) is the second most common subtype of stroke with high morbidity and mortality, which imposes a big burden on individuals and the whole society. These together prompted us to question whether VCAM1 has some association with neuron apoptosis during the pathological process of ICH. An ICH rat model was established and assessed by behavioral tests in order to explore the role of VCAM1 after ICH. Up-regulation of VCAM1 was observed in brain areas surrounding the hematoma following ICH by western blotting and immunohistochemistry. Immunofluorescence manifested VCAM1 was strikingly increased in neurons, but not in astrocytes and microglia. Furthermore, we detected that neuronal apoptosis marker active caspase-3 had co-localizations with VCAM1. At the same time, Bcl-2 was also co-localized with VCAM1. Taken together, our findings suggested that VCAM1 might be involved in the neuronal apoptosis and pathophysiology of ICH.

Upregulated expression of SSTR1 is involved in neuronal apoptosis and is coupled to the reduction of bcl-2 following intracerebral hemorrhage in adult rats.

Somatostatins are peptide hormones that regulate diverse cellular processes, such as neurotransmission, cell proliferation, apoptosis, and endocrine signaling as well as inhibiting the release of many hormones and other secretory proteins. SSTR1 is a member of the superfamily of somatostatin receptors possessing seven-transmembrane segments. Aberrant expression of SSTR1 has been implicated in several human diseases, including pseudotumor cerebri, and oncogenic osteomalacia. In this study, we investigated a potential role of SSTR1 in the regulation of neuronal apoptosis in the course of intracerebral hemorrhage (ICH). A rat ICH model in the caudate putamen was established and subjected to behavioral tests. Western blot and immunohistochemistry indicated a remarkable up-regulation of SSTR1 expression surrounding the hematoma after ICH. Double-labeled immunofluorescence showed that SSTR1 was mostly co-localized with neurons, and was rarely distributed in activated astrocytes and microglia. Additionally, SSTR1 co-localized with active-caspase-3 and bcl-2 around the hematoma. The expression of active-caspase-3 was parallel with that of SSTR1 in a time-dependent manner. In addition, SSTR1 knockdown specifically resulted in reduced neuronal apoptosis in PC12 cells. All our findings suggested that up-regulated SSTR1 contributed to neuronal apoptosis after ICH, which was accompanied with reduced expression of bcl-2.

FBP1 and p27kip1 expression after sciatic nerve injury: implications for Schwann cells proliferation and differentiation.

Far Upstream Element (FUSE) Binding Protein 1 (FBP1), first identified as a single-stranded DNA (ssDNA) binding protein that binds to the FUSE, could modulate c-myc mRNA levels and also has been shown to regulate tumor cell proliferation and replication of virus. Typically, FBP1 could active the translation of p27kip1 (p27) and participate in tumor growth. However, the expression and roles of FBP1 in peripheral system lesions and repair are still unknown. In our study, we found that FBP1 protein levels was relatively higher in the normal sciatic nerves, significantly decreased and reached a minimal level at Day 3, and then returned to the normal level at 4 weeks. Spatially, we observed that FBP1 had a major colocation in Schwann cells and FBP1 was connected with Ki-67 and Oct-6. In vitro, we detected the decreased level of FBP1 and p27 in the TNF-α-induced Schwann cells proliferation model, while increased expression in cAMP-induced Schwann cells differentiation system. Specially, FBP1-specific siRNA-transfected SCs did not show fine and longer morphological change after cAMP treatment and had a decreased motility compared with normal. At 3 days after cAMP treatment and SC/neuron co-cultures, p27 was transported to cytoplasm to form CDK4/6-p27 to participate in SCs differentiation. In conclusion, we speculated that FBP1 and p27 were involved in SCs proliferation and the following differentiation in the sciatic nerve after crush by transporting p27 from nucleus to cytoplasm.

LPS-stimulating astrocyte-conditioned medium causes neuronal apoptosis via increasing CDK11(p58) expression in PC12 cells through downregulating AKT pathway.

Activation of astrocytes in central nervous system inflammation leads to a disturbance of crosstalk between astrocytes and neurons, and that this may contribute to the death of neurons. CDK11(p58) is a member of the large family of p34cdc2-related kinases. It specifically expresses in G2/M phase of the cell cycle and is closely related to cell cycle arrest and apoptosis. Here, we show that astrocyte-conditioned medium stimulated by lipopolysaccharide upregulates CDK11(p58) expression and meanwhile causes neuronal apoptosis. CDK11(p58) knockdown in PC12 cells represses neuronal apoptosis. CDK11(p58) overexpression in PC12 cells promotes neuronal apoptosis. AKT signaling pathway is involved in CDK11(p58)-induced neuronal apoptosis process.

Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-κB activation.

The Notch signaling regulator Numblike (Numbl) is expressed in the brain, but little is known regarding its role in the pathophysiology of glial cells. In this paper, we report that Numbl expression was down-regulated in high-grade human glioma tissue samples and glioblastoma cell lines. To investigate the role of Numbl in glioma migration and invasion, we generated human glioma cell lines in which Numbl was either overexpressed or depleted. Overexpression of Numbl suppressed, while elimination of Numbl promoted, the migration and invasion of glioma cells. Numbl inhibited glioma migration and invasion by dampening NF-κB activity. Furthermore, Numbl interacted directly with tumor necrosis factor receptor-associated factor 5 (TRAF5), which signals upstream and is required for the activation of NF-κB, and committed it to proteasomal degradation by promoting K48-linked polyubiquitination of TRAF5. In conclusion, our data suggest that Numbl negative regulates glioma cell migration and invasion by abrogating TRAF5-induced activation of NF-κB.

The functional interaction between CDK11p58 and ß-1,4-galactosyltransferase I involved in astrocyte activation caused by lipopolysaccharide.

Glial cells are mediating the main activation of the central nervous system (CNS), being astrocytes the mayor glial cells in the brain. Glial activation may result beneficial since it could promote tissue repair and pathogen elimination. However, excessive glial activation mechanism can also have do harm to the tissue. ß-1,4-Galactosyltransferase I (ß-1,4-GalT-I) is a key inflammatory mediator that participates in the initiation and maintenance of inflammatory reaction in some diseases. Moreover, CDK11(p58) has been reported to be associated with ß-1,4-GalT-I. We have found that CDK11(p58) and ß-1,4-GalT-I are induced in lipopolysaccharide (LPS)-challenged rat primary astrocytes in a affinis dose- and time-dependent manner. CDK11(p58) regulates the expression of ß-1,4-GalT-I by interacting with it. After the knockdown of CDK11(p58) expression, the expression of ß-1,4-GalT-I decreases, and astrocyte activation downregulates. Inversely, the expression of ß-1,4-GalT-I increases, and astrocyte activation enhances due to the overexpression of CDK11(p58). Knockdown of ß-1,4-GalT-I reduces the activation potentiation caused by the overexpression of CDK11(p58), illustrating the function of CDK11(p58) to promote astrocyte activation depends on ß-1,4-GalT-I. The interaction between CDK11(p58) and ß-1,4-GalT-I to upregulate astrocyte activation is related to activating p38 and JNK pathways. These findings indicated that the functional interaction between CDK11(p58) and ß-1,4-GalT-I may play an important role during astrocyte activation after LPS administration.

Involvement of CLEC16A in activation of astrocytes after LPS treated.

CLEC16A, C-type lectin domain family 16, member A was recently found to be associated with inflation process in the autoimmune diseases. In this study, we elucidated the dynamic expression changes and localization of CLEC16A in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. CLEC16A expression was strongly induced in active astrocytes in inflamed cerebral cortex. In vitro studies indicated that the up-regulation of CLEC16A may be involved in the subsequent astrocyte activation following LPS challenge. And Knock-down of CLEC16A in cultured primary astrocytes by siRNA showed that CLEC16A was required for the activation of astrocytes induced by LPS. Collectively, these results suggested CLEC16A may be important in host defense in astrocyte-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammtion in CNS.

Changes of peroxisome proliferator-activated receptor-γ on crushed rat sciatic nerves and differentiated primary Schwann cells.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has been found to play an essential role in cell proliferation, but whether it was involved in Schwann cells differentiation has never been studied. We have found in sciatic nerve injury that expression of PPAR-γ decreases mainly in Schwann cells, and it was also increased in differentiated Schwann cells. Further, activated PPAR-γ by the endogenous ligand 15 d-PGJ(2) increased expressions of PPAR-γ level and Schwann cell differentiation, and this effect may be protected by its antagonist GDW9662. These results indicate that PPAR-γ could promote Schwann cell differentiation, which plays an important role in peripheral nerve injury and regeneration.

CDK11(p58) promotes rat astrocyte inflammatory response via activating p38 and JNK pathways induced by lipopolysaccharide.

In response to a variety of neural damages in the CNS, quiescent astrocytes become reactive astrocytes. Astrocytes are the major glial subtype and are important effectors that participate in the pathogenesis of numerous neural disorders, including trauma, stroke, aging, and developmental, genetic, idiopathic or acquired neurodegenerative diseases. CDK11(p58) (Cyclin-dependent kinases 11 protein 58/PITSLRE) is a p34cdc2-related protein kinase that plays an important role in normal cell cycle progression. In the process of LPS stimulus, the expression of CDK11(p58) in astrocytes was increased. Induced CDK11(p58) was parallel to astrocyte inflammatory response. Knockdown of CDK11(p58) by small-interfering RNAs (siRNAs) reduced the LPS-induced astrocyte inflammatory response, while overexpression CDK11(p58) enhanced the process. CDK11(p58) exerted its functions via activating p38 and JNK MAPK pathways. This study delineates that CDK11(p58) may be a significant regulatory factor for host defenses in central nervous system (CNS) inflammation.

Expressions of forkhead class box O 3a on crushed rat sciatic nerves and differentiated primary Schwann cells.

Forkhead box-containing protein, class O 3 a (FOXO3a), an Akt downstream target, plays an important role in peripheral nervous system. FOXO3a shares the ability to be inhibited and translocated from the nucleus on phosphorylation by proteins such as Akt/PKB in the PI3K signaling pathway. To elucidate the expression and possible function of FOXO3a in lesion and repair, we performed an acute sciatic nerve crush model and studied differential expressions of FOXO3a. We observed that expressions of FOXO3a in Schwann cells (SCs) of the peripheral nervous system and cAMP-induced differentiation were dynamically regulated. Western blot analysis showed FOXO3a level significantly decreased post injury. Moreover, Immunofluorescence double labeling suggested the changes were striking especially in SCs. In vitro, Western blot analysis showed that the expression of FOXO3a was decreased in cAMP-induced differentiated primary SCs. The FOXO3a siRNA-transfected SCs treated by cAMP promote differentiation of SCs through the PI3K/Akt pathway. The results indicate that FOXO3a plays an important role during differentiation of SCs.

[The role of PKC to TNF-alpha induced beta-1, 4-galactosyltransferase-I expression in endothelial cells].

AIM: To investigate the role of protein kinase C (PKC) to regulate the expression of beta-1, 4-galactosyltransferase-I(beta-1, 4-GalT-I)and the influence to cytoskeleton and adherence ability of human umbilical vein endothelial cells(HUVECs) when stimulated by tumor necrosis factor (TNF-alpha). METHODS: Cultured HUVECs were pretreated by various PKC inhibitors or PMA, an excitomotor of PKC respectively for 30 minutes, then stimulated by TNF-alpha for 4 hours, beta-1, 4-GalT-I expression were detected by RT-PCR and Western blot, expression of beta-1, 4-galactosylated carbohydrate chains and cytoskeleton were assayed by immunofluorescence, adherence ability of HUVECs was observed by endothelial-monocyte cell adherence test. RESULTS: Up-regulated expression of beta-1, 4-GalT-I and beta-1, 4-galactosylated carbohydrate chains in HUVECs stimulated by TNF-alpha were suppressed by PKC inhibitors and increased by PMA. F-actin and beta-1, 4-GalT-I were partly co-localized in HUVECs, PKC inhibitor inhibited the effect of TNF-alpha on the distribution of f-actin and beta-1, 4-GalT-I. Adherence ability of HUVECs enhanced by LPS was significantly suppressed by PKC inhibitor. CONCLUSION: PKC signal transduction pathway may participate in regulating beta-1, 4-GalT-I expression in endothelial cells (EC) stimulated by TNF-alpha, furthermore, polytypes of PKC may participate in this regulating process; PKC might regulate cytoskeleton reorganization and adherence ability of EC through beta-1, 4-GalT-I during inflammation.

SSeCKS promote beta-amyloid-induced PC12 cells neurotoxicity by up-regulating tau phosphorylation in Alzheimer's disease.

In Alzheimer's disease, beta-amyloid peptide (Abeta) could induce tau hyperphosphorylation which is the major cause of neuron apoptosis. However, the underlying mechanisms in the process remain unclear. In this study, Abeta-induced apoptosis and tau phosphorylation were investigated in differentiated PC12 cells. This Abeta-induced tau phosphorylation paralleled with the increase of expression and phosphorylation of Src-suppressed protein kinase C substrate (SSeCKS). By knocking down the expression of SSeCKS, Abeta-induced apoptosis and tau hyperphosphorylation in PC12 cells were partially rescued, and were increased further due to the overexpression of SSeCKS in PC12 cells. Also, the cell apoptosis and tau hyperphosphorylation were strongly decreased when the cells were pretreated with the protein kinase C inhibitor, Gö6983. In addition, Abeta-induced tau phosphorylation was also partially decreased due to the overexpression of SSeCKS in PC12cells. In summary, our data indicate that SSeCKS may play a critical role in Abeta-induced PC12 cells apoptosis through its phosphorylation.

Expression of beta-1,4-galactosyltransferase-I affects cellular adhesion in human peripheral blood CD4+ T cells.

beta-1,4-galactosyltransferase-I (beta-1,4-GalT-I) has two isoforms that differ only in the length of their cytoplasmic domains. In this study, we found that both the long and short isoforms of beta-1,4-GalT-I were expressed in human CD4(+) T lymphocytes, and localized in the cytoplasm and on the plasma membrane. The expression level of beta-1,4-GalT-I was increased in CD4(+) T cells after stimulation with interleukin (IL)-2, and was further increased after stimulation with IL-2+IL-12, but decreased after stimulation with IL-2+IL-4 when compared to stimulation with IL-2 alone. We also demonstrated that the cellular adhesion of CD4(+) T cells was significantly increased upon cytokine stimulation, and was inhibited by alpha-lactalbumin, indicating that the increase in adhesion was positively correlated with the expression and activity of long beta-1,4-GalT-I. Collectively, the data suggest that beta-1,4-GalT-I plays a role in the cellular adhesion of CD4(+) T cells.