Gossypetin targets the liver-brain axis to alleviate pre-existing liver fibrosis and hippocampal neuroinflammation in mice.

Liver fibrosis occurs in response to chronic damage and inflammation to the liver. Leaving untreated, it can lead to decreased liver function and can eventually progress to cirrhosis, a more advanced and irreversible state of liver damage. Clinical investigations showed that chronic liver disease associated with neurological symptoms including anxiety, depression, and cognitive decline. However, few therapeutic options are available for treating liver and related brain pathologies simultaneously. In this study, we aim to find therapeutic candidates that target the liver-brain axis. Gossypetin, a flavonoid from sedum, shows promising capability in treating liver and brain pathologies in CCl4-induced mouse model. Short term of gossypetin administration is sufficient to ameliorate impaired liver function and pre-existing liver fibrosis, suppress MKK3/6-p38 MAPK and p53 activation, and abolish the activation of hepatic stellate cells and Kupffer cells. Although we observe no neuronal loss in the brain of mice with liver fibrosis, we do observe astrogliosis and microglial activation in certain brain regions, especially the hippocampus. Brief gossypetin administration also shows potential in alleviating neuroinflammation in these regions. These results suggest that gossypetin can target the liver-brain axis and be a promising candidate for treating chronic liver fibrosis patients with neurological symptoms.

Curcumin inhibits propofol-induced autophagy of MN9D cells via Akt/mTOR/p70S6K signaling pathway.

The rapid rise in propofol dependency and abuse has highlighted limited resources for addressing substance abuse-related cognitive impairment, prompting the development of novel therapies. Dysregulated autophagy flow accelerates neuronal cell death, and interventions countering this dysregulation offer an appealing strategy for neuronal protection. Curcumin, a potent natural polyphenol derived from turmeric rhizomes, is renowned for its robust antineurotoxic properties and enhanced cognitive function. Utilizing CCK-8 and Ki67 fluorescent staining, our study revealed that curcumin treatment increased cell viability and proliferative potential in MN9D cells exposed to propofol-induced neurotoxicity. Furthermore, enzyme-linked immunosorbent assay and western blot analysis demonstrated the partial restoration of dopamine synthesis, secretion levels, and TH expression in damaged MN9D cells treated with curcumin. Scanning electrode microscope images displayed reduced autolysosomes and phagosomes in curcumin-treated cells compared to the propofol group. Immunoblotting revealed that curcumin mitigated the degradation of LC3I to LC3II and p62 induced by propofol stimulation, with green fluorescence expression of LC3 postcurcumin treatment resembling that following autophagy inhibitor HCQ treatment, indicating that modulating autophagy flow can alleviate propofol's toxic effects. Moreover, curcumin treatment upregulated the Akt/mTOR/p70S6K signaling pathway, suggesting that curcumin potentially curtails autophagy dysregulation in nerve cells by activating Akt/mTOR/p70S6K. In conclusion, our findings suggest that curcumin can ameliorate propofol abuse-induced neurotoxicity, partially through autophagy regulation and Akt/mTOR/p70S6K signaling activation.

Mcm5 Represses Endodermal Migration through Cxcr4a-itgb1b Cascade Instead of Cell Cycle Control.

Minichromosome maintenance protein 5 (MCM5) is a critical cell cycle regulator; its role in DNA replication is well known, but whether it is involved in the regulation of organogenesis in a cell cycle-independent way, is far from clear. In this study, we found that a loss of mcm5 function resulted in a mildly smaller liver, but that mcm5 overexpression led to liver bifida. Further, the data showed that mcm5 overexpression delayed endodermal migration in the ventral-dorsal axis and induced the liver bifida. Cell cycle analysis showed that a loss of mcm5 function, but not overexpression, resulted in cell cycle delay and increased cell apoptosis during gastrulation, implying that liver bifida was not the result of a cell cycle defect. In terms of its mechanism, our data proves that mcm5 represses the expression of cxcr4a, which sequentially causes a decrease in the expression of itgb1b during gastrulation. The downregulation of the cxcr4a-itgb1b cascade leads to an endodermal migration delay during gastrulation, as well as to the subsequent liver bifida during liver morphogenesis. In conclusion, our results suggest that in a cell cycle-independent way, mcm5 works as a gene expression regulator, either partially and directly, or indirectly repressing the expression of cxcr4a and the downstream gene itgb1b, to coordinate endodermal migration during gastrulation and liver location during liver organogenesis.

Zebrafish Minichromosome Maintenance Protein 5 Gene Regulates the Development and Migration of Facial Motor Neurons via Fibroblast Growth Factor Signaling.

Minichromosome maintenance protein 5 (MCM5), a member of the microchromosomal maintenance protein family, plays an important role in the initiation and extension of DNA replication. However, its role in neural development in zebrafish remains unclear. Here, we used morpholino (MO) and CRISPR/Cas9 to knock down mcm5 and investigated the developmental features of facial motor neurons (FMNs) in the hindbrain of zebrafish. We found that knockdown of mcm5 using mcm5 MO resulted in a small head, small eyes, and a blurred midbrain-hindbrain boundary, while MO injection of mcm5 led to decrease in FMNs and their migration disorder. However, the mutant of mcm5 only resulted in the migration defect of FMNs rather than quantity change. We further investigated the underlying mechanism of mcm5 in the development of hindbrain using in situ hybridization (ISH) and fgfr1a mRNA co-injected with mcm5 MO. Results from ISH showed that the fibroblast growth factor (FGF) signaling pathway was changed when the MCM5 function was lost, with the decrease in fgfr1a and the increase in fgf8, while that of pea3 had opposite trend. FMN development defects were rescued by fgfr1a mRNA co-injected with mcm5 MO. Our results demonstrated that FGF signaling pathway is required for FMN development in zebrafish. Specifically, mcm5 regulates FMN development during zebrafish growing.

Toxicological effects of propofol abuse on the dopaminergic neurons in ventral tegmental area and corpus striatum and its potential mechanisms.

This study was aimed at examining propofol- (a known anesthetic) induced emotion-related behavioral disorders in mice, and exploring the possible molecular mechanisms. A total of 60 mice were divided into two groups: control and propofol group. Mice were injected with propofol (150 mg/kg, ip) at 8:00 a.m. (once a day, lasting for 30 days). During the 30 days, loss of righting reflex (LORR) and return of righting reflex (RORR) of mice were recorded every day. At the 1st (T1) and 30th (T2) day of drug discontinuance (T2), 15 mice of each group were selected to perform the open field test; then the mice underwent perfusion fixation, and the midbrain and corpus striatum were separated for immunofluorescence assay with anti-tyrosine hydroxylase (Th) and anti- dopamine transporter (DAT) antibodies. Results showed that after propofol injection, LORR and RORR increased and decreased, respectively. Long-term use of propofol resulted in decreased activities of mice (activity trajectory, line crossing, rearing time, scratching times and defecating frequency). Immunofluorescence assay showed long-term use of propofol induced decrease of Th and DAT. Collectively, our present work suggested long-term abuse of propofol induces neuropsychiatric function impairments, and the possible mechanisms are related to dopamine dyssynthesis via down-regulating tyrosine hydroxylase and dopamine transporter.

Primary hyaline vascular Castleman disease of the kidney: case report and literature review.

BACKGROUND: Castleman's disease (CD) is an uncommon type of benign proliferation of the lymphoid tissue, characterized by local or systemic lymphadenopathy that most frequently appears in the mediastinum; involvement of the kidney is uncommon, and proliferation originating from the kidney is extremely rare. Herein, we report a rare case of hyaline vascular Castleman's disease (HV-CD) in a 56-year-old male patient and discuss its morphological characteristics and differential diagnoses including mantle cell lymphoma (MCL), follicular lymphoma (FL), and nodal marginal zone lymphoma (NMZL). CASE PRESENTATION: A right upper-middle renal mass was detected after physical examination in a 56-year-old man without any clinical symptoms and a previous partial resection of the right kidney. Microscopically, the lymphoid follicles were increased in number and had expanded mantle zones and atrophic germinal centers. Vascular proliferation and hyalinization in the interfollicular zones were observed. Immunohistochemical staining showed CD20-positive cells in the mantle zones; CD21 and CD35 were expressed in the dendritic cells, CD3 was positive in a small number of T cells, and CD38 and CD138 were positive in the plasma cells. Additionally, Ki-67 expression was positive in the follicle centers. In contrast, staining for Bcl-2 in the germinal centers and cyclin D1 were negative. The immunohistochemical analysis combined with the morphological results supported the diagnosis of HV-CD. The patient recovered well after surgery. CONCLUSIONS: Primarily renal HV-CD without lymph node hyperplasia or clinical symptoms is extraordinarily rare and different from the multicentric-type CD (MCD) with kidney involvement. Therefore, it is extremely important to improve the awareness of this diagnosis. Attention should be paid to the difference between HV-CD and common lymph node reactive hyperplasia, MCL, FL, NMZL, and so on. To avoid misdiagnosis as a renal malignant tumor requiring radical resection, distinguishing these diseases is crucial.

Aplnra/b Sequentially Regulate Organ Left-Right Patterning via Distinct Mechanisms.

The G protein-coupled receptor APJ/Aplnr has been widely reported to be involved in heart and vascular development and disease, but whether it contributes to organ left-right patterning is largely unknown. Here, we show that in zebrafish, aplnra/b coordinates organ LR patterning in an apela/apln ligand-dependent manner using distinct mechanisms at different stages. During gastrulation and early somitogenesis, aplnra/b loss of function results in heart and liver LR asymmetry defects, accompanied by disturbed KV/cilia morphogenesis and disrupted left-sided Nodal/spaw expression in the LPM. In this process, only aplnra loss of function results in KV/cilia morphogenesis defect. In addition, only apela works as the early endogenous ligand to regulate KV morphogenesis, which then contributes to left-sided Nodal/spaw expression and subsequent organ LR patterning. The aplnra-apela cascade regulates KV morphogenesis by enhancing the expression of foxj1a, but not fgf8 or dnh9, during KV development. At the late somite stage, both aplnra and aplnrb contribute to the expression of lft1 in the trunk midline but do not regulate KV formation, and this role is possibly mediated by both endogenous ligands, apela and apln. In conclusion, our study is the first to identify a role for aplnra/b and their endogenous ligands apela/apln in LR patterning, and it clarifies the distinct roles of aplnra-apela and aplnra/b-apela/apln in orchestrating organ LR patterning.

[Effect of Human Adipose Mesenchymal Stem Cells on Phenotype Polarization of Mice Microglia via TLR3/TRIF Signal Pathway].

OBJECTIVE: To explore the effect and mechanism of human adipose-derived mesenchymal stem cells (hADMSCs) on phenotypic polarization of microglia. METHODS: BV-2 microglia of C57/BL6 mice were co-cultured with hADMSCs+lipopolysaccharide (LPS), or cultured with LPS alone. Cell morphology was observed under an inverted microscope. The effect of hADMSCs on microglial proliferation was evaluated by CCK-8 assay. The impact of hADMSCs on microglia M1/M2 phenotype markers were detected using quantitative real-time PCR (RT-qPCR). The affect of hADMSCs on the proteins expression levels of Toll-like receptor 4 (TLR4)-TIR domain containing adaptor protein inducing interferon ß (TRIF) signaling pathway in BV-2 microglia was detected by using Western blot analysis. RESULTS: As compared with the LPS treatment, hADMSCs treatment had no obvious effect on microglia morphology, whereas showed significant inhibition on microglial proliferation activity (P<0.05). Simultaneously, hADMSCs treatment reduced expression of microglia M1 phenotype markers (P<0.05), and increased microglia M1 phenotype markers in gene levels (P<0.05). At the same time, protein expression levels of TRIF, TLR4, phosphorylated interferon regulatory factor 3 (P-IRF3) and interferon regulatory factor 3 (IRF3) in BV-2 microglia were decreased after hADMSCs treatment. CONCLUSION: hADMSCs can blockade the LPS-induced pro-inflammatory microglia M1 phenotype, whereas induces protective microglial M2 phenotype, which may be related to inhibition of the TLR4-TRIF signaling pathway.

Combined use of bFGF/EGF and all-trans-retinoic acid cooperatively promotes neuronal differentiation and neurite outgrowth in neural stem cells.

Neural stem cells (NSCs) as sources of new neurons in brain injuries or diseases are required to not only elicit neurons for neuronal repair, but also to enhance neurite outgrowth for neuronal network reestablishment. Various trophic or chemotropic factors have been shown to cooperatively improve NSC neurogenesis. However, effects of combined treatment of all-trans-retinoic acid (RA) with GF (Basic fibroblast growth factor and epidermal growth factor, bFGF/EGF) on neurogenesis of NSCs are poorly understood. To address this question, NSCs were isolated from the forebrains of embryonic mice, and treated with GF and RA either alone or in combination for differentiation in vitro. Neurons and astrocytes differentiated from NSCs were stained for MAP2 and GFAP separately by immunofluorescence. The results indicated that GF displayed superior efficacy in promoting neuronal differentiation, and RA showed better efficacy in advancing neurite outgrowth by increasing both neurite length and number. In addition, higher differentiation efficiency of neurons to astrocytes in RA or GF, or both acted at the early stage. However, more importantly, compared with RA alone, GF and RA in combination enhanced neuronal differentiation. Moreover, the combined use of GF and RA increased the length and number of neurites compared with GF, as well as the relative expression level of Smurf1. In addition, astrocytes induced by GF, RA, or both exhibited a radial glia-like morphology with long processes differing from serum effects, which might in part attribute to the total numbers of neurons. These findings for the first time unveil the roles of combined use of GF and RA on the neurogenesis of NSCs, suggesting that the use of this combination could be a comprehensive strategy for the functional repair of the nervous system through promoting neuronal differentiation, and advancing neurite outgrowth.

CD4+ αß T cell infiltration into the leptomeninges of lumbar dorsal roots contributes to the transition from acute to chronic mechanical allodynia after adult rat tibial nerve injuries.

BACKGROUND: Antigen-specific and MHCII-restricted CD4+ αß T cells have been shown or suggested to play an important role in the transition from acute to chronic mechanical allodynia after peripheral nerve injuries. However, it is still largely unknown where these T cells infiltrate along the somatosensory pathways transmitting mechanical allodynia to initiate the development of chronic mechanical allodynia after nerve injuries. Therefore, the purpose of this study was to ascertain the definite neuroimmune interface for these T cells to initiate the development of chronic mechanical allodynia after peripheral nerve injuries. METHODS: First, we utilized both chromogenic and fluorescent immunohistochemistry (IHC) to map αß T cells along the somatosensory pathways for the transmission of mechanical allodynia after modified spared nerve injuries (mSNIs), i.e., tibial nerve injuries, in adult male Sprague-Dawley rats. We further characterized the molecular identity of these αß T cells selectively infiltrating into the leptomeninges of L4 dorsal roots (DRs). Second, we identified the specific origins in lumbar lymph nodes (LLNs) for CD4+ αß T cells selectively present in the leptomeninges of L4 DRs by two experiments: (1) chromogenic IHC in these lymph nodes for CD4+ αß T cell responses after mSNIs and (2) fluorescent IHC for temporal dynamics of CD4+ αß T cell infiltration into the L4 DR leptomeninges after mSNIs in prior lymphadenectomized or sham-operated animals to LLNs. Finally, following mSNIs, we evaluated the effects of region-specific targeting of these T cells through prior lymphadenectomy to LLNs and chronic intrathecal application of the suppressive anti-αßTCR antibodies on the development of mechanical allodynia by von Frey hair test and spinal glial or neuronal activation by fluorescent IHC. RESULTS: Our results showed that during the sub-acute phase after mSNIs, αß T cells selectively infiltrate into the leptomeninges of the lumbar DRs along the somatosensory pathways responsible for transmitting mechanical allodynia. Almost all these αß T cells are CD4 positive. Moreover, the temporal dynamics of CD4+ αß T cell infiltration into the lumbar DR leptomeninges are specifically determined by LLNs after mSNIs. Prior lymphadenectomy to LLNs specifically reduces the development of mSNI-induced chronic mechanical allodynia. More importantly, intrathecal application of the suppressive anti-αßTCR antibodies reduces the development of mSNI-induced chronic mechanical allodynia. In addition, prior lymphadenectomy to LLNs attenuates mSNI-induced spinal activation of glial cells and PKCγ+ excitatory interneurons. CONCLUSIONS: The noteworthy results here provide the first evidence that CD4+ αß T cells selectively infiltrate into the DR leptomeninges of the somatosensory pathways transmitting mechanical allodynia and contribute to the transition from acute to chronic mechanical allodynia after peripheral nerve injuries.

Differentiation of rat adipose-derived mesenchymal stem cells into corneal-like epithelial cells driven by PAX6.

Corneal integrity, transparency and vision acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by corneal limbal stem cells (LSCs). Deficiency of CECs and/or LSCs is associated with numerous ocular diseases. Paired box (PAX)6 is an eye development-associated transcription factor that is necessary for cell fate determination and differentiation of LSCs and CECs. In the present study, the PAX6 gene was introduced into adipose-derived rat mesenchymal stem cells (ADMSCs) to investigate whether PAX6-transfected cells were able to transdifferentiate into corneal-like epithelial cells and to further verify whether the cells were suitable as a cell source for corneal transplantation. The ADMSCs were isolated from the bilateral inguinal region of healthy Sprague Dawley rats. The characteristics of ADMSCs were identified using flow cytometric analysis. After subculture, ADMSCs underwent transfection with recombinant plasmid containing either PAX6-enhanced green fluorescent protein (EGFP) complementary (c)DNA or EGFP cDNA (blank plasmid group), followed by selection with G418 and determination of the transfection efficiency. Subsequently, the morphology of the ADMSCs and the expression profiles of corneal-specific markers CK3/12 and epithelial-specific adhesion protein were determined. E-cadherin was detected using immunofluorescence staining and western blot analysis at 21 days following transfection. An MTT cell proliferation and a colony formation assay were performed to assess the proliferative activity and clonogenicity of PAX6-transfected ADMSCs. Finally, the PAX6-expressing ADMSCs were transplanted onto the cornea of a rabbits with limbal stem cell deficiency (LSCD). At 21 days after transfection, the ADMSCs with PAX6 transfection exhibited a characteristic flagstone-like appearance with assembled corneal-like epithelial cells, and concomitant prominent expression of the corneal-specific markers cytokeratin 3/12 and E-cadherin. Furthermore, the proliferation and colony formation ability of PAX6-overexpressing ADMSCs was significantly retarded. The transplantation experiment indicated that PAX6-reprogramed ADMSCs attached to and replenished the damaged cornea via formation of stratified corneal epithelium. Taken together, these results suggested that conversion of ADMSCs into corneal-like epithelium may be driven by PAX6 transfection, which makes ADMSCs a promising cell candidate for the treatment of LSCD.

Co-administration of biocompatible self-assembled polylactic acid-hyaluronic acid block copolymer nanoparticles with tumor-penetrating peptide-iRGD for metastatic breast cancer therapy.

The safe and efficient targeted delivery of chemotherapeutic drugs has remained a challenge in metastatic breast cancer therapy. Herein, we report a rational drug delivery strategy of co-administering tumor-penetrating peptide-iRGD with self-assembled amphiphilic block copolymer nanoparticles (HA-PLA) to inhibit tumor growth and lung metastasis in 4T1 breast cancer xenograft bearing mice through increasing drug accumulation in the tumors, inducing receptor-mediated tumor cell targeting without causing severe side effects. In vitro, HA-PLA displayed sustained and pH-sensitive release behavior. The cellular uptake of HA-PLA on high CD44-expressing 4T1 cells was significantly higher than the endocytosis on low CD44-expressing L929 fibroblast cells. In vivo, HA-PLA significantly extended the blood circulation time of DOX, displayed no "accelerated blood clearance (ABC) phenomenon" after repeated injection and decreased the side effects of DOX. When combined with iRGD, the drug distribution and penetration of HA-PLA in tumors were remarkably increased, resulting in better antitumor efficacy and the longest whole survival. In particular, the co-administration of iRGD with HA-PLA greatly increased drug distribution in the lung, which contributed to the effective inhibition of the lung metastasis of breast cancer. Therefore, co-administering iRGD with HA-PLA is a promising approach for metastatic breast cancer therapy.

Sex differences of steroid receptor coactivator-1 expression after spinal cord injury in mice.

OBJECTIVE: The neural functional recovery of female is often better than that of male after spinal cord injury (SCI). Evidences show that estrogen can attenuate inflammation and promote the neural survival and regeneration after SCI. SRC-1 is an essential initiation factor for the estrogen-regulated target gene transcription and plays a key role in regulating estrogen activity. However, it is not known whether and how SRC-1 mediates the neural regeneration promoted by estrogen after SCI. Study of the sex differences and changes of SRC-1 expression after SCI will be helpful to understand the above questions. METHODS: In this study, the sex differences of expressions of SRC-1 and cytoskeleton-associated protein Profilin-1 in normal and SCI mice were detected by immunohistochemistry at 1-, 3-, and 7 days after injury, respectively. RESULTS: Although the SRC-1 expression in female mice was lower than that in males under normal conditions, its expression in females was more dominant after SCI. The expression of Profilin-1 in both sexes increased first, and then decreased at 3 days after injury. However, there was a second increase in females at 7 days after injury. CONCLUSION: Our study suggests that the more SRC-1 expression in females after SCI may play a role in improving the efficiency of estrogen function and thus, promote regeneration better. SRC-1 may also participate in the regulation of Profilin-1 in spinal cord, which is important in the assembly and extension of the axonal cytoskeleton during regeneration after SCI.

TIR-Domain-Containing Adapter-Inducing Interferon-ß (TRIF) Is Essential for MPTP-Induced Dopaminergic Neuroprotection via Microglial Cell M1/M2 Modulation.

Dynamic changes of two phenotypes of microglia, M1 and M2, are critically associated with the neurodegeneration of Parkinson's disease. However, the regulation of the M1/M2 paradigm is still unclear. In the MPTP induced neurodegeneration model, we examined the concentration of dopamine (DA) related metabolites and the survival of tyrosine hydroxylase (TH) positive cells in WT and Trif-/- mice. In in vitro experiments, MN9D cells were co-cultured with BV2 cells to mimic the animal experiments. Inhibition of TRIF aggravated TH+ cell loss, and DA-related metabolites decreased. TRIF inhibition was able to interrupt the microglial M1/M2 dynamic transformation. More BV2 cells were activated and migrated across the membrane of transwell plates by siTRIF treatment. Also, TRIF interruption inhibits the transformation of BV2 cells from the M1 to M2 phenotype which played a beneficial role in neuronal degenerative processes, and increased MN9D apoptosis. Moreover, MPP+ treatment decreases the (DAT) dopamine transporter and TH synthesis by MN9D. Taken together, the current results suggest that TRIF plays a key switch function in contributing to the microglial M1/M2 phenotype dynamic transformation. The interruption of TRIF may decrease the survival of MN9D cells as well as DAT and TH protein production. The current study sheds some light on the PD mechanism research by innate inflammation regulation.

Spatial and Temporal Distribution of Dopaminergic Neurons during Development in Zebrafish.

As one of the model organisms of Parkinson's disease (PD) research, the zebrafish has its advantages, such as the 87% homology with human genome and transparent embryos which make it possible to observe the development of dopaminergic neurons in real time. However, there is no midbrain dopaminergic system in zebrafish when compared with mammals, and the location and projection of the dopaminergic neurons are seldom reported. In this study, Vmat2:GFP transgenic zebrafish was used to observe the development and distribution of dopaminergic neurons in real time. We found that diencephalons (DC) 2 and DC4 neuronal populations were detected at 24 h post fertilization (hpf). All DC neuronal populations as well as those in locus coeruleus (LC), raphe nuclei (Ra) and telencephalon were detected at 48 hpf. Axons were detected at 72 hpf. At 96 hpf, all the neuronal populations were detected. For the first time we reported axons from the posterior tubercle (PT) of ventral DC projected to subpallium in vivo. However, when compared with results from whole mount tyrosine hydroxylase (TH) immunofluorescence staining in wild type (WT) zebrafish, we found that DC2 and DC4 neuronal populations were mainly dopaminergic, while DC1, DC3, DC5 and DC6 might not. Neurons in pretectum (Pr) and telencephalon were mainly dopaminergic, while neurons in LC and Ra might be noradrenergic. Our study makes some corrections and modifications on the development, localization and distribution of zebrafish dopaminergic neurons, and provides some experimental evidences for the construction of the zebrafish PD model.

ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration.

Neurolytic celiac plexus block enhances skeletal muscle insulin signaling and attenuates insulin resistance in GK rats.

Non-insulin-dependent diabetes mellitus (NIDDM) is associated with chronic inflammatory activity and disrupted insulin signaling, leading to insulin resistance (IR). The present study investigated the benefits of neurolytic celiac plexus block (NCPB) on IR in a rat NIDDM model. Goto-Kakizaki rats fed a high-fat, high-glucose diet to induce signs of NIDDM were randomly divided into NCPB and control groups; these received daily bilateral 0.5% lidocaine or 0.9% saline injections into the celiac plexus, respectively. Following 14 and 28 daily injections, rats were subject to oral glucose tolerance tests (OGTTs) or sacrificed for the analysis of serum free fatty acids (FFAs), serum inflammatory cytokines and skeletal muscle insulin signaling. Compared with controls, rats in the NCPB group demonstrated significantly (P<0.05) lower baseline, 60-min and 120-min OGTT values, lower 120-min serum insulin, lower IR [higher insulin sensitivity index (ISI1) and lower ISI2) and lower serum FFAs, tumor necrosis factor-α, interleukin (IL)-1ß and IL-6. Conversely, NCPB rats exhibited higher basal and insulin-stimulated skeletal muscle glucose uptake and higher skeletal muscle insulin receptor substrate-1 (IRS-1) and glucose transporter type 4 expression. There were no differences between the groups in insulin receptor ß (Rß) or Akt expression; however Rß-Y1162/Y1163 and Akt-S473 phosphorylation levels were higher and IRS-1-S307 phosphorylation were lower in NCPB rats than in the controls. These results indicate that NCPB improved insulin signaling and reduced IR, possibly by inhibiting inflammatory cytokine release.

Distinct mechanisms determine organ left-right asymmetry patterning in an uncoupled way.

Disruption of Nodal in the lateral plate mesoderm (LPM) usually leads to left-right (LR) patterning defects in multiple organs. However, whether the LR patterning of organs is always regulated in a coupled way has largely not yet been elucidated. In addition, whether other crucial regulators exist in the LPM that coordinate with Nodal in regulating organ LR patterning is also undetermined. In this paper, after briefly summarizing the common process of LR patterning, the most puzzling question regarding the initiation of asymmetry is considered and the divergent mechanisms underlying the uncoupled LR patterning in different organs are discussed. On the basis of cases in which different organ LR patterning is determined in an uncoupled way via an independent mechanism or at a different time, we propose that there are other critical factors in the LPM that coordinate with Nodal to regulate heart LR asymmetry patterning during early LR patterning.

Small Nogo-66-binding peptide promotes neurite outgrowth through RhoA inhibition after spinal cord injury.

Abortive regeneration in the adult mammalian central nervous system (CNS) is partially mediated through CNS myelin proteins, among which Nogo-A plays an important role. Nogo-66, which is located at the C-terminus of Nogo-A, inhibits axonal regrowth through the Nogo-66/NgR signalling pathway. In this study, two small peptides were tested in a neurite outgrowth assay and spinal cord injury (SCI) model to examine the effects of these molecules on the inhibition of Nogo-66/NgR signalling. PepIV was selected from a phage display peptide library as a Nogo-66 binding molecule. And PepII was synthesized as a potential NgR antagonist. The results indicated that PepIV and PepII decrease the mRNA levels of the small GTPase RhoA and partially neutralize CNS myelin inhibition to cultured cerebellar granule cells (CGCs). Moreover, treatment with both peptides was propitious to maintaining residual axons after SCI, thereby promoting regeneration and locomotion recovery. Because RhoA plays a role in stabilizing the cytoskeleton in growth cones and axons, enhanced neurite outgrowth might reflect a decrease in RhoA expression through PepIV and PepII treatment. Moreover, PepIV induced lower RhoA mRNA expression compared with PepII. Therefore, PepIV could block Nogo-66/NgR signalling and reduce RhoA mRNA level, and then contribute to neuronal survival and axonal regrowth after SCI, showing its ability to reverse CNS myelin inhibition to regeneration. Furthermore, selected small peptide might cover some unknown active sites on CNS myelin proteins, which could be potential targets for improving neurite outgrowth after injury.

Identification of a NEP1-35 recognizing peptide that neutralizes CNS myelin inhibition using phage display library.

Nogo-A has been identified as an inhibitory molecule to neurite outgrowth after injury in adult mammalian central nervous system (CNS). The C-terminal fragment of Nogo-A, Nogo-66, inhibits axonal regrowth through NgR1 signaling. Residues 1-32 of Nogo-66 cover two regions that contribute most affinity of Nogo-66 to NgR1. It is unclear whether blocking the two regions with specific small ligands could neutralize the inhibition of Nogo-66. Therefore in this study we explored two phage display peptide libraries to screen small peptides that might bind Nogo-66. NEP1-35 containing 1-33 residues of Nogo-66 was taken as the target for panning. We found that phage-borne peptides with stronger affinity to NEP1-35 contained a relatively conserved motif, RRXXXXXXXRRX. Afterwards one identified peptide, NH(2)-RRQTLSHQMRRP-COOH was synthesized and tested in neurite outgrowth assay, in which this small molecule showed moderate ability to neutralize CNS myelin inhibition in vitro. Our results demonstrated that short peptides could act as adaptors to Nogo-66 and neutralize CNS myelin inhibition in vitro. Additionally, the results also suggested that phage display could help to discover novel small molecules with high affinity to CNS regrowth inhibitors, which might be able to promote CNS regeneration with fewer side effects since they could block only the corresponding regions of inhibitors.