microRNA-146a Promotes Growth of Acute Leukemia Cells by Downregulating Ciliary Neurotrophic Factor Receptor and Activating JAK2/STAT3 Signaling

PURPOSE: Acute leukemia (AL) is classified as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). This study aimed to investigate the effect of miR-146a on childhood AL and its underlying molecular mechanisms. MATERIALS AND METHODS: Bone marrow samples were obtained from 39 AL children and 10 non-cancer controls. The expressions of miR-146a and ciliary neurotrophic factor receptor (CNTFR) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in ALL and AML pediatric patients, as well as ALL (Jurkat) and AML (HL-60) cells. Correlations between miR-146a and clinical indicators were explored. A targeting relationship between miR-146a and CNTFR was detected by dual luciferase reporter gene assay. Cell proliferation, apoptosis, migration, and invasion of Jurkat and HL-60 cells were measured by MTT assay, flow cytometry, and transwell assay, respectively. LIF expression was detected by qRT-PCR in Jurkat and HL-60 cells. The expression of p-JAK2, JAK2, p-STAT3, and STAT3 in HL-60 cells was measured by Western blot. RESULTS: miR-146a was increased in ALL and AML pediatric patients, while CNTFR was decreased. miR-146a expression was associated with immunophenotype, karyotype, fusion gene, and SIL-TAL1. CNTFR was a target gene of miR-146a. miR-146a could promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis in Jurkat and HL-60 cells by downregulating CNTFR. Meanwhile, miR-146a inhibited the expression of LIF and activated JAK2/STAT3 pathway by downregulating CNTFR. CONCLUSION: miR-146a could promote the proliferation, migration, and invasion and inhibit the apoptosis of AL Jurkat and HL-60 cells by downregulating CNTFR and activating the JAK2/STAT3 pathway.

Delayed Treatment of Capsaicin Produces Partial Motor Recovery by Enhancing Dopamine Function in MPP⁺-lesioned Rats via Ciliary Neurotrophic Factor

Transient receptor potential vanilloid subtype 1 (TRPV1) on astrocytes prevents ongoing degeneration of nigrostriatal dopamine (DA) neurons in MPP⁺-lesioned rats via ciliary neurotrophic factor (CNTF). The present study determined whether such a beneficial effect of astrocytic TRPV1 could be achieved after completion of injury of DA neurons, rather than ongoing injury, which seems more relevant to therapeutics. To test this, the MPP⁺-lesioned rat model utilized here exhibited approximately 70~80% degeneration of nigrostriatal DA neurons that was completed at 2 weeks post medial forebrain bundle injection of MPP⁺. TRPV1 agonist, capsaicin (CAP), was intraperitoneally administered. CNTF receptor alpha neutralizing antibody (CNTFRαNAb) was nigral injected to evaluate the role of CNTF endogenously produced by astrocyte through TRPV1 activation on DA neurons. Delayed treatment of CAP produced a significant reduction in amphetamine-induced rotational asymmetry. Accompanying this behavioral recovery, CAP treatment increased CNTF levels and tyrosine hydroxylase (TH) activity in the substantia nigra pars compacta (SNpc), and levels of DA and its metabolites in the striatum compared to controls. Interestingly, behavioral recovery and increases in biochemical indices were not reflected in trophic changes of the DA system. Instead, behavioral recovery was temporal and dependent on the continuous presence of CAP treatment. The results suggest that delayed treatment of CAP increases nigral TH enzyme activity and striatal levels of DA and its metabolites by CNTF endogenously derived from CAP-activated astrocytes through TRPV1, leading to functional recovery. Consequently, these findings may be useful in the treatment of DA imbalances associated with Parkinson's disease.

PINK1 Deficiency Decreases Expression Levels of mir-326, mir-330, and mir-3099 during Brain Development and Neural Stem Cell Differentiation

PTEN-induced putative kinase 1 (PINK1) is a Parkinson's disease (PD) gene. We examined miRNAs regulated by PINK1 during brain development and neural stem cell (NSC) differentiation, and found that lvels of miRNAs related to tumors and inflammation were different between 1-day-old-wild type (WT) and PINK1-knockout (KO) mouse brains. Notably, levels of miR-326, miR-330 and miR-3099, which are related to astroglioma, increased during brain development and NSC differentiation, and were significantly reduced in the absence of PINK1. Interestingly, in the presence of ciliary neurotrophic factor (CNTF), which pushes differentiation of NSCs into astrocytes, miR-326, miR-330, and miR-3099 levels in KO NSCs were also lower than those in WT NSCs. Furthermore, mimics of all three miRNAs increased expression of the astrocytic marker glial fibrillary acidic protein (GFAP) during differentiation of KO NSCs, but inhibitors of these miRNAs decreased GFAP expression in WT NSCs. Moreover, these miRNAs increased the translational efficacy of GFAP through the 3'-UTR of GFAP mRNA. Taken together, these results suggest that PINK1 deficiency reduce expression levels of miR-326, miR-330 and miR-3099, which may regulate GFAP expression during NSC differentiation and brain development.

Preventive Effect of Different Compatibilities of Ramulus Cinnamomi and Radix Paeomlae alba in Guizhi Decoction on Cardiac Sympathetic Denervation Induced by 6-OHDA / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the preventive effect of different compatibilities of Ramulus Cinnamomi (RC) and Radix Paeomiae alba (RPA) in Guizhi Decoction (GZD) on neurotransmitters and their rate-limiting enzymes, and neurotrophic factors of cardiac sympathetic denervation model rats induced by 6-hydroxydopamine (6-OHDA).</p><p><b>METHODS</b>Totally 54 male Wistar rats were randomly divided into 6 groups, i.e., the blank control group, the model group, the methycobal group, the 2:1 (RC/RPA) Guishao group, the 1:2 Guishao group, and the 1:1 Guishao group, 9 in each group. Sympathetic denervation was induced by intraperitoneal injection of 6-OHDA for three successive days. Rats in the methycobal group and GZD groups were administered with corresponding decoction by gastrogavage 1 week before modeling (methycobal at the daily dose 0.15 mg/kg; GZD at the daily dose of 4.0, 5.5, 5.5 g crude drugs/kg for GZD 1:1, 1:2, and 2:1 groups). All medication lasted for 10 successive days. Levels of norepinephrine (NE), tyrosine hydroxylase (TH), choline acetyl-transferase (ChAT), nerve growth factor (NGF), growth associated protein43 (GAP-43) and ciliary neurotrophic factor (CNTF) in myocar- dial homogenates of right atrium and ventricular septum were detected by ELISA.</p><p><b>RESULTS</b>Compared with the blank control group, levels of NE, TH, TH/ChAT ratio, and GAP-43 in myocardial homogenates of right atrium and ventricular septum decreased in the model group, and level of NGF increased (P < 0.01, P < 0.05). Compared with the model group, levels of NE and GAP-43 increased in the right atrium and interventricular septum; NGF level of the ventricular septum decreased in the methycobal group and each GZD groups. TH and TH/ChAT ratio in the right atrium increased in the 2:1 Guishao group and the 1:2 Guishao group (P < 0.01, P < 0.05); NGF levels in the right atrium and interventricular septum decreased only in the 1:1 Guishao group (P < 0.01, P< 0.05). Compared with the methycobal group, levels of NE, TH, and GAP-43 in the right atrium and interventricular septum increased, and NGF levels in the right atrium and interventricular septum decreased in the 1:1 Guishao group (P < 0.05). Compared with the methycobal group, levels of NE and GAP-43 in interventricular septum increased in the 2:1 Guishao group (P < 0.05).</p><p><b>CONCLUSION</b>GZD (with the proportion between RC and RPA 2:1 and 1:1) could improve contents of neurotransmitters and their rate-limiting enzymes, as well as neurotrophic factors in cardiac sympathetic denervation model rats induced by 6-OHDA, alleviate cardiac sympathetic denervation induced by 6-OHDA, and maintain the balance of sympathetic-vagal nerve system.</p>

Effect of Draconis Sanguis-containing serum on NGF, BDNF, CNTF, LNGFR, TrkA, GDNF, GAP-43 and NF-H expressions in Schwann cells / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration.</p><p><b>METHOD</b>SD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later.</p><p><b>RESULT</b>Most of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P < 0.01). Whereas that of BDNF decreased significantly (P < 0.05), and so did that of TrkA, CNTF (P < 0.01), with no remarkable difference in NF-H-mRNA.</p><p><b>CONCLUSION</b>Traditional Chinese medicine Draconis Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.</p>

Light-induced retinal injury enhanced neurotrophins secretion and neurotrophic effect of mesenchymal stem cells in vitro / Lesão de retina induzida por luz aumentou a secreção de neurotrofinas e o efeito neurotrófico das células primordiais mesenquimais in vitro

PURPOSE: To investigate neurotrophins expression and neurotrophic effect change in mesenchymal stem cells (MSCs) under different types of stimulation. METHODS: Rats were exposed in 10,000 lux white light to develop light-induced retinal injury. Supernatants of homogenized retina (SHR), either from normal or light-injured retina, were used to stimulate MSCs. Quantitative real time for polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were conducted for analysis the expression change in basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in MSCs after stimulation. Conditioned medium from SHR-stimulated MSCs and control MSCs were collected for evaluation their effect on retinal explants. RESULTS: Supernatants of homogenized retina from light-injured rats significantly promoted neurotrophins secretion from MSCs (p<0.01). Conditioned medium from mesenchymal stem cells stimulated by light-injured SHR significantly reduced DNA fragmentation (p<0.01), up-regulated bcl-2 (p<0.01) and down-regulated bax (p<0.01) in retinal explants, displaying enhanced protective effect. CONCLUSIONS: Light-induced retinal injury is able to enhance neurotrophins secretion from mesenchymal stem cells and promote the neurotrophic effect of mesenchymal stem cells.

OBJETIVO: Investigar a expressão de neurotrofinas e mudança no efeito neurotrófico de células-tronco mesenquimais (MSCs) sob diferentes tipos de estimulação. MÉTODOS: Os ratos foram expostos em 10.000 lux de luz branca para desenvolver a lesão da retina induzida por luz. Os sobrenadantes de homogeneizado de retina (SHR) quer a partir de retina normal ou da lesada por luz, foram usados para estimular as células-tronco mesenquimais. O RT-PCR quantitativa e ELISA foram realizados para análise da alteração de expressão do fator básico de crescimento de fibroblastos (bFGF), do fator neurotrófico derivado do cérebro (BDNF) e do fator neurotrófico ciliar (CNTF) em MSCs após a estimulação. O meio condicionado de células-tronco mesenquimais estimuladas por SHR e controles foram coletadas para avaliação de seu efeito sobre os explantes de retina. RESULTADOS: SHR de retinas de rato lesadas por luz promoveram aumento significativo de secreção de neurotrofinas em MSCs (p<0,01). O meio condicionado de SHR lesado por luz reduziu significativamente a fragmentação do DNA de MSCs (p<0,01), elevação de Bcl-2 (p<0,01) e redução de bax (p<0,01) em explantes de retina, mostrando um aumento do efeito protetor. CONCLUSÕES: A lesão da retina induzidos pela luz é capaz de aumentar a secreção de neurotrofinas e promover o efeito neurotrófico de células-tronco mesenquimais.

Effects of Jinmaitong Capsule () on ciliary neurotrophic factor in sciatic nerves of diabetes mellitus rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effects of the Chinese medicine Jinmaitong Capsule (, JMT) on the pathomorphology of sciatic nerves, ciliary neurotrophic factor (CNTF), and the mRNA expressions of CNTF in rats with streptozotocin-induced diabetes mellitus (STZ-DM).</p><p><b>METHODS</b>The animal model was established by one time intraperitoneal injection of streptozotocin. The rats were simply divided by random into 5 groups including model group, low-dose JMT group (JL), medium-dose JMT group (JM), high-dose JMT group (JH) and neurotropin group. For each of the above 5 groups, a group of 10 normal Wistar rats matched in body weight, age and gender were set as normal group. Intragastric administrations were started after the animal model established. The JL group were administered with five times the JMT dose recommended for a human adult; the JM group were administered with ten times the JMT dose recommended for a human adult; the JH group were administered with twenty times the JMT dose recommended for a human adult. The neurotropin group was administered with ten times the neurotropin dose recommended for a human adult. All rats were given intragastric administration for 16 weeks and then killed. In the 4th, 8th, 12th, 16th week, body weight and blood glucose level were detected before and after the intervention. The morphologic changes of the sciatic nerves were observed by optical microscope and transmission electron microscope. The CNTFmRNA expressions were detected by real-time fluorescent quantitative polymerase chain protein, and the CNTF protein expressions were detected by immunohistochemical method.</p><p><b>RESULTS</b>The blood glucose levels of the STZ-DM rats were much higher than normal group (P<0.01), and there was no apparent difference between any treatment groups and the model group (P>0.05). Before and after the intervention in the 4th, 8th, 12th, 16th week, there were no significant differences in the body weight among all the groups (P>0.05). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium. The levels of CNTF and CNTF-mRNA expressions in the STZ-DM rats were both significantly decreased (P<0.01). The sciatic nerves of STZ-DM rats might have pathomorphological changes in axons, myelin sheaths, and interstitium.</p><p><b>CONCLUSION</b>JMT could improve the pathomorphology of sciatic nerves by increasing CNTF's and CNTF-mRNA expressions in sciatic nerve tissues, and promote the repair and regeneration of damaged nerve fibers.</p>

Neuroprotection in glaucoma: present and future / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To review the updated research on neuroprotection in glaucoma, and summarize the potential agents investigated so far.</p><p><b>DATA SOURCES</b>The data in this review were collected from PubMed and Google Scholar databases published in English up to September 2012, with keywords including glaucoma, neuroprotection, and retinal ganglion cells, both alone and in combination. Publications from the past ten years were selected, but important older articles were not excluded.</p><p><b>STUDY SELECTION</b>Articles about neuroprotection in glaucoma were selected and reviewed, and those that are cited in articles identified by this search strategy and judged relevant to this review were also included.</p><p><b>RESULTS</b>Although lowering the intraocular pressure is the only therapy approved as being effective in the treatment of glaucoma, increasing numbers of studies have discovered various mechanisms of retinal ganglion cells death in the glaucoma and relevant neuroprotective strategies. These strategies target neurotrophic factor deprivation, excitotoxic damage, oxidative stress, mitochondrial dysfunction, inflammation, activation of intrinsic and extrinsic apoptotic signals, ischemia, and protein misfolding. Exploring the mechanism of axonal transport failure, synaptic dysfunction, the glial system in glaucoma, and stem cell used in glaucoma constitute promising research areas of the future.</p><p><b>CONCLUSIONS</b>Neuroprotective strategies continue to be refined, and future deep investment in researching the pathogenesis of glaucoma may provide novel and practical neuroprotection tactics. Establishing a system to assess the effects of neuroprotection treatments may further facilitate this research.</p>

[Assessment of neurotrophic factors expression and cell proliferation in the coculture of neural and mesenchymal stem cells]

Neurotrophic factors are diffusible polypeptides that have critical roles in survival, proliferation and differentiation of stem cells. This study was done to assess the role of neurotrophic factors [CNTF, BDNF, GDNF, NT-3] expression and proliferation rate of neural stem cells [NSCs] in coculture with mesenchymal stem cells [MSCs]. In this experimental study, NSCs and MSCs were isolated from adult Wistar rat. Initially, NSCs was harvested from temporal lobe after mechanical digestion by a sterile flamed Pasteur pipette and enzymatic digestion with trypsin and Dnase. The cell suspension was cultivated in a flask with DMEM/F12 medium supplemented with 10% FBS 100U/ml Penicillin and 100 mg/ml Streptomycin. To obtain MSCs, bone marrow of femur and tibia bones were flashed out and cultured. MSCs and NSCs cocultured by transwell system in DMEM/F12 medium supplemented with 10% FBS 100U/ml Penicillin and 100 mg/ml Streptomycin. Haemocytometer, immunocytochemistry and RT-PCR methods were performed to identify and evaluate cell proliferation, purity levels and neurotrophic factors expression. There is no differences in NTFs profile of neurotrophic factors expression between coculture group and control NSCs, but interactions between MSCs and NSCs significantly promoted NSCs proliferation [P<0.05]. This study showed that coculture of NSCs with MSCs might be prfered in cell-therapy than NSC

Fibroblast growth factor-2 counteracts the effect of ciliary neurotrophic factor on spontaneous differentiation in adult hippocampal progenitor cells / 华中科技大学学报（医学）（英德文版）

Neural stem/progenitor cells (NSCs) can spontaneously differentiate into neurons and glial cells in the absence of mitogen fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) in medium and the spontaneous differentiation of NSCs is mediated partially by endogenous ciliary neurotrophic factor (CNTF). This study examined the relationship of FGF-2 and CNTF in the spontaneous differentiation of adult hippocampal progenitor cells (AHPs). AHPs were cultured in the medium containing different concentration of FGF-2 (1-100 ng/mL). Western blotting and immunofluorescence staining were applied to detect the expression of the astrocytic marker GFAP, the neuronal marker Tuj1, the oligodendrocytic marker CNPase and, Nestin, the marker of AHPs. The expression of endogenous CNTF in AHPs at early (passage 4) and late stage (passage 22) was also measured by Western blotting. The results showed that FGF-2 increased the expression of Nestin, dramatically inhibited the expression of GFAP and Tuj1 and slightly suppressed the expression of CNPase. FGF-2 down-regulated the expression of endogenous CNTF in AHPs at both early (passage 4) and late stage (passage 22). These results suggested that FGF-2 could inhibit the spontaneous differentiation of cultured AHPs by negatively regulating the expression of endogenous CNTF in AHPs.

Effects of bone marrow mesenchymal stem cell transplantation on retinal cell apoptosis in premature rats with retinopathy / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the effects of marrow mesenchymal stem cell (BMSC) transplantation on retinal cells apoptosis and changes to neurotrophin-3 (NT-3 and ciliary neurotrophic factor (CNTF) in rats with retinopathy of prematurity (ROP).</p><p><b>METHODS</b>Seven-day-old Sprague-Dawley rats were randomly divided into normal control (CON), ROP, BMSC transplantation (BMSCs were transplanted 5 days after oxygen conditioning) and phosphate buffered saline (PBS) groups. The ROP model was prepared according to the classic hyperoxygen method. Seven days after transplantation, TUNEL/DAPI, NT-3/API and CNTF/DAPI double-labeled immunofluorescence were used to examine the effects of BMSC transplantation on both the apoptosis of retinal cells and the expression of NT-3 and CNTF protein in the retinal cells of the ROP rats.</p><p><b>RESULTS</b>Seven days after BMSC transplantation, there were few TUNEL+ DAPI+ cells observed in the CON group. There were fewer TUNEL+DAPI+ cells observed in the BMSC group than in the ROP group (P<0.01), but there was no significant difference between the ROP and PBS groups (P>0.05). There were few NT-3+DAPI+ cells and CNTF+DAPI+ cells in the CON group. There were more NT-3+DAPI+ and CNTF+DAPI+ cells in the ROP group than in the CON group, but there was no significant difference between the ROP and CON groups (P>0.05). More NT-3+DAPI+ and CNTF+DAPI+ cells were observed in the BMSC group compared with the ROP group (P<0.01), and there was no significant difference in either NT-3+DAPI+ or CNTF+DAPI+ cells between the ROP and PBS groups (P>0.05).</p><p><b>CONCLUSIONS</b>BMSC transplantation therapy could alleviate the apoptosis of retinal cells in ROP rats, and its mechanisms might be associated with promoting the expression of NT-3 and CNTF protein in retinal cells.</p>

Expression of ciliary neurotrophic factor and its receptor in experimental obstructive nephropathy / 대한해부학회지

Ciliary neurotrophic factor (CNTF) is well known as a growth/survival factor of neuronal tissue. We investigated the expression of CNTF and its specific receptor alpha (CNTFRalpha) in a unilateral ureteral obstruction (UUO) model. Complete UUO was produced by left ureteral ligation in Sprague-Dawley rats. The animals were sacrificed on days 1, 3, 5, 7, 14, 21, and 28 after UUO. The kidneys were fixed, and processed for both immunohistochemistry and in situ hybridization. CNTF immunoreactivity in sham-operated kidneys was observed only in the descending thin limb (DTL) of the loop of Henle. In UUO kidneys, CNTF expression was induced in the S3 segment (S3s) of the proximal tubule from day 1, and progressively expanded into the entire S3s and a part of the convoluted proximal tubules, distal tubules (DT), and glomerular parietal epithelium up to day 7. Upregulated CNTF expression was maintained to day 28. From day 14, the inner medullary collecting duct showed weak CNTF immunoreactivity. The CNTFRalpha mRNA hybridization signal in sham-operated kidneys was weakly detected in the DTL, DT, medullary thick ascending limb, and in a few S3s cells. After UUO, CNTFRalpha mRNA expression increased progressively in both the renal cortex and the medulla up to day 7 and increased expression was maintained until day 28. The results suggest that the S3s may be the principal induction site for CNTF in response to renal injury, and that CNTF may play a role in chronic renal injury.

Bone marrow stromal cells transfected with ciliary neurotrophic factor gene ameliorates the symptoms and inflammation in C57BL/6 mice with experimental allergic encephalomyelitis / 南方医科大学学报

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18 percent (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87 percent, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91 percent over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Expression of ciliary neurotrophic factor after induction of ocular hypertension in the retina of rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Glaucoma is mainly characterized by the loss of retinal ganglion cells. Ciliary neurotrophic factor (CNTF) is believed to stimulate the regeneration of axons of retinal ganglion cells. The objective of our study was to detect the expression of CNTF in the retina of a rat glaucoma model with increased intraocular pressure (IOP).</p><p><b>METHODS</b>The rat glaucoma model was set up by electrocoagulating at least three episcleral and limbal veins. The location and the expression level of CNTF were detected at 1, 3, 7, 14, and 28 days post-surgery by immunohistochemistry, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), and Western blot analysis.</p><p><b>RESULTS</b>The rat glaucoma model with chronic, moderately elevated IOP was successfully produced. A minimum expression of CNTF was found in the ganglion cell layer of the retinas of the control group, and temporally increased expression and intensity of CNTF were found in the experimental retinas.</p><p><b>CONCLUSION</b>The expression of endogenous CNTF in the rat retina was found altered after the induction of ocular hypertension.</p>

Effects of high power microwave exposure on cholinergic neurotrophic factors protein in rabbit retina / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the changes of cholinergic neurotrophic factors (CNTF) protein at different time points and the distribution of CNTF in rabbit retina after exposure to high power microwave (HPM), in order to determine the changes rule of CNTF protein.</p><p><b>METHODS</b>The rabbits were irradiated by HPM (peak power 90 W/cm(2)) for 15 min respectively, and then killed at 0, 3, 6, 12, 24 and 72 h after irradiation. The changes of CNTF protein were investigated by immunohistochemistry and semi-quantity analysis.</p><p><b>RESULTS</b>CNTF protein was distributed in full retinal layers, special in the cell membrane and cytoplasm. HPM irradiation could immediately down-regulated CNTF protein expression at 0 h, up-regulated and arrived at peak level at 6 h (P<0.05 vs 0 h group), and then kept control level.</p><p><b>CONCLUSION</b>HPM may cause acute retinal injure and change the expression of CNTF protein in rabbit retina. These effects show the time-dependent feature. These results suggest that CNTF activation plays a central role in the retinal injures induced by HPM, and supplies a therapy method by using foreign-aid CNTF to remedy the retinal injure induced by HPM.</p>

The difference of nerve growth factor and ciliary neurotrophic factor between the sensitive and motor fibres regeneration / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) to promoting the sensitive and motor fibres regeneration.</p><p><b>METHODS</b>Thirty two Sprague-Dawley rats with 10 mm gap of sciatic nerve were operated and bridged with the new double channel nerve conduit in rhomboid shape. They were randomly divided into two groups, with each in twelve animals. In the first group, 200 micro1 of chitin for medical use was injected into the conduit, and in the second group the two branches of the conduit contained 100 microl of the chitin and 5 microl NGF or CNTF. At twelve and sixteen weeks after the operation, we evaluated the sensitive and motor fibres of regenerative nerve with Holmes, Acetylcholinesterase and Carbonic anhydrase staining.</p><p><b>RESULTS</b>After the twelve and sixteen weeks of the operation, there were not significant differences of the regenerative nerve fibres between two channels in the first groups. But in the second group, the number of the motor fibres of the regenerative nerve of CNTF branch channel was much more and distributed better than the others, while the number and density of sensitive fibres was inferior to NGF. The latency of compound muscle active potential and cortical somatosensory evoked potential of the nerve in the CNTF branch was shorter than the one in the NGF branch, but its amplitude was higher.</p><p><b>CONCLUSIONS</b>The CNTF could significantly promote the regeneration of motor fibres and the NGF could promote better for the regeneration of sensitive fibres.</p>

Prevalence of fatty liver in non-obese Japanese children with atopic dermatitis.

Fatty liver in non-obese Japanese children was observed in 3.2% of non-atopic children and in 17.6% of patients with atopic dermatitis in 2000. The prevalence of fatty liver in non-obese children aged 0-12 years was studied from 2001 to 2003. Subjects were either non-atopic children, or suffering from bronchial asthma, allergic rhinitis, or atopic dermatitis. Fatty liver was studied by abdominal ultrasound scans. The prevalence of fatty liver was increasing annually, and it reached to 12.5% in non-atopic children, 13.1% in patients with bronchial asthma, 13.7% in patients with allergic rhinitis, or 33.9% in patients with atopic dermatitis, in 2003. Since fatty liver in childhood may be a risk factor for lifestyle-related diseases in future, care should be taken to prevent it.

Purification and activity assay of HSA-AX15 (R13K) fusion protein expressed in Pichia pastoris / 生物工程学报

To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.

Construction of protease resistant mutein of human CNTF and its expression in Pichia pastoris / 生物工程学报

AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.