Phytohormone ethylene mediates oligogalacturonic acid-induced growth inhibition in tomato etiolated seedlings.

Plant growth and immunity are tightly interconnected. Oligogalacturonic acids (OGs) are pectic fragments and have been well investigated in plant immunity as a damage-associated molecular pattern. However, little is known regarding how OGs affect plant growth. Here, we reveal that OGs inhibit the growth of intact etiolated seedling by using the horticultural crop tomato as a model. This inhibitory effect is partially suppressed by the action of ethylene biosynthesis inhibitors, or the gene silencing of SlACS2, an essential rate-limiting enzyme for ethylene biosynthesis, suggesting that SlACS2-mediated ethylene production promotes OG-induced growth inhibition. Furthermore, OGs treatment elevates the SlACS2 protein phosphorylation, and its decrease by the kinase inhibitor K252a partially rescue OG-induced growth inhibition, indicating that SlACS2 phosphorylation involves in OG-induced growth inhibition. Moreover, the mitogen-activated protein kinase SlMPK3 could be activated by OGs treatment and can directly phosphorylate SlACS2 in vitro, and the bimolecular fluorescence complementation combining with the yeast two-hybrid assay shows that SlMPK3 interacts with SlACS2, indicating that SlMPK3 may participate in modulating the OG-induced SlACS2 phosphorylation and growth inhibition. Our results reveal a regulatory mechanism at both the transcriptional and post-transcriptional levels by which OGs inhibit the growth of intact plant seedlings.

The BDNF-TrkB signaling pathway is partially involved in the neuroprotective effects of hydrogen sulfide in Parkinson's disease.

Recent studies have demonstrated that hydrogen sulfide (H2S) has a neuroprotective effect in neurodegenerative diseases. It is possible that this effect is supported by brain-derived neurotrophic factor (BDNF). Our aim is to examine the effects of H2S on neural damage in Parkinson's disease (PD) and to reveal the role of the BDNF-TrkB pathway in its possible effect. PD model was created with 1-methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP). C57BL/6 breed male mice were randomly divided into six groups: control, K252a, MPTP, MPTP + K252a, MPTP + NaHS, and MPTP + NaHS + K252a. TrkB receptor antagonist K252a and sodium hydrosulfide (NaHS) as a H2S donor were administered intraperitoneally. An increase was observed in the motor behavior tests in MPTP group, but NaHS treatment shortened the time spent on the balance beam and pole tests. It was also noticed that the BDNF-pathway played a role in the shortening of this period. Mice that received NaHS were found to have less MPTP-induced cellular damage. A positive effect of BDNF was also detected in the protection of these neurons. BDNF levels in the SN were significantly increased in MPTP group, compared to control group. Tissue CBS levels decreased in the groups that received K252a, compared to MPTP group. The findings of the present study display that the BDNF-TrkB pathway partially plays a role in the protective effect of H2S in the experimental mouse model of PD. This effect is probably due to changes in intracellular signaling pathways, rather than TrkB receptor expression.

Involvement of the BDNF-TrkB-KCC2 pathway in neuropathic pain after brachial plexus avulsion.

INTRODUCTION: Brachial plexus avulsion significantly increased brain-derived neurotrophic factor (BDNF) release in the spinal cord. Here we investigated the involvement of the BDNF-TrkB-KCC2 pathway in neuropathic pain caused by BPA injury. We hypothesized that activation of BDNF-TrkB may inhibit neuronal excitability by downregulating KCC2 to maintain a high intracellular Cl-concentration. We established a neuropathic pain rat model by avulsion of the lower trunk brachial plexus, and investigated the effects of the TrkB-specific antibody K-252a on the expression of BDNF, TrkB, and KCC2. METHODS: We randomly divided 40 male SD rats into four groups. In the brachial plexus avulsion group, C8-T1 roots were avulsed from the spinal cord at the lower trunk level. In the K252a group, 5uL K252a was applied intrathecally daily for three days after avulsion. In the sham surgery group, expose only and without damage. The control group did not undergo any treatment. Mechanical hyperalgesia and cold allodynia were analyzed by electronic pain measuring instrument and acetone spray method at different time points on days 1, 3, 7, 10, 14, and 21 after surgery. At 21 days after surgery, the expression of BDNF and TrkB in dorsal horn neurons and GFAP in astrocytes were detected by immunohistochemistry at the C5-T1 segment of the spinal cord. The expression levels of BDNF, TrkB, and KCC2 in the C5-T1 spinal cord were measured by Western Blot at 7 and 21 days. RESULTS: Mechanical hyperalgesia and cold allodynia were significantly reduced in the K252a group compared with the brachial plexus avulsion group. Compared with the BPA group, BDNF, TrkB and GFAP were significantly decreased in the K252a group at 21 days after treatment by immunohistochemical test. In the WB test, the expressions of BDNF and TrkB in the K252a group were quantitatively detected to be decreased, while the expression of KCC2 was increased, which was obvious at 7 and 21 days. CONCLUSION: BDNF-TrkB-KCC2 pathway can significantly relieve neuropathic pain after BPA, and is a potential target for the treatment of neuropathic pain.

The antidepressant-like effects of Shen Yuan: Dependence on hippocampal BDNF-TrkB signaling activation in chronic social defeat depression-like mice.

The Shen Yuan prescription (SY) comprises Panax ginseng (GT) and Polygala tenuifolia (YT), elicited superior antidepressant activity compared with that of GT or YT alone. The aim of this paper is to elucidate the effects of SY treatment on chronic social defeat stress (CSDS)-induced depression-like symptoms and the related mechanism. Our results indicated that SY treatment reverses the depressive-like behaviors induced by CSDS as measured by the social interaction test, sucrose preference test, forced swim test, and tail suspension test. SY decreased the serum levels of CORT and increased hippocampal neurotransmitters (5-HT, DA, and NE) in CSDS mice. Meanwhile, SY upregulated the brain-derived neurotrophic factor (BDNF) signaling pathway and reversed the decreased hippocampal neurogenesis caused by CSDS. In addition, we found that the TrkB antagonist K252a fully blocked the SY effects on behavioral improvement and eliminated the promoting effects of SY on hippocampal neurogenesis and BDNF-TrkB signaling (including the downstream ERK and Akt pathways) activation, thus further demonstrating that BDNF-TrkB signaling was necessary for the SY effects. In conclusion, our study showed that SY acted as an antidepressant in mice exhibiting CSDS-induced depression-like symptoms, and its effect was facilitated by promoting hippocampal neurogenesis and BDNF signaling pathway activation.

2,3,5,4'-tetrahydoxystilbene-2-O-ß-D-glucoside eliminates staurosporine-induced cytotoxicity by restoring BDNF-TrkB/Akt signaling axis.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside (THSG) is the major active ingredient in Plygonum multiflorum that displays a great deal of health-benefits including anti-oxidation, anti-hyperlipidemia, anti-cancer, anti-inflammation and neuroprotection. However, it is unclear whether THSG exerts neuroprotective functions by regulating neurotrophic factors and their associated signaling pathways. In this study, hippocampal neurons were challenged with staurosporine (STS) to establish a neural damage model. We found that STS-induced cytotoxicity introduced significant morphological collapse and initiating cell apoptosis, along with the down regulation of BDNF and TrkB/Akt signaling axis. In contrast, neurons pretreated with THSG showed resistance to STS-induced toxicity and maintained cell survival. THSG rescued STS induced dysfunctions of BDNF and its associated TrkB/Akt signaling, and restored the expression of Bcl-2 and Caspase-3. However, inhibition of TrkB activity by K252a or Akt signaling by LY294002 abolished the neuroprotective effects of THSG. Therefore, BDNF and TrkB/Akt signaling axis is a promise target for THSG mediated neuroprotective functions.

Staurosporine and venetoclax induce the caspase-dependent proteolysis of MEF2D-fusion proteins and apoptosis in MEF2D-fusion (+) ALL cells.

MEF2D-fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5 % of acute lymphoblastic leukemia (ALL) cases. Their introduction to cells has been reported to transform cell lines or increase the colony formation of bone marrow cells, suggesting their survival-supporting ability, which prompted us to examine M-fusion-targeting drugs. To identify compounds that reduce the protein expression level of MEF2D, we developed a high-throughput screening system using 293T cells stably expressing a fusion protein of MEF2D and luciferase, in which the protein expression level of MEF2D was easily measured by a luciferase assay. We screened 3766 compounds with known pharmaceutical activities using this system and selected staurosporine as a potential inducer of the proteolysis of MEF2D. Staurosporine induced the proteolysis of M-fusion proteins in M-fusion (+) ALL cell lines. Proteolysis was inhibited by caspase inhibitors, not proteasome inhibitors, suggesting caspase dependency. Consistent with this result, the growth inhibitory effects of staurosporine were stronger in M-fusion (+) ALL cell lines than in negative cell lines, and caspase inhibitors blocked apoptosis induced by staurosporine. We identified the cleavage site of MEF2D-HNRNPUL1 by caspases and confirmed that its caspase cleavage-resistant mutant was resistant to staurosporine-induced proteolysis. Based on these results, we investigated another Food and Drug Administration-approved caspase activator, venetoclax, and found that it exerted similar effects to staurosporine, namely, the proteolysis of M-fusion proteins and strong growth inhibitory effects in M-fusion (+) ALL cell lines. The present study provides novel insights into drug screening strategies and the clinical indications of venetoclax.

Tea polyphenols attenuate staurosporine-induced cytotoxicity and apoptosis by modulating BDNF-TrkB/Akt and Erk1/2 signaling axis in hippocampal neurons.

Tea polyphenols (TP) are the major ingredients in tea beverages that display health-benefits including anti-oxidation, anti-inflammation, anti-aging, attenuating blood pressure and deflating. In this study, we investigated the neuroprotective effects of TP to attenuate staurosporine (STS)-induced cytotoxicity. Rat hippocampal neurons were isolated, cultured and incubated with STS to induce neurite collapse and apoptosis, however, the medication of TP eliminated these adverse effects and maintained the morphology of neurons. STS decreased the expression of pro-BDNF, downregulated the TrkB/Akt/Bcl-2 signaling axis and promoted the activation of Erk1/2 and caspase-3. In contrast, TP rescued the expression of pro-BDNF and antagonistically restored the biochemistry of aforementioned signaling effectors. Consistently, the activity of TP can be attenuated by the inhibition of TrkB or Akt by small chemicals K252a and LY294002. Therefore, BDNF-TrkB and Akt signaling axis is essential for TP-mediated neuroprotective effects. In summary, TP showed beneficial effects to protect neurons from exogenous insults such as STS-induced neural cytotoxicity and cell death.

Accessibility of axonal G protein coupled mu-opioid receptors requires conceptual changes of axonal membrane targeting for pain modulation.

The mechanisms of axonal trafficking and membrane targeting are well established for sodium channels, which are the principle targets for perineurally applied local anaesthetics. However, they have not been thoroughly investigated for G protein coupled receptors such as mu-opioid receptors (MOR). Focusing on these axonal mechanisms, we found that axonal MOR functionality is quite distinct in two different pain states, i.e. hindpaw inflammation and nerve injury. We observed axonal membrane MOR binding and functional G protein coupling exclusively at sites of CCI nerve injury. Moreover at these axonal membrane sites, MOR exhibited extensive co-localization with the membrane proteins SNAP and Na/K-ATPase as well as NGF-dependent enhanced lipid rafts and L1CAM anchoring proteins. Silencing endogenous L1CAM with intrathecal L1CAM specific siRNA, disrupting lipid rafts with the perineurial cholesterol-sequestering agent MßCD, as well as suppressing NGF receptor activation with the perineurial NGF receptor inhibitor K252a abrogated MOR axonal membrane integration, functional coupling, and agonist-elicited antinociception at sites of nerve injury. These findings suggest that local conceptual changes resulting from nerve injury are required for the establishment of functional axonal membrane MOR. Axonal integration and subsequent accessibility of functionally coupled MOR are of great relevance particularly for patients suffering from severe pain due to nerve injury or tumour infiltration.

Changes of behavior and depression-like classic indicators after hippocampal microinjection of K252a / 中国比较医学杂志

Objective To study the changes of behavior and depression-like classic indicators after hippocampal microinjection of K252a,and to establish a new animal model of depression.Methods SD rats were randomly divided into five groups,namely the control group,sham group,chronic stress depression model group,hippocampal of K252a microinjection group,and hippocampal microinjection K252a plus chronic stress group.Open field experiments,sucrose consumption test,and Morris water maze behavioral assay were used to assess the behavioral changes in the rats.ELISA was used to detect the plasma monoamine neurotransmitter,radioimmunoassay was used to determine the plasma CRH,ACTH,CORT contents,and western-blotting was performed to observe the protein expression of BDNF,CREB,ERK1/2,and BCL-2 in the hippocampus.Results Compared with the control group,the amount of activity,sugar consumption,learning and memory abilities were decreased(P<0.05 or P<0.01),also the serum monoamine neurotransmitters were decreased (P<0.01),HPA axis function was improved (P<0.01),and the expression of BDNF,CREB,ERK1/2,BCL-2 decreased in the CUMS group(P<0.05 or P<0.01),but there was no significant difference in the DMSO group.Compared with the DMSO group,the activity,consumption of sucrose,learning and memory ability were significantly decreased(P<0.05 or P<0.01),while the HPA axis function was increased (P<0.05 or P<0.01),the serum monoamine neurotransmitters decreased(P<0.05 or P<0.01),and the BDNF,CREB,ERK1/2,BCL-2 expressions in the hypocampus were significantly decreased(P<0.05 or P<0.01) in the K252a group and K252a + CUMS group.Compared with the CUMS group,the K252a group and K252a + CUMS group did not show significant changes in these parameters.Compared with the K252a group,these indicators were not significantly changed in the K252a + CUMS group.Conclusions The results of behavior,hematology,and molecular biology analysis show that this model has a great similarity to the classical model of CUMS in surface validity,construct validity,and functional validity.It may provide an alternative investigative technology platform for basic research and antidepressant drug screening.

Brain-derived Neurotrophic Factor Signaling Mediates the Antidepressant-like Effect of the Total Flavonoids of Alpiniae Oxyphyllae Fructus in Chronic Unpredictable Mild Stress Mice.

The present study verified the antidepressant-like effects of the total flavonoids of Alpinia oxyphylla Miq. (AOF) using the chronic unpredictable mild stresses paradigm and explored the mechanism that underlies antidepressant-like effects of AOF in mice. Previous research has shown that tropomyosin-related kinase B (TrkB) receptor-mediated extracellular regulated protein kinases (ERK) signaling pathways participate in depression pathophysiology. Therefore, we aimed to explore whether AOF improved depression-like behaviors by increasing activity of ERK pathways mediated by TrkB. Results showed that AOF significantly reduced the immobility time in the forced swimming test and increased the sucrose preference in sucrose preference test. In addition, decreased phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB)/CREB, pERK/ERK, and pTrkB/TrkB levels in the hippocampus induced by chronic unpredictable mild stresses were reversed by intragastric administration of AOF. Results suggested that AOF increased pCREB/CREB, pERK/ERK, and pTrkB/TrkB levels by acting on the TrkB receptor. To verify this hypothesis, mice were pretreated with the TrkB inhibitor K252a (or 0.1% dimethyl sulfoxide, intraperitoneally, 2 weeks), before the intragastric administration of AOF. This resulted in an absence of antidepressant-like effects, as well as no activation of pERK/pCREB/BDNF signaling pathways. Results demonstrated that AOF might exert antidepressant-like effects by targeting TrkB receptor-mediated pERK/pCREB/BDNF signal systems, which could help to identify the AOF receptor. Copyright © 2016 John Wiley & Sons, Ltd.

Danggui-Jakyak-San enhances hippocampal long-term potentiation through the ERK/CREB/BDNF cascade.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui-Jakyak-San (DJS), a traditional herbal prescription, has long been used to treat gerontological disorders due to insufficient blood supply. AIM OF THE STUDY: Previously, we reported that DJS increased hippocampal neurogenesis and enhanced learning and memory. However, the precise mechanism of DJS and its effects on learning and memory are still not well understood. In this study, we investigated the effect of DJS on hippocampal long-term potentiation (LTP), a cellular mechanism thought to underlie learning and memory. MATERIALS AND METHODS: To understand the effect of DJS on LTP, we used acute mouse hippocampal slices and delivered one train of high frequency stimulation (100 Hz, 100 pulses). Western blots were used to analyze the changes in protein levels induced by DJS. Morris water maze test was used to evaluate the effect of DJS on spatial long-term memory. RESULTS: DJS enhanced LTP in the Schaffer-collateral pathway of the hippocampus in a concentration-dependent manner. Extracellular signal-regulated kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) were activated by DJS. Moreover, brain-derived neurotropic factor (BDNF) was also increased by DJS. Blockade of ERK1/2 activation with PD198306 blocked the DJS-induced activation of the ERK1/2/CREB/BDNF cascade and LTP enhancement. In vivo, DJS improved spatial long-term memory and upregulated the hippocampal CREB/BDNF cascade. CONCLUSION: These results suggest that DJS enhances hippocampal LTP and spatial memory through the ERK/CREB/BDNF cascade.

Tanshinone II A, a multiple target neuroprotectant, promotes caveolae-dependent neuronal differentiation.

Neuron loss is one fundamental features of neurodegenerative diseases. Stimulating endogenous neurogenesis, especially neuronal differentiation, might potentially provide therapeutic effects to these diseases. In this study, tanshinone II A (TIIA), a multiple target neuroprotectant, was demonstrated to promote dose-dependent neuronal differentiation in three cell models of immortalized C17.2 neuronal stem cells, rat embryonic cortical neural stem cells (NSCs) and rat PC12 pheochromocytoma cells. In particular, TIIA exerted promising effects on NSCs even at the dose of 3 nM. In PC12 cells, TIIA activated mitogen-activated protein kinase 42/44 (MAPK42/44) and its downstream transcription factor, cAMP response element-binding protein (CREB). In addition, TIIA up-regulated the expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The MEK inhibitor and the antagonist to the receptors of NGF and BDNF could partially attenuate the differentiation effects, indicating that MAPK42/44 mediated BDNF and NGF signals were involved in TIIA's differentiation effects. Caveolin-1 (CAV-1), the major functional protein of membrane caveolae, plays critical roles in the endocytosis of exogenous materials. CAV1, which was activated by TIIA, might help TIIA transport across cell membrane to initiate its differentiation effects. It was proven by the evidences that suppressing the function of caveolin inhibited the differentiation effects of TIIA. Therefore, we concluded that TIIA promoted neuronal differentiation partially through MAPK42/44 mediated BDNF and NGF signals in a caveolae-dependent manner.

Role of TrkB during the postnatal development of the rat carotid body.

Brain-derived neurotrophic factor (BDNF) supports innervation of the carotid body by neurons projecting from the petrosal ganglion. Although carotid body glomus cells also express TrkB, BDNF's high affinity receptor, the role of BDNF in carotid body growth and O2 sensitivity has not been studied. Neonatal rats were treated with the TrkB antagonist K252a (100 µg kg(-1), i.p., b.i.d.) or vehicle on postnatal days P0-P6 and studied on P7. Carotid body volume was decreased by 35% after chronic K252a (P<0.001); a reduction in carotid body size was also elicited using the more selective TrkB antagonist ANA-12 (500 µg kg(-1), i.p., b.i.d.). In contrast, single-unit chemoafferent responses to 5% O2, measured in vitro, were unaffected by chronic K252a administration. Normoxic and hypoxic ventilation, measured by head-body plethysmography, were also normal after chronic K252a administration, but acute K252a administration produced a slower, deeper breathing pattern during the transition into hypoxia. These data suggest that BDNF regulates postnatal carotid body growth but does not influence the development of glomus cell O2 sensitivity.

Macranthol promotes hippocampal neuronal proliferation in mice via BDNF-TrkB-PI3K/Akt signaling pathway.

Macranthol is a lignans natural product isolated from Illicium dunnianum Tutch. Our previous study has demonstrated that macranthol exerted an antidepressant-like effect in mice, at least in part, by increasing expression of hippocampal brain-derived neurotrophic factor (BDNF) expression. However, the relationship between macranthol and BDNF downstream signaling pathway in the hippocampus remains unknown. The aim of this present study was to explore the mechanism of macranthol-modulated BDNF signaling pathway in a depression-like model of chronic unpredictable mild stress. Our results found that pharmacological inhibition of tropomyosin related kinase B (TrkB) with K252a abolished the improvement of macranthol on sucrose preference and immobility time, and attenuated the stimulatory effect of macranthol on hippocampal BDNF and phospho-Akt. Furthermore, K252a also reversed the improvement of macranthol on hippocampal Bcl-2, caspase-3 expression and hippocampal neuronal cell proliferation. Therefore, our findings verify that macranthol-mediated antidepressant-like action is associated with BDNF-TrkB and downstream activation of PI3K/Akt-Bcl-2/caspase-3 signaling pathway.

Maternal Voluntary Exercise during Pregnancy Enhances the Spatial Learning Acquisition but not the Retention of Memory in Rat Pups via a TrkB-mediated Mechanism: The Role of Hippocampal BDNF Expression.

OBJECTIVE(S): The effect of maternal voluntary exercise on hippocampal BDNF level in rat offspring was studied. In addition, the possible role of hippocampal BDNF receptors in maternal exercise induced enhancement of learning in the rat pups was investigated. MATERIALS AND METHODS: Pregnant rats have been randomly assigned to sedentary control or voluntary exercise groups. Each of the exercising pregnant rats was given access to a cage that was equipped with a running wheel until the end of their pregnancy. On post natal day (PND) 36, two groups consisted of 7 male rat pups in each group from sedentary or exercised mothers were sacrificed and the hippocampus was dissected for BDNF proteins level determination. Also, bilateral injection of K252a to the hippocampus was used to block the hippocampal BDNF action on PND59 in the rat pups. RESULTS: Voluntary exercise during pregnancy significantly increased the level of BDNF protein in the hippocampus of the rat pups on PND36 compared to the control group (P=0.048). Inhibiting BDNF action abolished the exercise-induced improvement of learning acquisition in offspring in training trials (P=0.0001). No difference was observed in the platform location latency and the time spent in the target in the probe test between two groups. Conclusion : This study demonstrates that voluntary exercise during pregnancy via a TrkB-mediated mechanism enhances the spatial learning acquisition, however, not the retention of memory in the rat pups.

The PPARγ agonist rosiglitazone prevents neuronal loss and attenuates development of spontaneous recurrent seizures through BDNF/TrkB signaling following pilocarpine-induced status epilepticus.

Hippocampal neuronal loss plays an important role in epileptogenesis, and it is considered a trigger of repeated spontaneous recurrent seizures (SRS). The BDNF/TrkB signaling pathway regulates neuronal plasticity in the CNS, and promotes epileptogenesis. Previous studies have shown that Peroxisome proliferator-activated receptor gamma (PPARγ) agonists exert neuroprotective effects by inhibiting oxidative stress and inflammation in epilepsy. In the present study, the PPARγ agonist rosiglitazone inhibited increases in BDNF and TrkB after status epilepticus (SE), and also prevented hippocampal neuronal loss. More importantly, our study showed that rosiglitazone suppressed SRS. However, the effects of rosiglitazone were significantly reversed by cotreatment with K252a, an antagonist of TrkB. Additionally, rosiglitazone did not affect the development and severity of SE. Thus, our data provide evidence that rosiglitazone exerts neuroprotective and antiepileptic effects involve BDNF/TrkB signaling. Our study also offers new perspectives for the treatment of epilepsy.

Effects of tyrosine kinase receptor B-brain-derived neurotrophic factor signal pathway on the secretion of vascular endothelial growth factor and matrix metalloproteinase-9 of neuroblastoma / 中国小儿急救医学

Objective To study the effects of tyrosine kinase receptor B-brain-derived neurotrophic factor (TrkB-BDNF) signal pathway on the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases-9(MMP-9) of neuroblastoma.Methods We used all-trans retinoic acid (ATRA) to induce the high expression of TrkB in the SH-SY5Y cell line,and then added the ectogenid BDNF to activate the TrkB-BDNF and its three downstream signal pathways.TrkB-BDNF signal pathway was inhibited by specific tyrosine kinase inhibitor K252a.The three downstream signal pathway was respectively inhibited by LY294002 (the phosphatidylinositol 3-hydroxy kinase (PI3 K) pathway inhibitor)、U73122 (the phospholipase C pathway inhibitor) 、U0126(the mitogen activated protein kinase pathway inhibitor).Enzyme linked immunosorbent assay was used to detect the concentration of VEGF and MMP-9 protein in the SY5Y cell culture supernatants.Results VEGF [(485.89 ± 109.99) pg/ml] and MMP-9 [(15.73 ± 1.72) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF were significantly higher than that of the control group and ATRA alone group(P ＜0.05).VEGF [(272.42 ±86.33) pg/ml]and MMP-9 [(5.25 ± 1.44) pg/ml] protein levels in the group of ATRA + BDNF + K252a were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(314.12 ±24.68) pg/ml] and MMP-9 [(4.91 ± 1.08) pg/ml] protein levels in the group of ATRA + BDNF + LY294002 were significantly lower than those of the ATRA + BDNF group(P ＜ 0.05) and had no significant difference compared with the control group and the ATRA alone group(P ＞0.05).VEGF [(444.08 ±64.49) pg/ml] and MMP-9 [(13.28 ±3.38) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA +BDNF + U73122 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).VEGF [(429.97 ± 19.95) pg/ml] and MMP-9 [(13.96 ± 4.45) pg/ml] protein levels in neuroblastoma cells cultured in serum-free media in the group of ATRA + BDNF + U0126 had no significant difference compared with the ATRA + BDNF group(P ＞ 0.05).Conclusion Activation of TrkB-BDNF signal pathway can increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.TrkB-BDNF signal pathway may be through activating its downstream PI3K pathway to increase the synthesis and secretion of VEGF and MMP-9 in human neuroblastoma cells.The synthesis and secretion of VEGF and MMP-9 can be inhibited by blocking the TrkB-BDNF signal pathway with K252a or blocking its downstream signal pathway PI3 K with LY294002.

Effect of NGF on Cultured Human Retinal Capillary Endothelial Cells(HRCEC) / 中山大学学报(医学科学版)

[Objective] To observe NGF on cultured human retinal vascular endothelial cells (HRCEC) proliferation. [Methods] The MTT assay was used to analyze the impact of culture HRCEC on different factors (NGF concentration groups, NGF + K252a concentration groups, bFGF group, bFGF + K252a groups, the normal culture medium groups) in normal and hypoxic condition. [Results] With the increase of NGF concentration (20,50,100 ng/mL), HRCEC significantly increased (normal condition: 0.254±0.033,0.696±0.029, 1.136±0.051; hypoxic condition: 0.422±0.036, 0.798±0.044, 1.376±0.052, P< 0.05). Compared NGF + K252a group with the same concentration of NGF (100 ng/ml) group, HRCEC reduced (P<0.05), with increasing the concentration of K252a (50,100,200 nmol/L), the trend of HRCEC decreasing is become more significant (normal condition:0.864±0.067, 0.496±0.025, 0.202±0.078; hypoxic condition:K252a 1.042±0.047,0.700±0.065, 0.401±0.078, P<0.05). [Conclusion] NGF can promote the proliferation of HRCEC, the effect could be specifically blocked by TrkA inhibitor K252a.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Inhibition effect of c-Met inhibitor on proliferation of lens epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To observe the inhibition of c-Met inhibitor on proliferation of lens epithelial cells (LECs).METHODS: Human's LECs were cultured and hepatocyte growth factor (HGF) and K252a were added to second passage of cells supplied with Dulbecco's modified eagle's medium (DMEM). MTT assay was used to examine the proliferation of LECs, and Western-blot was used to detect the expression change of Bcl-2 and Caspase-3.RESULTS: The photodensity (A) of HGF (50nmol/L) + K252a (30nmol/L) was not significantly different from that of DMEM control (P＞0.05). The expression of Bcl-2 and Caspase-3 were not significantly different from that in the control group.CONCLUSION: K252a, the inhibitor of c-Met, can effectively inhibit the proliferation of LECs.