Ginkgo biloba extract protects diabetic rats against cerebral ischemia­reperfusion injury by suppressing oxidative stress and upregulating the expression of glutamate transporter 1.

The current study aimed to evaluate the neuroprotective effect of Ginkgo biloba extract (GbE) on the progression of acute cerebral ischemia­reperfusion injury in diabetic rats, and to determine the molecular mechanism associated with this effect. Streptozotocin (STZ) induced diabetic rats were pretreated with GbE (50, 100 and 200 mg/kg/day; intragastric) for 3 weeks. During this period, body weight changes and fasting blood glucose levels were assessed each week. Following pretreatment, rats were subjected to suture occlusion of the middle cerebral artery for 30 min, which was followed by 24 h of reperfusion. Neurological deficits were subsequently evaluated at 2 and 24 h following reperfusion. Rats were sacrificed after 24 h reperfusion, and infarct volume and S100B content were measured to evaluate the neuroprotective effect of GbE. The results of the present study demonstrated that GbE pretreatment improved neurological scores, and reduced cerebral infarct volume and S100B content. Oxidative stress markers, including glutathione (GSH) and superoxide dismutase (SOD) were increased, and malondialdehyde (MDA) contents were reduced following GbE treatment. The levels of p­Akt, p­mTOR and glutamate transporter 1 (GLT1) were observed to be increased in GbE­pretreated rats. These results indicated that GbE pretreatment may serve a protective role against cerebral ischemia­reperfusion injury in diabetic rats by inhibiting oxidative stress reaction, upregulating the expression of Akt/mTOR and promoting GLT1 expression. In conclusion, the current study revealed the protective role and molecular mechanisms of GbE in diabetic rats with cerebral ischemia­reperfusion injury, and may provide novel insight into the future clinical treatment of this condition.

Dexmedetomidine protects against learning and memory impairments caused by electroconvulsive shock in depressed rats: Involvement of the NMDA receptor subunit 2B (NR2B)-ERK signaling pathway.

Cognitive impairment is a common adverse effect of electroconvulsive therapy (ECT) during treatment for severe depression. Dexmedetomidine (DEX), a sedative-anesthetic drug, is used to treat post-ECT agitation. However, it is not known if DEX can protect against ECT-induced cognitive impairments. To address this, we used chronic unpredictable mild stress (CUMS) to establish a model of depression for ECT treatment. Our Morris water maze and sucrose preference test results suggest that DEX alleviates ECT-induced learning and memory impairments without altering the antidepressant efficacy of ECT. To further investigate the underlying mechanisms of DEX, hippocampal expression of NR2B, p-ERK/ERK, p-CREB/CREB, and BDNF were quantified by western blotting. These results show that DEX suppresses over-activation of NR2B and enhances phosphorylation of ERK1/2 in the hippocampus of ECT-treated depressed rats. Furthermore, DEX had no significant effect on ECT-induced increases in p-CREB and BDNF. Overall, our findings suggest that DEX ameliorates ECT-induced learning and memory impairments in depressed rats via the NR2B-ERK signaling cascade. Moreover, CREB/BDNF seems not appear to participate in the cognitive protective mechanisms of DEX during ECT treatment.

The antidepressant-like effect of alarin is related to TrkB-mTOR signaling and synaptic plasticity.

Alarin is a newly derived neuropeptide from a splice variant of the galanin-like peptide gene. We previously showed that alarin has an antidepressant-like effect by increasing the activity of the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) pathways, mediated by the tropomyosin-related kinase B receptor in the unpredictable chronic mild stress (UCMS) mouse model. Administration of rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, prevents the rapid antidepressant-like effect induced by ketamine in animal models, indicating a vital role of mTOR in depression pathophysiology. mTOR is a target of the ERK and AKT pathways that regulates the initiation of protein translation via its downstream components: ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Therefore, we hypothesized that the antidepressant-like effects of alarin were achieved by activating ERK/AKT pathways, increasing the activity of mTOR and its downstream signaling components that contribute to protein synthesis required for synaptic plasticity. Our results suggest that intracerebroventricular administration of alarin significantly ameliorates depression-like behaviors in the UCMS mouse model. Furthermore, alarin restored UCMS-induced reductions of p70S6K and post-synaptic density 95 (PSD-95) mRNA levels, and of phospho-mTOR and phospho-4EBP1 in the prefrontal cortex, hippocampus, hypothalamus, and olfactory bulb. Additionally, alarin reversed the UCMS-induced downregulation of PSD-95 and synapsin I protein expression in these brain regions. Thus, the antidepressant-like effects of alarin may be mediated by restoring decreased activity of the mTOR signaling pathway and expression of synaptic proteins. Our findings help advance the understanding of depression pathophysiology.

Cytokines and glucocorticoid receptors are associated with the antidepressant-like effect of alarin.

Little is known about the physiological or pharmacological properties of alarin, a new neuropeptide belonging to the galanin family. We previously showed that alarin has an antidepressant-like effect and is associated with a decrease in the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis that is observed in patients with depression using unpredictable chronic mild stress (UCMS) mouse model of depression. However, the mechanisms underlying these effects have not been uncovered. Inflammatory cytokines are reportedly associated with depression. Animal studies and cytokine immune therapy in humans suggest that pro-inflammatory cytokines induce depressive symptomatology and potently activate the HPA axis, whereas anti-inflammatory cytokines may decrease activation. Thus, we first determined the levels of inflammatory cytokines in the blood and brain to evaluate whether the antidepressant-like effect of alarin in UCMS-treated mice is related to its regulation of these inflammatory cytokines. Pro-inflammatory cytokines disrupt the function and/or expression of glucocorticoid receptors (GRs), which mediate the negative feedback of glucocorticoids on the HPA axis to keep it from being overactivated. We next explored the expression level of GRs in the brains of mice subjected to UCMS and to the administration of alarin. We found that intracerebroventricular administration of alarin significantly ameliorated depression-like behaviors in the UCMS-treated mice. Alarin restored the UCMS-induced an increase in the levels of the pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α and a decrease in the anti-inflammatory cytokine IL-10 level in the blood, prefrontal cortex, hippocampus and hypothalamus. Alarin also reversed the UCMS-induced down-regulation of GR expression in these brain regions. Thus, the antidepressant-like effects of alarin may be mediated by restoring altered pro-inflammatory and anti-inflammatory cytokine levels and GR expression to decrease HPA axis hyperactivity. Our findings provide additional knowledge to interpret the pathophysiology of depression.

Dexmedetomidine prevents post-ischemic LTP via presynaptic and postsynaptic mechanisms.

Increasing evidence indicates that dexmedetomidine (DEX), a selective α2-adrenergic receptor agonist, has a neuroprotective effect against cerebral injury. However, it remains unknown whether and how DEX functionally prevents the pathological form of synaptic plasticity caused by ischemia in the hippocampal CA1 neurons. To address this issue, we analyzed the role of DEX using a model of brain ischemia (oxygen and glucose deprivation, OGD) referred to as post-ischemic LTP (i-LTP). We found that DEX could reduce i-LTP by selectively activating α2 receptors. To clarify its detailed mechanisms, the presynaptic and postsynaptic roles of DEX were investigated. The activation of the α2 receptors of DEX decreased the frequency spontaneous mEPSCs, which exerted its presynaptic mechanisms. In addition, DEX also decreased the amplitude of mEPSCs and prevented the depolarization of postsynaptic membranes during OGD treatment, which exerted its postsynaptic mechanisms. More importantly, our results indicate that postsynaptic ß receptors, not α1 receptors, participated in i-LTP. Therefore, these results demonstrated that decreasing ß receptors activation by DEX-medicated pre- and post-synaptic α2 receptors activation is responsible for i-LTP. Because of the NMDARs required for i-LTP, we further examined the critical roles of postsynaptic ß receptors downstream PKA regulation of NMDA receptor-mediated EPSCs (NMDA EPSC). We clarified that it is attributable to the direct effect of DEX on NMDA EPSC as mediated by PKA inactivation. These findings suggest that DEX can protect neurons from functional damage caused by a relatively mild degree of transient cerebral ischemia, and this effect is mediated by both presynaptic reduction of NE and glutamate release and postsynaptic suppression of NMDAR activation by ß receptors and downstream PKA regulation.

Antidepressant-like effects of alarin produced by activation of TrkB receptor signaling pathways in chronic stress mice.

Alarin is a newly identified member of the galanin family of neuropeptides. Until now, research on alarin is limited compared with other members of the galanin family. Unearthing the new biological effects of alarin and its unidentified receptor(s) interests us. We previously showed that alarin has an effect on depression-like behaviors, although the underlying mechanisms are not fully clarified. The present study verified the antidepressant-like effects of alarin using the unpredictable chronic mild stresses (UCMS) paradigm, and explored the mechanism that underlies antidepressant-like effects of alarin in mice. Previous research has shown that TrkB receptor-mediated ERK and AKT signaling pathways participate in depression pathophysiology. Therefore, we aimed to explore whether alarin improved depression-like behaviors by increasing activity of ERK and AKT pathways mediated by TrkB. Results showed that alarin significantly reduced immobility time in the forced swim test and latency to feed in the novelty suppressed feeding test. In addition, decreased p-ERK/ERK and p-AKT/AKT levels in the prefrontal cortex, hippocampus, olfactory bulb, and hypothalamus induced by UCMS were reversed by intracerebroventricular injection of alarin. Results suggested that alarin increased p-ERK/ERK and p-AKT/AKT 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, 3 days), followed by intracerebroventricular injection of alarin. This resulted in an absence of antidepressant-like effects, as well as no activation of ERK and AKT signaling pathways. Results demonstrate that alarin may exert antidepressant-like effects by targeting TrkB receptor-mediated ERK and AKT signal systems, which could help to identify the alarin receptor.

Dynamic metabolites profile of cerebral ischemia/reperfusion revealed by (1)H NMR-based metabolomics contributes to potential biomarkers.

Current metabolomic studies of ischemic brain mainly attach importance on a certain ischemic period, are lack of data about dynamic metabolites in ischemic stroke process, especially early period. Thus, in this study, (1)H NMR spectroscopy was used to investigate biochemical changes in the early stages of rats' focal cerebral ischemia reperfusion (I/R) injury. Serum samples of 0, 0.5, 1, 3, 6, 12, 24 h of reperfusion, based on multivariate data analyses, were tested to analyze the changing of metabolites during the early disease process. Partial least squares-discriminant analysis scores plots of the (1)H NMR data revealed clear differences among the experiment groups. Combination the results of loading plot and t-test, we found that 13 metabolites were changed significantly. Among that, malonic acid and glycine are the most noticeable variable metabolites. Dramatic changed malonic acid and glycine most probably served as biomarkers in this study. These findings help us understand the biochemical metabolite changes in early ischemic stroke stages, especially different periods. That may be conducive to distinguish at-risk individuals, benefit early diagnosis and understand the dynamic pathogenesis of early cerebral ischemia.

Alarin-induced antidepressant-like effects and their relationship with hypothalamus-pituitary-adrenal axis activity and brain derived neurotrophic factor levels in mice.

Alarin is a newly identified member of the galanin family of peptides. Galanin has been shown to exert regulatory effects on depression. Similar to galanin in distribution, alarin is also expressed in the medial amygdala and hypothalamus, i.e., regions interrelated with depression. However, it remains a puzzle whether alarin is involved in depression. Accordingly, we established the depression-like mouse model using behavioral tests to ascertain the possible involvement of alarin, with fluoxetine as a positive control. With the positive antidepressant-like effects of alarin, we further examined its relationship to HPA axis activity and brain-derived neurotrophic factor (BDNF) levels in different brain areas in a chronic unpredictable mild stress (CUMS) paradigm. In the acute studies, alarin produced a dose-related reduction in the immobility duration in tail suspension test (TST) in mice. In the open-field test, intracerebroventricular (i.c.v.) injection of alarin (1.0 nmol) did not impair locomotion or motor coordination in the treated mice. In the CUMS paradigm, alarin administration (1.0 nmol, i.c.v.) significantly improved murine behaviors (FST and locomotor activity), which was associated with a decrease in corticotropin-releasing hormone (CRH) mRNA levels in the hypothalamus, as well as a decline in serum levels of CRH, adrenocorticotropic hormone (ACTH) and corticosterone (CORT), all of which are key hormones of the HPA axis. Furthermore, alarin upregulated BDNF mRNA levels in the prefrontal cortex and hippocampus. These findings suggest that alarin may potentiate the development of new antidepressants, which would be further secured with the identification of its receptor(s).

Penehyclidine hydrochloride protects against oxygen and glucose deprivation injury by modulating amino acid neurotransmitters release.

Penehyclidine hydrochloride (PHC) is an anticholinergic agent, with only high degree of selectivity for M1 and M3 receptor subtypes. In this study, we investigated whether PHC could play a protective effect on hippocampal slice against oxygen and glucose deprivation (OGD), as well as related to the change of amino acid neurotransmitters release. Penehyclidine hydrochloride 2, 10, and 50 µM doses were adopted in the lactate dehydrogenase (LDH) leakage assay and triphenyl tetrazolium chloride (TTC) staining. The spontaneous miniature excitatory postsynaptic currents (mEPSCs) and amino acid neurotransmitters were detected by electrophysiology method and high-performance liquid chromatography (HPLC), respectively. Our study showed that PHC can lessen the LDH leakage ratio and tissue injury values according to TTC staining. Penehyclidine hydrochloride decreased the content of aspartate acid (Asp) and glutamate (Glu), and elevated the content of glycine (Gly) and gamma-aminobutyric acid (GABA). Ischemia increased the amplitude and frequency of the mEPSCs, but PHC obviously decreased the frequency and amplitude of mEPSCs. Thus, the study reveals the fact that PHC protects hippocampus slice against OGD injury by decreasing excitatory amino acids release and increasing inhibitory amino acids release.

A selective M1 and M3 receptor antagonist, penehyclidine hydrochloride, prevents postischemic LTP: involvement of NMDA receptors.

Our previous and other studies have confirmed that a selective M1 and M3 receptor antagonist, Penehyclidine hydrochloride (PHC), has neuroprotection activity in cerebral ischemia. However, the precise mechanisms of protection of PHC are still elusive. In this study we analyzed PHC-mediated neuroprotection on a model of brain ischemia (oxygen and glucose deprivation), named postischemic LTP (i-LTP). We found that the activation of NMDA receptor was required for the induction of i-LTP. Compared with scopolamine, PHC could prevent it due to selectively blocking M1 receptor, not M2 receptor, to decrease NMDAR activation. Our findings further showed that the inhibition of SK2 channels occluded the prevention of PHC on NMDAR activation. Furthermore, we confirmed that PHC exerted its roles through directly disinhibition of SK2 channels by blocking M1 receptor and subsequent restricting PKC activation. Moreover, our studies further revealed the critical roles of SK2 channels in i-LTP. Thus, the mechanisms of PHC in brain protection may be involved in suppression of NMDAR by regulation of SK2 channels. Our results obtained in effects of PHC on i-LTP further provided a better understanding of the therapy strategy during stroke and identified potential therapeutic targets to prevent development of ischemia.

(1)H NMR-based metabolomics exploring biomarkers in rat cerebrospinal fluid after cerebral ischemia/reperfusion.

In our study, metabolomics was used to investigate biochemical changes in the early stages of rats focal cerebral ischemia/reperfusion (I/R) injury. Cerebrospinal fluid (CSF) samples at 0, 0.5, 1, 3, and 6 h of reperfusion (n = 10), based on (1)H NMR spectroscopy and multivariate data analyses, were tested to analyze the changing of metabolites during the early disease process. Partial least squares-discriminant analysis scores plots of the (1)H NMR data revealed clear differences among the experiment groups. Combining the results of the loading plot and t-test, we found that twenty-seven metabolites were changed significantly (p < 0.05) in the CSF samples among the different groups. Among that, the potential biomarkers in CSF of ischemic rats were: acetic acid, 3-hydroxyisovaleric acid, 3-hydroxybutyric acid, choline, l-alanine, creatine, creatinine, glycine, pyruvic acid, glycerol, glutamic acid, d-fructose, l-lactic acid and acetone. These findings help us understand the biochemical metabolite changes in CSF of I/R rats in early stages. What's more, metabolomics may, therefore, have the potential to be developed into a clinically useful diagnostic tool of ischemic brain injury.

[The effect of oxymatrine on aging mice caused by D+ -galactose].

OBJECTIVE: To investigate the effect of oxymatrine (OMT) on aging mouse caused by D (+)-Galactose. METHODS: Healthy Kunming mouse were randomly divided into control, model and treated groups. The treated group was divided into three subgroups which were given oxymatrine 5 mg/kg, 10 mg/kg,20 mg/kg (OMT5, OMT10, OMT20). After 30 days of administration,the stand-jumping test and the Morris water maze test of each group were used to observe the learning and memory ability. After the behavior indicators test, 1-2 mL blood were got from ophthalmic vein plexus,and then got the serum by centrifuge. The super oxide dismutase (SOD) activity and malondialdehyde (MDA) content were determined by SOD and MDA kit. RESULTS: In the water maze test, the model group was significantly longer in the latency than that of the control group (P < 0.01), and the number of the crossing platforms was significantly reduced (P < 0.01), but compared with the model group, the OMT5, OMT10, OMT20 groups were significantly shorter in the latency to the end (P < 0.05, P < 0.05, P < 0.01), and the number of the crossing platforms was significantly increased (P < 0.05, P < 0.05, P < 0.01); In the stand-jumping test,the OMT groups all could extend the platform latency and reduce the error reaction times, the learning ability of the OMT groups was better than the model group, but the statistical tests failed (P > 0.05); In the serum biochemical indicators test,the SOD activity of the model group was significantly lower than that of the control group (P < 0.01), but the SOD activity of OMT5, OMTIO, OMT20 groups was significant higher than that of the model group (P < 0.05, P < 0.05, P < 0.01); The MDA content of the model group was significantly higher than that of the control group (P < 0.01) but the MDA content of OMT5, OMT1O, OMT20 groups was significant lower than that of the model group (P < 0.05, P < 0.05, P < 0.01). CONCLUSION: Oxymatrine could improve the learning and memory ability of aging mice caused by intraperitoneal injection D(+)-Galactose,and the anti-aging effect is related to the oxygen free radicals and reducing the lipid peroxidation.

Regulated hypoxia/reperfusion-dependent modulation of ERK1/2, cPLA2, and Bcl-2/Bax: a potential mechanism of neuroprotective effect of penehyclidine hydrochloride.

The activation of event-related kinase 1/2 (ERK1/2) and cytosolic phospholipaseA2 (cPLA2), which can aggravate hypoxia/reoxygenation (H/R) damage related to their downstream Bcl-2/Bax and Caspase-3 pathway, plays a key role in H/R. The M1 receptors could be responsible for activation of ERK1/2. Thus, it seems that the regulation of M1 receptors mediated the ERK1/2; cPLA2-mediated Bcl-2/Bax pathway may be a significant responsive signal in H/R. Penehyclidine hydrochloride (PHC) is an anticholinergic agent with high degree of selectivity for M1 and M3 receptor subtypes, it is reported that PHC has a protective effect against H/R damage. Here we hypothesize and demonstrate that PHC could downregulate the expression of pERK1/2, cPLA2, and Caspase-3, increased the ratio of Bcl-2/Bax. This study may widen the application of PHC and therapeutic agents of stroke.

Improvement of contractile function in isolated cardiomyocytes from ischemia-reperfusion rats by ginkgolide B pretreatment.

Ginkgo biloba extract is an important natural product for treatment of cerebral and cardiovascular diseases, whereas ginkgolide B (GB) is a main component of it. Its effects on ischemic heart and ventricular contractile function in Sprague-Dawley male rats are unclear yet. In the present study, we investigated the function of isolated hearts subjected to ischemia-reperfusion (IR) with or without GB pretreatment by using Millar transducer instruments. We also tested the left ventricular cardiomyocyte shortening amplitude after IR with different concentrations of GB pretreatment for 0.1, 1.0, 2.0, 5.0, and 10.0 microM. The infarct size was tested by triphenyltetrazolium chloride. The release of lactate dehydrogenase (LDH) in the coronary effluent was determined with LDH kit. The expressions of Bcl-2 and Bax were assessed by Western blotting. We found that GB improved the function of left ventricle from IR injury and decreased infarct size and the release of LDH. The cardiomyocyte shortening amplitude depended on different concentrations of GB, which increased significantly at 2.0 microM GB (P < 0.01). The expression of protein Bcl-2 was upregulated by GB and the ratio of Bax to Bcl-2 was decreased by GB. Our results showed that GB can partly prevent IR injury in rat heart.

Oestrogen changed cardiomyocyte contraction and beta-adrenoceptor expression in rat hearts subjected to ischaemia-reperfusion.

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, oestrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and oestrogen replacement on ventricular myocyte contractile function and expression of beta-adrenoceptors (beta-ARs). Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham operation (Sham). A subgroup of OVX rats received oestrogen (E2) replacement (40 microg kg(-1) day(-1)) for 4 weeks. Cardiomyocyte shortening was evaluated in basal conditions and in the presence of isoprenaline (ISO). The expression of beta-ARs was assessed by Western blotting. The presence of lactate dehydrogenase (LDH) activity in the coronary effluent was determined. Ovariectomy promoted body weight gain associated with reduced serum E2 and uterine weight, all of which were abolished by treatment with E2. Ovariectomy increased the amplitude of both basal and ISO-stimulated contractions, increased LDH release, upregulated beta1-AR expression and downregulated beta2-AR expression, all of which were restored by treatment with E2. A beta1-AR antagonist, CGP20712A, but not a beta2-AR antagonist, ICI118,551, significantly decreased the amplitude of ventricular myocyte shortening. Oestrogen decreased cardiomyocyte contraction and the expression of beta1-AR, and increased expression of beta2-AR, and all these effects were abolished by the E2 receptor antagonist, ICI182,780. These data suggest that oestrogen plays a cardioprotective role in female rat hearts subjected to ischaemia-reperfusion injury, and the effects of oestrogen are associated with decreased cardiomyocyte contraction and expression of beta1-AR, and increased expression of beta2-AR.

[Effect of gamma-hydroxybutyric acid receptor on focal cerebral ischemia-reperfusion injury in rats].

This study is to investigate the effect of gamma-hydroxybutyric acid receptor (GHBR) on focal cerebral ischemia-reperfusion injury in rats and its mechanism. NCS-356 (the agonist of GHBR) and NCS-382 (the antagonist of GHBR) were adopted as the tool medicine. The ripe male Sprague-Dawley rats weighing 240 - 280 g were randomly divided into seven groups: sham operation group (sham), ischemia-reperfusion group (Isc/R), NCS-356 160 microg x kg(-1) group (N1), NCS-356 320 microg x kg(-1) group (N2), NCS-356 640 microg x kg(-1) group (N3), NCS-382 640 microg x kg(-1) + NCS-356 640 microg x kg(-1) group (NCS-382 + N3), and nimodipine (Nim) 600 microg x kg(-1) group. The middle cerebral artery occlusion (MCAO) model referring to Longa's method with modifications was adopted. The effect of GHBR on behavioral consequence of MCAO rats was studied after 2 h of ischemia-reperfusion. After 24 h of ischemia-reperfusion, part of animals were used to measure the cerebral infarction volume by TTC staining; ischemic cortex of another part of animals were used to measure the content of intracellular free calcium by flow cytometry, the tNOS, iNOS activity and the content of NO by spectrophotometric method, the content of cGMP by radioimmunoassay. The neurological function score and infarction volume rate in Isc/R group rats increased significantly than that in sham group; The content of intracellular calcium ([Ca2+]) of cortex neuron and cGMP, the activities of tNOS and iNOS, and the content of NO in Isc/R group were higher than that in sham group obviously (P < 0.01); These consequence we mentioned of N1, N2, N3 and Nim group were lower than that of Isc/R. NCS-382 + N3 group could significantly antagonize the above effect of N3. Thus, NCS-356 has protective effects against ischemia-reperfusion brain injury by activating GHBR. The neuroprotective effect of GHBR is related with decreasing the content of [Ca2+]i, NO, cGMP and tNOS, iNOS activity in MCAO rats.

Fumagillin treatment of hepatocellular carcinoma in rats: an in vivo study of antiangiogenesis.

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats. METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg x d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats. RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046). CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity.

Prognostic significance of preoperative circulating vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma: a prospective study.

AIM: To investigate the prognostic value of vascular endothelial growth factor messenger RNA (VEGF mRNA) in the peripheral blood (PB) of patients with hepatocellular carcinoma (HCC) undergoing curative resection. METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA in the PB was determined prospectively in 50 controls and in 50 consecutive patients undergoing curative resection for HCC. RESULTS: Among the isoforms of VEGF mRNA, VEGF(165) and VEGF(121) were expressed. By multivariate analysis, a higher level of VEGF(165) in preoperative PB correlated with a risk of HCC recurrence with borderline significance (P=0.050) and significantly with recurrence-related mortality (P=0.048); while VEGF(121) did not. Other significant predictors of HCC recurrence included cellular dedifferentiation (P=0.033), an absent or incomplete capsule (P=0.020), vascular permeation (P=0.018), and daughter nodules (P=0.006). The other significant parameter of recurrence related mortality was cellular dedifferentiation (P=0.053). The level of circulating VEGF mRNA, however, did not significantly correlate with tumor size, cellular differentiation, capsule, daughter nodules, vascular permeation, necrosis and hemorrhage of tumors. CONCLUSION: The preoperative level of circulating VEGF mRNA, especially isoform VEGF(165), plays a significant role in the prediction of postoperative recurrence of HCC.

Is the vascular endothelial growth factor messenger RNA expression in resectable hepatocellular carcinoma of prognostic value after resection?

AIM: To study whether vascular endothelial growth factor messenger RNA (VEGF mRNA) in the hepatocellular carcinoma (HCC) tissues obtained after curative resection has a prognostic value. METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in liver tissues of 50 controls and in HCC tissues of 50 consecutive patients undergoing curative resection for HCC. RESULTS: Among the isoforms of VEGF mRNA, VEGF(165) and VEGF(121) were expressed. By multivariate analysis, a higher level of VEGF(165) in HCC tissue correlated with a significant risk of HCC recurrence (P=0.038) and significantly with recurrence-related mortality (P=0.045); while VEGF(121) did not. Other significant predictors of HCC recurrence included cellular dedifferentiation (P=0.033), an absent or incomplete capsule (P=0.020), vascular permeation (P=0.018), and daughter nodules (P=0.006). The other significant variables of recurrence related mortality consisted of vascular permeation (P=0.045), and cellular dedifferentiation (P=0.053). The level of VEGF mRNA in HCC tissues, however, did not significantly correlate with tumor size, cellular differentiation, capsule, daughter nodules, vascular permeation, necrosis and hemorrhage of tumors. CONCLUSION: The expression of VEGF mRNA, especially isoform VEGF(165), in HCC tissues, may play a significant and independent role in the prediction of postoperative recurrence of HCC.

[The protective effects of melatonin on global cerebral ischemia-reperfusion injury in gerbils].

AIM: To investigate the effects of melatonin (MT) on histology and behavioral tests during global cerebral ischemia-reperfusion in gerbils. METHODS: Global cerebral ischemia was induced by occluding the bilateral common carotid arteries for 10 min in gerbils. Three doses of MT were administrated intraperitoneally 30 min prior to the onset of ischemia. Locomotor activity was measured by using the open field method 3 and 7 days after the ischemic episode. T maze test was carried out 4, 5 and 6 days after ischemia to assess the working memory of gerbils. Neuronal damage was assessed in CA1 pyramidal layer of gerbil hippocampus and evaluated 7 days after ischemia. RESULTS: MT significantly reversed the locomotor activity increases, ameliorated learning and working memory deficit, and reduced the extent of CA1 hippocampal pyramidal cells injury after transient global cerebral ischemia in the Mongolian gerbil. CONCLUSION: MT provides significantly protective effect against both histological and behavioral consequences of global cerebral ischemia-reperfusion injury in gerbils.