Zhong Pei

Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction.

Cerebral infarction can cause secondary degeneration of thalamus and delay functional recovery. However, the mechanisms underlying secondary degeneration are unclear. The present study aimed to determine the occurrence and contribution of autophagy to the thalamic degeneration after cerebral infarction. Focal cerebral infarction was induced by distal middle cerebral artery occlusion (MCAO). Autophagic activation, Beclin 1 expression and amyloid β (Aβ) deposits were determined by immunofluorescence, immunoblot and electron microscopy. Secondary damage to thalamus was assessed with Nissl staining and immunofluorescence analysis. Apoptosis was determined using TUNEL staining. The contribution of autophagy to the secondary damage was evaluated by shRNA-mediated downregulation of Beclin 1 and the autophagic inhibitor, 3-methyladenine (3-MA). The potential role of Aβ in autophagic activation was determined with N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The results showed that the conversion of LC3-II, the formation of autophagosomes, and the levels of activated cathepsin B and Beclin 1 were significantly increased in the ipsilateral thalamus at 7 and 14 days after MCAO (p < 0.05 or 0.01). Both Beclin 1 knockdown and 3-MA treatment significantly reduced LC3-II conversion and autophagosome formation, which were accompanied by obvious decreases in neuronal loss, gliosis and apoptosis in the ipsilateral thalamus (p < 0.05 or 0.01). Additionally, DAPT treatment markedly reduced Aβ deposits, which coincided with decreases in LC3-II conversion and autophagosome formation (p < 0.01). These results suggest that inhibition of autophagy by Beclin 1 knockdown can attenuate the secondary thalamic damage after focal cerebral infarction. Furthermore, Aβ deposits may be involved in the activation of autophagy.

DL-3-n-butylphthalide protects endothelial cells against oxidative/nitrosative stress, mitochondrial damage and subsequent cell death after oxygen glucose deprivation in vitro

DL-3-n-butylphthalide (NBP) has been used for stroke treatment in China for years. Recently, we found that NBP can reduce the incidence of stroke and have protective action on cerebral microvessels, suggesting a direct action of NBP on endothelial cells. However, it is difficult to evaluate the direct action of NBP on endothelial cells in vivo because of the interactions of endothelial cells with other types of neuronal cells. Therefore, we investigated whether NBP protects against oxygen glucose deprivation (OGD)-induced cell injury in an immortalized human umbilical vein endothelial cells (HUVEC) in vitro. Cells were exposed to OGD, leading to endothelial damage. Endothelial injury was assessed by measuring MTT and the changes in chromatin morphology. Mitochondrial superoxide, mitochondrial membrane potential and mitochondrial morphology were assessed using MitoSOX Red. Rhodamine 123 and MitoTracker, respectively. Nitrosative stress was assessed by measuring the production of peroxynitrite. The activity of superoxide dismutase (SOD) is evaluated using SOD assay kit-WST. The expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was assessed at the protein level by immunofluorescence and Western blotting. NBP at doses between 0.01 and 100 micromol/L dose-dependently protected against OGD-induced cell death. In addition, NBP attenuated OGD-induced mitochondria superoxide, cellular formation of peroxynitrite, and decrease in SOD activity, mitochondria fragmentation and loss of mitochondrial membrane potential. In parallel, NBP enhanced OGD-induced HIF-1alpha expression. This study demonstrates that NBP can protect HUVEC against OGD-induced oxidative/nitrosative stress, mitochondrial damage and subsequent cell death. This protective effect is, at least in part, associated with its enhancement on OGD-induced HIF-1alpha expression.

Design and Synthesis of Novel Xyloketal Derivatives and Their Vasorelaxing Activities in Rat Thoracic Aorta and Angiogenic Activities in Zebrafish Angiogenesis Screen

A novel series of xyloketal derivatives (1-21) were designed and prepared. The majority of the compounds demonstrated vasorelaxation action on 60 mM KCl-induced contractions rat isolated aortic rings in a concentration-dependent manner, and the action is mediated by both endothelium-independent and endothelium-dependent mechanisms. Compounds 9, 12, 13, 14, 15, and 19 showed higher vasorelaxation activities comparing with the lead compound 3. In addition, these derivatives had potential protective action against oxLDL-induced endothelial oxidative injury and enhanced NO production in HUVECs without toxic effects. The NO release was completely inhibited by eNOS inhibitor L-NAME. Furthermore, 3 significantly promoted the angiogenesis in zebrafish in a concentration-dependent manner at 0.1, 1, and 10 muM. Compounds 9, 12, 14, 16, 20, and 21 exhibited stronger angiogenic activities than 3. Therefore, xyloketal derivatives are unique compounds with multiple pharmacological properties and may have potential implications in the treatment of cardiovascular diseases.

Longitudinal Investigations on the Anterograde and Retrograde Degeneration in the Pyramidal Tract following Pontine Infarction with Diffusion Tensor Imaging

Background: Secondary degeneration following supratentorial stroke has been detected by some studies using diffusion tensor imaging (DTI), but the anterograde and retrograde degeneration in pyramidal tract after pontine infarction and its potential clinical significance are not well understood. Methods: Fourteen patients with a recent pontine infarct underwent three DTIs at week 1, week 4, and week 12 after onset. Fourteen age- and gender-matched controls underwent DTI once. Mean diffusivity and fractional anisotropy (FA) were measured. Neurological deficit, motor deficit and life independence were assessed with the NIH Stroke Scale, Fugl-Meyer scale and Barthel index, respectively, 2 h before each DTI examination. Results: FA values at the ipsilateral medulla and the proximal portion of the pyramidal tract, including centrum semiovale, internal capsule and cerebral peduncle, significantly decreased progressively from week 1 to week 12 (p < 0.01). The NIH Stroke Scale decreased; Fugl-Meyer scale and Barthel index increased significantly over the time points (p < 0.01). The absolute values of percent reduction of FA value at the ipsilateral medulla and the proximal portion of pyramidal tract correlated negatively with the absolute values of percent reduction of the NIH Stroke Scale and percent increase of the Fugl-Meyer scale. Conclusions: Progressive anterograde and retrograde degeneration in pyramidal tract revealed by DTI may hinder the process of neurological recovery after a pontine infarct. To confirm the clinical significance, future studies with a longer observation period and a larger sample size of patients with more homogeneous pontine infarcts are still needed.

Reduction of β-Amyloid Deposits by γ-Secretase Inhibitor is Associated with the Attenuation of Secondary Damage in the Ipsilateral Thalamus and Sensory Functional Improvement after Focal Cortical Infarction in Hypertensive Rats

Abnormal β-amyloid (Aβ) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aβ deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aβ accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aβ, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aβ burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aβ deposits are associated with the secondary thalamic damage. Reduction of thalamic Aβ by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.

Autophagosomes accumulation is associated with β-amyloid deposits and secondary damage in the thalamus after focal cortical infarction in hypertensive rats

J. Neurochem. (2012) 120, 564–573.

Neurogenesis and angiogenesis within the ipsilateral thalamus with secondary damage after focal cortical infarction in hypertensive rats

Neurogenesis and angiogenesis in the subventricular zone and peri-infarct region have been confirmed. However, newly formed neuronal cells and blood vessels that appear in the nonischemic ipsilateral ventroposterior nucleus (VPN) of the thalamus with secondary damage after stroke has not been previously studied. Twenty-four stroke-prone renovascular hypertensive rats were subjected to distal right middle cerebral artery occlusion (MCAO) or sham operation. 5′-Bromo-2′-deoxyuridine (BrdU) was used to label cell proliferation. Rats were killed at 7 or 14 days after the operation. Neuronal nuclei (NeuN), OX-42, BrdU, nestin, laminin+, BrdU+/nestin+, BrdU+/NeuN+, nestin+/GFAP+(glial fibrillary acidic protein), and BrdU+/laminin+ immunoreactive cells were detected within the ipsilateral VPN. The primary infarction was confined to the right somatosensory cortex. Within the ipsilateral VPN of the ischemic rats, the number of NeuN+ neurons decreased, the OX-42+ microglia cells were activated, and BrdU+ and nestin+ cells were detected at day 7 after MCAO and increased in number at day 14. Moreover, BrdU+/nestin+ cells and BrdU+/NeuN+ cells were detected at day 14 after MCAO. In addition, the ischemic rats showed a significant increase in vascular density in the ipsilateral VPN compared with the sham-operated rats. These results suggest that secondary damage with neurogenesis and angiogenesis of the ipsilateral VPN of the thalamus occurs after focal cortical infarction.

Marine compound Xyloketal B protects PC12 cells against OGD-induced cell damage.

Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Recently, we have demonstrated that Xyloketal B is an antioxidant and can protect against oxidized low density lipoprotein (LDL)-induced cell injury. In the present study, we investigated whether Xyloketal B can protect against ischemia-induced cell injury in an in vitro oxygen glucose deprivation (OGD) model of ischemic stroke in PC12 cells. We found that Xyloketal B could directly scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and protect PC12 cells against OGD insult. Furthermore, Xyloketal B alleviated OGD-induced mitochondria superoxide, mitochondria fragmentation and GTPase dynamin-related protein 1 (Drp1) overexpression as well as reduction of mitochondrial membrane potential. All together, the present study demonstrates that Xyloketal B protects PC12 cells against OGD-induced cell injury and that the anti-oxidative property and protective action on mitochondria may account for its neuroprotective actions.

Enhanced angiogenesis with dl-3n-butylphthalide treatment after focal cerebral ischemia in RHRSP.

Appropriate restoration of blood flow via angiogenesis is critical for the recovery from ischemic stroke. Previously, we reported that treatment with dl-3n-butylphthalide (NBP) increases the number of local potent cerebral microvessels. However, the underlying mechanism remained unclear. The present study was conducted to test whether NBP enhances post-ischemic cerebral angiogenesis via vascular endothelial growth factor (VEGF) and hypoxia induced factor-1 alpha (HIF-1 alpha). Stroke-prone renovascular hypertensive rats (RHRSP) were used to create middle cerebral artery occlusion (MCAO) model. NBP was given 80 mg/kg per d for 10 consecutive days, starting 12, 24, 48 and 72 h respectively after MCAO. Neurological function was assessed daily and infarct volume as well as the expressions of CD31, VEGF, HIF-1 alpha and bFGF was detected 13 days after MCAO. The administration of NBP starting within 24 h after MCAO enhanced recovery of neurobehavioral function, reduced infarct volume, increased the quantity of CD31 positive vessels, and up-regulated expressions of VEGF and HIF-1 alpha. These findings suggest that treatment with NBP within 24 h post-ischemic stroke rescues brain tissue by enhancing angiogenesis associated with up-regulation of VEGF and HIF-1 alpha expressions.

Melatonin protects against rotenone-induced cell injury via inhibition of Omi and Bax-mediated autophagy in Hela cells

Abstract:  Parkinson’s disease is the second most common neurodegenerative disease, and environmental toxins such as rotenone play an important role in causing degeneration of dopaminergic neurons. Melatonin, a major secretory product of pineal, is recently reported to protect against rotenone‐induced cell death in animal models. Yet, the mechanism involved in this protection needs to be elucidated. Here, we report that rotenone treatment (0–100 μm) decreased cell survival of Hela cells in a dose‐dependent manner. At concentrations ranging from 0.1 to 100 μm, rotenone induced a dose‐dependent increase in the expression of microtubule‐associated protein 1 light chain 3 (LC3)‐II, a protein associated with the autophagosomal membrane. Knockdown of Bax or Omi using shRNA inhibited 1 μm rotenone‐induced autophagy. To determine whether melatonin would protect cells against rotenone‐induced cell death and autophagy, we pretreated Hela cells with 250 μm melatonin for 24 hr in the presence of rotenone. Melatonin inhibited Bax expression and the release of the omi/HtrA2 into the cytoplasm induced by 1 μm rotenone. Melatonin 250 μm treatment also suppressed cell death induced by 0.1–100 μm rotenone and protected against the formation of LC3‐II in cells exposed to 1 μm rotenone. This work demonstrates a novel role for melatonin as a neuroprotective agent against rotenone.