Zhaofu Chi

Ghrelin protects against cell death of hippocampal neurons in pilocarpine-induced seizures in rats

Ghrelin, a 28-amino-acid peptide, is mainly secreted by the stomach. Evidence has shown ghrelin to have neuroprotective effects. However, whether ghrelin protects hippocampal neurons against cell death in pilocarpine-induced seizures is unknown. We used Nissl staining to show that ghrelin attenuated the neuronal loss caused by pilocarpine-induced seizures in the hippocampus. Ghrelin exerted the protective action through regulating the phosphatidylinositol-3-kinase and Akt pathway. Moreover, ghrelin treatment reversed the decreased ratio of Bcl-2 to Bax induced by seizures while inhibiting the activated caspase-3. Ghrelin can inhibit hippocampal neuronal damage caused by pilocarpine-induced seizures, which might have therapeutic value in seizures.

Role of mitochondrial fission in neuronal injury in pilocarpine-induced epileptic rats

Mitochondrial fission has been reported to be involved in oxidative stress, apoptosis and many neurological diseases. However, the role of mitochondrial fission in seizures, which could induce oxidative stress and neuronal loss, remains unknown. In this study, we used pilocarpine to elicit seizures in rats. Meanwhile, we used mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of mitochondrial fission protein dynamin-related protein1 (Drp1), to suppress mitochondrial fission in epileptic model of rats in vivo. We found that mitochondrial fission was increased after seizures and the inhibition of mitochondrial fission by mdivi-1 significantly attenuated oxidative stress and reduced neuronal loss after seizures, shown by the decreased 8-hydroxy deoxyguanosine (8-oHdG) content, the increased superoxide dismutase (SOD) activity, the reduced expression of cytochrome c and caspase3 and the increased surviving neurons in the hippocampus. These results indicated that mitochondrial fission is up-regulated after seizures and the inhibition of mitochondrial fission is protective against neuronal injury in seizures, the underlying mechanism may be through the mitochondria/reactive oxygen species (ROS)/cytochrome c pathway.

Increased expressions of TLR2 and TLR4 on peripheral blood mononuclear cells from patients with Alzheimer's disease

BACKGROUND Toll-like receptors 2 (TLR2) and TLR4 are involved in the microglia-mediated inflammatory response, Aβ plaque formation and Aβ clearance in Alzheimers disease (AD). Our previous studies have shown that variants in the TLR2 and TLR4 genes are associated with the risk of AD. Therefore, we hypothesize that there may be significant changes in TLR2 and TLR4 expressions on peripheral blood mononuclear cells (PBMCs) from patients with AD when compared to healthy control subjects. METHODS Sixty patients with late-onset AD (LOAD) and 60 healthy controls matched for sex and age were recruited. Flow cytometry (FCM) was used to detect expressions of TLR2 and TLR4 proteins and real-time quantitative RT-PCR was performed to determine TLR2 and TLR4 mRNAs. RESULTS Compared with controls, expressions of TLR2 and TLR4 mRNAs were up-regulated in LOAD patients (TLR2/beta-actin mRNA: 0.390±0.204 versus 0.281±0.167, P<0.01; TLR4/beta-actin mRNA: 0.503±0.195 versus 0.322±0.183, P<0.01). The proteins levels were higher in LOAD patients than in controls (TLR2: 97.12±1.67% versus 41.07±18.44%, P<0.01, TLR4: 66.56±23.74% versus 14.83±4.31, P<0.01). In both cases, either AD or control group, TLR2 and TLR4 mRNAs expressions were positively correlated with the levels of proteins (TLR2: r=0.980 and 0.976,P<0.01; TLR4: r=0.938 and 0.970, P<0.01), respectively. There were significant negative correlations between TLR levels and MMSE score (TLR2: r=-0.32; P=0.01; TLR4: r=-0.29; P=0.02). In addition, CC genotype can increase the expression of TLR4 in AD patients. CONCLUSION This study gives the first evidence that expressions of TLR2 and TLR4 in PBMCs were markedly elevated in LOAD patients.

The role of p38 MAPK in valproic acid induced microglia apoptosis

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, induces apoptosis in microglia, but the underlying mechanism by which microglia apoptosis in response to VPA is not yet known. In this study, we found that the mitochondrial pathway played an important role in VPA-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. In addition, VPA increased the level of phospho-p38 mitogen-activated protein kinase (MAPK), but had no effects on phospho-ERK and phospho-JNK MAPKs. Moreover, p38 inhibitor SB203580 strongly inhibited VPA-induced apoptosis and caspase-3 activation. Taken together, our results clearly demonstrated that VPA could induce apoptosis of microglia via p38 MAPK and mitochondrial apoptosis pathway.

Glycogen synthase kinase-3 and p38 MAPK are required for opioid-induced microglia apoptosis

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. We previously reported that opioids inhibit cell growth and trigger apoptosis in lymphocytes. However, the underlying mechanism by which microglia apoptosis in response to opioids is not yet known. In this study, we show that morphine induces microglia apoptosis and caspase-3 activation in an opioid-receptor dependent manner. Morphine decreased the levels of microglia phosphorylated Akt (p-Akt) and p-GSK-3β (glycogen synthase kinase-3 beta) in an opioid-receptor dependent manner. More interestingly, GSK-3β inhibitor SB216763 significantly increases morphine-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. Moreover, co-treatment of microglia with SB216763 and morphine led to a significant synergistic effect on the level of phospho-p38 mitogen-activated protein kinase (MAPK). In addition, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly inhibited morphine-induced apoptosis and caspase-3 activation. Taken together, our data clearly demonstrates that morphine-induced apoptosis in microglial cells, which is mediated via GSK-3β and p38 MAPK pathways.

Resveratrol pre-treatment reduces early inflammatory responses induced by status epilepticus via mTOR signaling

Resveratrol is indicated to be involved in neuroprotection and anti-inflammation in epileptic rats. The molecular mechanism is still not fully understood. In this study, we investigated the role of resveratrol in nuclear factor-kappa B associated inflammatory responses induced by status epilepticus. Our data showed that seizures activated mammalian target of rapamycin (mTOR), increased the activity of nuclear factor-kappa B and promoted the expressions of inflammatory molecules including inducible nitric oxide synthase, cyclooxygenase-2 and interleukin-1β. Futhermore, resveratrol significantly inhibited the activation of nuclear factor-kappa B and the production of proinflammatory molecules via mTOR pathway. Additionally, we also proved that the inhibition of mTOR signal by resveratrol was mostly attributed to AMP-activated kinase (AMPK) activation. Altogether, our results suggest that resveratrol suppresses inflammatory responses induced by seizures partially via AMPK/mTOR pathway.

Autophagy is upregulated in rats with status epilepticus and partly inhibited by Vitamin E

Autophagy, a process of bulk degradation of cellular constituents through autophagosome-lysosomal pathway, is enhanced during oxidative stress. Whether autophagy is induced during status epilepticus (SE), which induces an excess production of reactive oxygen species (ROS) and leads to oxidative stress, is not established. We also sought to determine if pretreatment with Vitamin E reduced autophagy. We used pilocarpine to elicit SE in rats. The ratio of LC3 II to LC3 I and beclin 1 were used to estimate autophagy. We found that ratio of LC3 II to LC3 I and beclin 1 increased significantly at 2, 8, 16, 24 and 72 h, peaking at 24 h after SE onset. Pretreatment with Vitamin E partially inhibited autophagy by reducing LC3 II formation and de novo synthesis of beclin 1 at 24 h after seizures. These data show that autophagy is increased in rats with pilocarpine-induced SE, and Vitamin E have a partial inhibition on autophagy.

Ascorbic acid ameliorates seizures and brain damage in rats through inhibiting autophagy

Oxidative stress is a mechanism of cell death induced by seizures. Antioxidant compounds have neuroprotective effects due to their ability to inhibit free radical production. Autophagy is a process in which cytoplasmic components such as organelles and proteins are delivered to the lysosomal compartment for degradation, and plays an essential role in the maintenance of cellular homeostasis. The activity of autophagy is enhanced during oxidative stress. The objectives of this work were first to study the inhibitory action of antioxidant ascorbic acid on behavioral changes and brain damage induced by high doses of pilocarpine, then to study the effect of ascorbic acid on oxidative stress (MDA and SOD were used to estimate oxidative stress) and activated autophagy (beclin 1 was used to estimate autophagy) induced by seizures, aiming to further clarify the mechanism of action of this antioxidant compound. In order to determinate neuroprotective effects, we studied the effects of ascorbic acid (500 mg/kg, i.p.) on the behavior and brain lesions observed after seizures induced by pilocarpine (340 mg/kg, i.p., P340 model) in rats. Ascorbic acid injections prior to pilocarpine suppressed behavioral seizure episodes by increasing the latency to the first myoclonic, clonic and tonic seizure and decreasing the percentage of incidence of clonic and tonic seizures as well as the mortality rate. These findings suggested that oxidative stress can be produced and autophagy is increased during brain damage induced by seizures. In the P340 model, ascorbic acid significantly decreased cerebral damage, reduced oxidative stress and inhibited autophagy by reducing de novo synthesis of beclin 1. Antioxidant compound can exert neuroprotective effects associated with inhibition of free radical production and autophagy. These results highlighted the promising therapeutic potential of ascorbic acid in treatment for seizures.

Sirtuin 1 activation enhances the PGC-1α/mitochondrial antioxidant system pathway in status epilepticus

Sirtuin 1 (SIRT1) regulates numerous neuronal processes, including metabolism, antioxidation and aging, through activation of peroxisome proliferator-activated receptor coactivator 1-α (PGC-1α), an upstream regulator of mitochondrial biogenesis and function. However, the role of SIRT1 in the oxidative stress induced by seizures has yet to be elucidated. The present study aimed to investigate whether SIRT1 was involved in the activation of the PGC-1α/mitochondrial antioxidant system following status epilepticus (SE) in rats. The data demonstrated that SIRT1 expression and activity were enhanced in the rat hippocampus following SE. SIRT1 inhibition effectively blocked the SE-associated increase in PGC-1α and mitochondrial antioxidant enzymes, including superoxide dismutase 2 (SOD2) and uncoupling protein 2 (UCP2). Additionally, it was also demonstrated that the activation of SIRT1 enhanced mitochondrial electron transport chain complex I activity and increased ATP content. In conclusion, the present results suggest that SIRT1 activation may alleviate mitochondrial oxidative stress induced by seizures partially via PGC-1α signaling.

Vitamin E inhibits activated chaperone-mediated autophagy in rats with status epilepticus

Seizures and status epilepticus induce an excessive production of reactive oxygen species leading to oxidative stress. Vitamin E, a classic antioxidant, has a neuroprotective effect on rats with seizures by regulating reactive oxygen species production. The activity of chaperone-mediated autophagy, a selective pathway for the degradation of cytosolic proteins in lysosomes, is enhanced during oxidative stress. Whether chaperone-mediated autophagy is induced during status epilepticus is not established. To address this problem, we used pilocarpine to elicit status epilepticus in rats. Lysosome-associated membrane protein 2a was used to estimate chaperone-mediated autophagy. We showed that compared to control animals, lysosome-associated membrane protein 2a at lysosomal membranes increased significantly in rats at 8 h, 16 h, and 24 h after induction of status epilepticus, which directly correlated with chaperone-mediated autophagy activity. Since reactive oxygen species are believed to be important in the pathogenesis of status epilepticus and are essential for the process of chaperone-mediated autophagy, we also sought to determine if pretreatment with vitamin E reduced chaperone-mediated autophagy. Pretreatment with vitamin E reduced oxidative stress and partially inhibited chaperone-mediated autophagy in brain at 24 h after status epilepticus versus vehicle. Taken together, these data show that chaperone-mediated autophagy is increased in rats with pilocarpine-induced status epilepticus through upregulation of de novo synthesis of lysosome-associated membrane protein 2a. Antioxidants such as vitamin E may partially inhibit activated chaperone-mediated autophagy.