Shucai Ling

Autophagy Upregulation and Apoptosis Downregulation in DAHP and Triptolide Treated Cerebral Ischemia

It has previously been demonstrated that ischemic stroke activates autophagy pathways; however, the mechanism remains unclear. The aim of this study is to further investigate the role that autophagy plays in cerebral ischemia. 2, 4-diamino-6-hydroxy-pyrimidine (DAHP), for its nitric oxide synthase (NOS) inhibiting neuroprotective effect, and triptolide (TP), for its anti-inflammatory property, were selected to administer pre middle cerebral artery occlusion (MCAO). The drugs were administered 12 hours prior to MCAO. Both magnetic resonance imaging (MRI) and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the drugs reduce the area of infarction. Immunoblotting analysis revealed increases in Beclin-1 and myeloid cell leukelia-1(Mcl-1) in treated rats. This could be a contributing factor to the reduction in autophagy induced damage. Immunochemistry and western blot showed that mTOR expression in treated rats was marginally different 24 h after injury, and this could also be significant in the mechanism. Furthermore, terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) staining proved that the drugs are effective in reducing apoptosis. The upregulation of Beclin-1 and Mcl-1 and downregulation of Bcl-2, caspase-3, and the Bcl-2/Beclin-1 ratio infer that the neuroprotective effect of DAHP and TP act via the mediation of autophagy and apoptosis pathways.

Neuroprotective effects of DAHP and Triptolide in Focal Cerebral Ischemia via Apoptosis Inhibition and PI3K/Akt/mTOR pathway Activation

Triptolide (TP), one of the major active components of the traditional Chinese herb Tripterygium wilfordii Hook F, and 2, 4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of tetrahydrobiopterin (BH4) synthesis, have been reported to have potent anti-inflammatory and immunosuppressive properties. However, the protective effects of TP and DAHP on cerebral ischemia have not been reported yet. In this study, we investigated the neuroprotective effects of TP and DAHP in a middle cerebral artery occlusion (MCAO) rat model. Furthermore, we examined whether the neuroprotective effects of TP and DAHP were associated with the inhibition of apoptosis through suppressing BH4 and inducible NOS (iNOS) synthesis or the activation of the phosphoinositide-3-kinase/serine-threonine kinase Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway. Our results showed that pretreatments with TP (0.2 mg/kg) and DAHP (0.5 g/kg) significantly reduced ischemic lesion volume, water content, and neuronal cell death compared with the vehicle MCAO rats. In addition, compared with the MCAO group, TP, and DAHP pretreatment groups significantly reduced astrocyte numbers, caspase-3, cleaved caspase-3, and NF-κB up-regulation, while increased Bcl-2 expression. Moreover, protein expressions of PI3K, Akt, and mTOR increased, while extracellular signal-regulated protein kinases 1 and 2 (ERK1 and ERK2) phosphorylation decreased in both the TP-treated rats and DAHP-treated rats. These results demonstrate that TP and DAHP can decrease cell apoptosis in focal cerebral ischemia rat brains and that the mechanism may be related to the activation of the PI3K/Akt/mTOR pathway and inactivation of the ERK1/2 pathway. Thus our hypothesis was reached PI3K/Akt/mTOR and ERK1/2 pathways may provide distinct cellular targets for a new generation of therapeutic agents for the treatment of stroke, and TP and DAHP may be potential neuroprotective agents for cerebral ischemia/reperfusion injury.

17β-Estradiol Protects Human Eyelid-Derived Adipose Stem Cells against Cytotoxicity and Increases Transplanted Cell Survival in Spinal Cord injury

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b‐estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose‐derived stem cells (hEASCs) in vitro with hydrogen peroxide (H2O2)‐induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H2O2‐induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.

C16 peptide shown to prevent leukocyte infiltration and alleviate detrimental inflammation in acute allergic encephalomyelitis model

Integrins are important adhesion receptors for leukocytes binding to endothelial cellular adhesion molecules. Previous studies have suggested that blocking relevant integrins might prevent leukocyte infiltration and suppress clinical and pathological features of neuroinflammatory disease. Experimental autoimmune encephalomyelitis (EAE), a rodent model of Multiple sclerosis (MS), is characterized by chronic inflammatory disorder of the central nervous system in which circulating leukocytes enter the brain and spinal cord leading to inflammation, myelin damage and subsequent paralysis. To prove this hypothesis and explore a promising application for MS treatment, the effects of C16, an ανβ3 integrin-binding peptide, were tested in vitro and in vivo by transendothelial assay, electron microscopy observation, multiple histological and immunohistochemical staining. The results showed C16 inhibited transendothelial migration of the C8166-CD4 lymphoblast cells, and alleviated extensive spinal cord and brain infiltration of leukocytes and macrophages in the EAE model. Furthermore, a significant amelioration of astrogliosis and a dramatic decrease in demyelination and axonal loss were observed in C16 treated animals. The attenuating inflammatory progression may improve the regional environment and trigger further neuroprotective effects on myelin and axons, all this suggests that C16 peptide may be a promising therapeutic agent for multiple sclerosis.

Anti-neuroinflammatory and neurotrophic effects of combined therapy with annexin II and Reg-2 on injured spinal cord.

The present study was designed to investigate the neuroprotective effects of Ca2+-dependent phospholipid-binding protein annexin II and a secreted protein Reg-2 (regeneration gene protein 2) in spinal cord injury (SCI) model produced by contusion SCI at T9 using the weight drop method. The agents were delivered intrathecally with Alzet miniosmotic pumps. We found that annexin II and Reg-2 remarkably reduced neuronal death, attenuated tissue damage and alleviated detrimental inflammation in vivo; meanwhile, a significant increase in white matter sparing and myelination area was observed. The propriospinal axons and long-distance supraspinal pathways were protected by the treatments as revealed by retrograde tracing. Basso Beattie Bresnahan locomotor rating scores also revealed a measurable behavioral improvement. However, no evident behavioral improvements in locomotor performance were achieved by the combined treatment with annexin II and Reg-2, compared with the separate treatment with annexin II and Reg-2.

The neuroprotective effects of Reg-2 following spinal cord transection injury.

This study was designed to elucidate the potential neuroprotective effects of Reg‐2 (regeneration gene protein 2) in a rodent model of spinal cord transection injury at the ninth thoracic level. Reg‐2 at 100 and 500 μg, recombinant rat ciliary neurotrophic factor, or vehicle were delivered intrathecally using Alzet miniosmotic pumps. We found that Reg‐2 treatment significantly reduced neuronal death in the spinal cord. There was also an attenuation of inflammation at the injury site and an increase in white matter sparing and retained myelination. Retrograde tracing revealed that Reg‐2 protected axons of long descending pathways at 6 weeks post‐SCI, and the number of FluoroGold‐labeled neurons in spinal and supraspinal regions was also significantly increased. Immunofluorescent staining confirmed that the spared white matter contained neurofilament‐positive axons. Moreover, behavioral improvements were revealed by Basso Beattie Bresnahan locomotor rating scores and grid‐walk analysis. These results suggest that Reg‐2 might promote functional recovery by increasing axonal growth, inhibiting neuronal apoptosis, and attenuating spinal cord secondary injury after SCI. Anat Rec, 2010.

The Recent Updates of Therapeutic Approaches Against Aβ for the Treatment of Alzheimer's Disease

One of the main neuropathological lesions observed in brain autopsy of Alzheimers disease (AD) patients is the extracellular senile plaques mainly composed of amyloid‐beta (Aβ) peptide. Recently, treatment strategies have focused on modifying the formation, clearance, and accumulation of this potentially neurotoxic peptide. β‐ and γ‐secretase are responsible for the cleavage of amyloid precursor protein (APP) and the generation of Aβ peptide. Treatments targeting these two critical secretases may therefore reduce Aβ peptide levels and positive impact on AD. Vaccination is also an advanced approach against Aβ. This review focuses on recent advances of our understanding of this key peptide, with emphasis on Aβ peptide synthesis, accumulation and neurotoxicity, and current therapies including vaccination and two critical secretase inhibitors. MicroRNAs (miRNAs) are a class of conserved endogenous small noncoding RNAs, known to regulate the expression of complementary messenger RNAs, involved in AD development. We therefore address the relationship of miRNAs in the brain and Aβ generation, as a novel therapeutic approach to the treatment of AD while also providing new insights on the etiology of this neurological disorder. Anat Rec, 2011.

Effects of Reg-2 on Survival of Spinal Cord Neurons In Vitro

Regeneration gene protein 2 (Reg‐2) is a small secreted protein expressed in motor and sensory neurons of spinal cord during developmental stages and following injury of peripheral nerves. Reg‐2 appears to act as a neurotrophic factor and protects injured neurons from death during regeneration. To illustrate these potential protective effects in vitro, we investigated the blocking effects of Reg‐2 antibodies on the survival of primary cultured spinal cord neurons and astrocytes, as well as on neurite outgrowth. In addition, the effects of Reg‐2 in neuron injury models induced by peroxide and mitochondrial poisoning were assessed. Our results showed that Reg‐2 antibody markedly reduced survival and neurite outgrowth from neurons, whereas astrocyte survival was unaffected. Addition of Reg‐2 into the culture medium had no effect on neuron survival or neurite outgrowth. However, the addition of the Reg‐2 into culture media after peroxide treatment or cellular hypoxia insult induced by mitochondrial poisoning can reduce lactate dehydrogenase release levels and cell death. Thus, the data suggests that Reg‐2 is essential for the survival and neurite outgrowth of developing spinal cord neurons but not the survival of glial cells, and that Reg‐2 plays protective effects on spinal cord neurons against injury in vitro. Anat Rec, 2010.

Identification of a microRNA regulator for axon guidance in the olfactory bulb of adult mice

Semaphorin3A (sema3a), mainly localized in the olfactory neuron layer and periglomerular layer, is essential for the normal arrangement of axons in the olfactory bulb both in embryonic and adult mice functioning through its dynamic spatiotemporal expression. The regulators that can modulate the expression of sema3a by direct interaction, however, are unknown. In order to find the regulators of sema3a in the olfactory bulb, we focused on microRNAs, well-known post-transcriptional regulators. We found that axon guidance is the main molecular and biological process ongoing in the steady-state olfactory bulb of the adult mouse by screening the abundant microRNAs and exploring their functions in the olfactory bulb via our customized microRNA arrays, Gene Ontology and Kyoto Encyclopedia of Genes annotation. Furthermore, we traced the expression of three candidate regulators (miR-30c, miR-200b, and miR-429) and sema3a by the quantitative real-time polymerase chain reaction and immunohistochemistry. The results showed that only miR-30c expression corresponded inversely with sema3a. Finally, miR-30c was verified to be a specific regulator of sema3a by dual luciferase reporter assay in vitro. Taken together, our results suggested that miR-30c is a potential regulator in axon-guidance by suppressing the expression of sema3a, which will give new insights in elucidating the mechanism of architectonic and functional maintenance of the olfactory bulb.

Correlation Between the Distribution of SP and CGRP Immunopositive Neurons in Dorsal Root Ganglia and the Afferent Sensation of Preputial Frenulum

The aim of this study was to explore the distribution of substance P (SP) and calcitonin gene‐related peptide (CGRP) immunoreactive nerve terminals in the penis prepuce and the preputial frenulum. The possible correlation between SP‐ and CGRP‐immunopositive neurons in dorsal root ganglia (DRG) and the afferent sensation of the penile preputial frenulum is also discussed. Immunohistochemistry showed SP‐ and CGRP‐positive nerve terminals in the epidermal basal layer of the prepuce and frenulum in adult human males. The majority of the nerve terminals presented as bundles of different lengths and a few as enlarged nodosities. The density of SP‐ and CGRP‐immunopositive nerve terminals in the preputial frenulum was significantly higher than those in the penis prepuce (P<0.01). Fluoro‐Gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in Sprague‐Dawley rats. SP and CGRP immunofluorescence labeling was employed to detect the distribution of SP‐ and CGRP‐immunoreactive neurons in DRG. FG retro‐labeled neurons were localized in L6‐DRG and S1‐DRG. All the FG/SP and FG/CGRP double‐labeled neurons were medium or small‐sized. One‐third of the FG‐labeled neurons were SP‐immunoreactive, and a half of them CGRP‐immunoreactive in L6‐DRG and S1‐DRG, respectively. The FG/SP/CGRP‐labeled neurons accounted for one fifth of the FG retro‐labeled neurons. Taken together, these data suggest that the SP‐ and CGRP‐immunopositive nerve fibers may participate in the transmission of afferent sensation in the preputial frenulum. Anat Rec, 2011.