Zhengquan Yu

Hydrogen Sulfide Ameliorates Early Brain Injury Following Subarachnoid Hemorrhage in Rats.

Increasing studies have demonstrated the neuroprotective effect of hydrogen sulfide (H2S) in central nervous system (CNS) diseases. However, the potential application value of H2S in the therapy of subarachnoid hemorrhage (SAH) is still not well known. This study was to investigate the potential effect of H2S on early brain injury (EBI) induced by SAH and explore the underlying mechanisms. The role of sodium hydrosulfide (NaHS), a donor of H2S, in SAH-induced EBI, was investigated in both in vivo and in vitro. A prechiasmatic cistern single injection model was used to produce experimental SAH in vivo. In vitro, cultured primary rat cortical neurons and human umbilical vein endothelial cells (HUVECs) were exposed to OxyHb at concentration of 10xa0μM to mimic SAH. Endogenous production of H2S in the brain was significantly inhibited by SAH. The protein levels of the predominant H2S-generating enzymes in the brain, including cystathionineb-synthase (CBS) and 3-mercaptopyruvate sulfur transferase (3MST), were also correspondingly reduced by SAH, while treatment with NaHS restored H2S production and the expressions of CBS and 3MST. More importantly, NaHS treatment could significantly attenuate EBI (including brain edema, blood–brain barrier disruption, brain cell apoptosis, inflammatory response, and cerebral vasospasm) after SAH. In vitro, H2S protects neurons and endothelial function by functioning as an antioxidant and antiapoptotic mediator. Our results suggest that NaSH as an exogenous H2S donor could significantly reduce EBI induced by SAH.

Attenuation of Early Brain Injury and Learning Deficits Following Experimental Subarachnoid Hemorrhage Secondary to Cystatin C: Possible Involvement of the Autophagy Pathway

Cystatin C (CysC) is a cysteine protease inhibitor and previous studies have demonstrated that increasing endogenous CysC expression has therapeutic implications on brain ischemia, Alzheimer’s disease, and other neurodegenerative disorders. Our previous reports have demonstrated that the autophagy pathway was activated in the brain after experimental subarachnoid hemorrhage (SAH), and it may play a beneficial role in early brain injury (EBI). This study investigated the effects of exogenous CysC on EBI, cognitive dysfunction, and the autophagy pathway following experimental SAH. All SAH animals were subjected to injections of 0.3xa0ml fresh arterial, nonheparinized blood into the prechiasmatic cistern in 20xa0s. As a result, treatment with CysC with low and medial concentrations significantly ameliorated the degree of EBI when compared with vehicle-treated SAH rats. Microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosomes, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased in the cortex 48xa0h after SAH and were further up-regulated after CysC therapy. By ultrastructural observation, there was a marked increase in autophagosomes and autolysosomes in neurons of CysC-treated rats. Learning deficits induced by SAH were markedly alleviated after CysC treatment with medial doses. In conclusion, pre-SAH CysC administration may attenuate EBI and neurobehavioral dysfunction in this SAH model, possibly through activating autophagy pathway.

The Neuroprotection of Lysosomotropic Agents in Experimental Subarachnoid Hemorrhage Probably Involving the Apoptosis Pathway Triggering by Cathepsins via Chelating Intralysosomal Iron.

Abstractα-Lipoic acid-plus (LAP), an amine derivative of α-lipoic acid (LA), could protect cells against oxidant challenges via chelating intralysosomal iron. However, the application of LAP in experimental subarachnoid hemorrhage (SAH) is still not well known. This study was designed to evaluate the potential neuroprotection of LAP on the early brain injury (EBI) and the underlying mechanisms in a rat model of SAH. The SAH models were induced in Sprague–Dawley rats. LA and LAP were oral administration and lasted for 72xa0h once a day. The brain tissue samples were obtained for assay at 72xa0h after SAH. In experiment 1, we found that lysosome amounts in neurons decreased significantly in SAH group, and LAP (100xa0mg/kg) could stabilize lysosomal membrane markedly based on lysosomal-associated membrane protein-1 (LAMP-1) expression in neurons by immunofluorescence. Hence, the LAP dosages of 100 and 150xa0mg/kg were applied in experiment 2. Firstly, Western blot analysis showed that the protein levels of cathepsin B/D, caspase-3, Bax, ferritin, and heme-oxygenase-1 (HO-1) markedly increased after SAH, which were further confirmed by double immunofluorescence staining and reversed by LA and LAP treatments. In addition, LA and LAP also reduced oxidative stress and iron deposition in brain tissue. Furthermore, LA and LAP significantly ameliorated brain edema, blood–brain barrier injury, cortical apoptosis, and neurological behavior impairment induced by SAH. Finally, it is noteworthy that LAP exerted more significant effects than LA on these parameters as described above. LAP probably exerted neuroprotective effects via targeting lysosomes and chelating intralysosomal iron in EBI post-SAH in rats.

Hyperacetylation of Histone H3K9 Involved in the Promotion of Abnormally High Transcription of the gdnf Gene in Glioma Cells

The mechanism underlying abnormally high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene in glioma cells is not clear. In this study, to assess histone H3K9 acetylation levels in promoters I and II of the gdnf gene in normal human brain tissue, low- and high-grade glioma tissues, normal rat astrocytes, and rat C6 glioblastoma cells, we employed chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), real-time PCR, and a pGL3 dual fluorescence reporter system. We also investigated the influence of treatment with curcumin, a histone acetyltransferase inhibitor, and trichostatin A (TSA), a deacetylase inhibitor, on promoter acetylation and activity and messenger RNA (mRNA) expression level of the gdnf gene in C6 cells. Compared to normal brain tissue, H3K9 acetylation in promoters I and II of the gdnf gene increased significantly in high-grade glioma tissues but not in low-grade glioma tissues. Moreover, H3K9 promoter acetylation level of the gdnf gene in C6 cells was also remarkably higher than in normal astrocytes. In C6 cells, curcumin markedly decreased promoter II acetylation and activity and GDNF mRNA expression. Conversely, all three measurements were significantly increased following TSA treatment. Our results suggest that histone H3K9 hyperacetylation in promoter II of the gdnf gene might be one of the reasons for its abnormal high transcription in glioma cells.

Changes in Transcriptional Factor Binding Capacity Resulting from Promoter Region Methylation Induce Aberrantly High GDNF Expression in Human Glioma

Glial cell line-derived neurotrophic factor (GDNF), which belongs to transforming growth factor β superfamily, plays important roles in glioma pathogenesis. Gdnf mRNA is aberrantly increased in glioma cells, but the underlying transcription mechanism is unclear. Here, we found that although the base sequence in the promoter region of the gdnf gene was unchanged in glioma cells, there were significant changes in the methylation level of promoter region I (Pu2009<u20090.05) in both high- and low-grade glioma tissues. However, the methylation degree in promoter region II was notably decreased in low-grade glioma tissue compared to normal brain tissue (Pu2009<u20090.05), and the demethylation sites were mainly located in the enhancer region. Conversely, methylation was markedly increased in high-grade glioma tissue (Pu2009<u20090.05), and the sites with decreased methylation level were mainly located in the silencer region. The binding capacities of several transcriptional factors, such as activating protein 2, specificity protein 1, ETS-related gene 2, and cAMP response element binding protein, which specifically bind to regions with altered methylation status decreased along with the pathological grade of glioma, and the differences between high-grade glioma and normal brain tissue were significant (Pu2009<u20090.05). The results suggest that changes in transcriptional factor binding capacity are due to changes in promoter region methylation and might be the underlying mechanism for aberrantly high gdnf expression in glioma.

Dopamine content in the striatum and expression changes of Bad and Bcl-2 in elderly rats with abnormal behavior

To determine the dopamine (DA) content in the striatum and the expression changes of the apoptosis-associated proteins Bad and Bcl-2 in the substantia nigra compacta (SNc) in elderly rats with abnormal behavior. Fifty three Sprague–Dawley rats were divided into three groups: adult, age-motorplus (normal behavior) and aged-motorminus (abnormal behavior) using the hanger test. The DA content in the striatum and the expression of tyrosine hydroxylase (TH), Bad and Bcl-2 in the SNc were measured by HPLC/MS (high performance liquid chromatogram–mass spectra) and Western Blot. (1) The results from the hanger test demonstrated that the scores and latency of aged-motorminus group were lower than the age-motorplus group. (2) Results from HPLC-MS showed that, compared with the age-motorplus and adult group, the content of DA in elderly rat striata decreased significantly, with a statistically significant difference. (3) The Western Blot demonstrated that, compared with the adults, the expression of TH in elderly rats significantly decreased, but the difference was not significant between the aged-motorminus group and the age-motorplus group. Compared with the age-motorplus and the adult group, the expression of Bad increased but Bcl-2 decreased in the aged-motorminus group. The decrease in TH content in the SNc correlated with the aging of rats. The decrease in DA content in the striatum may correlate with the abnormal behavior in elderly rats, which could be ascribed to the variations in Bad and Bcl-2.

Egr-1 participates in abnormally high gdnf gene transcription mediated by histone hyperacetylation in glioma cells.

Abnormally high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene in glioma cells is related to the hyperacetylation of histone H3 lysine 9 (H3K9) in its promoter region II, but the mechanism remains unclear. There are three consecutive putative binding sites for the transcription factor early growth response protein 1(Egr-1) in promoter region II of the gdnf gene, and Egr-1 participates in gdnf gene transcription activation. Here we show that the acetylation level of H3K9 at Egr-1 binding sites in gdnf gene promoter region II in rat C6 astroglioma cells was significantly higher than that in normal astrocytes, and the binding capacity was also significantly higher. In C6 astroglioma cells, gdnf gene transcription significantly decreased after Egr-1 knock-down. In addition, the deletion or mutation of the Egr-1 binding site also significantly down-regulated the activity of promoter region II of this gene in vitro. When curcumin decreased the acetylation level of H3K9 at the Egr-1 binding site, the binding of Egr-1 to promoter region II and GDNF mRNA levels significantly decreased. In contrast, trichostatin A treatment significantly increased H3K9 acetylation at the Egr-1 binding site, which significantly increased both the binding of Egr-1 with promoter region II and GDNF mRNA levels. In this context, knocking down Egr-1 significantly reduced the elevation in gdnf gene transcription. Collectively, our results demonstrate that the hyperacetylation of H3K9 at Egr-1 binding sites in promoter region II of the gdnf gene can up-regulate the binding of Egr-1 to increase gdnf gene transcription in glioma cells.

A new alternative NF-ΚB Pathway mediated the neuroprotection of GDNF on 6-OHDA-induced DA neurons neurotoxicity

Glial cell line-derived neurotrophic factor (GDNF) is a potent protective factor for dopaminergic (DA) neurons, but the signaling mechanisms underlying the effect of GDNF on these neurons remain obscure. Here, both our in vivo and in vitro studies demonstrate that the majority of DA neurons express the NF-κB-inducing kinase (NIK), which is the essential kinase for mediating activation of the new alternative NF-κB signaling pathway. Additionally, we also show that GDNF induced the time/dose-dependent phosphorylation of IκB kinase α (IKKα) and p100, facilitated the processing of p100 to p52 and accelerated the translocation of NF-κB dimmers into the nuclei of DA neurons. We furtherly found that the dimmer which translocate into the nucleus was RelA/p52 not RelB/p52. Meanwhile, the attenuation of 6-OHDA-induced DA neuronal apoptosis due to GDNF was reversed subsequent to the inhibition of p100 expression by RNAi while the neuroprotective effect of GDNF on injured DA neurons was strengthened by the overexpression of p100. Our data, therefore, indicate that a new alternative NF-κB signaling pathway, which was not the classic pathway but different from the non-canonical pathway, exists in DA neurons and mediates the neuroprotective effect of GDNF on these neurons.

Effect of Intranigral Injection of GDNF and EGF on the Survival and Possible Differentiation Fate of Progenitors and Immature Neurons in 6-OHDA-Lesioned Rats

We investigated the survival and the possible differentiation fate of the progenitors and immature neurons in the pars compacta of the substantia nigra (SNc) by intranigral injection of a glial cell line-derived neurotropic factor (GDNF) or glial cell line-derived neurotropic factor plus epidermal growth factor (EGFxa0+xa0GDNF) in 6-hydroxydopamine (6-OHDA)-lesioned rats. First, we performed behavioral tests by postural asymmetry and forelimb akinesia on the rats injected with 6-OHDA in striatum at day 7, and selected the qualified model according to the results. Then, intranigral GDNF or EGFxa0+xa0GDNF treatment was administered in the qualified PD model rats. On day 21, behavioral tests were performed with these rats; and then the rats were sacrificed for analyses of β-tubulin isotype-III (Tuj1), nestin, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) by immunohistochemistry and Western blotting. The results indicated that GDNF could promote the survival of the progenitor cells and immature neurons in rat SNc following 6-OHDA lesion. Moreover, EGF is capable of enhancing the survival effect of GDNF on the progenitor cells and immature neurons in SNc. On day 21, rapid functional recovery from the lesion-induced behavioral asymmetries was observed in the GDNF or EGFxa0+xa0GDNF-treated rats, and the numbers of TH-positive neurons increased in SNc, suggesting that the rats might generate new dopaminergic neurons. Thus, our study provides the new insight that the progenitors and immature neurons in SNc of 6-OHDA-lesioned rats might be able to differentiate toward the dopaminergic neurons fate subsequent to treatment with GDNF or EGFxa0+xa0GDNF.

Neuroprotection Exerted by Netrin-1 and Kinesin Motor KIF1A in Secondary Brain Injury following Experimental Intracerebral Hemorrhage in Rats

Binding of extracellular netrin-1 to its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5H2 (UNC5H2), inhibits apoptosis mediated by these receptors. A neuron-specific kinesin motor protein, KIF1A, has been shown to participate in netrin-1 secretion. This study aimed to identify the roles of netrin-1 and KIF1A in secondary brain injury after intracerebral hemorrhage (ICH) and the potential mechanisms. An autologous blood ICH model was established in adult male Sprague-Dawley rats, and cultured neurons were exposed to OxyHb to mimic ICH conditions in vitro. Mouse recombinant netrin-1, expression vectors encoding KIF1A, and KIF1A-specific siRNAs were administered intracerebroventricularly. After ICH, protein levels of netrin-1, DCC, and UNC5H2 increased, while protein levels of KIF1A decreased. Levels of UNC5H2 and DCC bound to netrin-1 increased after ICH but were significantly lower than the increase in total amount of protein. Administration of recombinant netrin-1 attenuated neuronal apoptosis and degeneration in ICH rats. Moreover, KIF1A overexpression increased concentrations of netrin-1 in cerebrospinal fluid and cell culture supernatant and exerted neuroprotective effects via netrin-1 and its receptor pathways. KIF1A plays a critical role in netrin-1 secretion by neurons. An increase in protein levels of netrin-1 may be a neuroprotective strategy after ICH. However, this process is almost completely abolished by ICH-induced loss of KIF1A. An exogenous increase of KIF1A may be a potential strategy for neuroprotection via the netrin-1 pathway.