LncRNAGAS5 sponges miRNA-221 to promote neurons apoptosis by up-regulated PUMA under hypoxia condition.

Objectives: Long noncoding RNAs (lncRNAs) play substantial roles in cerebral ischemia. Growth arrest-specific 5 (GAS5) was reported to be involved in stroke. In the present study, we aimed to investigate the roles of GAS5 in cerebral condition and unveil the underlying mechanism.Method: Transient focal ischemia was induced by intraluminal occlusion of the right Middle cerebral artery occlusion (MCAO) and 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to evaluate the volume of cerebral infarction. RT-qPCR was applied to evaluate the level of GAS5 and miR-221. Fluorescence activated Cell Sorting (FACS) and Terminal deoxynucleotidyl transferased (TUNEL)  were used for detection of apoptosis. Western blotting was applied for protein level. Luciferase assay was applied to reveal the underlying relationship between GAS5 and miR-221 or p53-upregulated modulator of apoptosis (PUMA) and miR-221.Results: The results indicated that GAS5 was up-regulated in MCAO rats and in vitro hypoxia cell model while miR-221 expression was decreased in vitro hypoxia cell model. GAS5 promoted cells apoptosis, while miR-221 inhibited cell apoptosis through regulation of PUMA and downstream JNK/H2AX signaling. Moreover, GAS5 and miR-221 have direct interaction and PUMA was the target of miR-221, indicating that GAS5 regulated PUMA through sponging miR-221.Conclusions: the present study revealed that GAS5 aggravated cell apoptosis in hypoxia condition via miR-221/PUMA axis, which may provide potential targets for the treatment of stroke.

Biochanin A Reduces Inflammatory Injury and Neuronal Apoptosis following Subarachnoid Hemorrhage via Suppression of the TLRs/TIRAP/MyD88/NF-κB Pathway.

Inflammatory injury and neuronal apoptosis participate in the period of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Suppression of inflammation has recently been shown to reduce neuronal death and neurobehavioral dysfunction post SAH. Biochanin A (BCA), a natural bioactive isoflavonoid, has been confirmed to emerge the anti-inflammatory pharmacological function. This original study was aimed at evaluating and identifying the neuroprotective role of BCA and the underlying molecular mechanism in an experimental Sprague-Dawley rat SAH model. Neurobehavioral function was evaluated via the modified water maze test and modified Garcia neurologic score system. Thus, we confirmed that BCA markedly decreased the activated level of TLRs/TIRAP/MyD88/NF-κB pathway and the production of cytokines. BCA also significantly ameliorated neuronal apoptosis which correlated with the improvement of neurobehavioral dysfunction post SAH. These results indicated that BCA may provide neuroprotection against EBI through the inhibition of inflammatory injury and neuronal apoptosis partially via the TLRs/TIRAP/MyD88/NF-κB signal pathway.

Roles of Pannexin-1 Channels in Inflammatory Response through the TLRs/NF-Kappa B Signaling Pathway Following Experimental Subarachnoid Hemorrhage in Rats.

Background: Accumulating evidence suggests that neuroinflammation plays a critical role in early brain injury after subarachnoid hemorrhage (SAH). Pannexin-1 channels, as a member of gap junction proteins located on the plasma membrane, releases ATP, ions, second messengers, neurotransmitters, and molecules up to 1 kD into the extracellular space, when activated. Previous studies identified that the opening of Pannexin-1 channels is essential for cellular migration, apoptosis and especially inflammation, but its effects on inflammatory response in SAH model have not been explored yet. Methods: Adult male Sprague-Dawley rats were divided into six groups: sham group (n = 20), SAH group (n = 20), SAH + LV-Scramble-ShRNA group (n = 20), SAH + LV-ShRNA-Panx1 group (n = 20), SAH + LV-NC group (n = 20), and SAH + LV-Panx1-EGFP group (n = 20). The rat SAH model was induced by injection of 0.3 ml fresh arterial, non-heparinized blood into the prechiasmatic cistern in 20 s. In SAH + LV-ShRNA-Panx1 group and SAH + LV-Panx1-EGFP group, lentivirus was administered via intracerebroventricular injection (i.c.v.) at 72 h before the induction of SAH. The Quantitative real-time polymerase chain reaction, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, immunofluorescence staining, and western blotting were performed to explore the potential interactive mechanism between Pannexin-1 channels and TLR2/TLR4/NF-κB-mediated signaling pathway. Cognitive and memory changes were investigated by the Morris water maze test. Results: Administration with LV-ShRNA-Panx1 markedly decreased the expression levels of TLR2/4/NF-κB pathway-related agents in the brain cortex and significantly ameliorated neurological cognitive and memory deficits in this SAH model. On the contrary, administration of LV-Panx1-EGFP elevated the expressions of TLR2/4/NF-κB pathway-related agents, which correlated with augmented neuronal apoptosis. Conclusion: Pannexin-1 channels may contribute to inflammatory response and neurobehavioral dysfunction through the TLR2/TLR4/NF-κB-mediated pathway signaling after SAH, suggesting a potential role of Pannexin-1 channels could be a potential therapeutic target for the treatment of SAH.

Fisetin alleviates early brain injury following experimental subarachnoid hemorrhage in rats possibly by suppressing TLR 4/NF-κB signaling pathway.

Early brain injury (EBI) determines the unfavorable outcomes after subarachnoid hemorrhage (SAH). Fisetin, a natural flavonoid, has anti-inflammatory and neuroprotection properties in several brain injury models, but the role of fisetin on EBI following SAH remains unknown. Our study aimed to explore the effects of fisetin on EBI after SAH in rats. Adult male Sprague-Dawley rats were randomly divided into the sham and SAH groups, fisetin (25mg/kg or 50mg/kg) or equal volume of vehicle was given at 30min after SAH. Neurological scores and brain edema were assayed. The protein expression of toll-like receptor 4 (TLR 4), p65, ZO-1 and bcl-2 was examined by Western blot. TLR 4 and p65 were also assessed by immunohistochemistry (IHC). Enzyme-linked immunosorbent assay (ELISA) was performed to detect the production of pro-inflammatory cytokines. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) was perform to assess neural cell apoptosis. High-dose (50mg/kg) fisetin significantly improved neurological function and reduced brain edema at both 24h and 72h after SAH. Remarkable reductions of TLR 4 expression and nuclear factor κB (NF-κB) translocation to nucleus were detected after fisetin treatment. In addition, fisetin significantly reduced the productions of pro-inflammatory cytokines, decreased neural cell apoptosis and increased the protein expression of ZO-1 and bcl-2. Our data provides the evidence for the first time that fisetin plays a protective role in EBI following SAH possibly by suppressing TLR 4/NF-κB mediated inflammatory pathway.

Expression of Cytoplasmic Gelsolin in Rat Brain After Experimental Subarachnoid Hemorrhage.

Convincing evidence indicates that apoptosis contributes to the unfavorable prognosis of subarachnoid hemorrhage (SAH), a significant cause of morbidity and case fatality throughout the world. Gelsolin (GSN) is a Ca(2+)-dependent actin filament severing, capping, and nucleating protein, as well as multifunctional regulator of cell structure and metabolism, including apoptosis. In the present study, we intended to investigate the expression pattern and cell distribution of GSN in rat brain after experimental SAH. GSN expression was examined in sham group and at 3, 6, 12 h, day 1 (1 day), 2, 3, 5, and 7 days after SAH by Western blot analysis as well as real-time polymerase chain reaction. Immunohistochemistry and immunofluorescence were performed to detect the localization of GSN. The level of GSN protein expression was significantly decreased in SAH group and reached a bottoming point on 1 day after SAH. GSN mRNA level was significantly decreased in SAH groups in comparison with the sham group, and reached a minimum value at 12 h after SAH. Immunohistochemistry showed that GSN was constitutively and obviously expressed in the cortex of the normal rat brain and significantly decreased in the rat cortex after SAH. In addition, immunofluorescence results revealed that GSN expression could be found in both neurons and microglias, as well as in glialfibrillary acidic protein-positive astrocytes. The decreased expression of GSN could mainly be found in neurons and astrocytes as well, and GSN-positive microglias showed different cell morphological characteristics. Interestingly, the protein and gene levels of GSN seemed to be constant in the rat hippocampus of sham and SAH groups. These findings suggested a potential role of GSN in the pathophysiology of the brain at the early stage of SAH.

Expression and Cell Distribution of SENP3 in Brain Tissue After Traumatic Brain Injury in Mice: A Pilot Study.

SUMO-specific proteases 3 (SENP3) is a member of the small ubiquitin-like modifier-specific protease family and deconjugates SUMO2/3 from protein substrates. To date, the expression and function of SENP3 in traumatic brain injury (TBI) are unclear. The present study examined dynamic changes in SENP3 expression in the cerebral cortex and in its cellular localization, using an acute TBI model in adult mice. SENP3 expression was examined at 3, 6, 12, 24 h, 3, and 5 days after TBI using Western Blot analysis and quantitative real-time PCR. Immunohistochemistry and immunofluorescence were examined to detect SENP3 localization. Western Blot indicated that SENP3 protein levels gradually increased from 3 h after TBI and peaked at 24 h. Quantitative real-time PCR demonstrated a gradual increase in SENP3 expression, which peaked 12 h after TBI and declined subsequently. Immunohistochemical staining demonstrated that SENP3-positive cells were observed in both the sham and 24 h post-TBI groups. However, robust expression of SENP3 was seldom observed in the sham group, while it was notably enhanced after TBI. Furthermore, immunofluorescence results revealed that the expression of SENP3 increased more significantly in neurons at day 1 after TBI compared with sham group and less significantly in astrocytes and microglia. Moreover, the SENP3-positive cells that were co-expressed with NeuN also expressed caspase-3, indicating a potential correlation between SENP3 and apoptosis after TBI. Collectively, our results showed obvious up-regulation of SENP3 expression in the brain after TBI, especially in the neurons. However, the full role of SENP3 and its therapeutic potential in TBI needs further investigation.

Constriction and dysfunction of pial arterioles after regional hemorrhage in the subarachnoid space.

Increasing evidence indicates that poor outcomes after brain hemorrhage, especially after subarachnoid hemorrhage (SAH), can be attributed largely to dysfunction of the cerebral microcirculation. However, the cause of this dysfunction remains unclear. Here, we investigated changes in the cerebral microcirculation after regional hemorrhage in the subarachnoid space using the closed cranial window technique in mice. A single pial arteriole on the surface of the brain was punctured to induce a regional hemorrhage in the subarachnoid space. Physiological parameters were monitored during the procedure, and microvessel diameter was measured after hemorrhage. The vasoreactivity of the arterioles in response to hypercapnia as well as to topical application of the vasodilator acetylcholine (ACh) and S-nitroso-N-acetyl-penicillamine (SNAP) were assessed. The constriction of pial arterioles was detected without changes in other physiological parameters. Decreased reactivity of pial arterioles to all of the applied vasodilatory stimuli was observed after hemorrhage. Our results indicate that regional hemorrhage in the subarachnoid space can induce the vasospasm of microvessels and also reduce the vasoreactivity of pial arterioles.

Baincalein alleviates early brain injury after experimental subarachnoid hemorrhage in rats: possible involvement of TLR4/NF-κB-mediated inflammatory pathway.

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) largely contributes to unfavorable outcomes. Hence, effective therapeutic strategies targeting on EBI have recently become a major goal in the treatment of SAH patients. Baicalein is a flavonoid that has been shown to offer neuroprotection in kinds of brain injury models. This study investigated the effects of baicalein on EBI in rats following SAH. SAH was inducted in male Sprauge-Dawley rats by injection of fresh non-heparinized arterial blood into the prechiasmatic cistern. Baicalein (30 or 100 mg/kg) or vehicle were administrated 30 min after injury. Neurological deficit, brain edema, blood-brain barrier (BBB) permeability and neural cell apoptosis were assessed. To explore the further mechanisms, the change of toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) signaling pathway and the levels of apoptosis associated proteins were also examined. Our study showed that treatment with baicalein (30 mg/kg) significantly improved neurological function at 24h after SAH and reduced brain edema at both 24h and 72 h after SAH. Baicalein also significantly reduced neural cell death, BBB permeability. These changes were associated with the remarkable reductions of TLR4 expression, IκB-α degradation, NF-κB translocation to nucleus, as well as the expressions of matrix metalloproteinase-9, tight junctions protein, interleukin-1ß and tumor necrosis factor- ɑ. These findings suggest that baicalein may ameliorate EBI after SAH potentially via inhibition of inflammation-related pathway.

Ghrelin alleviates early brain injury after subarachnoid hemorrhage via the PI3K/Akt signaling pathway.

Early brain injury (EBI) plays a key role in the pathogenesis of subarachnoid hemorrhage (SAH). Although the neuroprotective effects of ghrelin have been demonstrated in several studies, whether ghrelin reduces EBI after SAH remains unknown. In this study, we hypothesized that treatment with ghrelin would attenuate EBI after SAH, and that this protection would be mediated, at least in part, by activation of the PI3K/Akt signaling pathway. Adult male Sprague-Dawley rats (n=100) were randomly divided into the following groups: control group (n=20), SAH group (n=20), SAH+vehicle group (n=20), SAH+ghrelin group (n=20) and SAH+ghrelin+LY294002 group (n=20). The rats were injected with autologous blood (0.3mL) into the prechiasmatic cistern to induce SAH. Ghrelin (80µg/kg, IP), or an equal volume of vehicle, was administered immediately after surgery. The PI3K inhibitor, LY294002, was applied to manipulate the proposed pathway. Mortality, neurological scores, brain edema, cell apoptosis, and the expression of p-Akt, and cleaved caspase-3 proteins were assayed after 24h SAH. Ghrelin significantly improved neurological function and reduced neuronal apoptosis and brain edema at 24h after SAH. The level of p-Akt, expressed mainly in neurons, was markedly up-regulated. Additionally, the level of cleaved caspase-3 was decreased by ghrelin treatment. The beneficial effects of ghrelin in SAH rats were partially suppressed by LY294002. These results demonstrate that ghrelin may reduce EBI after SAH, via a mechanism involving the PI3K/Akt signaling pathway.

Astaxanthin offers neuroprotection and reduces neuroinflammation in experimental subarachnoid hemorrhage.

BACKGROUND: Neuroinflammation has been proven to play a crucial role in early brain injury pathogenesis and represents a target for treatment of subarachnoid hemorrhage (SAH). Astaxanthin (ATX), a dietary carotenoid, has been shown to have powerful anti-inflammation property in various models of tissue injury. However, the potential effects of ATX on neuroinflammation in SAH remain uninvestigated. The goal of this study was to investigate the protective effects of ATX on neuroinflammation in a rat prechiasmatic cistern SAH model. METHODS: Rats were randomly distributed into multiple groups undergoing the sham surgery or SAH procedures, and ATX (25 mg/kg or 75 mg/kg) or equal volume of vehicle was given by oral gavage at 30 min after SAH. All rats were sacrificed at 24 h after SAH. Neurologic scores, brain water content, blood-brain barrier permeability, and neuronal cell death were examined. Brain inflammation was evaluated by means of expression changes in myeloperoxidase, cytokines (interleukin-1ß, tumor necrosis factor-α), adhesion molecules (intercellular adhesion molecule-1), and nuclear factor kappa B DNA-binding activity. RESULTS: Our data indicated that post-SAH treatment with high dose of ATX could significantly downregulate the increased nuclear factor kappa B activity and the expression of inflammatory cytokines and intercellular adhesion molecule-1 in both messenger RNA transcription and protein synthesis. Moreover, these beneficial effects lead to the amelioration of the secondary brain injury cascades including cerebral edema, blood-brain barrier disruption, neurological dysfunction, and neuronal degeneration. CONCLUSIONS: These results indicate that ATX treatment is neuroprotective against SAH, possibly through suppression of cerebral inflammation.

Haplotype analysis of single nucleotide polymorphisms in anti-Müllerian hormone gene in Chinese PCOS women.

PURPOSE: Anti-Müllerian hormone (AMH) inhibits FSH-stimulated follicle growth and aromatase activity. The three fold higher serum AMH in PCOS patients may account for the increased number of small follicles and androgen level. We attempted to determine whether polymorphisms in AMH gene were associated with PCOS in Chinese han population. METHODS: A case-control study involving 475 PCOS patients and 512 normoovulatory women was conducted. Rs10407022 and rs8112524 were two tagging SNPs selected according to the HapMap database. Taqman assay was used for rs8112524 genotyping, and PCR-RFLP method for rs10407022. RESULTS: No significant difference was found in genotypic or allelic distributions of both of the two SNPs, rs10407022 and rs8112524, between PCOS women and controls. LH level and progesterone level were significantly higher in rs8112524 AA genotype in PCOS group (P = 0.012, 0.014 respectively). TA haplotype might enhance susceptibility to PCOS (P = 0.013, OR = 4.996, 95 % CI = 2.001-5.251), and GA haplotype might be protective (P = 0.000, OR = 0.117, 95 % CI = 0.049-0.417). CONCLUSIONS: Although individual TagSNPs in AMH gene do not affect susceptibility to PCOS, haplotypes of the two SNPs were associated with PCOS risk, as TA haplotype might enhance susceptibility to PCOS and GA inversely.

Microsatellite polymorphism in the fibrillin 3 gene and susceptibility to PCOS: a case-control study and meta-analysis.

The D19S884 marker at the fibrillin 3 gene has been analysed as a candidate location for polycystic ovary syndrome (PCOS) mainly in Caucasian descendants. A case-control study was performed with 272 PCOS women and 271 controls to test the hypothesis that variants in the D19S884 marker increase susceptibility to PCOS in Chinese women and a meta-analysis was undertaken to clarify whether there is an allele consistently contributing to the susceptibility. The association analysis showed that PCOS women were significantly different from controls in the distribution of D19S884 allele frequencies. Instead of the well-known A8 allele, the most common allele in Chinese population was proved to be A7, and the allele frequencies of A7 were statistically different between cases and controls (P=0.037). The meta-analysis of A8 and A7 only identified A8 as a significant allelic association at the D19S884 marker in all combined samples (A8: OR 1.391, 95% CI 1.169-1.654; A7: OR 1.154, 95% CI 0.894-1.490). In conclusion, the association study showed a potential association of the D19S884 marker with PCOS in Chinese Han women and the meta-analysis identified that A8 may increase susceptibility to PCOS. Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age, and it affects an estimated 15% of women worldwide based on the Rotterdam criteria. Many studies in Caucasian descendants suggested that variants of the D19S884 marker at the fibrillin 3 gene are associated with the risk of this syndrome. Here we performed a case-control study with 272 PCOS women and 271 controls to investigate whether variants in the D19S884 marker increase susceptibility to PCOS in Chinese women. We also carried out a meta-analysis of some relevant studies to find a more reliable result. Our association analysis showed that PCOS women were significantly different from controls in the distribution of D19S884 allele frequencies, and instead of the well-known A8 (the letter 'A' represents 'allele'), the most common allele in Chinese population was proved to be A7, whose allele frequencies were statistically different between cases and controls. The meta-analysis of A8 and A7 only identified A8 as a significant allelic association at the D19S884 marker in all combined samples. In conclusion, our association study showed a potential association of the D19S884 marker with PCOS in Chinese Han women and the meta-analysis identified that A8 may increase susceptibility to PCOS.