Expression, purification and functional identification of the modified hEGF protein.

Human epidermal growth factor (hEGF) plays an important role in the growth and division of epithelial cells and has good application prospects in skin-related injuries and diseases. Weak skin penetration and rapid clearance of hEGF in skin via the mononuclear phagocyte system have restricted the application of hEGF. To overcome these shortcomings, the recombinant gene TAT-hEGF-CD47 was constructed in our experiments, and the fusion protein TAT-hEGF-CD47 was expressed, purified and renatured. The cell proliferation-promoting function, skin penetration and concentration of TAT-hEGF-CD47 in skin after its application were determined. The results showed that TAT-hEGF-CD47 effectively promoted human skin fibroblast and skin epithelial cell proliferation, and the proliferation-promoting effect was positively correlated with the TAT-hEGF-CD47 concentration. After administration to the skin, TAT-hEGF-CD47 effectively penetrated the epidermal layer of the skin because of the TAT domain and stayed in the skin for a long time because the CD47 fragment slowed its clearance via the mononuclear phagocytic system. In conclusion, TAT-hEGF-CD47 exhibits high cell proliferation-promoting activity, high skin penetration efficiency and long retention time in skin and has laid the foundation for its wide application in skin repair, ulcer, diabetes and even cancer treatments.

Modeling Emission Flow Pattern of a Single Cruising Vehicle on Urban Streets with CFD Simulation and Wind Tunnel Validation.

Transportation has become one of the primary sources of urban atmospheric pollutants and it causes severe diseases among city residents. This study focuses on assessing the pollutant dispersion pattern using computational fluid dynamics (CFD) numerical simulation, with the effect and results validated by the results from wind tunnel experiments. First, the wind tunnel experiment was carefully designed to preliminarily assess the flow pattern of vehicle emissions. Next, the spatiotemporal distribution of pollutant concentrations around the motor vehicle was modeled using a CFD numerical simulation. The pollutant concentration contours indicated that the diffusion process of carbon monoxide mainly occurred in the range of 0-2 m above the ground. Meanwhile, to verify the correctness of the CFD simulation, pressure distributions of seven selected points that were perpendicular along the midline of the vehicle surface were obtained from both the wind tunnel experiment and the CFD numerical simulation. The Pearson correlation coefficient between the numerical simulation and the wind tunnel measurement was 0.98, indicating a strong positive correlation. Therefore, the distribution trend of all pressure coefficients in the numerical simulation was considered to be consistent with those from the measurements. The findings of this study could shed light on the concentration distribution of platoon-based vehicles and the future application of CFD simulations to estimate the concentration of pollutants along urban street canyons.

Knockdown of TRIM32 Protects Hippocampal Neurons from Oxygen-Glucose Deprivation-Induced Injury.

Tripartite motif 32 (TRIM32) is a member of TRIM family that plays a potential role in neural regeneration. However, the biological function of TRIM32 in cerebral ischemia reperfusion injury has not been investigated. In the present study, we evaluated the expression level of TRIM32 in hippocampal neurons following oxygen-glucose deprivation/reperfusion (OGD/R). The results showed that TRIM32 expression was significantly elevated in hippocampal neurons subjected to OGD/R as compared to the neurons cultured in the normoxia condition. To further evaluate the role of TRIM32, hippocampal neurons were transfected with TRIM32 small interfering RNA (si-TRIM32) to knock down TRIM32. We found that knockdown of TRIM32 improved cell viability of OGD/R-stimulated hippocampal neurons. Generation of reactive oxygen species was decreased, while contents of superoxide dismutase and glutathione peroxidase were increased after si-TRIM32 transfection. Knockdown of TRIM32 suppressed cell apoptosis, as proved by the increased bcl-2 expression along with decreased bax expression and caspase-3 activity. We also found that TRIM32 knockdown enhanced OGD/R-induced activation of Nrf2 signaling pathway in hippocampal neurons. Furthermore, siRNA-Nrf2 was transfected to knock down Nrf2. SiRNA-Nrf2 transfection reversed the protective effects of TRIM32 knockdown on neurons. These data suggested that knockdown of TRIM32 protected hippocampal neurons from OGD/R-induced oxidative injury through activating Nrf2 signaling pathway.

Monocytes in the Peripheral Clearance of Amyloid-ß and Alzheimer's Disease.

Aging societies have high incidence rates of Alzheimer's disease (AD). AD is diagnosed at later disease stages and has a poor prognosis, and effective drugs and treatments for AD are lacking. The molecular mechanism of AD is not clear, and current research focuses primarily on amyloid-ß (Aß) deposition and tau protein hyperphosphorylation. Aß deposition is the most frequently hypothesized initiating factor of AD, and Aß clearance during the pathogenesis of AD may be an optional strategy to suppress AD development. Monocytes play important roles in the peripheral clearance of Aß. Therefore, the present review summarizes our current knowledge of the potential roles of infiltrating macrophages, circulating monocytes, and Kupffer cells in the peripheral clearance of Aß in AD.

The neuroprotective effect of hyperoxygenate hydrogen-rich saline on CO-induced brain injury in rats.

This study was designed to investigate the neuroprotective effect of hyperoxygenate hydrogen-rich saline (HOHS) against brain injury induced by carbon monoxide (CO) poisoning in rats. A rat model of CO poisoning was established by administering CO via intraperitoneal injection to male Sprague-Dawley rats. Forty-eight adult male rats were randomly divided into the following groups: normal control group (NG), CO poisoning group (CO), HOS treatment group (hyperoxygenated solution, HOS) and HOHS treatment group (HOHS). After CO poisoning, the carboxyhemoglobin (COHb) contents in the blood of rats in all the CO poisoning groups were increased significantly. However, HOS and HOHS significantly decreased COHb contents, furthermore, the HOHS group had lower COHb contents than the HOS group. Arterial oxygen partial pressure (PaO2) and arterial oxygen saturation (SaO2) results showed that HOS and HOHS could improve the oxygenation of the rats with CO poisoning. Compared with the CO group, the HOS group and the HOHS group had persistently neuroprotective effect on CO-induced brain injury, as assessed by modified neurological severity score (mNSS), furthermore, the HOHS group had better neurological functional recovery than the HOS group. The neuronal apoptosis induced by CO was also evaluated. Except the NG group, all the CO-poisoning groups had varying degrees of neuronal apoptosis. There was lesser degree of neuronal apoptosis in both the HOS group and the HOHS group than that in the CO group. Moreover, the HOHS group had more minor degree of neuronal apoptosis than the HOS group. Compared with the CO group, the free radicals production in the HOS group and the HOHS group were significantly inhibited. In addition, there were significantly difference in the free radicals production between the HOS group and the HOHS group. We could conclude that HOHS exerted a stronger neuroprotective effect against CO-induced brain injury than HOS, and the neuroprotective mechanism of HOHS may be related with inhibition of both neuronal apoptosis and free radicals.

MicroRNA-770 affects proliferation and cell cycle transition by directly targeting CDK8 in glioma.

BACKGROUND: MicroRNAs play crucial roles in tumorigenesis and tumor progression. miR-770 has been reported to be downregulated in several cancers and affects cancer cell proliferation, apoptosis, metastasis and drug resistance. However, the role and underlying molecular mechanism of miR-770 in human glioma remain unknown and need to be further elucidated. METHODS: The expression of miR-770 in glioma tissues and cell lines was measured by quantitative real-time PCR (qRT-PCR) to explore the association of miR-770 expression with clinicopathological characteristics. The expression of CDK8 was detected by qRT-PCR and Western blotting in glioma tissues. A target prediction program and a dual-luciferase reporter assay were used to confirm that CDK8 is a target gene of miR-770. MTT and cell counting assays were used to assess the effect of miR-770 on glioma cell proliferation. The cell cycle distribution and apoptosis were examined by flow cytometry. CDK8 siRNA and overexpression were used to further confirm the function of the target gene. RESULTS: We demonstrated that miR-770 expression was downregulated in human glioma tissues and cell lines. The overexpression of miR-770 inhibited glioma cell proliferation and cell cycle G1-S transition and induced apoptosis. The inhibition of miR-770 facilitated cell proliferation and G1-S transition and suppressed apoptosis. miR-770 expression was inversely correlated with CDK8 expression in glioma tissues. CDK8 was confirmed to be a direct target of miR-770 by using a luciferase reporter assay. The overexpression of miR-770 decreased CDK8 expression at both the mRNA and protein levels, and the suppression of miR-770 increased CDK8 expression. Importantly, CDK8 silencing recapitulated the cellular and molecular effects observed upon miR-770 overexpression, and CDK8 overexpression eliminated the effects of miR-770 overexpression on glioma cells. Moreover, both exogenous expression of miR-770 and silencing of CDK8 resulted in suppression of the Wnt/ß-catenin signaling pathway. CONCLUSIONS: Our study demonstrates that miR-770 inhibits glioma cell proliferation and G1-S transition and induces apoptosis through suppression of the Wnt/ß-catenin signaling pathway by targeting CDK8. These findings suggest that miR-770 plays a significant role in glioma progression and serves as a potential therapeutic target for glioma.

TAT-PEP Enhanced Neurobehavioral Functional Recovery by Facilitating Axonal Regeneration and Corticospinal Tract Projection After Stroke.

Paired immunoglobulin-like receptor B (PirB) has been identified as a new receptor for myelin-associated inhibitory (MAI) proteins, which may play important role in axonal regeneration and corticospinal tract (CST) projection associated with neurobehavioral function recovery after stroke. Here, we found that the expression of PirB was increased in the cortical penumbra from 1 to 28 days after transient focal cerebral ischemic reperfusion of rats. Then, transactivator of transcription-PirB extracellular peptide (TAT-PEP) was generated that might block the interactions between MAIs and PirB. The results showed that TAT-PEP displayed high affinity for MAIs and ameliorated their inhibitory effect on neurite growth. Furthermore, TAT-PEP can widely distribute in the penumbra after intraperitoneal injection. Then, we found that TAT-PEP enhanced neurite growth and alleviated growth cone collapse after oxygen glucose deprivation (OGD) injury. In addition, TAT-PEP promoted long-term neurobehavioral functional recovery through enhancing axonal regeneration and CST projection. Finally, the observations demonstrated that POSH/RhoA/growth-associated protein 43 (GAP43) as PirB-associated downstream signaling molecules played important role in neurobehavioral functional recovery after stroke. Moreover, the underlying mechanism associated with TAT-PEP-mediated promoting axonal regeneration and CST projection was by intervening in the expression of POSH, RhoA, and GAP43. These studies suggest that TAT-PEP may represent an attractive therapeutic strategy against stroke.

Inhibition of PirB Activity by TAT-PEP Improves Mouse Motor Ability and Cognitive Behavior.

Paired immunoglobulin-like receptor B (PirB), a functional receptor for myelin-associated inhibitory proteins, plays an important role in axon regeneration in injured brains. However, its role in normal brain function with age has not been previously investigated. Therefore in this study, we examined the expression level of PirB in the cerebral cortex, hippocampus and cerebellum of mice at 1 month, 3 months and 18 months of age. The results showed that the expression of PirB increased with age. We further demonstrated that overexpression of PirB inhibited neurite outgrowth in PC12 cells, and this inhibitory activity of PirB could be reversed by TAT-PEP, which is a recombinant soluble PirB ectodomain fused with TAT domain for blood-brain barrier penetration. In vivo study, intraperitoneal administration of TAT-PEP was capable of enhancing motor capacity and spatial learning and memory in mice, which appeared to be mediated through regulation of brain-derived neurotrophic factor (BDNF) secretion. Our study suggests that PirB is associated with aging and TAT-PEP may be a promising therapeutic agent for modulation of age-related motor and cognitive dysfunctions.

Overexpression of MicroRNA-216a Suppresses Proliferation, Migration, and Invasion of Glioma Cells by Targeting Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 5.

Increasing studies have suggested that microRNAs (miRNAs) are involved in the development of gliomas. MicroRNA-216a has been reported to be a tumor-associated miRNA in many types of cancer, either as an oncogene or as a tumor suppressor. However, little is known about the function of miR-216a in gliomas. The present study was designed to explore the potential role of miR-216a in gliomas. We found that miR-216a was significantly decreased in glioma tissues and cell lines. Overexpression of miR-216a significantly suppressed the proliferation, migration, and invasion of glioma cells. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) was identified as a target gene of miR-216a in glioma cells by bioinformatics analysis, dual-luciferase reporter assay, real-time quantitative polymerase chain reaction, and Western blot analysis. Moreover, miR-216a overexpression inhibited the Wnt/ß-catenin signaling pathway. The restoration of LGR5 expression markedly reversed the antitumor effect of miR-216a in glioma cells. Taken together, these findings suggest a tumor suppressor role for miR-216a in gliomas, which inhibits glioma cell proliferation, migration, and invasion by targeting LGR5. Our study suggests that miR-216a may serve as a potential therapeutic target for future glioma treatment.

Parthenolide attenuates cerebral ischemia/reperfusion injury via Akt/GSK-3ß pathway in PC12 cells.

Parthenolide (PN), a sesquiterpene lactone isolated from the herbal medicine feverfew (Tanacetum parthenium), was reported to possess neuroprotective activity. However, the neuroprotective effect of PN against cerebral ischemia/reperfusion (I/R) injury remains unclear. Therefore, the aim of the present study was to explore the neuroprotective effects of PN against oxygen-glucose deprivation (OGD)-induced apoptosis in PC12 cells and the underlying mechanisms. Our results demonstrated that PN ameliorated OGD/R-evoked neuronal injury and oxidative stress in PC12 cells. In addition, PN notably decreased HIF-1α expression, as well as inhibited apoptosis in PC12 cells after OGD/R. Furthermore, PN pretreatment significantly enhanced the phosphorylation of Akt and GSK-3ß in PC12 cells exposed to OGD/R. In conclusion, the present study demonstrated that PN exhibits a neuroprotective effect against OGD/R through activation of the Akt/GSK-3ß signaling pathway. Our findings suggest that PN has the potential to serve as a novel therapeutic agent for cerebral I/R injury.

PirB Overexpression Exacerbates Neuronal Apoptosis by Inhibiting TrkB and mTOR Phosphorylation After Oxygen and Glucose Deprivation Injury.

Previous studies have proven that paired immunoglobulin-like receptor B (PirB) plays a crucial suppressant role in neurite outgrowth and neuronal plasticity after central nervous system injury. However, the role of PirB in neuronal survival after cerebral ischemic injury and its mechanisms remains unclear. In the present study, the role of PirB is investigated in the survival and apoptosis of cerebral cortical neurons in cultured primary after oxygen and glucose deprivation (OGD)-induced injury. The results have shown that rebarbative PirB exacerbates early neuron apoptosis and survival. PirB gene silencing remarkably decreases early apoptosis and promotes neuronal survival after OGD. The expression of bcl-2 markedly increased and the expression of bax significantly decreased in PirB RNAi-treated neurons, as compared with the control- and control RNAi-treated ones. Further, phosphorylated TrkB and mTOR levels are significantly downregulated in the damaged neurons. However, the PirB silencing markedly upregulates phosphorylated TrkB and mTOR levels in the neurons after the OGD. Taken together, the overexpression of PirB inhibits the neuronal survival through increased neuron apoptosis. Importantly, the inhibition of the phosphorylation of TrkB and mTOR may be one of its mechanisms.

MicroRNA-25 Negatively Regulates Cerebral Ischemia/Reperfusion Injury-Induced Cell Apoptosis Through Fas/FasL Pathway.

MicroRNA-25 (miR-25) has been reported to be a major miRNA marker in neural cells and is strongly expressed in ischemic brain tissues. However, the precise mechanism and effect of miR-25 in cerebral ischemia/reperfusion (I/R) injury needs further investigations. In the present study, the oxygen-glucose deprivation (OGD) model was constructed in human SH-SY5Y and IMR-32 cells to mimic I/R injury and to evaluate the role of miR-25 in regulating OGD/reperfusion (OGDR)-induced cell apoptosis. We found that miR-25 was downregulated in the OGDR model. Overexpression of miR-25 via miRNA-mimics transfection remarkably inhibited OGDR-induced cell apoptosis. Moreover, Fas was predicted as a target gene of miR-25 through bioinformatic analysis. The interaction between miR-25 and 3'-untranslated region (UTR) of Fas mRNA was confirmed by dual-luciferase reporter assay. Fas protein expression was downregulated by miR-25 overexpression in OGDR model. Subsequently, the small interfering RNA (siRNA)-mediated knockdown of Fas expression also inhibited cell apoptosis induced by OGDR model; in contrast, Fas overexpression abrogated the protective effects of miR-25 on OGDR-induced cells. Taken together, our results indicate that the upregulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through downregulating Fas/FasL, which will provide a promising therapeutic target.

CD147 is increased in HCC cells under starvation and reduces cell death through upregulating p-mTOR in vitro.

Transarterial chemoembolization (TACE) is the standard of care for treatment of intermediate hepatocellular carcinoma (HCC), however, key molecules involved in HCC cell survival and tumor metastasis post-TACE remain unclear. CD147 is a member of the immunoglobulin superfamily that is overexpressed on the surface of HCC cells and is associated with malignant potential and poor prognosis in HCC patients. In this study, using an Earle's Balanced Salt Solution medium culture model that mimics nutrient deprivation induced by TACE, we investigated the regulation of CD147 expression on HCC cells under starvation conditions and its functional effects on HCC cell death. During early stages of starvation, the expression of CD147 was considerably upregulated in SMMC7721, HepG2 and HCC9204 hepatoma cell lines at the protein levels. Downregulation of CD147 by specific small interfering RNA (siRNA) significantly promoted starvation-induced cell death. In addition, CD147 siRNA-transfected SMMC7721 cells demonstrated significantly increased levels of both apoptosis and autophagy as compared to cells transfected with control siRNA under starvation conditions, whereas no difference was observed between the two treatment groups under normal culture conditions. Furthermore, silencing of CD147 resulted in a remarkable downregulation of phosphorylated mammalian target of rapamycin (p-mTOR) in starved SMMC7721 cells. Finally, the combined treatment of starvation and anti-CD147 monoclonal antibody exhibited a synergistic HCC cell killing effect. Our study suggests that upregulation of CD147 under starvation may reduce hepatoma cell death by modulating both apoptosis and autophagy through mTOR signaling, and that CD147 may be a novel potential molecular target to improve the efficacy of TACE.

Vasoactive intestinal peptide protects against ischemic brain damage induced by focal cerebral ischemia in rats.

Vasoactive intestinal peptide (VIP) exerts neuroprotective effects under various neurotoxic conditions in vitro. In the present study, we investigated the effects of VIP on transient ischemic brain damage. Focal cerebral ischemia was induced using middle cerebral artery occlusion (MCAO) for 120 min in the adult rat brain. Either a single intracerebroventricular injection of VIP or saline was given at the beginning of reperfusion. Forty-eight hours after MCAO, the rats were sacrificed for evaluation of the infarct volume and histological analysis. ELISA was performed to assay levels of serum S100B before being sacrificed. We also evaluated the blood-brain barrier (BBB) permeability using Evans blue dye injection method. In contrast to the cases treated with vehicle, the infarct volume was significantly (P<0.05) reduced, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining and immunoreactivity for S100B were also significantly (P<0.05) decreased in the ischemic hemisphere with VIP treatment. In addition, the elevations of serum S100B were significantly (P<0.01) attenuated in VIP-treated rats compared with those of control rats. Treatment with VIP did not result in a significant reduction of Evans blue leakage, although it tended to be lower than that in the control rats. Our data suggest that treatment with VIP reduces brain damage in ischemic rats, and this effect may be associated with the attenuation of apoptosis and S100B expression.

[Neuroprotective effects of vasoactive intestinal peptide against rat cerebral ischemia].

OBJECTIVE: To explore the neuroprotective effect of vasoactive intestinal peptide (VIP) in rat ischemic brain injury. METHODS: VIP was administered via intracerebroventricular injection in SD rats prior to focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery. The infarct volume was assessed with TTC staining, and immunohistochemistry was performed to analyze the S100beta expression in the cerebral tissue, with the serum concentrations of S100beta detected by double-antibody sandwich enzyme-linked immunosorbent assay. RESULTS: After VIP injection, the relative infarct volume in the rats with cerebral ischemia was significantly reduced by 32.3% as compared with the volume in the control group on day 1 (P<0.05), and the number of S100beta-positive cells was significantly decreased in the cerebral tissue (P<0.05). The injection also resulted in significantly decreased serum S100beta concentrations in the rats (P<0.05). CONCLUSION: VIP injection can reduce the infarct volume in rats with focal cerebral ischemia, suggesting the neuroprotective effect of VIP in brain ischemia possibly by reducing S100beta overexpression.