Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury.

The current study explores the potential function and the underlying mechanisms of endothelial cell-derived R-spondin 3 (RSPO3) neuroprotection against ischemia/reperfusion-induced neuronal cell injury. In both neuronal cells (Neuro-2a) and primary murine cortical neurons, pretreatment with RSPO3 ameliorated oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced neuronal cell death and oxidative injury. In neurons RSPO3 activated the Akt, Erk and ß-Catenin signaling cascade, but only Erk inhibitors reversed RSPO3-induced neuroprotection against OGD/R. In mouse embryonic fibroblasts (MEFs) and neuronal cells, RSPO3-induced LGR4-Gab1-Gαi1/3 association was required for Erk activation, and either silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. In mice, middle cerebral artery occlusion (MCAO) increased RSPO3 expression and Erk activation in ischemic penumbra brain tissues. Endothelial knockdown or knockout of RSPO3 inhibited Erk activation in the ischemic penumbra brain tissues and increased MCAO-induced cerebral ischemic injury in mice. Conversely, endothelial overexpression of RSPO3 ameliorated MCAO-induced cerebral ischemic injury. We conclude that RSPO3 activates Gαi1/3-Erk signaling to protect neuronal cells from ischemia/reperfusion injury.

Oxygen glucose deprivation/re-oxygenation-induced neuronal cell death is associated with Lnc-D63785 m6A methylation and miR-422a accumulation.

Oxygen glucose deprivation/re-oxygenation (OGD/R) induces neuronal injury via mechanisms that are believed to mimic the pathways associated with brain ischemia. In SH-SY5Y cells and primary murine neurons, we report that OGD/R induces the accumulation of the microRNA miR-422a, leading to downregulation of miR-422a targets myocyte enhancer factor-2D (MEF2D) and mitogen-activated protein kinase kinase 6 (MAPKK6). Ectopic miR-422a inhibition attenuated OGD/R-induced cell death and apoptosis, whereas overexpression of miR-422a induced significant neuronal cell apoptosis. In addition, OGD/R decreased the expression of the long non-coding RNA D63785 (Lnc-D63785) to regulate miR-422a accumulation. Lnc-D63785 directly associated with miR-422a and overexpression of Lnc-D63785 reversed OGD/R-induced miR-422a accumulation and neuronal cell death. OGD/R downregulated Lnc-D63785 expression through increased methyltransferase-like protein 3 (METTL3)-dependent Lnc-D63785 m6A methylation. Conversely METTL3 shRNA reversed OGD/R-induced Lnc-D63785 m6A methylation to decrease miR-422a accumulation. Together, Lnc-D63785 m6A methylation by OGD/R causes miR-422a accumulation and neuronal cell apoptosis.

Echogenicity Changes in Brainstem Raphe Detected by Transcranial Parenchymal Sonography and Clinical Characteristics in Parkinson's Disease.

Background: Decreased brainstem raphe (BR) echogenicity detected by transcranial parenchymal sonography (TCS) is associated with depression in psychiatric and neurologic diseases. However, previous studies focusing on the relationship between motor and non-motor symptoms and echogenicity changes in BR in patients with PD yielded controversial results. Objectives: To investigate the relationship between echogenicity changes in BR detected by TCS and motor and a series of non-motor symptoms in patients with PD. Methods: Consecutive PD patients were recruited from the Second Affiliated Hospital of Soochow University. Demographic information and Motor and non-motor symptoms for all subjects were collected. TCS was used to detect the echogenicity changes in BR in PD patients. Results: One hundred and thirty-five consecutive patients with PD were enrolled in the study. The BR abnormal rate was significantly higher in PD patients with anxiety (p = 0.003) or depression (p = 0.022) than patients without. Spearman correlation analyses showed that Hamilton Rating Scale for Depression(HRSD) (r = 0.274, p = 0.002) and Parkinson's Disease Questionnaire 39-item(PDQ-39) (r = 0.208, p = 0.034) scores were positively correlated with abnormal BR echogenicity. Multivariate logistic regression analyses showed that HRSD and HAMA scores were associated with BR hypoechogenicity, the corresponding odds ratios (confidence intervals) were 1.07 (95% CI, 1.01-1.13) and 1.10(1.01-1.18), respectively. However, the PDQ-39 score was not associated with BR hypoechogenicity. Conclusion: The abnormal reduction in BR echogenicity detected by TCS is associated with depression and anxiety, but not motor symptoms in PD patients.

MicroRNA-6862 inhibition elevates sphingosine kinase 1 and protects neuronal cells from MPP+-induced apoptosis.

MPP+ (1-methyl-4-phenylpyridinium)-induced dopaminergic neuronal cell apoptosis is associated with sphingosine kinase 1 (SphK1) inhibition. We here tested the potential effect of microRNA-6862 (miR-6862), a novel SphK1-targeting miRNA, on MPP+-induced cytotoxicity in neuronal cells. MiR-6862 locates in the cytoplasm of SH-SY5Y neuronal cells. It directly binds to SphK1 mRNA. In SH-SY5Y cells and HCN-2 cells, ectopic overexpression of miR-6862 decreased SphK13'-untranslated region luciferase reporter activity and downregulated its expression. miR-6862 inhibition exerted opposite activity and elevated SphK1 expression. In neuronal cells, MPP+-induced cell death was significantly inhibited through miR-6862 inhibition. Conversely, ectopic overexpression of miR-6862 or CRISPR/Cas9-induced SphK1 knockout augmented MPP+-induced apoptosis in the neuronal cells. Importantly, antagomiR-6862 failed to inhibit MPP+-induced apoptosis in SphK1-knockout SH-SY5Y cells. These results suggest that inhibition of miR-6862 induces SphK1 elevation and protects neuronal cells from MPP+-induced cell death.

Systems-Based Interactome Analysis for Hematopoiesis Effect of Angelicae sinensis Radix: Regulated Network of Cell Proliferation towards Hemopoiesis.

OBJECTIVE: To explore the molecular-level mechanism on the hematopoiesis effect of Angelicae sinensis Radix (ASR) with systems-based interactome analysis. METHODS: This systems-based interactome analysis was designed to enforce the workflow of "ASR (herb)→compound→target protein→internal protein actions→ending regulated protein for hematopoiesis". This workflow was deployed with restrictions on regulated proteins expresses in bone marrow and anemia disease and futher validated with experiments. RESULTS: The hematopoiesis mechanism of ASR might be accomplished through regulating pathways of cell proliferation towards hemopoiesis with cross-talking agents of spleen tyrosine kinase (SYK), Janus kinase 2 (JAK2), and interleukin-2-inducible T-cell kinase (ITK). The hematopoietic function of ASR was also validated by colony-forming assay performed on mice bone marrow cells. As a result, SYK, JAK2 and ITK were activated. CONCLUSION: This study provides a new approach to systematically study and predict the therapeutic mechanism for ASR based on interactome analysis towards biological process with experimental validations.

Poor nighttime sleep is positively associated with dyskinesia in Parkinson's disease patients.

BACKGROUND: Dyskinesia is a troublesome complication of long-term dopaminergic medications in Parkinson's disease (PD) patients. Many factors are reported to be associated with dyskinesia in PD. OBJECTIVE: To investigate the association between sleep quality and dyskinesia in patients with PD. METHODS: Four hundred twenty-five patients with PD were enrolled in this study. Demographic information was collected. Unified Parkinson's Disease Rating Scale (UPDRS) and Hoehn and Yahr (H-Y) stage scale were also performed. Epworth Sleepiness Scale (ESS) and Pittsburgh Sleep Quality Index (PSQI) were applied to evaluate daytime sleepiness and overall nighttime sleep quality, respectively, in PD patients. RESULTS: Patients with dyskinesia tended to have a longer duration of disease, higher daily levodopa-equivalent dose (LED), H-Y stage, UPDRS II and PSQI score, and a higher percentage of levodopa treatment than those without dyskinesia. After adjusting for age, sex, age at onset of PD, disease duration, UPDRS I, UPDRS II, UPDRS III, cigarette smoking, use of different antiparkinsonian drugs, phenotype, daily LED, and restless leg syndrome (RLS), PSQI score was still associated with dyskinesia, with corresponding ORs 1.111 (95% CI, 1.004-1.229) as a continuous variable, and 2.469 (95% CI, 1.051-5.800) as a categorical variable, respectively. Further analysis of PSQI components showed that subjective sleep quality and sleep latency were associated with dyskinesia in PD patients. CONCLUSIONS: Our data showed that poor nighttime sleep is positively associated with dyskinesia in PD patients. Attention to the management of nighttime sleep quality may be beneficial to dyskinesia in patients with PD.

Odor selectivity of hyposmia and cognitive impairment in patients with Parkinson's disease.

OBJECTIVE: Hyposmia is one of the earliest non-motor features of Parkinson's disease (PD) and can precede the onset of motor symptoms by years. Most of the current olfactory detection tests are targeted at Western populations. The exact relationship between hyposmia and cognitive impairment is unknown. The purpose of the study was to find bromines that can effectively identify olfactory dysfunction and investigate the relationship between hyposmia and cognitive function in early, non-demented, drug-naïve patients with PD in the People's Republic of China. METHODS: Sixty-three early, non-demented, drug-naïve patients with PD and 55 healthy controls were enrolled in the study. The T&T olfactometer and a Chinese version of Montreal Cognitive Assessment (MoCA) were applied to assess subjects' olfactory and cognitive functions. Patients with PD also completed the Modified Unified Parkinson's disease-rating scale (UPDRS) and Hoehn and Yahr (H&Y) scale. RESULTS: Patients with PD had lower scores of visuospatial and executive function (p=0.000), attention (p=0.03), and delayed recall (p=0.001) than controls. ß-phenylethyl alcohol (floral smell, smell of rose petals) and isovaleric acid (smell of sweat, stuffy socks) were more sensitive for identifying hyposmia in patients with PD than three other odors. Multivariate logistic regression analysis showed that impaired visuospatial and executive function was associated with hyposmia (p=0.013), but was independent of other PD-associated variables. CONCLUSION: Hyposmia was common in early, non-demented, drug-naïve PD patients. ß-Phenylethyl alcohol and isovaleric acid were more superior for identifying hyposmia in early non-demented Chinese patients with PD. Hyposmia was associated with impaired visuospatial and executive function in patients with PD. Further prospective studies that apply a series of neuropsychological tests and functional magnetic resonance imaging methods in large samples in multicenter studies are needed to confirm our findings and to investigate the relationship between hyposmia and cognitive function with disease progression in patients with PD.

Parkinson's disease patients with pain suffer from more severe non-motor symptoms.

Non-motor symptoms, including pain, depression, sleep disorder, and olfactory dysfunction, occur frequently in patients with Parkinson's disease (PD), even before the onset of motor symptoms. Although studies have examined the correlation between pain and depression or sleep disorder in PD, few studies have investigated the correlation between pain and a range of other non-motor symptoms of PD. PD patients (n = 142) with or without pain were included in the study. PD severity was evaluated with the Unified Parkinson's Disease Rating Scale (UPDRS) and the Hoehn and Yahr (H/Y) staging scale. Pain severity was analyzed with the Visual Analog Scale. The Hamilton Rating Scale for Depression (HRSD; 24 items), Montreal Cognitive Assessment Beijing Version (MoCA), and non-motor questionnaire (NMSQT) measured symptoms of depression, cognitive function, and non-motor symptoms. The incidence of pain was 47.9% in patients with PD, most of whom had moderate pain levels. Patients with pain showed higher HRSD, UPDRS, H/Y, and NMSQT scores and lower MoCA scores compared to those of patients without pain. HRSD and NMSQT scores were closely related with pain (P < 0.001). Non-motor symptoms were more prominent in patients with pain compared to that of controls and PD patients without pain.

[Depression associated with movement disorders].

OBJECTIVE: To explore the incidence of depression in movement disorders and associated factors. METHODS: A total of 121 Parkinson's disease (PD) patients from August 2010 to June 2011 and 62 ET patients from July 2009 to June 2010 were recruited. All of them were outpatients of our hospital. PD patients received the unified Parkinson's disease rating scale (UPDRS) -motor examination and a modified Hoehn and Yahr scale to stage the severity of their disorders while 62 essential tremor (ET) patients were evaluated with tremor rating scale for tremor-motor examination (items 1 - 15 of rating scale). All participants completed Hamilton depression rating scale (24 items) to measure the presence and degree of symptoms of depression. RESULTS: It was found that 56.2% of PD patients and 53.2% of ET patients were depressed (HAMD score of 8 or higher). Among them, the rates of mild depression were 38.9% and 35.5%, moderate depression 15.7% and 17.7% and severe depression 1.7% and 0% in PD and ET patients respectively. No significant differences existed between each group. The factor scores of cognitive impairment were 1.81 ± 1.86 and 1.04 ± 1.07 in PD and ET patients while those of sense of despair were 2.95 ± 1.97 and 4.09 ± 2.08 in each group. The differences had statistical significance in two factor scores of two groups (P < 0.05). No differences in anxiety/somatization, lose weight, day-and-night changes, block and sleep disorders between two groups. For PD patients, the motor score of UPDRS-III was positively correlated with total HAMD score (r = 0.511, P < 0.01). Similarly, for ET patients, tremor rating scale for tremor-motor subscale score and HAMD score were positively correlated (r = 0.828, P < 0.01). CONCLUSION: As two common movement disorders, PD and ET have a high incidence of depression. The severity of depression is similar in two groups. There is no significant intra-group difference in severity and frequency of depression. The most common symptoms are anxiety somatization, anhedonia, working difficulty, slowness and sleep disturbance. The depression and motor symptoms are positively correlated. Like the non-motor symptoms in PD, we should also pay attention to the non-motor symptoms in ET.

[Effect of Budesonide on Smad4, PDGF-A and PAI-1 in a rat model of pulmonary fibrosis].

AIM: To observe the effect of Budesonide (BUD) on TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue in pulmonary fibrosis rats. METHODS: Forty-five adult female Wistar rats were randomly divided into normal solution (NS) group, BUD group and bleomycin (BLM) group. 9 g/L NaCl solution was instilled into the tratracheaes in NS group, and BLM were used in BUD group and BLM group. NS group and BLM group were inhaled with 9 g/L NaCl solution once everyday at day 0-6, and BUD group were used with BUD. Five rats in every group were killed at 7th, 14th 28th day. The appearances of alveolitis and fibrosis were displayed in HE and Masson staining. The expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue were detected by immunohistochemistry. RESULTS: The extent of the alveolitis in BUD group at 7th, 14th day were significantly lower than that in BLM group(P<0.05). The fibrosis and the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissues at 7th, 14th, 28th day, BUD group were significantly lower than BLM group(P<0.05). CONCLUSION: After inhaled BUD, the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue could be decreased, and the extent of alveolitis and pulmonary fibrosis could be improved in bleomycin-induced pulmonary fibrosis rats.

[Methylprednisolone enhances the expression of Toll-like receptor 2 on macrophages in induced sputum from patients with chronic cough].

OBJECTIVE: To explore the relationship between airway inflammation and Toll-like receptor 2 (TLR2) expression on macrophages in induced sputum from chronic cough patients, and to observe the effect of methylprednisolone on this receptor. METHODS: Eighty-six outpatients with chronic cough were enrolled in this study from the respiratory department in the Affiliated Hospital of Luzhou Medical College from September 2008 to March 2010. These diseases were diagnosed according to the guideline of Chinese Society of Respiratory Diseases in 2005, excluding severe liver, heart and kidney pathology and pregnant women. Among 86 cases, 31 were cough variant asthma (CVA), 14 upper airway cough syndrome (UACS), 16 eosinophilic bronchitis (EB) and 25 gastroesophageal reflux (GERC). Twenty healthy volunteers were included as controls. Induced sputum was collected and cell counts were analyzed. TLR2 expression on macrophages was determined by immunofluorescence staining. Cytokine levels were measured with enzyme linked immunosorbent assay (ELISA). RESULTS: (1) The total inflammatory cells [(3.4 ± 1.1) × 10(6)/L, (2.6 ± 0.6) × 10(6)/L, (2.6 ± 0.6) × 10(6)/L, (2.7 ± 0.5) × 10(6)/L] and neutrophils (0.52 ± 0.06, 0.58 ± 0.06, 0.52 ± 0.04, 0.54 ± 0.06) in induced sputum from CVA, UACS, EB and GERC were significantly increased (t value = 5.27, 4.09, 3.16, 3.92 and 3.62, 3.49, 4.82, 6.17 respectively, all P < 0.01), with decreased macrophages (0.35 ± 0.04, 0.40 ± 0.06, 0.38 ± 0.04, 0.43 ± 0.05) in comparison with the control group (t value = 4.76, 5.24, 4.57, 3.18, all P < 0.01). There was a significant increase in eosinophils (0.118 ± 0.054, 0.099 ± 0.034) in CVA and EB group (t value = 4.46, 3.87, 5.19 and 3.51, 4.06, 4.38 respectively, all P < 0.01). (2) The concentration of IL-8 [(7.0 ± 1.2) µg/L, (10.2 ± 3.1) µg/L, (6.3 ± 1.9) µg/L, (7.7 ± 2.5) µg/L] and TNF-α [(30 ± 11) ng/L, (46 ± 16) ng/L, (21 ± 5) ng/L, (33 ± 15) ng/L] from induced sputum in CVA, UACS, EB and GERC groups were significantly increased, compared with the control group(t value = 4.58, 3.67, 4.26, 5.39 and 5.16, 3.97, 4.59, 3.34 respectively, all P < 0.01). (3) The concentration of IL-8 was positively correlated with neutrophils in each group (R value = 0.628, 0.816, 0.769 and 0.733 respectively, all P < 0.05). The concentration of TNF-α was also positively correlated with neutrophils in each group (R value = 0.579, 0.865, 0.730 and 0.755 respectively, all P < 0.05). (4) The expression of TLR2 (56 ± 15, 38 ± 7, 65 ± 17, 27 ± 4) on macrophages in CVA, UACS, EB and GERC was significantly decreased. The expression of TLR2 (75 ± 17, 53 ± 16, 94 ± 30, 41 ± 5) on macrophages was enhanced by methylprednisolone. CONCLUSIONS: The expression of TLR2 on macrophages from induced sputum in CVA, UACS, EB and GERC was decreased, but could be enhanced by methylprednisolone, suggesting that TLR2 might play a regulatory role in airway inflammatory response.

Preoperative serum retinol-binding protein 4 is associated with the prognosis of patients with hepatocellular carcinoma after curative resection.

PURPOSE: Metabolic syndrome and insulin resistance have been linked to increased risk of occurrence and mortality of hepatocellular carcinoma (HCC). Recently, retinol-binding protein 4 (RBP4) was clarified as a specific serological marker of insulin resistance. The aim of this study was to determine whether serum RBP4 could be used as a potential marker for predicting prognosis in patients with HCC after curative resection. METHODS: Western immunoblotting and Enzyme-linked immunosorbent assay were used to measure the RBP4 expression in cell lines, supernatant, and serum. Serum RBP4 levels were compared with clinicopathological features and outcomes of patients with HCC. Furthermore, we investigated the impact of serum RBP4 level, serum C-peptide level, and HOMA-IR on overall survival (OS) and disease-free survival (DFS) of patients with HCC in the training cohort (156 patients with HCC), and then were validated in the validation cohort (105 patients with HCC). RESULTS: RBP4 protein overexpressed in HCC cell lines compared with normal liver cell line (P < 0.001) and correlated with metastatic potential. Serum RBP4 levels were associated with OS [hazard ratio (HR) = 2.208, P < 0.001] and DFS (HR = 1.878, P = 0.029) of patients with HCC. By multivariate analysis, the serum RBP4 level was identified as an independent factor for OS (HR = 2.170, P = 0.004) and DFS (HR = 1.769, P = 0.037) of patients with HCC. The prognostic value of serum RBP4 level was confirmed in the validation cohort. CONCLUSIONS: The serum RBP4 level is potential to be a useful prognostic factor for HCC after curative resection.

Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation.

The role of astrocytes in microglia-induced neuronal death remains controversial. In this study, astrocytes and astrocyte-derived conditioned media (ACM) supported the survival of dopaminergic neurons, and the former was more effective than the latter. In the presence of astrocytes, low concentrations of LPS enhanced the survival of dopaminergic neurons, while high concentrations attenuated survival. LPS dramatically induced astrocytes to secrete IL-6 in a dose-dependent manner with no effect on secretion of GDNF. Neuron-astrocyte cultures had highest secretion of GDNF, followed by ACM-treated neuron-enriched cultures. After neuron-astrocyte cultures treated with IL-6-neutralizing antibody, both effects of the enhanced and attenuated survival of dopaminergic neurons were abolished. Our results indicate that astrocytes play a protective role in the LPS-induced damage of dopaminergic neurons in certain circumstances, and the interaction between astrocytes and dopaminergic neurons may enhance the protective effect of astrocytes. Suitable activation of astrocytes increases the protective effect while excessive activation attenuates it, and IL-6 might mediate this dual action. The underlying mechanisms related to the secretion of GDNF and proinflammatory factors warrant further investigation.

[Prevalence of mite hypersensitivity in children with asthma].

By skin prick test, three kinds of mite allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis) were tested in a group of asthma children in Jinhua area from Oct 2005 to Sep 2006. The positive rate to allergen from D. pteronyssinus and D. farinae was 80.6% and 77.8% respectively, higher than that of Blomia tropicalis (61.1%) (chi2 = 21.39, P < 0.05). Cases positive to Blomia tropicalis allergen showed 100% and 95.9% positive reaction to D. pteronyssinus and D. farinae respectively. The results demonstrated that the important allergens for children's asthma are from D. pteronyssinus and D. farinae, while the with the former two mite allergens. Blomia tropicalis allergen is supposed to have cross-reactivity

[Purification and identification of recombinant varicella-zoster virus glycoprotein E].

OBJECTIVE: To purify and identify recombinant Varicella-Zoster Virus Glycoprotein E. METHODS: The recombinant plasmid pGEX-VZVgE was induced by isopropyl-beta-D-thiogalactoside (IPTG), the fusion protein was purified with affinity chromatography column; then the purified fusion protein was cleaved by thrombin, and the product's antigenicity was examined by Western blot. RESULTS: The product of pGEX-VZVgE induced by IPTG was separated from the mixture proteins by the affinity chromatography column, the expressed fusion protein's relative molecular mass was about 98 x 10(3). After cleavage, the obtained VZV Glycoprotein E's relative molecular mass was about 72 x 10(3); the purified fusion protein and VZV Glycoprotein E were single band by SDS-PAGE. The available antigenicity of Glycoprotein E was confirmed by Western blot. CONCLUSION: Purification of VZV Glycoprotein E with affinity chromatography is an effective method. It provides a foreground for studies on the application of VZV gE.