Evidence for A1 and A3 receptors mediating adenosine-induced intracellular calcium release in the dorsal root ganglion neurons by using confocal microscopy imaging

Adenosine exerts a key role in analgesia. In the present study, adenosine-induced Ca²⁺ responses were revealed by using confocal microscopy imaging in the rat dorsal root ganglia (DRG) neurons in vitro. Our results showed that adenosine could evoke increases in the intracellular Ca²⁺ concentration in the DRG neurons. In addition, by application of selective receptor antagonists, two types of receptors, A₁R and A₃R, were identified to be involved in the adenosine-induced Ca²⁺ release from intracellular stores in neurons. Altogether, these results suggest that confocal microscopy imaging combined with fluorescent dyes could help to detect the analgesic-induced ion signaling in single cell.     

shRNA-mediated GSTP1 gene silencing enhances androgen-independent cell line DU145 chemosensitivity

Purpose

Design short hairpin RNA (shRNA) interference sequence to silence glutathione S-transferase P1 (GSTP1) gene of androgen-independent prostate cancer cell line DU145, and then to explore its effect on sensitivity to chemotherapeutics.

Methods

Target sequence was picked up to form the shRNA. DU145 cell was divided into five groups according to the shRNA added for transfection: shRNA255, shRNA554, shRNA593, negative-shRNA and blank group. Fluorescence microscope was used to pick up the shRNA with the highest transfection ratio. Western blotting and RT-PCR were taken to pick up the shRNA with the best gene silencing result. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and terminal de-oxynucleotidyl transferase-mediated dUTP nick end-labeling assay were used to detect survival ratio and apoptosis ratio of DU145 administered of fluorouracil (5-FU) or paclitaxel (PA) at different concentrations before and after shRNA transfection.

Results

Three different shRNA oligonucleotides (shRNA255; shRNA554; shRNA593) targeting the coding sequence of GSTP1 mRNA and one negative control shRNA were constructed. The transfection ratio of shRNA554 (76.2 ± 0.68 %) was higher than that of shRNA255 (63.3 ± 1.04 %) (P < 0.01) or shRNA593 (72.7 ± 0.33 %) (P < 0.01). After transfection of shRNA554, the mRNA and protein of level were the lowest, P < 0.01. The survival ratio of DU145 administered with 5-FU of different concentrations (30, 60, 120, 240 μg/ml) declined after transfection (P < 0.01). Besides, the apoptosis ratio increased after transfection (P < 0.01). Similarly the survival ratio of DU145 administered with PA of different concentrations (0.2, 2, 10, 20 μg/ml) declined (P < 0.01) and the apoptosis ratio increased (P < 0.01) after transfection.

Conclusions

The gene GSTP1 silence via shRNA transfection to androgen-independent prostate cancer cell line DU145 enhances the sensitivity to chemotherapeutics.     

The risk factors of neurologic deficits of one-stage posterior vertebral column resection for patients with severe and rigid spinal deformities

Purpose

To determine the risk factors of neurologic deficits during PVCR correction, so as to help improve safety during and after surgery.

Methods

A consecutive series of 76 patients with severe and rigid spinal deformities who were treated with PVCR at a single institution between October 2004 and July 2011 were included in our study. Of the 76 patients, 37 were male and 39 female, with an average age of 17.5 years (range 10–48 years). There were 52 adolescent patients (with an age <18 years) and 24 adult patients (with an age ≥18 years). Preoperatively, postoperatively and 6 months after surgery, we performed systemically neurologic function evaluations of each patients through meticulous physical examination. Any new abnormality or deterioration in evaluation of neurologic function than preoperative is reckoned postoperative neurologic deficits. Ten variables that might affect the safety of neurologic deficits during PVCR procedures, including imaging factors, clinical factors and operational factors, were analyzed using univariate analysis. Then the variables with statistical difference were analyzed by using multi-factor unconditional logistic regression analysis.

Results

No patient in this series had permanent paraplegia and nerve root injury due to operation. Change of neurologic status was found in six patients after surgery. Results of single-factor comparison demonstrated that the following seven variables were statistically different (P < 0.05): location of apex at main curve (X ₃), Cobb angle at the main curve at the coronal plane (X ₄), scoliosis associated with thoracic hyperkyphosis (X ₅), level of vertebral column resected (X ₆), number of segmental vessels ligated (X ₇), preexisting neurologic dysfunction (X ₈), and associated with intraspinal and brain stem anomalies (X ₉). The multi-factor unconditional logistic regression analysis revealed that X ₈ (OR = 49.322), X ₉ (OR = 18.423), X ₅ (OR = 11.883), and X ₆ (OR = 8.769) were independent and positively correlated with the neurologic deficit.

Conclusions

Preexisting neurologic dysfunction, associated with intraspinal and brain stem anomalies, scoliosis associated with thoracic hyperkyphosis and level of vertebral column resected are independent risk factors for neurologic deficits during PVCR procedure.     

Decreased expression of Gab2 in patients with temporal lobe epilepsy and pilocarpine‐induced rat model

Growth factor receptor bound protein‐2 associated binding protein‐2 (Gab2) is widely expressed in the central nervous system, and participates in multiple signaling pathways. Recent studies showed that Gab2 was involved in the pathogenesis of Alzheimer's disease (AD). Gab2 reduces tau phosphorylation levels and is associated with cellular apoptosis and differentiation. However, whether Gab2 was also involved in the pathogenesis of epilepsy, remains unknown. This study aimed to investigate the expression pattern of Gab2 protein in brains with temporal lobe epilepsy (TLE) and in pilocarpine‐induced rat model of TLE. Western blot, immunohistochemistry, and immunofluorescence were used to assess the location and the expression level of Gab2 in the neocortex of the temporal lobe in patients with TLE and in rat model of epilepsy. Results showed that Gab2 protein was expressed mainly in the membranes and cytoplasm of neurons in the cortex and hippocampus. Gab2 protein expression was remarkably reduced in temporal neocortex of TLE patients. In hippocampus and adjacent cortex in rat epilepsy model, Gab2 expression was decreased at different time points after kindling compared with the controls, and the lowest level of Gab2 expression occurred at 1 week. Thus, significant reductions of Gab2 protein in both TLE patients and epilepsy rats suggest that Gab2 may play an important role in the pathogenesis of TLE. Synapse 68:168–177, 2014. © 2013 Wiley Periodicals, Inc.     

颅内微小动脉瘤的血管内治疗简

目的探讨血管内介入治疗颅内微小动脉瘤的疗效并进行分析。方法回顾性分析34例微小动脉瘤（共35个动脉瘤）病人的临床资料,均采用血管内栓塞治疗,其中栓塞加支架辅助治疗3例。结果动脉瘤完全填塞28个,瘤颈残留7个。术中无动脉瘤破裂,术后脑梗死8例。30例病人术后3个月复查DSA,残颈无增大表现而继续临床观察。随访34例,时间12个月,完全康复30例,遗留轻微神经功能障碍4例。结论对于已破裂的微小动脉瘤,在血管内介人治疗过程中,通过采取相应技巧,预防出血及缺血性并发症,可获得较好的临床疗效。     

Immunoglobulin A nephropathy in horseshoe kidney: Case reports and literature review

Horseshoe kidney is the most common congenital renal fusion anomaly. Immunoglobulin A nephropathy is a common glomerulonephritis worldwide. However, the co‐occurrence of these diseases had not been reported in the literature. We report the first two cases with the occurrence of immunoglobulin A nephropathy in horseshoe kidney. The first case was a 26‐year‐old male with hypertension and proteinuria (1.4 g/24 h), his pathological finding was primary immunoglobulin A nephropathy. The second case was a 15‐year‐old female who presented with recurrent peliosis on bilateral lower extremities, haematuria and proteinuria (1.7 g/24 h). Her renal biopsy finding was Henoch–Schonlein purpura nephritis (secondary immunoglobulin A nephropathy). In both cases, renal biopsy was performed by experienced doctors under ultrasonic guidance at the renal upper pole and no postoperative complications were observed. After they were treated based on the renal pathological findings for 6 months, urine protein excretion decreased significantly and blood pressure and serum creatinine stabilized. It is possible that immunoglobulin A nephropathy occurs in a horseshoe kidney patient. Renal biopsy may be valuable and viable for horseshoe kidney patients with heavy proteinuria to identify pathologic type of glomerulopathy and to guide treatment, if renal biopsy is performed by experienced doctors at the renal upper pole under renal ultrasonic guidance.     

胆管癌组织中信号转导及转录激活因子3信号通路相关基因的表达与意义

目的 探讨胆管癌组织中信号转导及转录激活因子3（STAT3）信号通路相关基因Survivin及环氧化酶2（COX-2）的表达及其与胆管癌患者临床病理特征和预后的关系.方法 收集2007年9月至2012年7月安徽医科大学附属省立医院收治的43例胆管癌患者的癌组织标本,收集同期行胆肠内引流术和胆管修复的12例肝内外胆管结石和胆管损伤患者的正常胆管组织标本作为对照.应用免疫组织化学染色检测胆管癌及正常胆管组织中STAT3、磷酸化STAT3（p-STAT3）、Survivin及COX-2蛋白的表达,并分析其与胆管癌患者临床病理特征和预后的关系.采用门诊或电话方式对胆管癌患者进行随访,随访时间截至2014年3月.计数资料采用x2检验,相关性分析采用Spearman检验.采用Kaplan-Meier法绘制生存曲线,生存分析采用Log-rank检验.结果 STAT3、p-STAT3、Survivin、COX-2蛋白在胆管癌组织中阳性表达率分别为69.8％ （30/43）、65.1％（28/43）、72.1％ （31/43）、79.1％（34/43）,在正常胆管组织中阳性表达率分别为41.7％（5/12）、8.3％（1/12）、16.7％（2/12）、41.7％（5/12）.p-STAT3、Survivin、COX-2蛋白在胆管癌组织中的阳性表达率显著高于正常胆管组织,两组比较,差异有统计学意义（x2=12.136,9.811,4.679,P＜0.05）.p-STAT3、Survivin、COX-2蛋白在胆管癌组织中的高表达与肿瘤局部浸润转移有关（x2=14.700,5.959,4.075,P＜0.05）;p-STAT3蛋白阳性表达与肿瘤神经侵犯有关（x2=10.384,P＜0.05）;Survivin、COX-2蛋白表达与胆管癌患者肿瘤神经侵犯无关（x2=2.718,3.024,P＞0.05）;p-STAT3、Survivin、COX-2蛋白表达与胆管癌患者性别、年龄、肿瘤位置、分化程度、肿瘤直径无关（x2=0.148,0.720,1.835,1.040,0.236;0.001,0.009,0.029,1.863,0.197;0.433,0.686,0.002,2.974,0.029,P＞0.05）.Survivin及COX-2蛋白的表达与p-STAT3呈正相关（r=0.524,0.583,P＜0.05）.43例胆管癌患者获得随访,随访时间为6个     

(4-[6-(4-isopropoxyphenyl) pyrazolo [1,5-a]pyrimidin- 3-yl] quinoline) is a novel inhibitor of autophagy

Background and Purpose

Autophagy is an important intracellular degradation system, which is related to various diseases. In preliminary experiments we found that D4-[6-(4-isopropoxyphenyl)pyrazolo [1,5-a]pyrimidin-3-yl] quinoline (DMH1) inhibited autophagy responses. However DMH1 also inhibits the signalling pathway activated by bone morphogenetic protein-4 (BMP4). The aim of the present study was to elucidate the inhibitory effects of DMH1 on autophagy and the underlying mechanisms.

Experimental Approach

The effects of DMH1 on autophagy responses were evaluated in cultures of different cell types and with different stimuli to induce autophagy, using Western blots, transmission electron microscopy and fluorescent microscopy.

Key Results

DMH1 inhibited starvation-induced autophagy in cardiomyocytes, HeLa and MCF-7 cells, without involving the signalling pathway of BMP4. DMH1 inhibited aminoimidazole carboxamide ribonucleotide (AICAR)- and rapamycin-induced autophagy in HeLa and MCF-7 cells. DMH1 reversed starvation- and AICAR-induced inhibition of Akt, mammalian target of rapamycin (mTOR) and p70S6 kinase (S6K), and reversed rapamycin-induced inhibition of mTOR and S6K. DMH1 reversed starvation-induced decrease of the phosphorylated form of glycogen synthase kinase-3 in MCF-7 and HT29 cells. Activation of Akt and inhibition of autophagy induced by DMH1 were antagonized by an Akt specific inhibitor or by small interfering RNA for Akt in HeLa cells.

Conclusion and Implications

DMH1 inhibited cellular autophagy responses in a range of cell types and the underlying mechanisms include activation of the Akt pathway.     

Lithocholic acid feeding results in direct hepato-toxicity independent of neutrophil function in mice

Lithocholic acid (LCA) supplementation in the diet results in intrahepatic cholestasis and bile infarcts. Previously we showed that an innate immune response is critical for cholestatic liver injury in the bile duct ligated mice. Thus, the purpose of this study was to investigate the role of neutrophils in the mechanism of liver injury caused by feeding mice a diet containing LCA. C57BL/6 mice were given control or 1% LCA containing diet for 24–96 h and then examined for parameters of hepatotoxicity. Plasma ALT levels were significantly increased by 48 h after LCA feeding, which correlated with both neutrophil recruitment to the liver and upregulation of numerous pro-inflammatory genes. The injury was confirmed by histology. Deficiency in intercellular adhesion molecule-1 (ICAM-1) expression or inhibition of neutrophil function failed to protect against the injury. Bile acid levels were quantified in plasma and bile of LCA-fed mice after 48 and 96 h. Only the observed biliary levels of taurochenodeoxycholic acid and potentially tauro-LCA caused direct cytotoxicity in mouse hepatocytes. These data support the conclusion that neutrophil recruitment occurs after the onset of bile acid-induced necrosis in LCA-fed animals, and is not a primary mechanism of cell death when cholestasis occurs through accumulation of hydrophobic bile acids.     

Perfluorooctanoic acid stimulates breast cancer cells invasion and up-regulates matrix metalloproteinase-2/-9 expression mediated by activating NF-κB

Perfluorooctanoic acid (PFOA) is widely used because of its stain-resistant and water-repellant properties. This study aimed to explore the molecular mechanisms undergoing the stimulation effects of PFOA on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Trans-well filter assay showed that PFOA exposure (≥5 nM) evidently enhanced the invasion ability of the breast cancer cells MDA-MB-231. Luciferase reporter assay, quantitative real-time PCR, western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after PFOA treatment (P < 0.05 each). Western blotting revealed that PFOA could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the breast cancer cells invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that PFOA can stimulate breast cancer cells invasion and up-regulate matrix metalloproteinase-2/-9 expression mediated by activating NF-κB, which deserves more environmental health concerns.