Immunohistochemical analysis of TrkA neurotrophin receptor expression in human non-neuronal carcinomas

The Trk family of tyrosine protein kinase receptors plays a significant role in the development and maintenance of neural tissues. It has been recently shown that Trk receptors are also expressed by a wide range of normal non-neuronal tissues in humans in a cell type-specific manner. In the present study, the expression patterns of TrkA in 337 non-neuronal invasive carcinomas of 15 different human tissues were investigated immunohistochemically. Overall, 133 (39%), 101 (30%) and 103 (31%) tumors exhibited strong, moderate and no TrkA Immunoreactivity, respectively. Esophageal and thyroid carcinomas expressed high levels of TrkA, whereas the levels in gastric and colon cancers were low. TrkA expression was detected not only in carcinomas originating from TrkA-positive normal counterpart tissues, Including the esophagus, breast, lung and uterus, but also in those from TrkA-negative tissues/cells of the thyroid, liver and ovary. Immunostaining for nerve growth factor-β, the specific ligand for TrkA, in esophageal and breast carcinomas demonstrated its immunoreactivity in stromal fibroblasts and some TrkA-expressing tumor cells. These results suggest that paracrine/autocrine regulation via stromal/tumoral NGF-tumoral TrkA interaction may be involved In the growth of certain non-neuronal carcinomas.     

Effect of amyloid peptides on the increase in TrkA receptor expression induced by nicotine in vitro and in vivo

The ability of nicotine to induce a cytoprotective or neuroprotective action occurs through several down-stream mechanisms. One possibility is that the drug increases the expression of tyrosine kinase A (TrkA) nerve growth factor (NGF) receptors. Certain β-amyloid peptides (e.g., Aβ1–42) have been shown to bind with high affinity to α7 nicotinic receptors and thus interfere with a potentially neurotrophic influence. Treatment of differentiated PC-12 cells with nicotine produced a concentration-dependent increase in cell-surface TrkA receptors that occurred concomitantly with cytoprotection. The effect of nicotine was blocked by either of the α7 receptor antagonists α-bungarotoxin (α-BTX) or methyllycaconatine. The cytoprotective action of nicotine also was inhibited by pretreatment with 10–100 nM Aβ1–42. Nicotine also was administered (four injections of 30 μg, spaced evenly over 24 h) to rats by direct injection into a lateral cerebral ventricle. Brain TrkA expression was increased significantly in hippocampus and entorhinal cortex (up to 32% above control), with no changes found in cerebral cortex or hypothalamus. The nicotine-induced increases in TrKA expression in hippocampus and entorhinal cortex were significantly inhibited by 10 μg α-BTX or by 10 nmol Aβ1–42. Therefore, physiologically relevant concentrations of Aβ1–42 can prevent nicotine-induced TrkA receptor expression in brain regions containing cholinergic neurons susceptible to the neurotoxicity associated with Alzheimer’s disease.     

Expression of cannabinoid receptors and neurotrophins in human gliomas

Recent studies have shown an anti-tumour activity of cannabinoid receptors CB1 and CB2 in gliomas. This effect was mediated by neurotrophins in breast and prostate carcinoma, while in gliomas this relationship has not yet been considered. The aim of this study was to investigate the expression of cannabinoid receptors CB1 and CB2, neurotrophin NGF and NT-3 and their receptors TrkA and TrkC in glioma and endothelial cells. The analysis was performed in 14 gliomas and 2 non-tumour brain specimens by immunohistochemistry and real-time quantitative-polymerase chain reaction (RTQ-PCR). Gliomas showed a weak immunoreactivity for CB1 and CB2 in tumour and in endothelial cells, and for NGF/TrkA mainly in tumour cells, while a moderate/diffuse immunoreactivity was found for NT-3/TrkC. CB2 was expressed on 3 out of 6 low-grade gliomas and in all high-grade gliomas. Non-tumour brain tissues were weakly positive in astrocytes and endothelium for CB1, CB2, NT-3 and TrkC and negative for NGF and TrkA. By RTQ-PCR, gliomas showed low mRNA levels of NGF/TrkA and moderate levels of CB1, NT-3 and TrkC. CB2 mRNA expression was low or absent. A potential role of cannabinoids, particularly of CB2 agonists devoid of psychotropic side effects, in glioma therapy could have a basis in glioblastomas, because they were all positive, though weakly, to CB2. The presence of neurotrophins and their receptors, mainly NT-3 and TrkC, suggests a possible role of these pathways in glioma growth/invasion, but further investigations are required to verify this hypothesis and a potential relationship between cannabinoids and neurotrophins.     

大鼠脊髓损伤后NGF及其受体TrkA在运动神经元及神经胶质细胞表达的变化

为探讨脊髓损伤后运动神经元及神经胶质细胞内神经生长因子(NGF)及其高亲和力受体(TrkA)表达的变化,用改良Allen重击法损伤SCI组动物T12脊髓,按伤后存活时间再将动物分为脊髓损1 d组、2 d组和5 d组。各组动物的脊髓切片经ABC法免疫组织化学染色,用光镜观察TrkA及NGF在脊髓前角运动神经元表达的变化和胶质纤维酸性蛋白(GFAP)及NGF免疫反应阳性胶质细胞的反应性增生程度,并进行图像分析。结果显示:脊髓损伤后前角运动神经元TrkA及NGF的表达随脊髓损伤后动物存活时间的延长逐渐上调;脊髓白质和灰质内尤其是皮质脊髓束内GFAP及NGF阳性胶质细胞明显增生;与此同时,室管膜细胞内亦可见明显的NGF免疫反应产物。上述结果表明,脊髓损伤可刺激脊髓前角运动神经元表达TrkA及NGF,通过自分泌维持受损神经元的存活;损伤部位反应性增生的胶质细胞亦可产生NGF,通过旁分泌作用于脊髓前角运动神经元或皮质脊髓束的轴突末梢,以维持运动神经元的存活及促进皮质脊髓束的再生;适时补充外源性神经营养素或改变损伤局部的微环境将有利于受损脊髓的修复和再生。     

TrkA对哮喘小鼠下呼吸道气道阻力及IL-1β表达的上调作用

目的研究NGF的特异性高亲合力受体TrkA对哮喘小鼠下呼吸道气道阻力及IL-1β的表达的调节作用,探讨TrkA介导的信号通路在哮喘发病机制中的作用。方法BALB/c小鼠30只,按随机数字表法均分为正常对照组、哮喘组、TrkA抗体阻断组。利用An iRes2005肺功能仪测小鼠气道阻力,利用免疫组织化学方法测定IL-1β表达,M etamoph图象分析系统对结果进行分析。结果哮喘小鼠吸气阻力和呼气阻力明显高于正常组小鼠(P<0.01),TrkA组吸气阻力和呼气阻力明显低于哮喘组。免疫组织化学结果显示TrkA抗体阻断组肺(0.324±0.013)、C7-T5节段脊神经节(0.301±0.065)及对应的脊髓后角(0.216±0.019)IL-1β免疫阳性产物平均光密度值明显低于哮喘组小鼠(0.796±0.025、0.745±0.016、0.528±0.011,P<0.01)。结论NGF介导的TrkA通路的激活可上调IL-1β的表达,介导哮喘气道高反应和炎症反应。     

NGF is involved in oral ovalbumin‐induced altered colonic contractility in rats: evidence from the blockade of TrkA receptors with K252a

Background Nerve growth factor (NGF)‐mucosal mast cell (MMC) interaction has been implicated in the remodeling of enteric circuitries and associated functional changes. We investigated the involvement of NGF and its receptor TrkA in the altered colonic contractile activity observed in the model of oral ovalbumin (OVA)‐induced MMC hyperactivity in rats. We also studied the role of colonic MMCs as a source of NGF. Methods Rats received oral OVA, alone or with the TrkA antagonist K252a. Colonic co‐expression of NGF/TrkA and rat mast cell protease II (RMCPII) (double immunofluorescence), RMCPII content (ELISA) and expression of NGF, Brain‐derived neurotrophic factor (BDNF) and TrkA/B (QT‐PCR) were assessed. Colonic contractile activity was determined in vivo and in vitro. Key Results TrkA, but not NGF, was localized in colonic MMCs (RMCPII‐positive). Oral ovalbumin exposure increased colonic RMCPII levels but did not change the percentage of TrkA‐positive MMCs. Neither OVA nor K252a, alone or combined, altered NGF, BDNF or TrkA/B expression. Spontaneous colonic activity in vivo and in vitro was altered by OVA, an effect prevented by K252a. Electrical stimulation‐induced contractile responses in vivo and carbachol responses in vitro were increased by OVA in a K252a‐independent manner. In OVA‐treated animals, inhibition of NO synthesis with l ‐NNA significantly enhanced spontaneous colonic activity in vitro, a response completely prevented by K252a. Conclusions & Inferences These results suggest that NGF‐TrkA‐dependent pathways are implicated in colonic contractile alterations observed during OVA exposure in rats. NGF‐TrkA system might represent a potential target for treatment of gastrointestinal disorders characterized by colonic motor alterations.     

Effects of LNG-IUS on nerve growth factor and its receptors expression in patients with adenomyosis

The levonorgestrel-releasing intrauterine system (LNG-IUS) is effective in the treatment of dysmenorrhea associated with adenomyosis. However, the mechanism of pain relief of LNG-IUS in patients with adenomyosis is unclear. We aimed to investigate the effects of LNG-IUS on the expression of nerve growth factor (NGF) and its receptors, NGFR p75 and TrkA in patients with adenomyosis. Endometrial and myometrial tissues were prepared from 17 LNG-IUS-treated patients and 15 hormonally untreated patients who had undergone hysterectomies for adenomyosis. Immunohistochemistry with antibodies against NGF, NGFR p75, and TrkA, was performed. The expression of NGF, NGFR p75, and TrkA in endometrium and myometrium of LNG-IUS-treated patients was significantly decreased compared to those of hormonally untreated patients. Our findings may indicate that the suppression of NGF and its receptors by LNG-IUS is another possible mechanism of relieving pain in patients with adenomyosis.     

天麻素注射液通过NGF/TrkA通路减轻大鼠脑缺血所致肺损伤

目的 基于神经生长因子（NGF）/酪氨酸激酶受体A（TrkA）通路探讨天麻素注射液治疗局灶性脑缺血致肺损伤大鼠的作用机制。方法 将40只SD大鼠随机分为4组：正常组、假手术组、模型组和天麻素治疗组（10只/组）。采用栓塞大鼠右侧大脑中动脉复制局灶性脑缺血模型,造模成功后,用天麻素注射液腹腔注射10 mg/kg,1次/d,持续14 d;假手术组和模型组大鼠不予治疗。治疗结束后,测定大鼠肺组织湿/干重比值,HE染色检测肺组织病理变化,ELISA法检测动脉血液中炎性因子（IL-10、TNF-α）的含量,Western blot检测肺组织NF-κB p65、TNF-α的表达,免疫组织化学染色法及蛋白质免疫印迹试验检测肺组织NGF、TrkA的表达。结果 与正常组和假手术组相比,模型组呈现出明显的炎症性肺损伤病理表型,肺湿/干重比值增加（P<0.01）,动脉血中TNF-α浓度增高（P<0.01）,肺组织内NF-κB p65（P<0.01）、TNF-α（P<0.01）、NGF（P<0.05）和TrkA（P<0.05）蛋白表达水平上升;与模型组相比,天麻素治疗组肺部炎性病理变化减轻,肺湿/干重比值降低（P<0.05）,动脉血中TNF-α浓度降低（P<0.01）,IL-10浓度增高（P<0.01）,肺组织内NF-κB p65和TNF-α蛋白表达水平降低（P<0.05）,肺组织内NGF和TrkA蛋白表达水平增加（P<0.05）。结论 脑缺血可引起炎症性肺损伤,NGF/TrkA通路可能参与了炎症反应的发生过程;天麻素注射液可减轻脑缺血大鼠的肺损伤,其机制可能与NGF/TrkA通路激活抗炎途径有关。     

神经生长因子在异丙酚对大鼠海马神经元缺氧/复氧损伤中的作用及机制

目的探讨不同浓度的异丙酚预处理对大鼠海马神经元缺氧/复氧损伤的保护及神经生长因子(Nerve growth factor,NGF)的作用。方法取离体培养的大鼠海马神经元,随机分成7组:对照组(Con组);CoC l2组(CoC l2组):加入300μM CoC l2处理1 h,然后更换正常的培养基培养24 h,之后更换无血清的培养基培养;脂肪乳剂组(Intralipid,Intra组):加入10%脂肪乳剂90μL预处理1 h后加入300μM CoC l2;异丙酚组(prop组)培养孔中加入10、20、50、100μM浓度的异丙酚预处理1 h后,同CoC l2组。MTT法测定细胞增殖,流式细胞技术测定细胞的凋亡。为进一步研究不同浓度的异丙酚对NGF及其受体TrkA表达的影响,本研究将大鼠海马神经元分为6组,分别为对照组,CoC l2组,10、20、50、100μM浓度异丙酚组,用RT-PCR检测NGF mRNA和TrkA mRNA表达。为探讨NGF/TrkA的作用,将细胞随机分为4组:对照组,CoC l2组,50μM异丙酚组,1.0μmol/L K252a组。结果脂肪乳剂组与CoC l2组比较,细胞活性差异无统计学意义(P>0.05),50μM异丙酚预处理可以明显增加大鼠海马神经元的增殖能力,减少凋亡(P<0.01),50μM异丙酚预处理上调NGF mRNA和TrkA mRNA的表达(P<0.05或P<0.01),10、20、100μM异丙酚组与缺氧/复氧组相比差异无统计学意义(P>0.05),异丙酚对海马神经元的保护作用被K252a抑制(P<0.01)。结论 50μM异丙酚预处理对缺氧/复氧后的大鼠海马神经元有保护作用,NGF及其受体TrkA在异丙酚的预处理中起到重要的作用。     

人参皂苷Rg1对痴呆模型大鼠行为学及酪氨酸蛋白激酶A表达的影响

目的探讨人参皂苷Rg1对痴呆模型大鼠学习记忆能力的影响及其对酪氨酸蛋白激酶A（TrkA）阳性细胞的保护作用。方法采用切断成年Wistar大鼠左侧穹窿海马伞（FF）的方法，建立隔-海马胆碱能系统损害的痴呆模型。分为模型组、治疗组和假手术对照组3组。治疗组经腹腔内注射Rg1（5mg／kg），模型组和假手术对照组给予生理盐水。利用Morris水迷宫测定其学习记忆能力，利用免疫组化及图像分析测定大鼠内侧隔核（MS）和斜角带垂直支（VDB）TrkA阳性细胞数及OD值。结果治疗组大鼠的学习记忆成绩优于模型组（P〈0．05），其MS和VDB的TrkA阳性细胞数及OD值与模型组相比也有改善（P〈0．05）。结论人参皂苷Rg1对中枢胆碱能系统有保护作用，能改善学习记忆能力。