獐牙菜苦苷可减轻糖尿病大鼠的周围神经痛:基于抑制NOXS/ROS/NLRP3通路实验

目的 基于NOXS/ROS/NLRP3信号通路探讨獐牙菜苦苷对糖尿病周围神经痛（DPN）大鼠的治疗作用及分子机制。方法 采用链脲佐菌素(STZ)法构建大鼠模型,造模成功后第1、7、14天分别按大鼠体质量腹腔注射干预制剂。将32只SD大鼠随机分为4组：空白对照组、DPN模型组、獐牙菜苦苷治疗组、NOXS抑制剂治疗组。空白对照组：给予10 mL/kg生理盐水;DPN模型组：给予10 mL/kg生理盐水;獐牙菜苦苷治疗组：给予5 mg/kg獐牙菜苦苷;NOXS抑制剂治疗组：给予10 mL/kg二苯基氯化碘盐。在给药完毕30 min后,对各组大鼠进行触觉过敏试验,采用ELISA检测NOXS/ROS/NLRP3以及炎性因子表达水平,采用Western blot方法检测各组大鼠脊髓组织的NOXS/ROS/NLRP3表达水平。结果 与空白对照组比较,DPN模型组大鼠在造模后出现痛觉过敏（P<0.001）,促炎因子TNF-α以及IL-6表达升高（P<0.001）,抑炎因子TGF-β表达下降（P<0.001）,NOXS/ROS/NLRP3通路各因子表达升高（P<0.001）。獐牙菜苦苷干预后有效减轻触觉过敏（P<0.001）,且抑制促炎因子TNF-α（P=0.03）以 及IL-6（P=0.002）的表达,促进抑炎因子TGF-β的表达（P=0.04）;并且下调NOXS（P<0.001）、ROS（P<0.001）以及NLRP3（P= 0.002）通路各因子的表达水平。獐牙菜苦苷对炎性因子及NOXS/ROS/NLRP3通路各因子的表达影响与NOXS抑制剂治疗组间差异无统计学意义（P>0.05）。结论 獐牙菜苦苷有效缓解触觉过敏,治疗DPN,通过抑制NOXs/ROS/NLRP3信号通路表达,纠正DPN炎性因子失衡是其分子机制之一。     

赖氨酰氧化酶在眼部疾病的研究进展

赖氨酰氧化酶(LOX)是细胞外具有铜结合部位的氨基氧化酶,大量研究已经证实LOX可能参与许多疾病的病理发生过程。目前LOX导致疾病发展的常见变异的潜在机制仍然未知,但潜在的作用可能在于通过组织特异性的可变剪接或表达调控来影响LOX的生物学活性。但无论是在正常或病理情况下,关于眼组织中LOX的信息很少。近年有相关研究发现LOX可能参与许多眼部疾病的发生和发展,现就以近年来LOX参与的眼部疾病的研究进展做一综述。     

燃煤型氟中毒大鼠诱导型一氧化氮合酶蛋白表达、血清睾酮与生精细胞凋亡的相关性研究

目的:研究燃煤型氟中毒大鼠睾丸组织诱导型一氧化氮合酶(iNOS)蛋白表达、血清睾酮(T)与生精细胞凋亡的相互关系,探索燃煤型氟中毒对睾丸生殖功能损伤的机制。方法:将40只雄性SD大鼠随机分为4组:对照组、低氟组、中氟组、高氟组。各染毒组喂饲含不同比例的燃煤型氟中毒病区煤烘玉米的饲料,建立雄性大鼠燃煤型氟中毒模型。分别于染毒4和6个月后以股动脉放血法处死大鼠,各时间点每组处死大鼠5只。采用RIA法测定大鼠血清T水平;检测血清中超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量、iNOS活性;原位缺口末端标记(TUNEL)法检测大鼠睾丸生精细胞凋亡情况并计算凋亡指数(AI);免疫组织化学染色SP法观察iNOS蛋白表达的变化。结果:成功建立雄性大鼠氟中毒模型。大鼠血清T水平在4和6个月时低氟组[(28.37±0.19)nmol/L、(14.68±0.36)nmol/L]、中氟组[(25.85±0.23)nmol/L、(9.08±0.31)nmol/L]、高氟组[(23.63±0.29)nmol/L、(4.89±0.54)nmol/L]较对照组[(29.77±0.43)nmol/L、(29.97±0.36)nmol/L]均降低(P0.05或P0.01),且各染氟组间差异有统计学意义(P0.05或P0.01)。睾丸生精细胞AI在4和6个月时低氟组(8.83±1.64、10.40±2.70)、中氟组(17.24±3.96、22.20±3.96)、高氟组(44.21±7.85、49.60±4.77)较对照组(0.46±0.11、0.54±0.11)升高(P均0.01),且各染氟组间差异有统计学意义(P0.05或P0.01)。睾丸组织iNOS蛋白表达的OD值在4和6个月时低氟组(0.073 3±0.008 5、0.108 4±0.012 6)、中氟组(0.142 5±0.027 2、0.207 7±0.020 4)、高氟组(0.527 5±0.079 1、0.717 3±0.065 6)较对照组(0.001 3±0.000 2、0.001 6±0.000 1)增加(P0.05或0.01),且各染氟组间差异有统计学意义(P0.05或P0.01)。iNOS蛋白表达量与生精细胞AI呈正相关(r=0.962,P0.01);血清T水平与生精细胞AI呈负相关(r=-0.744,P0.01)。结论:血清T水平改变和睾丸组织iNOS蛋白表达调控改变可能参与燃煤型氟中毒对睾丸生殖功能损害的过程。     

蛇足石杉HsCAO2基因的克隆、生物信息学分析及载体构建

目的 为筛选高活性的蛇足石杉铜胺氧化酶（copper-containing amine oxidase,CAO）,通过生物工程方法实现石杉碱甲（huperzine A,HupA）的合成、为加快新药创制进度提供理论依据。方法 基于蛇足石杉转录组测序得到的CAO序列片段,利用cDNA末端快速扩增技术（rapid amplification of cDNA ends,RACE）技术,扩增、拼接并验证获得该基因的全长cDNA序列,并将其命名为HsCAO2。结果 通过RACE技术得到HsCAO2的cDNA全长为2696 bp,CDS为2199 bp,编码732个氨基酸。氨基酸序列同源性比对发现,HsCAO2具有保守的氨基酸位点和Cu²⁺结合位点;进化树分析结果表明,HsCAO2与蛇足石杉中的另一个CAO（HsCAO1）同源性最高,达91.67%;蛋白质结构分析结果表明,HsCAO2也可能在生物体里以同源二聚体的形式存在。在此基础上,进一步分别构建了超表达载体pOX-HsCAO2和体外蛋白表达载体pET-28a （+）-HsCAO2。结论 HsCAO2很可能是蛇足石杉中的一个新的功能性CAO,且超表达载体和体外蛋白表达载体的成功构建为进一步研究其功能奠定了基础。     

MAOA/B deletion syndrome in male siblings with severe developmental delay and sudden loss of muscle tonus

Deletion of the monoamine oxidase (MAO)-A and MAO-B was detected in two male siblings and in their mother. The approximately 800-kb deletion, extending from about 43.0 MB to 43.8 MB, was detected by array comparative genomic hybridization analysis. The MAOA and MAOB genes were included in the deletion, but the adjacent Norrie disease gene, NDP, was not deleted. The boys had short stature, hypotonia, severe developmental delays, episodes of sudden loss of muscle tone, exiting behavior, lip-smacking and autistic features. The serotonin levels in their cerebrospinal fluid were extremely elevated. Another set of siblings with this deletion was reported previously. We propose recognition of MAOA/B deletion syndrome as a distinct disorder.     

Feedforward and feedback connections and their relation to the cytox modules of V2 in Cebus monkeys

To study the circuitry related to the ventral stream of visual information processing and its relation to the cytochrome oxidase (CytOx) modules in visual area V2, we injected anterograde and retrograde cholera toxin subunit B (CTb) tracer into nine sites in area V4 in five Cebus apella monkeys. The injection site locations ranged from 2° to 10° eccentricity in the lower visual field representation of V4. Alternate cortical sections, cut tangentially to the pial surface or in the coronal plane, were stained for CTb immunocytochemistry or for CytOx histochemistry or for Nissl. Our results indicate that the V4‐projecting cells and terminal‐like labeling were located in interstripes and thin CytOx‐rich stripes and avoided the CytOx‐rich thick stripes in V2. The feedforward projecting cell bodies in V2 were primarily located in the supragranular layers and sparsely located in the infragranular layers, whereas the feedback projections (i.e., the terminal‐like labels) were located in the supra‐ and infragranular layers. V4 injections of CTb resulted in labeling of the thin stripes and interstripes of V2 and provided an efficient method of distinguishing the V2 modules that were related to the ventral stream from the CytOx‐rich thick stripes, related to the dorsal stream. In V2, there was a significant heterogeneity in the distribution of projections: feedforward projections were located in CytOx‐rich thin stripes and in the CytOx‐poor interstripes, whereas the feedback projections were more abundant in the thin stripes than in the interstripes. J. Comp. Neurol. 522:3091–3105, 2014. © 2014 Wiley Periodicals, Inc.     

NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke

Although the innate immune response to induce postischemic inflammation is considered as an essential step in the progression of cerebral ischemia injury, the role of innate immunity mediator NLRP3 in the pathogenesis of ischemic stroke is unknown. In this study, focal ischemia was induced by middle cerebral artery occlusion in NLRP3^−/−, NOX2^−/−, or wild-type (WT) mice. By magnetic resonance imaging (MRI), Evans blue permeability, and electron microscopic analyses, we found that NLRP3 deficiency ameliorated cerebral injury in mice after ischemic stroke by reducing infarcts and blood–brain barrier (BBB) damage. We further showed that the contribution of NLRP3 to neurovascular damage was associated with an autocrine/paracrine pattern of NLRP3-mediated interleukin-1β (IL-1β) release as evidenced by increased brain microvessel endothelial cell permeability and microglia-mediated neurotoxicity. Finally, we found that NOX2 deficiency improved outcomes after ischemic stroke by mediating NLRP3 signaling. This study for the first time shows the contribution of NLRP3 to neurovascular damage and provides direct evidence that NLRP3 as an important target molecule links NOX2-mediated oxidative stress to neurovascular damage in ischemic stroke. Pharmacological targeting of NLRP3-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.     

Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases

Activation of microglial NADPH oxidase (NOX2) plays a critical role in mediating neuroinflammation, which is closely linked with the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease (PD). The inhibition of NOX2‐generated superoxide has become an effective strategy for developing disease‐modifying therapies for PD. However, the lack of specific and potent NOX2 inhibitors has hampered the progress of this approach. Diphenyleneiodonium (DPI) is a widely used, long‐acting NOX2 inhibitor. However, due to its non‐specificity for NOX2 and high cytotoxicity at standard doses (µM), DPI has been precluded from human studies. In this study, using ultra‐low doses of DPI, we aimed to: (1) investigate whether these problems could be circumvented and (2) determine whether ultra‐low doses of DPI were able to preserve its utility as a potent NOX2 inhibitor. We found that DPI at subpicomolar concentrations (10^?14 and 10^?13 M) displays no toxicity in primary midbrain neuron‐glia cultures. More importantly, we observed that subpicomolar DPI inhibited phorbol myristate acetate (PMA)‐induced activation of NOX2. The same concentrations of DPI did not inhibit the activities of a series of flavoprotein‐containing enzymes. Furthermore, potent neuroprotective efficacy was demonstrated in a post‐treatment study. When subpicomolar DPI was added to neuron‐glia cultures pretreated with lipopolysaccharide, 1‐methyl‐4‐phenylpyridinium or rotenone, it potently protected the dopaminergic neurons. In summary, DPI's unique combination of high specificity toward NOX2, low cytotoxicity and potent neuroprotective efficacy in post‐treatment regimens suggests that subpicomolar DPI may be an ideal candidate for further animal studies and potential clinical trials. GLIA 2014;62:2034–2043     

The ontogeny of drug metabolism enzymes and implications for adverse drug events

Profound changes in drug metabolizing enzyme (DME) expression occurs during development that impacts the risk of adverse drug events in the fetus and child. A review of our current knowledge suggests individual hepatic DME ontogeny can be categorized into one of three groups. Some enzymes, e.g., CYP3A7, are expressed at their highest level during the first trimester and either remain at high concentrations or decrease during gestation, but are silenced or expressed at low levels within one to two years after birth. SULT1A1 is an example of the second group of DME. These enzymes are expressed at relatively constant levels throughout gestation and minimal changes are observed postnatally. ADH1C is typical of the third DME group that are not expressed or are expressed at low levels in the fetus, usually during the second or third trimester. Substantial increases in enzyme levels are observed within the first one to two years after birth. Combined with our knowledge of other physiological factors during early life stages, knowledge regarding DME ontogeny has permitted the development of robust physiological based pharmacokinetic models and an improved capability to predict drug disposition in pediatric patients. This review will provide an overview of DME developmental expression patterns and discuss some implications of the data with regards to drug therapy. Common themes emerging from our current knowledge also will be discussed. Finally, the review will highlight gaps in knowledge that will be important to advance this field.