温阳解郁颗粒调节BDNF/TrkB/ERK信号通路保护皮质酮诱导损伤型海马神经细胞的作用机制研究＊

目的 观察温阳解郁颗粒（Wenyang Jieyu granule，WYJY）对皮质酮（Corticosterone，CORT）诱导损伤型小鼠海马神经细胞（TH22 cell）的保护作用，基于脑源性神经营养因子（Brain derived neurotrophic factor， BDNF）/酪氨酸激酶B（Tyrosine kinase B， TrkB）/细胞外信号调节蛋白激酶（Extra cellular regulated protein kinases， ERK）信号通路探讨WYJY保护海马神经细胞的作用机制。方法 体外构建小鼠海马神经细胞皮质酮诱导损伤模型，以不同浓度的WYJY和氟西汀（Fluoxetine，FXT）含药血清作用于模型细胞，细胞增殖-毒性检测（Cell Counting Kit-8， CCK-8）法分析细胞活性，倒置显微镜下观察给药前后细胞形态结构的改变，采用蛋白免疫印迹法（Western Blot）、实时荧光定量PCR（Quantitative real-time PCR， qPCR）法检测神经细胞内凋亡因子（BCL2-Associated X， Bax）、抗凋亡因子（B-cell lymphoma-2， Bcl-2）、BDNF、Trkb、ERK以及丝氨酸/苏氨酸激酶（Phospho-p90RSK， RSK）、环磷腺苷效应元件结合蛋白（cAMP-response element binding protein， CREB）蛋白表达水平以及相关基因的表达水平。结果 在浓度为459.5 μmol·L^-1的CORT作用24 h后，HT22细胞的活性抑制率达到50%，在此条件作用下细胞形态结构损伤明显，凋亡程度严重，细胞上清中BDNF的含量显著减少（P<0.05），细胞内凋亡相关因子Bax/Bcl-2的比值明显升高（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平明显降低（P<0.01）；以5%的浓度为2.85 g·kg^-1的WYJY和10%的FXT含药血清作用于受损的HT22细胞后，HT22细胞存活率明显提升（P<0.01），细胞结构的损伤明显改善，细胞凋亡程度减轻，细胞外BDNF的含量显著升高（P<0.05），细胞内Bax/Bcl-2比值显著下调（P<0.01），BDNF、Trkb、ERK、RSK、CREB磷酸化蛋白表达水平和mRNA表达水平显著提升（P<0.05，P<0.01）。结论 温阳解郁颗粒可有效保护高浓度CORT造成的小鼠海马神经细胞损伤。调控BDNF/Trkb/ERK通路，放大CREB信号传导，影响Bcl-2、BDNF水平，可能是其保护海马神经元，发挥抗抑郁疗效的重要机制。     

大补元煎通过上调BDNF/TrkB/CREB信号通路改善APP/PS1双转基因痴呆小鼠海马突触可塑性

目的:观察大补元煎对APP/PS1痴呆小鼠海马突触可塑性及脑源性神经营养因子(BDNF)/酪氨酸蛋白激酶受体B(TrkB)/环磷酸腺苷反应元件结合蛋白(CREB)信号通路的作用,并探讨其改善突触可塑性的可能机制。方法:将APP/PS1小鼠36只分为模型组、多奈哌齐组(6.5×10~(-4)g·kg~(-1)·d~(-1))和大补元煎组(13.2 g·kg~(-1)·d~(-1)),野生鼠12只设为正常组,正常组和模型组给予等体积生理盐水,各组连续灌胃30 d。应用Morris水迷宫检测各组小鼠的学习记忆能力,应用尼氏染色和高尔基染色观察海马区神经元和突触的病理形态变化,应用免疫荧光(IF)观察海马突触后致密蛋白95(PSD95)及突触素(SYN)的蛋白表达水平,采用蛋白免疫印迹法(Western blot)检测海马中BDNF,TrkB,CREB及磷酸化CREB(p-CREB)的蛋白表达水平。结果:与空白组比较,模型组小鼠平台潜伏期和游泳总路程增加(P0.01),穿越平台次数和目标象限停留时间减少(P0.01),小鼠海马CA3区神经元胞内尼氏体减少或消失,小鼠海马CA3区神经元及树突分支数量、树突棘密度减少(P0.01),小鼠海马SYN,PSD95,BDNF,TrkB及p-CREB的蛋白表达水平减少(P0.01)。与模型组比较,多奈哌齐组和大补元煎组小鼠平台潜伏期和游泳总路程减少(P0.05,P0.01),穿越平台次数和目标象限停留时间增加(P0.05,P0.01),小鼠海马CA3区神经元胞内尼氏体数量增多,小鼠海马CA3区神经元及树突分支数量,树突棘密度增加(P0.05,P0.01),小鼠海马SYN,PSD95,BDNF,TrkB及p-CREB的蛋白表达水平增加(P0.05,P0.01)。结论:大补元煎改善APP/PS1双转基因小鼠突触可塑性的机制可能与其上调小鼠海马中BDNF/TrkB/CREB信号通路有关。     

全膝关节置换术前应用双下肢全长摄影对不同体位下肢负重轴线的影响

目的探讨全膝关节置换术前应用双下肢全长摄影对不同体位下肢负重轴线的影响。方法选择在我院接受全膝关节置换术患者138例,均拍摄两组双下肢全长正位片,一组以骨盆正中,下肢呈中立位为标准,另一组以膑骨正中,胫骨覆盖腓骨小头内1/3为标准方案。比较两种体位下的股骨胫骨机械轴夹角等数据,并评估两者图像质量。术后随访,比较术前术后膝关节功能。结果骨盆正中以及髌骨正中下摄影获得的股骨侧弓角(FBA)、机械股骨远端外侧角(mLDFA)以及近端内侧角(mMPTA)比较,差异均有统计学意义(P<0.05),且前者明显高于后者,但机械股胫角(mFTA)比较差异无统计学意义(P>0.05);两种体位下患者摄影图像质量比较差异无统计学意义(P>0.05);患者术后疼痛、功能、活动度、屈曲畸形和稳定性评分较术前均显著上升(P<0.05)。结论骨盆正中体位的双下肢全长摄影在全膝关节置换术的应用可正确展示患者本身该有的下肢力线,减小人为操作不当引起的误差,同时可以优化下肢力线在不同摄影体位的角度测量。     

围产期PFOS暴露对幼鼠海马BDNF/TrkB/CREB信号通路影响

目的 探讨观察母孕鼠围产期全氟辛烷磺酸盐(perfluorooctane sulphonate,PFOS)暴露对幼鼠海马组织BDNF/TrkB/CREB信号通路关键基因表达的影响。 方法 20只昆明种雌性小鼠随机分为对照组和低、中、高剂量组,从孕鼠怀孕第2 d开始(gestation day2, GD2)到幼鼠出生后21 d(postnatal day21,PND21)分别给予低、中、高剂量组孕鼠PFOS剂量为 0.1、1.0、5.0 mg/(kg·bw)灌胃染毒,对照组给予等体积的0.05% Tween-20水溶液。灌胃量为0.1 ml/10 (g·bw)。PND 21 d处死幼鼠,收集脑组织,分离海马及皮层。HE染色观察脑组织常规病理改变,实时荧光定量PCR(Quantitative Real-time PCR,QPCR)检测海马组织中BDNF、TrkB、CREB、Syn1及Syp的mRNA表达水平。 结果 与对照组相比较,低、中、高三个剂量组对幼鼠的死亡率和体质量的影响差异无统计学意义(P>0.05)。但是在高剂量组幼鼠海马组织出现空泡,海马BDNF、TrkB、CREB mRNA水平显著降低,分别由对照组的(0.98±0.11)、(1.03±0.09)、(1.08±0.12)下降到(0.22±0.21)、(0.71±0.14)、(0.37±0.26),并在中、高剂量组引起了幼鼠突触相关蛋白Syn1和Spy的mRNA水平显著降低,分别由对照组的(1.10±0.09)、(0.97±0.08)下降到中剂量的(0.41±0.23)、(0.71±0.17)和高剂量的(0.39±0.19)、(0.63±0.19),差异均有统计学意义(P<0.05)。 结论 PFOS损伤海马BDNF/TrkB/CREB信号通路可能是PFOS神经发育毒性之一。     

Brain‐derived neurotrophic factor but not vesicular zinc promotes TrkB activation within mossy fibers of mouse hippocampus in vivo

The neurotrophin receptor, TrkB receptor tyrosine kinase, is critical to central nervous system (CNS) function in health and disease. Elucidating the ligands mediating TrkB activation in vivo will provide insights into its diverse roles in the CNS. The canonical ligand for TrkB is brain‐derived neurotrophic factor (BDNF). A diversity of stimuli also can activate TrkB in the absence of BDNF, a mechanism termed transactivation. Zinc, a divalent cation packaged in synaptic vesicles along with glutamate in axons of mammalian cortical neurons, can transactivate TrkB in neurons and heterologous cells in vitro. Yet the contributions of BDNF and zinc to TrkB activation in vivo are unknown. To address these questions, we conducted immunohistochemical (IHC) studies of the hippocampal mossy fiber axons and boutons using an antibody selective for pY816 of TrkB, a surrogate measure of TrkB activation. We found that conditional deletion of BDNF resulted in a reduction of pY816 in axons and synaptic boutons of hippocampal mossy fibers, thereby implicating BDNF in activation of TrkB in vivo. Unexpectedly, pY816 immunoreactivity was increased in axons but not synaptic boutons of mossy fibers in ZnT3 knockout mice that lack vesicular zinc. Marked increases of BDNF content were evident within the hippocampus of ZnT3 knockout mice and genetic elimination of BDNF reduced pY816 immunoreactivity in these mice, implicating BDNF in enhanced TrkB activation mediated by vesicular zinc depletion. These findings support the conclusion that BDNF but not vesicular zinc activates TrkB in hippocampal mossy fiber axons under physiological conditions. J. Comp. Neurol. 522:3885–3899, 2014. © 2014 Wiley Periodicals, Inc.     

Chronic Administration of Bacopa Monniera Increases BDNF Protein and mRNA Expressions: A Study in Chronic Unpredictable Stress Induced Animal Model of Depression

Objective

The present study aimed to investigate whether graded doses of Bacopa Monniera (BM) extract could produce antidepressant-like effects in chronic unpredictable stress (CUS) induced depression in rats and its possible mechanism(s).

Methods

Rats were subjected to an experimental setting of CUS. The effect of BM extract treatment in CUS-induced depression was examined using behavioral tests including the sucrose consumption, open field test and shuttle box escape test. The mechanism underlying the antidepressant-like action of BM extract was examined by measuring brain-derived neurotrophic factor (BDNF) protein and mRNA expression in brain tissues of CUS-exposed rats.

Results

Exposure to CUS for 4 weeks caused depression-like behavior in rats, as indicated by significant decreases in sucrose consumption, locomotor activity and escape latency. In addition, it was found that BDNF protein and mRNA levels in the hippocampus and frontal cortex were lower in CUS-treated rats, as compared to controls. Daily administration of the graded doses of BM extract during the 4-week period of CUS significantly suppressed behavioral changes and attenuated the CUS-induced decrease in BDNF protein and mRNA levels in the hippocampus and frontal cortex.

Conclusion

The results suggest that BM extract alleviates depression induced by CUS. Present study also confirms that 80-120 mg/kg doses of BM extract have significantly higher antidepressant-like activity.     

A dopamine D1 receptor agonist improved learning and memory in morphine-treated rats

ABSTRACT

Objective: The objective of this article is to study the role of the dopamine (DA) D1 receptor in the midbrain periaqueductal grey (PAG) on learning and memory in morphine-addicted rats.

Methods: DA D1 receptor agonist SKF81297 and D1 receptor antagonist SCH SCH23390 were administrated into the PAG, respectively, and the learning and memory behavioral changes of morphine addicted rats were detected by water maze. Western blot and immunohistochemistry were used to detect glutamate decarboxylase 67 (GAD67) and tyrosine receptor kinase B (TrkB) in PAG.

Results: D1 receptor agonist shortened the latency to platform and increased the number of platform crossings, indicating improved learning and memory ability of morphine addict rat. D1 receptor agonist increased GAD67 expression and decreased TrkB in PAG.

Conclusion: (1) The PAG is involved in the learning and memory changes of the addicted rats; (2) the activation of DA D1 receptor will increase the GAD67, reduce the damage to peripheral neurons, and improve the learning and memory of the addicted rats; and (3) D1 receptor agonists further reduced TrkB expression in morphine-addicted rats, whereas TrkB levels deviated from changes in rat behavior.     

Brain-derived neurotrophic factor mediates neuroprotection against Aβ-induced toxicity through a mechanism independent on adenosine 2A receptor activation

Abstract

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival through TrkB-FL activation. The activation of adenosine A_2A receptors (A_2AR) is essential for most of BDNF-mediated synaptic actions, such as synaptic plasticity, transmission and neurotransmitter release. We now aimed at evaluating the A_2AR influence upon BDNF-mediated neuroprotection against Aβ_25–35 toxicity in cultured neurons. Results showed that BDNF increases cell survival and reduces the caspase-3 and calpain activation induced by amyloid-β (Aβ) peptide, in a mechanism probably dependent on PLCγ pathway. This BDNF-mediated neuroprotection is not affected by A_2AR activation or inhibition. Moreover neither activation nor inhibition of A_2AR, per se, significantly influenced Aβ-induced neuronal death on calpain-mediated cleavage of TrkB induced by Aβ. In conclusion, these results suggest that, in opposition to the fast synaptic actions of BDNF, the neuroprotective actions of this neurotrophin against a strong Aβ insult do not require the activation of A_2AR.     

Management of panurethral strictures

IntroductionPan-urethral stricture, involving the penile and bulbar urethra, is a common urological problem on the South Asian subcontinent. It represents a particularly difficult challenge to manage and there is a relative paucity of literature on the subject. In India, Lichen Sclerosus (LS) is the most common cause of pan-urethral stricture, followed by iatrogenic causes.2 stage surgery is not scientific in lichen sclerosus as this is a disease of genital skin. We present our experience of pan-urethral stricture repair using a single-stage, one-sided dissection, dorsal onlay repair with oral mucosa graft.Subjects and methodsWe retrospectively reviewed the records of 318 consecutive men undergoing management of pan-urethral stricture from June 1995 to December 2014. The median age was 44.6 years and the mean stricture length 14 cm. The median follow-up was 59 months. The strictures were approached through a perineal incision, limiting dissection to only one side of the urethra. The penis was invaginated to provide access to the entire length of anterior urethra in a single-stage, and two oral mucosal grafts were dorsally placed.ResultsThe outcome was considered a success if the patient needed no further instrumentation, including dilation or urethrotomy. The overall success rate was 84.90%, with a success rate of 89.39% in primary urethroplasty, and 57.85% in patients who had previous failed urethroplasty. Most recurrent strictures occurred at the proximal end of the graft.ConclusionsRepair of pan-urethral stricture in a single-stage, with one-sided dissection and dorsal onlay of oral mucosa, is a minimally invasive technique that is simple, fast, safe, effective and reproducible in the hand of any surgeon.     

A randomized,placebo-controlled trial of emricasan in patients with NASH and F1-F3 fibrosis

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