1-磷酸鞘氨醇受体1-3(S1P1-3)在去势雄性大鼠阴茎海绵体组织中的表达

目的:探讨1-磷酸鞘氨醇受体1-3(S1P1-3)在去势雄性大鼠阴茎海绵体内的表达,及其与NOS/NO/c GMP、Rho A/Rho激酶等信号通路的关系。方法:18只8周龄健康雄性SD大鼠,随机分为去势组、对照组及去势后睾酮替代组(替代组)各6只,去势组和替代组大鼠切除双侧睾丸、附睾,替代组大鼠去势术后给予生理剂量丙酸睾酮3 mg/(kg·d)皮下注射4周,对照组为假手术组,去势组及对照组大鼠术后给予等量植物油皮下注射4周,12周龄时,测定各组大鼠阴茎海绵体内压/平均动脉压(ICPmax/MAP)、采用免疫组化和Western印迹分析S1P1-3、e NOS、P-e NOS、ROCK1、ROCK2在阴茎海绵体内的表达变化。结果:去势组大鼠血清睾酮水平[(0.41±0.04)nmol/L]显著低于对照组[(16.01±1.02)nmol/L]及替代组[(15.84±1.32)nmol/L](P0.01),而替代组与对照组睾酮水平无显著差异。去势组ICPmax/MAP比值在0 V、3 V和5 V电刺激盆神经节时(0.088±0.014、0.323±0.014、0.432±0.012)均显著低于对照组(0.155±0.011、0.711±0.010、0.819±0.024)及替代组(0.153±0.012、0.696±0.017、0.763±0.027)(P0.01),而对照组与替代组无显著差异。去势组S1P1、e NOS、P-e NOS的蛋白表达量[以目的蛋白占内参GAPDH的百分率表示:S1P1(49.99±3.39)%,e NOS(46.82±3.81)%,P-e NOS(45.42±4.35)%]显著低于对照组[S1P1(72.57±3.06)%,e NOS(89.76±3.98)%,P-e NOS(82.53±8.92)%]和替代组[S1P1(71.77±4.43)%,e NOS(87.19±4.23)%,P-e NOS(79.82±7.38)%](P0.01),去势组S1P2、S1P3、ROCK1、ROCK2蛋白表达量[以目的蛋白占内参GAPDH的百分率表示:S1P2(82.35±4.13)%,S1P3(61.03±5.14)%,ROCK1(74.50±4.02)%,ROCK2(69.83±5.75)%]显著高于对照组[S1P2(41.67±1.68)%,S1P3(31.66±2.67)%,ROCK1(35.69±5.56)%),ROCK2(39.85±7.17)%]和替代组[S1P2(42.80±3.87)%,S1P3(32.25±4.22)%,ROCK1(38.06±5.21)%,ROCK2(42.36±4.44)%](P0.01)。结论:雄激素缺乏导致大鼠ICPmax/MAP显著降低,可能与阴茎海绵体内S1P1表达下调、抑制e NOS/NO/c GMP信号通路,S1P2、S1P3表达升高、激活Rho A/Rho激酶信号通路有关。     

芬戈莫德对血管紧张素Ⅱ介导的慢性肾脏病的影响

目的通过建立血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)灌注的大鼠肾损害模型,使用药物芬戈莫德(Fingolimod,FTY720)进行干预,来评估该药物对大鼠肾功能的影响,并探讨FTY720对AngⅡ灌注大鼠肾脏炎症改变的作用及可能涉及的机制,从而为肾脏疾病的治疗提供新的策略。方法 36只SPF级雄性SD大鼠随机分为3组:(1)模型组12只,即AngⅡ灌注组,皮下植入含AngⅡ的渗透性微量泵,持续灌注28 d(AngⅡ:400 ng·kg~(-1)·min~(-1));(2)干预组12只,即AngⅡ+FTY720干预组,在模型组的基础上,FTY720以0.5 g·kg~(-1)·d~(-1)灌胃28 d;(3)对照组12只,以等量的生理盐水灌胃28 d。每周称体质量,留24 h尿液,并于第28天处死各组大鼠,经心脏采血,并留取肾脏标本。放射免疫法检测各组大鼠血液中AngⅡ及肾匀浆组织中AngⅡ的浓度。对尿液及血液样本进行尿蛋白与血生化分析。光镜下观察各组大鼠肾组织病理学改变并行Jablonski评分。免疫印迹法检测炎症因子TNF-α、IL-6在肾脏中的表达。结果 (1)AngⅡ可导致模型组大鼠血肌酐、尿素氮水平升高,尿蛋白排泄明显增加,炎症因子TNF-α、IL-6在肾脏组织中的表达水平升高;(2)干预组的肾脏病理损伤较模型组减轻,且Jablonski评分明显低于模型组,血肌酐、尿素氮水平明显降低,尿蛋白明显减少,肾脏组织中炎症因子TNF-α、IL-6水平明显降低,差异具有统计学意义(P0.05)。结论 FTY720可减轻AngⅡ诱导的大鼠肾脏的病理损伤,可能是通过减少炎症因子的表达发挥了减轻尿蛋白,改善肾功能的作用。     

FTY720对大鼠急性脊髓损伤后RhoA表达的影响

目的观察大鼠急性脊髓损伤(spinal cord injury,SCI)后芬戈莫德(fingolimod,FTY720)对RhoA表达的影响,进一步探讨FTY720治疗SCI的可能机制。方法采用改良Allen’s法建立大鼠急性SCI模型。雌性SD大鼠75只,随机分为假手术组(n=15)、损伤组(n=30)和FTY720组(n=30)。FTY720组伤后30min一次性腹腔注射FTY7200.5mg/kg,假手术组和损伤组以相同方法给予浓度为0.9%的生理盐水。分别于伤后1天、3天、7天、14天及21天,应用RT-PCR和Western印迹法分别检测RhoA mRNA和RhoA蛋白的表达。结果损伤组RhoA mRNA和蛋白的表达于伤后3天开始明显增加,7天达到高峰,21天仍维持高水平表达;在同一时间点,与FTY720组和假手术组比较,差异有统计学意义(P0.05)。FTY720组伤后1天、3天和7天的RhoA表达高于假手术组(P0.05)。假手术组各时间点RhoA表达维持在较恒定水平。结论大鼠急性SCI后RhoA表达增加,FTY720可能通过抑制SCI后RhoA的表达,以达到减轻继发性SCI、促进脊髓神经功能恢复的目的。     

携带S1P3 siRNA慢病毒载体改善自发性高血压大鼠勃起功能

目的:探讨携带S1P3 siRNA慢病毒载体对自发性高血压大鼠(SHR)勃起功能的改善作用。方法:5只12周龄健康雄性WKY大鼠为A组,随机将12周龄健康雄性SHR大鼠分组(每组5只):B1组:携带S1P3 siRNA慢病毒转染的SHR;B2组:空慢病毒载体(GFP)转染的SHR;C组:SHR大鼠对照组。B1组阴茎海绵体中部注射20μl携带S1P3 siRNA慢病毒(2×10~8TU/ml),B2组注射GFP 20μl (2×10~8TU/ml),每侧10μl。1周后测各组大鼠ICPmax/MAP值,免疫组化、Western印迹、RT-qPCR检测S1P3、ROCK1、ROCK2、eNOS mRNA及蛋白在各组大鼠阴茎海绵体中的表达。结果:各组大鼠体重、血清T水平无明显差异。A、B1、B2、C组大鼠在0、3、5 V电压刺激下ICPmax/MAP分别为:0V:0.16±0.01、0.15±0.01、0.10±0.00、0.11±0.01,3 V:0.55±0.03、0.55±0.01、0.22±0.01、0.22±0.01,5 V:0.82±0.02、0.79±0.03、0.43±0.01、0.42±0.02,C、B2组ICPmax/MAP较A、B1组显著降低(P0.05)。免疫组化结果显示,S1P3、ROCK1、ROCK2在A、B1组大鼠阴茎海绵体组织中表达水平与B2、C组相比明显减少(P0.05),eNOS则明显增加(P0.05);Western印迹结果显示,与B2、C组相比,A、B1组大鼠阴茎海绵体组织中S1P3、ROCK1、ROCK2蛋白表达显著减低(P0.05),eNOS显著升高(P0.05);A、B1、B2、C组S1P3基因相对表达量分别为:0.72±0.04、0.71±0.07、1.00±0.06、1.00±0.10,ROCK1:0.99±0.05、1.08±0.16、1.85±0.44、2.02±0.38,ROCK2:1.00±0.03、1.08±0.16、2.16±0.78、2.46±0.69,eNOS:1.04±0.15、0.81±0.23、0.32±0.08、0.32±0.04,S1P3、ROCK1、ROCK2在A组和B1组中的表达水平与C组、B2组相比明显减少(P0.05),eNOS明显增加(P0.05)。结论:携带靶向S1P3 siRNA慢病毒载体抑制阴茎海绵体组织内S1P3基因的表达,并下调RhoA/Rho激酶信号通路,从而改善SHR大鼠勃起功能。     

携带siRNA的慢病毒载体抑制自发性高血压大鼠阴茎海绵体平滑肌细胞S1PR3的表达

目的:筛选出能够高效特异性沉默自发性高血压大鼠(SHR)阴茎海绵体平滑肌细胞(CCSMC)内1-磷酸鞘氨醇受体3(S1PR3)基因表达的siRNA慢病毒载体,并观察其对SHR CCSMC ROCK1、ROCK2、e NOS表达的影响。方法:以大鼠S1PR3基因mRNA序列作为干扰靶点,设计并合成3对靶向S1PR3的siRNA序列(siRNA1、siRNA2、siRNA3)及1对阴性对照序列,构建并包装成慢病毒载体。体外培养SHR CCSMC及魏-凯二氏大鼠(WKY)CCSMC,随机分为A组(SHR对照组)、B组(携带阴性对照病毒的SHR CCSMC转染组)、C~E组(分别携带靶向S1PR3基因siRNA 1~3号靶点慢病毒的SHR CCSMC转染组),F组(WKY对照组),以感染复数(MOI)=60转染SHR CCSMC,转染后观察细胞绿色荧光蛋白(GFP)表达情况,并用RT-PCR和Western印迹检测转染后细胞中S1PR3、ROCK1、ROCK2、e NOS mRNA及蛋白的表达情况。结果:经基因测序证明慢病毒载体构建成功。荧光显微镜下观察B~E组细胞转染效率均80%。与A组相比,B组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显改变(P均0.05),C、D、E、F组的S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均较A组显著下降(P均0.05),其中E组抑制作用最为明显,使S1PR3 mRNA及蛋白表达的抑制效率分别为(34.2±2.9)%、(77.7±4.7)%;ROCK1 mRNA及蛋白表达的抑制效率分别为(33.3±1.4)%、(51.1±7.3)%;ROCK2 mRNA及蛋白表达的抑制效率分别为(30.8±3.6)%、(58.32±5.5)%。A组e NOS mRNA及蛋白表达分别与B、C、D、E比较无明显差异(P均0.05),但较F组显著降低(P0.05);与F组比较,E组S1PR3、ROCK1、ROCK2 mRNA及蛋白的表达均无明显改变(P均0.05),A、B、C、D组的S1PR3、ROCK1、ROCK2 mRNA及蛋白表达均显著高于F组(P均0.05),A、B、C、D、E组e NOS mRNA及蛋白表达均较F组显著降低(P均0.05)。结论:本研究构建的3条携带不同位点的S1PR3基因的siRNA慢病毒载体均能够显著抑制SHR CCSMC内S1PR3基因的表达,并能有效抑制SHR CCSMC中上调的Rho A/Rho激酶信号通路,其中携带siRNA3慢病毒载体的抑制效率最高。     

背根神经节S1PR1在瑞芬太尼诱发切口痛大鼠痛觉过敏中的作用

目的评价背根神经节鞘氨醇-1-磷酸-1受体(S1PR1)在瑞芬太尼诱发切口痛大鼠痛觉过敏中的作用。方法取鞘内置管和尾静脉置管成功的雄性SD大鼠48只, 体质量260～280 g, 2～3月龄, 采用随机数字表法分为6组(n=8):对照组(C组)、S1PR1拮抗剂组(F组)、瑞芬太尼组(R组)、瑞芬太尼+ S1PR1拮抗剂组组(R+F组)、瑞芬太尼+切口痛组(R+I组)和瑞芬太尼+切口痛+ S1PR1拮抗剂组(R+I+F组)。C组尾静脉输注生理盐水0.1 ml·kg-1·min-1 60 min;R组尾静脉输注瑞芬太尼1.0 μg·kg-1·min-1 60 min;F组鞘内注射FTY720 3 noml, 10 min后尾静脉输注生理盐水1.0 μg·kg-1·min-1 60 min;R+F组鞘内注射FTY720 3 nmol, 10 min后尾静脉输注瑞芬太尼1.0 μg·kg-1·min-1 60 min;R+I组建立切口痛模型的同时尾静脉输注瑞芬太尼1.0 μg·kg-1·min-1 60 min;R+I+F组鞘内注射FTY720 3 nmol, 10 min后建立切口痛模型,...     

1-磷酸鞘氨醇受体在不同退变程度髓核组织中的表达变化

目的 :比较不同退变程度人椎间盘髓核组织中3种1-磷酸鞘氨醇受体(S1PR1/2/3)表达水平的差异,探讨椎间盘中S1PR表达水平与椎间盘退变的关系。方法:收集腰椎间盘退行性病变患者手术切除的椎间盘组织,其中轻度退变(Pfirrmann分级Ⅳ级)22例,严重退变(Pfirrmann分级Ⅴ级)14例;同时取6例无椎间盘退变患者(单纯腰椎椎体骨折,Pfirrmann分级Ⅱ级)手术切除的椎间盘组织作为对照组;通过HE染色以及Saf-O染色观察不同退变程度椎间盘的组织学变化,免疫组化检测不同退变程度组织中的S1PR表达水平;Ⅱ型胶原酶消化分离提取原代髓核细胞,通过Real-time PCR、Western-bolt检测不同退变程度椎间盘髓核细胞中S1PR的表达水平,并通过细胞免疫化学方法对S1PR进行定位。结果:HE染色及Saf-O染色结果显示退变椎间盘的纤维环出现破损,髓核细胞形成明显的集落,细胞外基质减少。免疫组化结果显示正常和轻度退变的髓核组织中3种受体(S1PR1/2/3)都有表达,严重退变的组织中表达极弱;Real-time PCR结果显示对照组髓核细胞中S1PR1/2/3的m RNA表达水平分别是严重退变组的5.34±0.52倍、7.25±0.04倍、1.92±0.06倍,轻度退变组S1PR1/2/3的m RNA表达水平分别是严重退变组的4.35±2.45倍、4.96±3.44倍、2.19±0.82倍;Western-blot发现对照组和轻度退变组髓核细胞中S1PR1/2/3均有表达,严重退变组表达水平较低;免疫细胞化学显示S1PR主要集中在髓核细胞的细胞质和细胞膜上。结论:髓核组织中主要表达S1PR1/2/3,在严重退变的髓核组织和细胞中其表达水平明显下降,S1P及其受体可能参与椎间盘髓核组织的退变过程。     

SphK1/S1P信号通路在肾脏疾病中的作用

鞘氨醇-1-磷酸（sphingosine 1-phosphate,S1P）具有很高的生物学活性,它参与细胞的增殖、凋亡、代谢等多个生理过程,是治疗多种疾病的一个潜在靶点。鞘氨醇激酶1(sphingosine kinase-1,SphK1)是一种广泛存在的脂类激酶,是维持体内S1P水平的关键调节酶,其在生物体内参与癌症、感染和炎症等诸多疾病的发生发展。目前已证实SphK1和S1P受体在肾脏中表达,并且多种肾脏疾病的发生发展与之相关,因此SphK1和S1P在肾脏疾病中作用受到人们越来越多的关注。本文就SphK1/S1P信号通路在肾脏疾病中的作用的研究现状进行综述,重点介绍SphK1/S1P信号通路及其在多种肾脏疾病进展中的作用。     

黄芪多糖对实验性精索静脉曲张大鼠睾丸HO-1表达的影响及意义

目的 研究黄芪多糖对实验性精索静脉曲张(Varicocele,VC)大鼠睾丸组织中血红素加氧酶-1(HO-l)表达的影响,探讨黄芪有效成分治疗VC的作用及机制.方法 青春期雄性SD大鼠70只,随机分为VC造模组(n=60)和假手术组(n=10,C组).VC造模组大鼠按灌胃药物随机分为黄芪多糖实验组(n=40,A组)和生理盐水对照组(n=20,B组).造模后分别于4周、8周取A、B组和C组大鼠左侧睾丸,用免疫组化方法检测睾丸组织中HO-1的表达.结果 HO-1在A组、B组睾丸组织中的阳性表达率分别为91.6％和94.1％,其表达强度明显高于C组(P＜0.01).A组HO-1表达强度低于B组(P＜0.05).结论 实验性VC大鼠左侧睾丸组织中存在HO-1表达上调.黄芪多糖可下调VC大鼠睾丸组织HO-1表达强度,减少睾丸组织氧化损伤,可能对VC不育有一定治疗作用.     

血清S1P与绝经后2型糖尿病患者骨密度和骨代谢指标之间的相关性研究

目的探讨血清1-磷酸鞘氨醇(S1P)与绝经后2型糖尿病患者骨密度(bone mineral density,BMD)和骨代谢指标之间的相关性。方法选取2018年2月至2019年12月期间在海口市妇幼保健院就诊的绝经后2型糖尿病女性,收集患者一般临床资料和获取其血液标本,检测生化指标、S1P和髋部、腰椎骨密度。结果最终选取130名血糖控制较好的绝经后2型糖尿病女性参与本研究,年龄为(59.3±8.9)岁,血糖为(8.75±1.5)mmol/L;S1P平均浓度为(6.46±0.78)μmol/L。相关分析表明S1P与腰椎(L1~4)、全髋和股骨颈BMD呈显著负相关(P均<0.05)。多步逐步回归分析表明,血清S1P和Ⅰ型胶原交联C末端肽(β-CTX)与腰椎(L1~4)、全髋和股骨颈BMD密切相关;而血清S1P和β-CTX是各部位BMD独立危险因素。结论1-磷酸鞘氨醇与绝经后2型糖尿病女性骨密度和β-CTX水平密切相关。     

Effect of sphingosine 1-phosphate on morphological and functional responses in endothelia and venules after scalding injury

Objective

The uncontrolled increase of vascular permeability is the major obstacle in treatment of severe burns. Sphingosine 1-phosphate (S1P) has emerged as an important modulator of EC barrier function. This study was designed to explore the effect of S1P on morphological alteration in cultured endothelial cells (ECs) after burned plasma stimulation, and second to investigate the hyper-permeability response in intact vessels after scalding injury.

Methods

The distribution of VE-cadherin and F-actin was observed by double staining in primary cultured human umbilical vein endothelial cells (HUVECs) with immunofluorescence and fluorescent probes; respectively. Permeability changes were measured by a fluorescence ratio technique in isolated venules from rat skin. Burned plasma was obtained from a third-degree scald covering 30% of the total body surface area.

Results

The intervention with burned plasma on injured rats cultured HUVECs caused a significant disruption of intercellular adherens junction labeled by VE-cadherin staining, accompanied by the formation of F-actin stress fibers in the cells. S1P prevented or reversed these burned plasma-induced morphological alterations in cultured endothelial cells. The inhibition of S1P synthesis with N,N-dimethylsphingosine (DMS) mimicked the burned plasma-evoked redistribution of VE-cadherin and reorganization of F-actin. Venules isolated from burned rats demonstrated similar endothelial cytoskeleton changes with cultured cells under the influence of S1P or DMS. Both pre- and post-burn application of S1P attenuated increased permeability in isolated and perfused skin venules after burned plasma stimulation.

Conclusion

Our results indicate that S1P plays a role in maintaining basal vascular barrier function and could be protective in burn injury by enhancing the endothelial barrier function.     

Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats

Background

Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types.

Methods

To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry.

Results

There was a significant decrease in wound area in diabetic rats treated with 125 and 60 μg/wound pcDNA3-FLAG-SPK1 (P < 0.001–0.01). The mean sizes of the wounds were 0.67 ± 0.15 cm², 0.83 ± 0.18 cm², and 1.09 ± 0.23 cm² in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2 ± 5.7% and 66 ± 7.3% in test group of 125 and 60 μg/wound respectively, and 55.4 ± 9.9% in diabetic control group (P < 0.001–0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats.

Conclusions

SPK1 gene therapy may represent a novel approach to cutaneous wound healing.     

The sphingosine‐1‐phosphate pathway is upregulated in response to partial urethral obstruction in male rats and activates RhoA/Rho‐kinase signalling

OBJECTIVES

To examine the effect of partial urethral obstruction (PUO) on the sphingosine‐1‐phosphate (S1P, a bioactive lipid shown to modulate smooth muscle, SM) pathway in the bladders of male rats, and to determine the effect of PUO on the RhoA/Rho‐kinase (ROK) pathway, and whether there is a molecular cross‐talk with the S1P pathways associated with bladder overactivity (S1P1‐S1P3, where S1P1 is associated with nitric oxide‐mediated SM relaxation, and S1P2 and S1P3 receptors are associated more with SM contraction via the ROK pathway).

MATERIALS AND METHODS

In all, 20 male rats were divided into two groups and underwent PUO or a sham operation (control). After 2 weeks all rats were killed humanely and bladder specimens used for in vitro organ‐bath physiological contractility studies, and for mRNA and protein analyses of major S1P/ROK pathway constituents via real‐time polymerase chain reaction and Western blotting, respectively. In addition, early‐passage SM cells were transfected with recombinant sphingosine kinase (SPHK, the enzyme that converts sphingosine to S1P).

RESULTS

Bladders from PUO rats had greater mRNA expression of the S1P2 and S1P3 receptors, as well as SPHK1, than the sham controls (4.78, 2.04 and 2.72 times, respectively). PUO rats also had significantly greater expression of RhoA and ROKα (1.76 and 2.19 times, respectively). Western blotting and organ‐bath contractility studies showed similar changes at the protein and in vitro functional level, with an increased contractility of bladder strips from PUO rats to exogenous S1P. Transfection of SPHK into isolated SM cells increased ROK expression.

CONCLUSIONS

We show for the first time that the S1P signalling pathway is significantly upregulated in response to PUO in male rats at both the molecular and in vitro functional levels, correlating with an activation of the RhoA/ROK pathway. Further, we provide novel data that SPHK overexpression increases ROK expression in vitro, suggesting a novel hypothesis of S1P‐induced bladder overactivity in the mechanism for PUO‐induced bladder dysfunction and the S1P signalling pathway as a possible therapeutic target for bladder overactivity.     

盐酸戊乙奎醚预处理对小鼠反复缺血?再灌注脑损伤时细胞外信号调节激酶1/2的影响

目的探讨盐酸戊乙奎醚预处理对小鼠反复缺血-再灌注(ischemia reperfusion, IR)脑损伤时细胞外信号调节激酶1/2(ERK1/2)的影响。方法 SPF级成年C57BL/6J小鼠96只,6～8周龄,体重16～25 g,按照随机数字表法分为三组:盐酸戊乙奎醚组(PHC组)、IR组和假手术组(Sham组),每组32只。Sham组腹腔注射等容量生理盐水,30 min后分离双侧颈总动脉但不夹闭;IR组腹腔注射等容量生理盐水,30 min后分离并夹闭双侧颈总动脉,建立反复IR脑损伤模型;PHC组腹腔注射盐酸戊乙奎醚1.0 mg/kg,30 min后采用双侧颈总动脉夹闭术建立反复IR脑损伤模型。采用Morris水迷宫实验评估术前及术后学习记忆能力,随后处死小鼠,留取海马组织并测定其湿/干重比(W/D)。采用伊文斯蓝(EB)法检测血脑屏障的通透性。采用逆转录-聚合酶链式反应(RT-PCR)测定海马组织ERK1/2 mRNA表达量,Western blot法测定海马组织磷酸化ERK1/2(p-ERK1/2)蛋白含量。结果与Sham组比较,术后3、7 d IR组逃避潜伏期和游泳距离明显延长(P0.05)。与IR组比较,术后3、7 d PHC组逃避潜伏期和游泳距离明显缩短(P0.05)。与Sham组比较,IR组海马组织W/D和脑组织EB含量明显升高P0.05)。与IR组比较,PHC组海马组织W/D和脑组织EB含量明显降低(P0.05)。与Sham组比较,IR组海马组织ERK1/2 mRNA表达量和p-ERK1/2蛋白含量明显升高(P0.05)。与IR组比较,PHC组海马组织ERK1/2 mRNA表达量和p-ERK1/2蛋白含量明显降低(P0.05)。结论盐酸戊乙奎醚预处理可通过抑制海马组织ERK 1/2激活而减轻小鼠反复缺血-再灌注脑损伤并改善其学习记忆能力。     

Effect of Seed of Cassia tora Extract in the Prevention of Remote Renal Reperfusion Injury

ObjectiveRenal ischemia/reperfusion (I/R) injury is characterized by the acute deterioration of renal function during ischemia and renal inflammation. Cassia tora has various effects, including antioxidant, antidiabetic, and hypolipidemic properties. In the present study, we investigated whether C tora has a renoprotective effect on I/R-induced acute kidney injury in rats.MethodsWe fed Sprague-Dawley rats either C tora (100 mg/kg/d) or saline. One week later, ischemia was induced by bilateral renal pedicle occlusion for 30 minutes, followed by reperfusion. Rats were randomized into 3 major groups, which were treated as follows: 1. the sham operation group; 2. the I/R group; and 3. the I/R-C tora group.ResultsCompared to the sham group, the I/R group had higher levels of blood urea nitrogen and serum creatinine in serum and lower expression of catalase, superoxide dismutase, glutathione peroxidase, antioxidant, and nitric oxide. Compared to the I/R group, the I/R-C tora group had higher expression of catalase, superoxide dismutase, glutathione peroxidase, antioxidant, and nitric oxide, as well as lower levels of blood urea nitrogen and creatinine in serum.ConclusionsThese results suggest that C tora has significant therapeutic effects in ischemic renal injury.     

绿色荧光蛋白转基因大鼠骨髓间充质干细胞在急性脊髓损伤大鼠中的迁移和分化

目的观察绿色荧光蛋白转基因大鼠来源的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)在急性脊髓损伤大鼠脊髓组织中的迁移和分化情况。方法全骨髓贴壁培养法培养BMSCs,Allen法制作脊髓损伤大鼠模型,共30只大鼠。于造模1周后进行干预,随机选取造模成功的24只大鼠并随机分为脊髓损伤组、假移植组(生理盐水注射)和细胞移植组(BMSCs注射),每组8只。于移植前、移植后7 d、14 d、21 d、28 d、35 d及42 d进行BBB(Basso,BeattieBresnahan locomotor rating scale)评分。HE染色、免疫荧光观察脊髓损伤的组织修复和BMSCs的迁移分化情况。结果从第14天始,细胞移植组大鼠的BBB评分较脊髓损伤组和假移植组大鼠高,差异具有统计学意义(P0.05)。HE染色显示细胞移植组大鼠的脊髓结构相对完整,液化和囊泡区缩小,炎性细胞减少。免疫荧光显示BMSCs聚集于脊髓损伤处,且能分化为神经元、神经胶质细胞和神经前体细胞。结论 BMSCs能向脊髓损伤部位迁移和聚集,并分化为相应的神经细胞促进脊髓功能恢复。     

细胞程序性坏死抑制剂Nec?1可减轻神经病理性痛大鼠的痛觉过敏

目的观察RIP1和RIP3在坐骨神经慢性压迫性损伤(chronic constriction injury, CCI)痛大鼠脊髓水平的表达变化,探讨程序性坏死特异性抑制剂Necrostatin-1(Nec-1)在神经病理性痛中的保护作用。方法雄性8周龄SD大鼠60只,随机分为三组:假手术组(Sham组)、坐骨神经CCI组(CCI组)、坐骨神经CCI+Nec-1组(CN组),每组20只。在术前1周、术后1、3、5、7、10和14 d分别测定大鼠机械缩足反射阈值(mechanical withdrawal threshold, MWT)和热刺激缩足反射潜伏期(thermal withdrawal latency, TWL)。每组取10只大鼠于术后7 d处死,采用Western blot法检测脊髓中RIP1、RIP3蛋白含量,采用透射电镜观察脊髓组织超微结构的变化。结果与Sham组比较,术后1、3、5、7、10和14 d CCI组MWT明显降低(P0.05),术后3、5、7、10和14 d TWL明显缩短(P0.05),术后7 d脊髓RIP1和RIP3蛋白含量明显升高(P0.05),脊髓中可见多处细胞核膜崩解,线粒体肿胀、嵴断裂,神经髓鞘出现明显的分离。与CCI组比较,术后7、10和14 d CN组MWT明显升高(P0.05)、TWL明显延长(P0.05),术后7 d脊髓RIP1和RIP3蛋白含量明显降低(P0.05),脊髓细胞核膜完整,线粒体稍有肿胀、嵴清晰,神经髓鞘分离不明显。结论 RIP1和RIP3介导的信号通路参与大鼠神经病理性痛的发生,给予Nec-1治疗可以减轻神经病理性痛大鼠的痛觉过敏症状。     

Gait analysis combined with the expression of TGF-β1, TGF-β3 and CREB during Achilles tendon healing in rat

PurposeTo observe the changes of gait behavior and the expression of wound healing factors of transforming growth factor-β1 (TGF-β1), TGF-β3 and cAMP response element binding protein-1 (CREB-1) during the healing of Achilles tendon in a rat model, and to investigate whether gait analysis can be used to evaluate the tendon healing.MethodsAchilles tendon of 40 healthy male Sprague-Dawley rats were transected and sutured to establish the Achilles tendon injury (ATI) model. They were randomly divided into 4 groups based on the observational time point at 1, 2, 4 and 6 weeks after injury (n = 10 for each group). Before modeling, 9 rats were randomly selected for CatWalk gait analysis, which contained step cycle, single stance time and average speed. Data were recorded as the normal controls. After then, ATI models were established in the left hind limbs of the all 40 rats (ATI group), while the right hind limbs were only cut and sutured without injury of the Achilles tendon (sham operation group). At 1, 2, 4 and 6 weeks after injury, the gait behavior of the corresponding group of rats (n = 9) as observed and recorded by CatWalk platform. After then, the rats were sacrificed and Achilles tendon of both limbs was harvested. The tendon healing was observed by gross anatomy and histological examination, and the protein and mRNA expression of TGF-β1, TGF-β3, CREB-1 were observed by immunohistochemistry and qPCR. The results of tendon gross grading were analyzed by Wilcoxon rank sum test, and other data were analyzed by one-way analysis of variance among multiple groups.ResultsCompared with normal controls, all gait indexes (step cycle, single stance time and average speed) were greatly affected following ATI, which however improved with time. The step cycle was significantly lower at 1, 2 and 4 weeks after ATI (compared with normal controls, all p < 0.05), but almost returned to the normal level at 6 weeks ((0.694 ± 0.102) vs. (0.503 ± 0.094) s, p > 0.05). The single stance time of the ATI group was significantly shorter at 1 and 2 weeks after operation ((0.078 ± 0.010) s at 1 week, (0.078 ± 0.020) s at 2 weeks, all p < 0.001) and revealed no significant difference at 4 weeks (p = 0.120). The average speed of ATI group at 1, 2, 4, 6 weeks was significantly lower than that in the normal control group (all p < 0.001).Gross observation showed that the grade of local scar adhesion in ATI group increased significantly at 2, 4 and 6 weeks, compared with the sham operation group (all p < 0.001). Extensive adhesion was formed at 6 weeks after ATI. The results of HE staining showed that the number of fibroblast increased gradually and arranged more orderly in ATI group at 1, 2 and 4 weeks (all p < 0.001), and decreased at 6 weeks, but it was still significantly higher than that of the sham operation group (p < 0.001). Immunohistochemistry showed that the positive expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at 4 time points (all p < 0.05), which reached the peak at 2 weeks after operation and decreased at 4 weeks (p = 0.002, p < 0.001, p = 0.041, respectively). The results of qPCR suggested that the mRNA expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at all-time points (all p < 0.05), which reached the peak at 2 weeks after operation, decreased at 4 weeks, and significantly decreased at 6 weeks (all p < 0.001).ConclusionGait behavior indexes are associated with Achilles tendon healing. The study gives an insight of TGF-β1, TGF-β3, CREB-1 changes in the coursing of Achilles tendon healing and these cytokines may be able to be used to regulate the Achilles tendon healing.     

低温通过冷诱导RNA结合蛋白/硫氧还蛋白1通路减轻心肺复苏后神经元氧化应激损伤

目的 探讨心跳骤停心肺复苏后脑损伤(BIC)模型中,治疗性低温(TH)对神经元氧化应激损伤的作用及相关机制.方法 雄性SD大鼠60只,7～10周,体重280～320 g.采用随机数字表法分为五组:假手术组(S组)、BIC组、低温治疗组(TH组)、沉默冷诱导RNA结合蛋白(CIRP)后低温治疗组(THC组)和空载后低温治...