腺苷A2受体激动剂对大鼠肝脏缺血再灌注损伤时细胞凋亡的影响

目的:研究腺苷A2受体激动剂对大鼠缺血再灌注损伤（I/R）时肝细胞凋亡的影响。方法:通过门静脉插管，分别用60 mL 4℃ HTK保存液和含有腺苷A2受体激动剂CGS21680（30 μg/100 mL）HTK保存液灌注大鼠肝脏, 然后切取肝脏4℃ HTK液中保存24h，Krebs-Henseleit缓冲液常温连续再灌注45 min。检测灌注期间丙氨酸氨基转移酶（ALT）和谷氨酸乳酸脱氢酶(GLDH)及门静脉压力（PVP）的变化。灌注结束时检测灌洗液中细胞凋亡、脂质过氧化物（LPO）和胆汁分泌量的变化。结果:与对照组相比, CGS21680组大鼠灌注液中ALT，GLDH 和PVP明显降低(均P<0.01)， 胆汁分泌量明显增加 (P<0.01), LPO含量明显降低(P<0.05)，细胞凋亡明显减少(P<0.05)。结论:腺苷A2受体激动剂可减轻离体大鼠肝脏I/R损伤和细胞凋亡的发生率，其作用机制可能与氧自由基的减少有关。     

腺苷2A受体激活抑制大鼠T淋巴细胞功能

目的研究激活腺苷2A受体对大鼠T淋巴细胞体外活化、增殖及细胞毒效应的影响。方法植物血凝素(phytohemagglutinin,PHA)刺激小鼠脾脏来源的T淋巴细胞,并以终浓度为0.01μmol/L、0.1μmol/L、1μmol/L及10μmol/L的腺苷2A受体激动剂CGS21680与其共培养。荧光抗体染色结合流式细胞术检测T淋巴细胞活化抗原CD69及CD25的表达及细胞增殖情况,ELISA法检测T淋巴细胞分泌IL-2及INF-γ的水平。结果各浓度的CGS21680均可明显抑制T淋巴细胞表面CD25及CD69分子的表达(P0.05,P0.01),抑制T淋巴细胞增殖(P0.01),也对IL-2及INF-γ的分泌产生明显抑制作用(P0.01)。结论激活腺苷2A受体能有效地抑制小鼠T淋巴细胞的体外活化、增殖及细胞毒作用。     

低氧后处理活化腺苷A_(2a)受体抑制皮瓣微血管内皮细胞凋亡

目的:本研究致力于探求腺苷A_(2a)在低氧后处理抗凋亡中的作用。方法:从人上睑皮肤组织中分离培养原代人皮肤微血管内皮细胞,经6h低氧培养后,置于正常氧环境培养12h;或者经过5min常氧/5min低氧,3次循环,即低氧后处理,之后再常氧培养11.5h。结果:细胞凋亡比例和细胞凋亡相关蛋白(Bax/Bcl-2和caspase-3)表达量增高,均证明低氧/复氧可引起严重的损伤(P0.05)。低氧后处理和选择性A_(2a)受体激动剂——CGS-21680均可降低前两者(P0.05)。同时,低氧后处理和CGS-21680均明显活化了腺苷A_(2a)受体(P0.05)。数据分析显示,凋亡的增加量与A_(2a)受体蛋白的表达增加量呈显著负相关(r2=0.8177,P0.0001)。结论:低氧后处理通过活化皮肤微血管内皮细胞表面的A_(2a)受体发挥抑制凋亡的作用。     

缺血预处理对大鼠肝脏缺血再灌注损伤保护作用机制的研究

目的　探讨缺血预处理 (IPC)保护作用的发生机制。方法　建立大鼠部分肝脏热缺血再灌注模型。IPC采用肝脏缺血 10min ,再灌注 10min。结果　IPC后肝组织中腺苷和NO水平明显升高 ,与对照组相比差异显著 (P <0 0 1) ,但IPC前应用腺苷A2 受体拮抗剂后NO的升高被抑制 (P<0 0 1)。缺血再灌注 (I/R) 2h后血清中TNF α、AST、ALT、LDH及W/D水平和假手术组相比明显增加 ,而IL 10含量降低 (P <0 0 1) ;IPC、I/R前加入腺苷、IPC前应用腺苷A1受体拮抗剂显著地降低TNF α释放和AST、ALT、LDH及W /D水平 ,提高IL 10含量 ,与I/R组相比差异显著 (P <0 0 1) ;但IPC前应用腺苷A2 受体拮抗剂 (IPC +A2 antag)和NO合成酶抑制剂NAME并没有能像IPC组那样有效降低TNF α、AST、ALT、LDH及W /D的水平 ,提高IL 10的含量 (P <0 0 1) ;而IPC前给IPC+A2 antag组提供NO前体精氨酸又获得和IPC组同样的结果 (P >0 0 5 )。结论　IPC引起细胞外腺苷水平升高 ,腺苷A2 受体活化 ,介导了NO合成增加 ,最终通过抑制效应器TNF α的释放、增加IL 10的合成来实现对缺血组织的保护作用。     

腺苷受体2A调控神经肽Y抑制髓核细胞凋亡及基质降解的机制研究

目的探索腺苷受体2A(A2AR)调控神经肽Y(NPY)的机制及其在椎间盘退行性病变中对髓核细胞凋亡和细胞外基质的影响。方法采用随机数字表分组法将30只雄性SD大鼠分为5组, 每组6只。S组为对照组, 即穿刺大鼠L5～6椎间盘后注射生理盐水;M组为模型组, 即通过椎间盘穿刺联合椎间盘内注射肿瘤坏死因子α构建大鼠椎间盘退变模型;A2AR-小干扰RNA(siRNA)组、NC-siRNA组和CGS-21680组则为构建椎间盘退变模型基础上分别向椎间盘内注射A2AR的siRNA、阴性对照(NC) siRNA和A2AR激动剂CGS-21680。采用蛋白质印迹法和实时荧光定量聚合酶链反应检测A2AR、NPY、B细胞淋巴瘤/白血病-2相关X蛋白(bax)、Ⅱ型胶原(Col-Ⅱ)、蛋白激酶A(PKA)和环磷酸腺苷反应元件结合蛋白(CREB)等蛋白在椎间盘中的表达。组间比较采用单因素方差分析。结果在M组中, A2AR(1.72±0.50比0.95±0.18, F=11.49, P>0.05)、NPY(0.40±0.03比0.20±0.04, F=22.08, P<0.05)和bax(0.62±...     

腺苷对犬心肺联合移植供肺缺血-再灌注损伤的保护作用

目的 研究腺苷(ADO)对犬心肺联合移植供肺缺血-再灌注(I/R)损伤的保护作用.方法 建立犬心肺联合移植模型,将实验分为心肺联合移植对照组(对照组)和腺苷组,观察恢复血流灌注后30、60、90和120 min时两组犬动脉血氧分压(PaO2)的变化;测定缺血前10 min、缺血后10 min、120 min、再灌注后10 min、60 min供肺组织一氧化氮(NO)含量;在恢复血流灌注60 min后,取供肺组织为标本,检测肺组织中髓过氧化物酶(MPO)活力、丙二醛(MDA)含量、超氧化物歧化酶(SOD)含量以及肺组织湿干重比(W/D);HE染色光学显微镜下观察病理变化.结果 (1)在各时间点腺苷组PaO2值较对照组明显升高(P＜0.05);(2)与缺血前比较,缺血后120min、再灌注后10min和60min的肺组织NO含量均显著降低(P＜0.05),而腺苷组缺血后120 min、再灌注后10 min和60 min肺组织NO含量显著高于对照组(P＜0.05);(3)腺苷组肺组织中MPO活力、MDA含量、肺组织W/D显著低于对照组,SOD含量显著高于对照组(P＜0.05);(4)病理切片观察显示对照组的肺组织呈严重的炎性细胞浸润及肺水肿形成,而腺苷组的病变程度较对照组轻微.结论 在心肺联合移植过程中应用ADO,对供肺的缺血-再灌注损伤有保护作用.     

17β-雌二醇、辛伐他汀和V-G溶液对内皮细胞合成NO的影响

目的 观察17β雌二醇(E2)、辛伐他汀和异搏定一硝酸甘油复合溶液(V-C)在短时间内对体外培养的人脐带静脉内皮细胞(HUVECs)合成一氧化氮(NO)功能的影响.方法 HUVECs分别用17β-E2(10-5mol/L)、辛伐他汀(10-9mol/L)和V-G溶液孵育30、60、120min后应用试剂盒测定NO的生成量,内皮细胞的总一氧化氮合酶(tNoS)活性,并用RT-PCR方法测定对内皮细胞一氧化氮合酶(eNOS)mRNA表达的影响.结果 17β-E2、辛伐他汀和V-G溶液作用30、60和120 min后可以使NO的生成量增加(P<0.05);17β-E2和辛伐他汀在作用30、60min时能够增加tNOS活性,但作用时120mintNOS的活性降低(P<0.05);V-G溶液在作用30、60和120 min时均能够增加HUVECs的tNOS活性(P<0.05);17β-E2、辛伐他汀和v-G溶液在作用120min后对eNOS的mRNA水平无影响(P>0.05).结论 短时间内17β-E2、辛伐他汀和v-G溶液能够影响内皮细胞的tNos,增加内皮细胞的NO合成功能,V-G溶液的保护效果要优于前两者.     

一氧化氮在人参皂甙Rb1预处理减轻糖尿病大鼠心肌缺血再灌注损伤中的作用

目的 评价一氧化氮(NO)在人参皂甙Rb1预处理减轻糖尿病大鼠心肌缺血再灌注损伤中的作用.方法 成年雄性SD大鼠,体重220～280g,腹腔注射1%链脲佐菌素-柠檬酸盐缓冲液65 mg/kg制备糖尿病模型.取糖尿病模型制备成功的大鼠40只,随机分为4组(n=10):假手术组(S组)、缺血再灌注组(IR组)、人参皂甙Rb1预处理组(R组)和L-NAME+人参皂甙Rb1预处理组(LR组).IR组、R组和LR组采用结扎左冠状动脉前降支30 min,再灌注120 min的方法制备大鼠心肌缺血再灌注模型;S组只穿线.LR组于缺血前25 min时静脉注射一氧化氮合酶抑制剂N-硝基-L-精氨酸甲酯10 mg/kg;R组和LR组于缺血前10 min时静脉注射人参皂甙Rb1 40mg/kg;S组和IR组给予等容量生理盐水.再灌注120 min时,颈动脉采集血样,测定血清肌酸激酶(CK)和乳酸脱氢酶(LDH)的活性.然后处死大鼠,取心肌组织,计算心肌梗死范围,测定内皮型一氧化氮合酶(eNOS)表达、MDA和NO的含量以及SOD活性,光镜下观察病理结果.结果 与S组比较,IR组、R组和LR组血清CK和LDH的活性升高,心肌梗死范围增大,IR组和LR组心肌eNOS表达下调,MDA含量升高,SOD活性和NO含量降低(P＜0.05);与IR组和LR组比较,R组血清CK和LDH的活性降低,心肌梗死范围减小,心肌eNOS表达上调,MDA含量降低,SOD活性和NO含量升高(P＜0.05);IR组和LR组各指标比较差异无统计学意义(P＞0.05).结论人参皂甙Rb1预处理可通过激活eNOS,促进NO生成,抑制心肌细胞脂质过氧化反应,从而减轻糖尿病大鼠心肌缺血再灌注损伤.     

腺苷A_(2A)受体基因敲除小鼠瘢痕增生中TGF-β的表达及意义

目的 探讨腺苷A_(2A)受体基因敲除小鼠在瘢痕形成中的作用及其机制.方法 4周大腺苷A_(2A)受体基因敲除小鼠和同窝野生型小鼠各12只,制作瘢痕模型,利用HE染色观察瘢痕组织厚度、横截面积变化情况.采用比色氯胺T法测量组织羟脯氨酸含量,利用Western免疫印迹测量TGF-β表达.结果 野生型组小鼠瘢痕增生明显,其厚度、横截面积比自身对照增加倍数显著大于腺苷A_(2A)受体基因敲除组小鼠(P<0.01),腺苷A_(2A)受体基因敲除小鼠瘢痕增生轻,羟脯氨酸含量与同窝野生型组小鼠瘢痕含量相比差异有统计学(P<0.01),野生型组小鼠瘢痕TGF-β表达比自身对照增加倍数显著大于腺苷A_(2A)受体基因敲除小鼠组(P<0.01).结论 腺苷A_(2A)受体基因敲除小鼠瘢痕TGF-β表达降低,瘢痕增生显著减轻.     

腺苷A_1受体激动剂后处理对兔心肌缺血-再灌注损伤的影响

目的 探讨腺苷A1受体激动剂(2-氯环戊腺苷,CCPA)后处理对兔心肌缺血-再灌注损伤的保护作用.方法 32只兔随机均分为四组:假手术组(S组,开胸后仅行左冠状动脉套线而不阻断160min)、缺血-再灌注组(IR组,行左冠状动脉前降支阻断40 min,再灌注120 min)、缺血后处理组(lPC组,结扎左冠状动脉前降支40 min,再通30 s,结扎30 s,重复3次,再灌注120 min)和CCPA后处理组(APC组,结扎左冠状动脉前降支40 min,开放左冠状动脉1 min内予以静推CCPA100 μg/kg,冉灌注120 min).分别于左冠状动脉前降支阻断前20 min(T1)、左冠状动脉前降支阻断20 min(T2)、左冠状动脉前降支阻断40 min(T3)、心肌再灌注1 h(T4)和心肌再灌注2 h(T5)抽取颈内动脉血测定血清心肌肌钙蛋白I(cTnI)含量.再灌注末抽血离心测定超氧化物歧化酶(SOD)、丙二醛(MDA),测定心肌梗死面积.结果 和IR组相比,IPC组和APC组再灌注各个时间点cTnI均降低(P<0.05);IPC组和APC组梗死面积均小于IR组(P<0.05);IPC组和APC组血清中sOD的活性高于IR组,MDA的含量低于IR组(P<0.05).结论 腺苷A1受体激动剂后处理具有类似缺血后处理的心肌保护作用.其机制可能是通过减少氧自山基的生成,增强心肌抗氧化能力.     

Attenuation of experimental subarachnoid hemorrhage--induced cerebral vasospasm by the adenosine A2A receptor agonist CGS 21680

OBJECT: Impaired endothelium-dependent relaxation is present in vasospastic cerebral vessels after subarachnoid hemorrhage (SAH) and may result from deficient production of endothelial nitric oxide synthase (eNOS) or increased production and/or activity of inducible NOS (iNOS). Accumulating evidence demonstrates that adenosine A2A receptors increase the production of NO by human and porcine arterial endothelial cells, which in turn leads to vasodilation. This study was designed to examine the effects of an adenosine A2A receptor agonist, (2(4-[2-carboxyethyl]phenyl)ethylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680), in the prevention of SAH-induced vasospasm. METHODS:. Experimental SAH was induced in Sprague-Dawley rats by injecting 0.3 ml of autologous blood into the cisterna magna of each animal. Intraperitoneal injections of CGS 21680 or vehicle were administered 5 minutes and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging measurements of cross-sectional areas of the basilar artery (BA) 48 hours after SAH. Expression of eNOS and iNOS in the BA was also evaluated. Prior to perfusion-fixation, there were no significant differences among animals in the control and treated groups in any physiological parameter that was recorded. The CGS 21680 treatment significantly attenuated SAH-induced vasospasm. Induction of iNOS mRNA and protein in the BA by the SAH was significantly diminished by administration of CGS 21680. The SAH-induced suppression of eNOS mRNA and protein was also relieved by the CGS 21680 treatment. CONCLUSIONS: This is the first evidence that adenosine A2A receptor agonism is effective in preventing SAH-induced vasospasm without significant complications. The beneficial effect of adenosine A2A receptor agonists may be, at least in part, related to the prevention of augmented expression of iNOS and the preservation of normal eNOS expression following SAH. Adenosine A2A receptor agonism holds promise in the treatment of cerebral vasospasm following SAH and merits further investigation.     

Adenosine A1 but Not A2a Receptor Agonist Reduces Hyperalgesia Caused by a Surgical Incision in Rats: A Pertussis Toxin-sensitive G Protein-dependent Process

Background: Activation of A1 adenosine receptors (A1Rs) causes antinociception after nerve injury and inflammation. However, the role of A2a adenosine receptors (A2aRs) for pain processing is less clear. In the current study, the authors investigated the role of spinal adenosine A1Rs and A2aRs for the maintenance of mechanical hyperalgesia in an animal model for postoperative pain.

Methods: Rats with intrathecal catheters were anesthetized and underwent plantar incision. Spontaneous pain behavior and withdrawal threshold to punctuate stimulation were measured before and after administration of intrathecal R-phenylisopropyl-adenosine (R-PIA; A1R agonist), 2-w p-2-carbonyl-ethyl-phenylethylaminox-5X-N-ethylcarboxami-doadenosine (CGS21680; A2aR agonist), or vehicle. In separate groups of animals, the effects of pertussis toxin, forskolin, glibenclamide, 4-aminopyridine, tetraethylammonium, apamin, charybdotoxin, or margatoxin on R-PIA-induced antinociception were examined.

Results: Intrathecal administration of 5 nmol R-PIA but not 10 nmol CGS21680 decreased nonevoked spontaneous pain behavior. Furthermore, intrathecal administration of R-PIA but not of CGS21680 increased withdrawal thresholds after incision. Pretreatment with pertussis toxin and administration of forskolin, glibenclamide, 4-aminopyridine, and tetraethylammonium inhibited R-PIA-induced antinociception. In addition, intrathecal administration of apamin, charybdotoxin, or margatoxin did not modify mechanical hypoalgesia mediated by R-PIA.     

高糖与高尿酸通过醛糖还原酶通路协同作用加重内皮细胞损伤

目的寻找高血糖与高尿酸血症协同加重内皮细胞损伤的共同作用靶点和分子机制,为糖尿病合并高尿酸血症患者心血管疾病的保护提供干预靶点。 方法用人脐静脉内皮细胞系(HUVEC-C)给予高糖(30 mmol/L,HG)、高尿酸(600 μmol/L,UA)和高糖(HG)＋高尿酸(UA)联合培养48 h。利用10 μmol/L阿司他丁阻断醛糖还原酶(AR)的活性。实时定量PCR检测内皮型一氧化氮合成酶(eNOS)和AR mRNA的表达;Western blot检测还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2 (NOX2)、NOX4、eNOS和AR蛋白的表达;共聚焦显微镜检测细胞内ROS的活性;一氧化氮(NO)试剂盒检测NO活性。 结果与单独HG组和UA组相比,HG＋UA共培养组明显降低eNOS mRNA水平和蛋白表达,减少NO产生,增加内皮细胞胞内ROS活性;HG＋UA共培养明显上调AR mRNA水平和蛋白表达。应用AR抑制剂能够明显增加HG＋UA组内皮细胞eNOS mRNA水平和蛋白表达,增加内皮细胞NO的分泌水平。AR抑制剂能够明显下调HG＋UA组内皮细胞NOX4的蛋白表达,对NOX2无影响;降低细胞内ROS的含量。 结论高血糖和高尿酸协同作用通过激活醛糖还原酶途径下调内皮细胞eNOS的表达,增加ROS活性,减少NO的产生,加重内皮细胞功能障碍;而抑制醛糖还原酶,能够通过抑制NOX4表达,阻断这种协同损伤作用。     

异丙酚对高血压大鼠胸主动脉内皮型一氧化氮合酶和诱导型一氧化氮合酶表达的影响

目的 评价异丙酚对高血压大鼠胸主动脉内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)表达的影响.方法 SD大鼠,雌雄各半,体重240～ 280 g,采用皮下注射去氧皮质酮的方法制备高血压模型,采用随机数字表法,将64只造模成功的大鼠随机分为4组(n＝16):高血压组(H组)、小剂量异丙酚组(P1组)、中剂量异丙酚组(P2组)和大剂量异丙酚组(P3组).P1组、P2组和P3组分别静脉输注异丙酚20、30、40 mg·kg-1·h-13 h,H组给予等容量生理盐水.分别于给药前、给药1h、3h时记录平均动脉压(MAP).给药3h时处死大鼠,摘眼球法采集血样,硝酸还原酶法测定血清一氧化氮(N0)浓度,取胸主动脉,采用RT-PCR和Western blot法测定eNOS mRNA、iNOS mRNA及其蛋白表达水平.结果 与H组比较,P1组、P2组和P3组给药3h时MAP降低,血清NO浓度升高,主动脉eNOS mRNA及其蛋白表达上调,主动脉iNOS mRNA及其蛋白表达下调,且呈剂量依赖性(P<0.05或0.01).结论 异丙酚降低高血压大鼠血压的机制与下调iNOS表达,上调血管内皮细胞eNOS表达,促进NO释放有关.     

Thrombus-induced endothelial dysfunction: Hemoglobin and fibrin decrease nitric oxide bioactivity without altering eNOS

BACKGROUND: Arterial thrombosis is associated with endothelial dysfunction. The purpose of this study was to determine which components of thrombus induce endothelial dysfunction and to determine their effect on endothelial nitric oxide synthase (eNOS) activity and expression. MATERIALS AND METHODS: Human aortic endothelial cells were grown to confluence. Group 1 consisted of control cells exposed to media. Group 2 was exposed to cellular components of thrombus, including erythrocytes (RBCs) (1.5, 3, and 6 x 10(4) cells/ml) and platelets (0.5, 1, and 2 x 10(5) platelets/ml). Group 3 was exposed to extracellular thrombus components, including hemoglobin (1.25, 2.5, and 5.0 g/dl), thrombin (0.4, 4.0, and 10.0 units/ml), and fibrin. The exposure time was 20 h. Nitric oxide (NO) levels were measured following exposure. Global cellular integrity was evaluated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) conversion. eNOS mRNA expression was measured using semiquantitative PCR and eNOS protein activity evaluated via a l-citrulline conversion assay. Studies were done in replicates of six and expressed as percentages of controls. RESULTS: NO levels decreased following exposure to hemoglobin (1.25 g/dl = 59 +/- 5%, 2.5 g/dl = 38 +/- 3%, 5 g/dl = 37 +/- 6%, P < 0.001). MTS metabolism was likewise suppressed (1.25 g/dl = 57 +/- 2%, 2.5 g/dl = 55 +/- 3%, 5 g/dl = 44 +/- 6%, P < 0.001). Fibrin diminished NO levels (37 +/- 5%, P < 0.01) and MTS metabolism (49 +/- 5%, P < 0.01). RBCs and platelets had no effect on NO production or MTS metabolism (P > 0.05). Thrombin's effect was concentration dependent, inhibiting nitric oxide production at low doses while stimulating it at high doses (P < 0.01). All thrombin doses enhanced MTS metabolism (P < 0.05). eNOS activity was not altered by fibrin, thrombin, or hemoglobin exposure (P > 0.05). Moreover, eNOS mRNA expression was unaffected (P > 0.05). CONCLUSIONS: Intact cellular components of thrombus do not cause endothelial dysfunction in vitro. However, free hemoglobin and fibrin diminish NO bioactivity, likely via scavenging. Thrombin affects NO levels in a concentration-dependent manner. Neither eNOS expression nor eNOS activity is diminished, indicating that thrombus does not cause permanent changes in NO generation capability.     

Adenosine A1 but not A2a receptor agonist reduces hyperalgesia caused by a surgical incision in rats: a pertussis toxin-sensitive G protein-dependent process

BACKGROUND: Activation of A1 adenosine receptors (A1Rs) causes antinociception after nerve injury and inflammation. However, the role of A2a adenosine receptors (A2aRs) for pain processing is less clear. In the current study, the authors investigated the role of spinal adenosine A1Rs and A2aRs for the maintenance of mechanical hyperalgesia in an animal model for postoperative pain. METHODS: Rats with intrathecal catheters were anesthetized and underwent plantar incision. Spontaneous pain behavior and withdrawal threshold to punctuate stimulation were measured before and after administration of intrathecal R-phenylisopropyl-adenosine (R-PIA; A1R agonist), 2-w p-2-carbonyl-ethyl-phenylethylaminox-5X-N-ethylcarboxami-doadenosine (CGS21680; A2aR agonist), or vehicle. In separate groups of animals, the effects of pertussis toxin, forskolin, glibenclamide, 4-aminopyridine, tetraethylammonium, apamin, charybdotoxin, or margatoxin on R-PIA-induced antinociception were examined. RESULTS: Intrathecal administration of 5 nmol R-PIA but not 10 nmol CGS21680 decreased nonevoked spontaneous pain behavior. Furthermore, intrathecal administration of R-PIA but not of CGS21680 increased withdrawal thresholds after incision. Pretreatment with pertussis toxin and administration of forskolin, glibenclamide, 4-aminopyridine, and tetraethylammonium inhibited R-PIA-induced antinociception. In addition, intrathecal administration of apamin, charybdotoxin, or margatoxin did not modify mechanical hypoalgesia mediated by R-PIA. CONCLUSIONS: Spinal A1Rs but not A2aRs play an important role in the maintenance of nonevoked and evoked pain behaviors after an incision. Furthermore, A1R-induced spinal antinociception is mediated by interactions with pertussis toxin-sensitive G proteins. In addition, the opening of adenosine triphosphate-sensitive K channels but not of calcium-activated potassium channels and voltage-gated Kv1.3 or Kv1.6 channels contribute to the antinociceptive effect of A1R agonists.     

NOSTRIN在无精子症患者睾丸组织中的表达

目的:通过测定无精子症患者睾丸组织中内皮型一氧化氮合酶运输介导物(NOSTRIN)和内皮型一氧化氮合酶(eNOS)的表达,探讨其与无精子症发病的相关性。方法:采用免疫组化法检测17例特发性无精子症患者(病例组)和10例正常男性(正常组)睾丸组织中NOSTRIN的定位和定量表达;RT-PCR检测睾丸组织中NOSTRINmRNA的表达;分光光度法测定睾丸组织中eNOS的活性;应用硝酸还原酶法测定睾丸组织上清液中NO代谢产物亚硝酸基/亚硝基(NO2-/NO3-)水平。结果:NOSTRIN在睾丸组织中表达于生精细胞、支持细胞以及血管内皮细胞,NOSTRIN在特发性无精子症患者睾丸中的表达水平显著低于正常男性;病例组患者睾丸组织中NOSTRINmRNA呈弱表达(0.312±0.076),明显低于正常组(0.793±0.082,P<0.01);病例组患者睾丸组织eNOS活性[(33.727±3.58)U/mg]与正常组[(17.69±3.84)U/mg]比较显著增高(P<0.01);病例组患者睾丸中NO2-/NO3-水平为[(48.56±8.49)μmol/L],与正常组[(25.37±9.61)μmol/L]比较显著升高(P<0.01);病例组睾丸组织中NOSTRIN表达水平与eNOS活性呈负相关(r=-0.57,P<0.01),与NO代谢产物NO2-/NO3-同样呈负相关(r=-0.61,P<0.01)。结论:无精子症患者睾丸组织中NOSTRIN表达水平降低,eNOS活性增强,NO-/NO-水平升高,这可能与无精子症的发生有着相关性。     

Direct Measurements of Nitric Oxide Release in Relation to Expression of Endothelial Nitric Oxide Synthase in Isolated Porcine Mitral Valves

The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT‐PCR. Results show that bradykinin increases NO release from mitral valves (ΔBradykinin: 33.71 ± 10.41 nm NO, P < 0.001, n = 10), whereas N‐nitro‐l ‐arginine methyl esther (l ‐NAME) decreases NO release when compared with basal level (Δl ‐NAME: 82.69 ± 15.66 nm NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.     

Protective effect of adenosine A2A receptor activation in small-for-size liver transplantation

The aim of the present study was to investigate the potential role of adenosine A(2A) receptor (A(2A)R) activation in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed by using 40% (range: 36-46%) liver grafts. Recipients were given either saline (control group) or CGS 21680 (2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride, a selective A(2A)R agonist), or CGS 21680+ ZM 241385 (a selective A(2A)R antagonist) immediately after reperfusion for 3 h. Compared with control group, CGS 21680 used at both low dose (0.05 microg/kg/min) and high dose (0.5 microg/kg/min) increased the survival rate from 16.7% (2/12) to 83.3% (10/12) and 66.7% (8/12), respectively. These effects correlated with improved liver function and preserved hepatic architecture. CGS 21680 effectively decreased neutrophil infiltration, suppressed pro-inflammatory (TNF-alpha, IL-1beta and IL-6) expression, promoted expression of antiapoptotic molecules, and inhibited apoptosis. The effects of CGS 21680 were prevented when ZM 241385 was co-administrated. In conclusion, the present study showed that A(2A)R activation alleviated portal hypertension, suppressed inflammatory response, reduced apoptosis, and potentiated the survival of small-for-size liver grafts. Our findings provide the rationale for a novel therapeutic approach using A(2A)R activation to maximize the availability of small-for-size liver grafts.     

Effects of polydeoxyribonucleotide on the histological damage and the altered spermatogenesis induced by testicular ischaemia and reperfusion in rats

The effects of polydeoxyribonucleotide (PDRN), an agonist of the A2A adenosine receptors which when activated positively influences sperm activity, were tested in an experimental testicular ischaemia/reperfusion injury model. Anaesthetized male Sprague-Dawley rats were subjected to testicular torsion-induced ischaemia, followed by reperfusion (TI/R). Immediately after detorsion, randomized animals, including SHAM, received intraperitoneal injections of: (i) vehicle (1 mL/kg 0.9% NaCl solution); (ii) PDRN (8 mg/kg); (iii) DMPX (3,7-dimethyl-1-propargilxanthine, 0.1 mg/kg); or (iv) PDRN (8 mg/kg) + DMPX (0.1 mg/kg). Animals were euthanized at 1, 7 and 30 days following reperfusion. Vascular endothelial growth factor (VEGF) expression is normally associated with adenosine A2A receptor stimulation. After treatment, VEGF mRNA/protein expression quantified by qPCR and Western blot, vascular endothelial growth factor receptor-1 (VEGFR1) and endothelial nitric oxide synthase (eNOS) mRNA measured by qPCR, VEGF and VEGFR1 assessed using immunohistochemical methods, histological staining and spermatogenic activity were all analysed. Testis ischaemia-reperfusion (TI/R) injury caused increases in VEGF mRNA and protein, VEGFR1 and eNOS mRNA, histological damage and reduced spermatogenic activity. Immunostaining showed a lower expression of VEGF in germinal epithelial cells and a strong expression of VEGFR1 in Leydig cells after TI/R. PDRN administration increased significantly VEGF message/protein, VEGFR1 and eNOS message, decreased histological damage and ameliorated spermatogenic activity. PDRN might be useful in the management of testicular torsion.