大鼠睾丸内一氧化氮合酶的表达

目的 :阐明一氧化氮合酶 (NOS)在睾丸内的表达 ,探讨一氧化氮 (NO)在睾丸生殖功能中的作用。　方法 :取雄性SD大鼠睾丸于 4 %多聚甲醛中固定 ,常规石蜡切片。用免疫组化ABC法检测成年大鼠睾丸NOS的表达和定位。　结果 :内皮型NOS(eNOS)、神经型NOS(nNOS)、诱导型NOS(iNOS)在睾丸间质细胞内均有表达 ,精曲小管周围肌样细胞、血管内皮细胞和平滑肌细胞内仅为eNOS免疫反应阳性 ,而血管外膜纤维内仅有nNOS表达。免疫反应物质主要定位于细胞质 ,胞核为阴性。在生精细胞内 3种NOS免疫反应均为阴性。　结论 :3种NOS在睾丸内均有表达 ,不同的NOS分布部位有一定区别     

雷公藤多甙对大鼠精子凋亡的影响

目的:研究雷公藤多甙(GTW)对大鼠精子凋亡的影响。方法:将SD雄性大鼠16只均分成2组,雷公藤多甙组和羧甲基纤维素钠对照组均为剂量20 mg/(kg.d)灌胃,6周后取大鼠附睾精子测定其凋亡率、膜脂流动性、一氧化氮(NO)、丙二醛(MDA)及超氧化物歧化酶(SOD)含量的变化。结果:灌胃6周后,GTW组精子的凋亡率、NO和MDA的含量显著增加(与对照组相比,P<0.01),精子膜脂流动性(P<0.05)和SOD含量(P<0.01)降低。结论:雷公藤多甙可致大鼠精子膜脂质过氧化损伤、膜结构受损以及膜流动性下降,精子凋亡率升高。     

N-硝基-L-精氨酸甲酯对大鼠隐睾生殖细胞凋亡的保护作用

目的探讨一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸甲酯(L-NAME)对大鼠隐睾生殖细胞凋亡的保护作用.方法用22 d SD雄性大鼠复制单侧隐睾模型.实验分假手术组、隐睾组、隐睾+L-NAME组[术后腹腔注射L-NAME,10 mg/(kg·d)],每组大鼠各10只.术后7 d,用生物素-dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡,用硝酸还原酶法测定睾丸组织中NO含量,用化学比色法测定睾丸组织中NOS活性.结果术后第7 d,与假手术组睾丸相比,隐睾组睾丸发生凋亡的生殖细胞数显著增加,隐睾+L-NAME组睾丸发生凋亡的生殖细胞数比隐睾组显著减少(P＜0.01),隐睾+L-NAME组睾丸组织中NO含量及NOS活性与隐睾组相比显著降低(P＜0.01).结论隐睾组织中NO和NOS升高是隐睾生殖细胞凋亡增加的病理机制之一,L-NAME通过抑制NOS活性、减少NO的产生来降低睾丸组织生殖细胞的凋亡发挥其保护作用.     

Ν-硝基-L-精氨酸甲酯对大鼠隐睾生殖细胞凋亡的保护作用

目的 :探讨一氧化氮合酶 (NOS)抑制剂N 硝基 L 精氨酸甲酯 (L NAME)对大鼠隐睾生殖细胞凋亡的保护作用。　方法 :用 2 2dSD雄性大鼠复制单侧隐睾模型。实验分假手术组、隐睾组、隐睾 +L NAME组 [术后腹腔注射L NAME ,10mg/(kg·d) ],每组大鼠各 10只。术后 7d ,用生物素 dUTP/酶标亲和素测定法检测睾丸生殖细胞凋亡 ,用硝酸还原酶法测定睾丸组织中N0含量 ,用化学比色法测定睾丸组织中NOS活性。　结果 :术后第 7d ,与假手术组睾丸相比 ,隐睾组睾丸发生凋亡的生殖细胞数显著增加 ,隐睾 +L NAME组睾丸发生凋亡的生殖细胞数比隐睾组显著减少 (P <0 .0 1) ,隐睾 +L NAME组睾丸组织中NO含量及NOS活性与隐睾组相比显著降低 (P<0 .0 1)。　结论 :隐睾组织中NO和NOS升高是隐睾生殖细胞凋亡增加的病理机制之一 ,L NAME通过抑制NOS活性、减少NO的产生来降低睾丸组织生殖细胞的凋亡发挥其保护作用。     

异丙酚对大鼠肺一氧化氮合酶活性的影响

目的 了解异丙酚对大鼠肺一氧化氮合酶(NOS)活性的影响,探讨异丙酚对肺血管、支气管扩张作用的机理。方法40只SD大鼠,随机分为异丙酚组(n=20)、对照组(n=20),分别腹腔注射等容积异丙酚(1ml·kg-1,即100mg·kg-1)和生理盐水(10ml·kg-1)。异丙酚组待鼠翻正反射消失后,经尾缘静脉泵以异丙酚10mg·kg-1·h-1,20min后处死,对照组鼠腹腔注射20min后处死。检测支气管肺泡灌洗液NO水平、肺组织匀浆中NOS酶活性、NO水平及内皮型NOS(eNOS)、神经型NOS(nNOS)在肺内的表达与分布(免疫组化法)。结果 异丙酚组支气管灌洗液和肺组织匀浆中NO水平均明显高于对照组(P<0.01),肺组织匀浆中NOS酶活性也明显大于对照组(P<0.01)。异丙酚组肺血管内皮细胞nNOS和eNOS、支气管粘膜上皮细胞nNOS染色表达强阳性。结论 异丙酚可以刺激肺中NOS活性,升高肺内内源性NO水平,在异丙酚的扩张肺血管、支气管中发挥一定的作用。     

雷公藤多甙对小鼠生育的影响及肉苁蓉干预作用的研究

目的:研究使用不同时间和剂量的雷公藤多甙(GTW)对昆明小鼠怀孕率的影响及肉苁蓉干预和停用GTW后的自愈倾向。方法:将90只成年雄性昆明鼠随机分为9组(每组10只)。对照组给予1%羧甲基纤维素(CMC)溶液;GTW各组分别给予45.0、30.0、22.5、15.0、7.5mg/(kg·d)GTW混悬液;肉苁蓉各组分别给予30.0、7.5mg/(kg·d)GTW混悬液;停药组给予30.0mg/(kg·d)GTW混悬液。以上各组先灌胃20d,20d后与成年雌鼠1∶2合笼7d。第28d起GTW各组按原剂量继续给药;肉苁蓉各组给原剂量GTW混悬液的同时分别给10.0、2.5g/(kg·d)肉苁蓉煎液;停药组停GTW,改为1%CMC溶液。20d后再次合笼7d。每次合笼后观察雌鼠怀孕率。结果:随着GTW给药时间的延长和剂量的增加,雌鼠怀孕率呈下降趋势。GTW45.0mg/(kg·d)组给药40d后怀孕率降为0%(P<0.01)。肉苁蓉10.0g/(kg·d)组怀孕率由20d时的25%上升到40d时的55%。停药组怀孕率由25%上升到56%,与对照组比较,差异无显著性(P>0.05)。结论:GTW对雄性小鼠的生殖毒性呈时间和剂量依赖性。肉苁蓉可以改善GTW对雄鼠生殖系统的抑制作用,提高雌鼠的怀孕率。停用GTW20d,怀孕率可以基本恢复正常。     

前列腺Ⅰ号水丸对慢性细菌性前列腺炎大鼠组织内菌落数量和NO、NOS浓度的影响

目的:探讨前列腺I号水丸对慢性细菌性前列腺炎(CMP)大鼠组织内菌落数量、一氧化氮(NO)浓度和一氧化氮合酶(NOS)活力的影响。方法:健康成年雄性Wistar大鼠120只,体重250~300 g,随机分为6组,分别为空白对照组、模型对照组、阳性对照组、水丸高剂量组、水丸中剂量组、水丸低剂量组,每组20只。前列腺注射大肠杆菌(107个/ml)建立CMP大鼠动物模型。造模成功1个月后,用前列腺水丸高[4.4 g/(kg.day)]、中[2.2 g/(kg.day)]、低[1.1 g/(kg.day)]剂量灌胃治疗,阳性对照组用美满霉素[0.018 g/(kg.day)]灌胃,模型组和空白组用生理盐水灌胃。连续治疗35 d后,计算大鼠前列腺组织匀浆菌落数。采用亚硝酸盐还原酶法检测前列腺组织中NO变化并计算NOS活力的变化。结果:前列腺Ⅰ号水丸高、中、低剂量组大鼠组织内菌落数的水平和前列腺组织内NO含量及NOS活力明显低于模型组,与模型组比较差异显著(P<0.01)。结论:前列腺Ⅰ号水丸对慢性细菌性前列腺炎模型大鼠有一定治疗作用。其作用机制可能与抑制前列腺内细菌生长以及降低前列腺组织内NO含量和NOS活力有关。     

大鼠前列腺增生模型阴茎海绵体内nNOS、eNOS表达研究

目的 分析引起阴茎勃起的主要神经递质NO合成的限速酶nNOS和eNOS在BPH模型大鼠阴茎海绵体中的表达.试探讨BPH引起ED的可能因素.方法 20只雄性SD大鼠随机分为正常对照组和BPH模型组.通过去势后丙酸睾酮注射建立BPH模型,10只老年大鼠为老年对照组.4周后处死大鼠,分析不同组前列腺湿质量与前列腺指数,并在光镜下观察前列腺组织病理学改变.应用免疫组化方法研究不同组间大鼠阴茎海绵体内nNOS和eNOS阳性表达情况并比较组间差异.结果 BPH模型组中前列腺湿质量和前列腺指数与正常对照组比较差异有统计学意义;显微镜下BPH模型组前列腺组织呈明显增生表现,大鼠BPH造模成功.免疫组化方法显示BPH模型组、老年对照组大鼠阴茎海绵体内的nNOS及eNOS阳性细胞表达率明显降低;nNOS及eNOS阳性表达与正常对照组的比较差异均有统计学意义.结论 nNOS及eNOS是调控阴茎勃起的神经递质NO合成的关键酶,BPH模型组的阴茎海绵体内nNOS、eNOS表达明显减少或活性降低,导致NO释放减少可能是BPH引起勃起功能障碍的重要原因.     

一氧化氮合酶同功异构酶在大鼠睾丸中的表达和定位

目的 :了解一氧化氮 ( NO)在睾丸中的作用。方法 :运用免疫组织化学方法观察 3种一氧化氮合酶 ( NOS)同功异构酶在大鼠生后 4、7、1 4、3 0、60 d睾丸中的分布。结果 :( 1 )生后 4、7、1 4d大鼠睾丸未见 3种 NOS免疫阳性反应 ;( 2 )生后 3 0 d少数精母细胞及生后 60 d生精小管腔面精子和间质细胞呈 NOS1阳性 ;( 3 )生后 3 0 d少数精母细胞、支持细胞和管周类肌细胞呈 NOS2阳性 ,而生后 60 d NOS2阳性反应见于睾丸间质细胞、管周类肌细胞、支持细胞、极少数精母细胞和不成熟精子头部 ;( 4)生后 3 0 d睾丸内少数精母细胞和血管壁呈 NOS3阳性 ,生后 60 d NOS3阳性反应仅见于血管壁。结论 :NO可能参与精子发生、睾酮的分泌过程 ,并调节睾丸内的血流。     

强精片对不育模型SD大鼠细胞凋亡通路Fas/FasL的影响

目的:观察强精片对不育模型SD大鼠精液质量及Fas/Fas L通路的影响。方法:70只雄性SD大鼠随机分为空白组20只与雷公藤多甙片(GTW)灌胃造模组50只,灌胃3周后两组各取10只进行模型验证。证明模型成功后,将40只模型组大鼠,随机分为模型组、强精片低、中、高剂量组(剂量分别为GTW 20 mg/(kg·d)、GTW 20 mg/(kg·d)+强精片58 mg/(kg·d)、GTW 20 mg/(kg·d)+强精片105 mg/(kg·d)、GTW 20 mg/(kg·d)+强精片233 mg/(kg·d)),灌胃4周后取血处死,检测精液质量及睾丸组织Fas L凋亡因子表达。结果:模型组大鼠精子浓度[(10.94±3.58)×10~6/ml]、活力[(9.31±5.79)%]、活率[(24.03±6.93)%]降低明显,与空白组[(71.99±9.72)×10~6/ml]比较差异显著(P0.01);强精片各组在用药4周后精子浓度、活力、活率均有提高,其中强精片高[(59.66±4.53)×10~6/ml、(35.45±5.21)%、(61.97±9.75)%]、中剂量组[(40.89±4.90×10~6/ml、(24.41±4.79)%、(60.06±10.62)%]与模型组间存在极显著差异(P0.05或P0.01),并降低模型大鼠睾丸内凋亡因子Fas L表达(P0.05或P0.01)。结论:雷公藤多甙可能通过上调Fas/Fas L信号通路,诱导生精细胞凋亡,导致精子浓度、活力、活动率等指标降低,造成不育。而中药强精片可能通过降低睾丸内Fas L凋亡因子表达,提高大鼠生殖功能。     

萝卜硫素对肾病综合征大鼠肾脏损伤保护的机制研究

目的研究萝卜硫素对肾病综合征大鼠肾脏损伤的保护作用及其对一氧化氮合酶表达的影响。方法采用5 mg/kg阿霉素诱导肾病综合征大鼠模型,将大鼠分为对照组、模型组、萝卜硫素组及阳性药物组,30 mg/kg萝卜硫素和6 mg/kg阳性药物贝那普利分别灌胃治疗大鼠6周,HE染色检测肾脏组织病理学变化,BCA法检测24 h尿蛋白量,ELISA法检测了血浆白蛋白水平,分光光度法测定尿液NO含量;Western blot法检测肾组织内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)和神经型一氧化氮合酶(nNOS)的表达。结果相对于对照组大鼠,模型组大鼠24 h尿蛋白量明显升高(P<0.05),血浆白蛋白明显降低(P<0.05),肾脏病理损伤严重,尿液中NO含量显著降低(P<0.05),eNOS、iNOS及nNOS酶表达水平亦显著降低(P<0.05);相比于模型组大鼠,萝卜硫素和阳性药物治疗组大鼠24 h尿蛋白量明显降低(P<0.05),血浆白蛋白明显升高(P<0.05),肾脏病理损伤明显改善,尿液中NO含量显著升高(P<0.05),eNOS、iNOS及nNOS酶表达水平亦显著增高(P<0.05)。结论萝卜硫素能够诱导肾病综合征大鼠eNOS、iNOS及nNOS的表达,提升大鼠体内NO水平,降低尿蛋白含量,改善肾脏损伤程度。     

增精颗粒对大鼠附睾精子质量的影响

目的 :观察中药增精颗粒 (ZJG)对不育大鼠附睾精子质量的影响 ,探讨ZJG改善附睾精子质量的机制。　方法 :将 4 0只雷公藤多甙 (GTW )实验性SD雄性大鼠不育模型随机分成 4组 ,每组 10只 ,包括不育组 [GTW 2mg/(ml·10 0g) ]和ZJG高、中、低剂量组 [高 :ZJG0 .6 7g/(ml·10 0g) +GTW 2mg/(ml·10 0 g) ,中 :ZJG0 .33g/(ml·10 0g) +GTW 2mg/(ml·10 0 g) ,低 :ZJG0 .17g/(ml·10 0 g) +GTW 2mg/(ml·10 0g) ],另设正常组 (对照 ) 10只(等量的 1%羧甲基纤维素钠溶液 )。整个实验周期 6周 ,由 3周造模期和 3周用药期组成。ZJG各组按指定的剂量用药 3周 (实验第 4 3d)观察大鼠附睾精子质量、附睾腺管壁厚度及主要性腺器官系数的变化。　结果 :ZJG高、中、低剂量组附睾精子密度分别为 (5 9.6± 3.72 )、(6 3.3± 5 .70 )、(6 9.7± 6 .91)× 10 6/ml,精子活率分别为 (6 5 .4±6 .33) %、(6 9.3± 10 .96 ) %和 (72 .6± 9.6 1) % ,畸形率分别为 (5 2 .3± 7.4 7) %、(4 6 .2± 7.73) %和 (33.2± 7.97) % ,与不育组 [精子密度 (13.1± 6 .81)× 10 6/ml,精子活率 (7.6± 5 .87) % ,畸形率 (77.2± 8.75 ) % ]相比 ,ZJG各组差异均有显著性 (P <0 .0 5 ) ,与正常组 [精子密度 (75 .6± 10 .82 )× 10 6/ml,精子?     

The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(-/-) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(-/-) mice showed significant mechanical repression of RANKL expression (p<0.05). Mechanical strain still increased NO production in the absence of eNOS, and was abolished by SMTC, a specific nNOS inhibitor. nNOS mRNA and protein expression were increased by strain in eNOS(-/-) but not in WT cells, revealing that nNOS was mechanically sensitive. When NO synthesis was blocked with either SMTC or siRNA targeting nNOS in eNOS(-/-) cells however, strain still was able to suppress RANKL expression by 34%. This indicated that strain suppression of RANKL can also occur through non-NO dependent pathways. While our results confirm the importance of NO in the mechanical control of skeletal remodeling, they also suggest alternative signaling pathways by which mechanical force can produce anti-catabolic effects on the skeleton.     

Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance

Studies from our laboratory using acute pharmacologic blockade of nitric oxide synthase (NOS) activity have suggested that nitric oxide (NO) has an important role in regulating carbohydrate metabolism. We now report on insulin sensitivity in mice with targeted disruptions in endothelial NOS (eNOS) and neuronal NOS (nNOS) genes compared with their wild-type (WT) counterparts. Mice underwent hyperinsulinemic-euglycemic clamp studies after a 24-h fast, during an insulin infusion of 20 mU x kg(-1) x min(-1). Glucose levels were measured at baseline and every 10 min during the clamp. Insulin levels were measured at baseline and at the end of the clamp study. Glucose infusion rates (GIRs) during the last 30 min of the clamp study were in a steady state. Tritiated glucose infusion was used to measure rates of endogenous glucose output (EGO) both at baseline and during steady-state euglycemia. Glucose disposal rates (GDRs) were computed from the GIR and EGO. Fasting and steady-state glucose and insulin levels were comparable in the 3 groups of mice. No differences in fasting EGO were noted between the groups. GIR was significantly reduced (37%, P = 0.001) in the eNOS knockout (KO) mice compared with the WT mice, with values for the nNOS mice being intermediate. EGO was completely suppressed in the nNOS and WT mice during insulin infusion, but not in the eNOS mice. Even so, the eNOS mice displayed significantly reduced whole-body GDRs compared with those of the WT mice (82.67+/-10.77 vs. 103.67+/-3.47 mg x kg(-1) x min(-1), P = 0.03). eNOS KO mice are insulin resistant at the level of the liver and peripheral tissues, whereas the nNOS KO mice are insulin resistant only in the latter. These data indicate that NO plays a role in modulating insulin sensitivity and carbohydrate metabolism and that the eNOS isoform may play a dominant role relative to nNOS.     

Effect of severe aortic banding above the renal arteries on nitric oxide synthase isotype expression

BACKGROUND: Severe aortic stenosis above the renal arteries leads to a reduction in renal perfusion, increased renin secretion, and elevation of arterial blood pressure above the stenotic site. Nitric oxide (NO) plays an important role in regulation of renal and systemic vascular resistance, renal blood flow, and Na(+) handling. Abdominal aortic banding provides an excellent model for simultaneous testing of the effects of increased and decreased pressure, flow, and shear stress in the same animal. METHODS: We studied protein expressions of endothelial NO synthase (eNOS), inducible NOS (iNOS), and neuroneal NOS (nNOS) isotypes in the renal cortex, renal medulla, heart, brain, and aorta segments above and below the stenosis site three weeks after abdominal aortic banding above the renal arteries. The results were compared with those obtained in the sham-operated controls. NOS isotype proteins were measured by Western blot. RESULTS: Compared with the control group, the banded group showed significant up-regulations of eNOS, iNOS, and nNOS in renal cortex and medulla. Likewise, heart eNOS, brain nNOS, and thoracic aorta eNOS proteins were significantly increased in the banded group. However, eNOS and iNOS expressions were unchanged in the aorta segment below the stenotic site. Likewise, iNOS expression in the heart and thoracic aorta remained unchanged in the banded animals. No significant difference was found in creatinine clearance or urinary protein excretion between the two groups. CONCLUSIONS: These findings clearly demonstrate the up-regulatory action of increased pressure on eNOS expression in the thoracic aorta and heart and of nNOS expression in the brain. These data further show up-regulation of all NOS isotypes in the kidney, which must have helped to mitigate the associated hypoperfusion.     

Novel nitric oxide signaling mechanisms regulate the erectile response

Nitric oxide (NO) is a physiologic signal essential to penile erection, and disorders that reduce NO synthesis or release in the erectile tissue are commonly associated with erectile dysfunction. NO synthase (NOS) catalyzes production of NO from L-arginine. While both constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms mediate penile erection, nNOS is widely perceived to predominate in this role. Demonstration that blood-flow-dependent generation of NO involves phosphorylative activation of penile eNOS challenges conventional understanding of NO-dependent erectile mechanisms. Regulation of erectile function may not be mediated exclusively by neurally derived NO: Blood-flow-induced fluid shear stress in the penile vasculature stimulates phosphatidyl-inositol 3-kinase to phosphorylate protein kinase B, which in turn phosphorylates eNOS to generate NO. Thus, nNOS may initiate cavernosal tissue relaxation, while activated eNOS may facilitate attainment and maintenance of full erection.     

Modulation of nitric oxide synthase isoenzymes inreperfused skeletal muscle

Objective:To investigate the modulation of nitric oxide synthase(NOS)isoenzymes in skeletal muscle during 3h ischemia/reperfusion (I/R,3h ischemia followed by 3h reperfusion).Methods:The extensor digitorum longuses(EDLs) from 20 adult rats were divided into 4 groups:the normal,the sham operation,the ischemia(3h),and the ischemia/reperfusion group.One normal EDL from each rat was used as the non-operated control,and the opposite ones are distributed into the 3 remaining groups.All the samples were studied with Western blotting technique and immunohistochemistry staining.Results:Three sizes or protein bands verified with the proteins of relative molecule to be of 155000,140000 and 135000,were detected in the EDL homogenate by Western blotting,which were comparable with the positive controls for nNOS,eNOS and iNOS,respectively.Immunostaining demonstrated that nNOS was present in the muscle fiber,with a similar location of the muscle stria,eNOS was found apparently in microvascular endothelia,but not found in muscle fibers,and iNOS was found in the leukocytes around the muscle fiber and some endothelia cells.Immunostaining paralleled the Western blotting results.Conclusions:It suggests that the constitutive nNOS and eNOS protein can be regulated by I/R,and I/R results in a down regulation of nNOW and up-regulation of eNOS and iNOS in reperfused skeletal muscle.The fact that nNOS is present around stria suggests that nNOS may have a close relationship with muscle function.The localization of eNOS in endothelial cell indicates its role in regulating blood supply of the muscle.Based on these findings,it is possible that No produced by distinct NOS may play a different role in I/R injury.