淫羊藿苷对小鼠骨溶解模型OPG/RANKL基因及其蛋白表达的影响

目的:研究不同剂量的淫羊藿苷对钛颗粒诱导小鼠骨溶解模型中OPG/RANKL基因及其蛋白表达的影响,探讨淫羊藿苷治疗关节置换术后骨溶解的作用机制.方法:取成年BALB/C小鼠40只,随机分为假手术组、阴性对照组、淫羊藿苷低剂量组及高剂量组,每组10只,除假手术组外,其余建立骨溶解模型后,各组按各自药物及剂量给予每日1次灌胃,共8周.停药次日取颅骨组织及外周血,运用real-time PCR及ELISA技术测定OPG/RANKL基因及其蛋白表达情况.结果:与阴性对照组相比,高剂量组OPG基因及蛋白表达显著上升(P＜0.01),RANKL基因及蛋白表达显著下降(P＜0.01);低剂量组无显著差异.结论:淫羊藿苷可以改变OPG/RANKL基因及蛋白表达量从而抑制骨溶解,这可能是淫羊藿苷治疗关节置换术后骨溶解的作用机制之一.     

淫羊藿苷缓解腹部皮瓣缺血再灌注损伤模型大鼠的作用及机制研究

摘要 目的：研究淫羊藿苷缓解腹部皮瓣缺血再灌注损伤（IRI）模型大鼠的作用及机制。方法：取30只SD级大鼠作为研究对象,将其按照随机数字表法分作假手术组、模型组以及淫羊藿苷组,每组各10只。其中模型组和淫羊藿苷组大鼠均制作大鼠腹部皮瓣IRI模型,假手术组以及模型组大鼠予以生理盐水腹腔注射,淫羊藿苷组大鼠则予以淫羊藿苷腹腔注射。对比各组大鼠皮瓣存活面积及存活率、血清炎症因子以及氧化应激指标水平、皮瓣组织中p38丝裂原活化蛋白激酶（p38 MAPK）信号通路相关蛋白表达情况。结果：模型组、淫羊藿苷组大鼠的皮瓣存活面积及存活率均低于假手术组,但淫羊藿苷组大鼠的皮瓣存活面积及存活率均高于模型组（P＜0.05）。模型组、淫羊藿苷组大鼠血清白细胞介素-10（IL-10）均低于假手术组,但淫羊藿苷组高于模型组;模型组、淫羊藿苷组大鼠血清肿瘤坏死因子-?琢（TNF-?琢）均高于假手术组,但淫羊藿苷组低于模型组（P＜0.05）。模型组、淫羊藿苷组大鼠血清超氧化物歧化酶（SOD）、谷胱甘肽（GSH）水平均低于假手术组,但淫羊藿苷组大鼠血清SOD、GSH水平均高于模型组;模型组、淫羊藿苷组大鼠血清丙二醛（MDA）水平均高于假手术组,但淫羊藿苷组大鼠血清MDA水平低于模型组（P＜0.05）。模型组、淫羊藿苷组大鼠皮瓣组织p38 MAPK、丝裂原活化蛋白激酶磷酸酶-2（MKP-2）相对表达量均高于假手术组,但淫羊藿苷组皮瓣组织p38 MAPK相对表达量低于模型组,而MKP-2相对表达量高于模型组（P＜0.05）。结论：淫羊藿苷可通过调控p38 MAPK信号通路缓解炎症反应及氧化应激,发挥减轻腹部皮瓣IRI的作用。     

不同品种淫羊藿黄酮类有效成分的主成分分析及对肾阳虚大鼠保护作用的研究

采用HPLC测定不同品种淫羊藿黄酮类即淫羊藿苷、朝藿定A、朝藿定B、朝藿定C 4种有效成分含量,结合主成分分析对不同品种淫羊藿进行质量分析与评价,并考察淫羊藿对大鼠肾阳虚保护作用。将实验动物随机分为阳虚模型对照组(模型组)、金匮肾气丸干预组(阳性组)、淫羊藿低(125 mg/kg)、中(250 mg/kg)、高(500 mg/kg)剂量组。四周后测定肾阳虚大鼠血清中尿酸(UA)、肌酐(CRE)和尿素氮(BUN)。主成分分析结果显示,主成分1淫羊藿苷、朝藿定C和主成分2朝藿定B是影响样品质量评价的主要因子。12批淫羊藿药材整体质量较好。药理学结果显示,与空白组相比,大鼠血清中UA、CRE和BUN含量显著降低。上述结果表明:本方法同时测定淫羊藿黄酮类4种有效成分,为建立淫羊藿质量评价提供参考;淫羊藿对氢化可的松诱导的肾阳虚具有明显的保护作用,具有量效关系。     

淫羊藿苷对小鼠力竭游泳时间及力竭恢复小鼠血清生化指标的影响

为探讨淫羊藿苷(icariin,ICA)对小鼠力竭游泳时间及力竭恢复小鼠血清生化指标的影响,对小鼠以不同剂量的ICA灌胃12 d,进行一次性力竭游泳,恢复24 h后,用超微量微孔板分光光度计测定小鼠血清的丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)活性和丙二醛(MDA)、血尿素氮(BUN)、血糖(SG)含量,用聚丙烯酰胺凝胶电泳分析乳酸脱氢酶(LDH)同工酶.结果表明,ICA中剂量组小鼠力竭游泳时间明显延长(P＜0.05),血清的ALT、AST活性和MDA、BUN含量以及LDH总活力与对照组相比都有显著性降低(P＜0.05),SG无显著性差异(P＞0.05).结果说明,淫羊藿苷中剂量组具有较强的抗疲劳作用.     

淫羊藿苷对大鼠正畸牙齿压力区牙周组织RANKL和Wnt3a表达的影响

目的:观察不同剂量的淫羊藿苷对大鼠正畸牙齿移动时压力区牙周组织中RANKL和Wnt3a表达的影响。方法:将24只健康雄性SD大鼠随机分为4组,根据淫羊藿苷灌胃的剂量分为生理盐水组(对照组)、1 mg/kg淫羊藿苷组、3 mg/kg淫羊藿苷组、5mg/kg淫羊藿苷组(实验组),使用50 g力近中移动左侧上颌第一磨牙。通过免疫组化方法检测压力区牙周组织中RANKL和Wnt3a蛋白的表达。结果:生理盐水组上颌第一磨牙压力区牙根和牙槽骨表面粗糙,牙周膜间隙变窄,可见骨吸收陷窝和破骨细胞,不同剂量淫羊藿苷组牙周膜间隙趋于恢复正常,骨吸收陷窝出现明显减少。RANKL和Wnt3a在生理盐水组和淫羊藿苷组的压力区牙周组织中都有表达。与生理盐水组比较,不同剂量淫羊藿苷组压力区牙周组织中RANKL的表达均显著降低,Wnt3a的表达均明显增加,且RANKL的表达随淫羊藿苷剂量的增加而逐渐减少(P0.05),Wnt3a的表达随淫羊藿苷剂量的增加明显增加(P0.05)。结论:不同剂量淫羊藿苷能减少正畸时牙齿移动过程中压力区牙周组织中RANKL的表达,增加Wnt3a的表达,且作用与其剂量具有一定的相关性。     

淫羊藿苷对RSV感染诱发哮喘小鼠体内PGD2水平的影响

摘要 目的：观察淫羊藿苷对RSV感染诱发哮喘小鼠血清及支气管肺泡灌洗液中前列腺素D2（Prostaglandin D2,PGD2）表达水平的影响,以期为哮喘治疗寻找新的靶点。方法：30只Balb/c小鼠随机均分为三组：即正常组,OVA/RSV-YYH组（即淫羊藿苷治疗组）及OVA/RSV-非YYH组（即未经淫羊藿苷治疗组）。卵蛋白致敏RSV感染诱发小鼠哮喘模型成功建立后,予以淫羊藿苷2.5 mg连续腹腔注射治疗2周,比较治疗前后肺功能检测结果、支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）中细胞分类计数、血清及BALF中PGD2表达水平、肺组织病理学变化。结果：淫羊藿苷治疗后,哮喘小鼠肺功能较治疗前明显改善,差异有统计学意义（P<0.05）;PGD2水平较前明显下降,差异有统计学意义（P<0.05）;各分类白细胞计数较前明显减少,差异有统计学意义（P<0.01）,气道管壁增厚及管腔狭窄现象较前明显改善,肺组织炎症细胞浸润较前减少。结论：淫羊藿苷可有效降低RSV感染诱发哮喘小鼠体内炎性介质PGD2水平,从而改善气道重塑,减轻小鼠的哮喘症状,它可能是以后哮喘治疗的一个新的靶点。     

淫羊藿总黄酮对肾阳虚大鼠下丘脑-垂体-肾上腺轴钙调蛋白基因表达的影响

目的:从分子水平探讨肾阳虚证病理改变及淫羊藿总黄酮的作用及其机理,为开发药物的临床新用途提供理论依据.方法:用大剂量外源糖皮质激素建立肾阳虚大鼠动物模型,以实时荧光定量RT-PCR技术,测定各组大鼠下丘脑-垂体-肾上腺轴CaM mRNA的表达,以及淫羊藿总黄酮对其的影响.结果:肾阳虚大鼠下丘脑、肾上腺CaMmRNA水平升高,垂体组织CaM mRNA水平没有显著变化,淫羊藿总黄酮能够降低下丘脑组织CaM mRNA水平,但对肾上腺影响不显著.结论:大鼠肾阳虚证时下丘脑中CaM mRNA水平升高,淫羊藿总黄酮可使其降低.     

大蒜素对小鼠脾细胞雌激素和雄激素受体影响的实验研究

本研究旨在探讨大蒜素对小鼠脾细胞雌激素和雄激素受体的影响。将健康ICR清洁小鼠,按性别随机分成三组实验组和三组对照组。实验组分别连续灌服大蒜素14、21和28 d,对照组同期分别灌服等量生理盐水。采用RT-PCR法检测雌性各组小鼠脾细胞雌激素受体(ERα和ERβ)的mRNA表达水平和雄性各组小鼠脾细胞雄激素受体(AR)的mRNA水平。实验结果显示,雌性小鼠连续灌服大蒜素第14和21 d时实验组ERαmRNA表达水平明显高于对照组(P0.05),第14 d时ERβmRNA表达水平明显高于对照组(P0.05)。雄性小鼠连续灌服大蒜素第14、21和28 d时实验组AR mRNA表达水平均明显高于对照组(P0.05)。综上提示大蒜素可增强小鼠脾细胞雌激素受体(ERα和ERβ)和雄激素受体(AR)mRNA的表达水平。     

淫羊藿黄酮对被动吸烟大鼠骨组织肿瘤坏死因子-α和白介素1-α表达的影响

目的:探讨淫羊藿黄酮对被动吸烟大鼠骨组织肿瘤坏死因子-α白介素1-α达的影响.方法:选用2月龄Sprague-Dawley(SD)大鼠60只,随机分为6组,每组10只,各组雌雄各半,雌雄分笼饲养.A组:空白对照组,常规方法饲养,不给予被动吸烟;B组:被动吸烟组,不给予灌服药物;C组:被动吸烟+钙组.灌服高效钙(75mg·kg-1·d-1)+维生素D3 21 IU·kg-1·d-1;D组:被动吸烟+低剂量,灌服淫羊藿黄酮(75mg·kg-1·d-1);E组:被动吸烟+中剂量,灌服淫羊藿黄酮(150mg·kg-1·d-1);F组:被动吸烟+高剂量,灌服淫羊藿黄酮(300mg·kg-1·d-1).按"密室熏烟法"给予实验组(B组、C组、D组、E组、F组)大鼠被动吸烟4个月.实验动物干预完毕后取出胫骨,制备标本,RT-PCR法检测TNF-αmRNA和IL-1mRNA表达,并应用曲线拟合分析其相关性.结果:应甩淫羊藿黄酮明显抑制TNF-αmRNA和IL-1-αmRNA在骨组织中的表达,其中TNF-αmRNA表达中A组、D组、E组、F组与B组比较,差异有统计学意义(P<0.05),C组与B组比较,差异无统计学意义(P0.05),D组、E组、F组与A组比较,差异无统计学意义(P0.05),D组、E组、F组随淫羊藿黄酮剂量的增加而降低,但组间差异无统计学意义(P0.05);其它各组比较无统计学意义(P0.05);而IL-1-αmRNA表达中A组、C组、D组、E组、F组与B组比较,差异有统计学意义(P<0.05),D组、E组、F组与A组比较,差异无统计学意义(P0.05),D组、E组、F组随淫羊藿黄酮剂量的增加而降低,但组间差异无统计学意义(P0.05);其它各组比较无统计学意义(P0.05).二者在淫羊藿黄酮干预下的相关性从二者的曲线拟合图中,TNF-αIL-1-α表达的相互作用关系经曲线拟合为Y=0.82115+0.09343X-0.01781X2.结论:淫羊藿黄酮可抑制被动吸烟大鼠骨组织TNF-αIL-1-α表达.     

淫羊藿苷对体外牙周膜细胞增殖、成骨分化以及成骨和破骨因子表达的影响

目的:明确淫羊藿苷对于体外分离培养的人牙周膜细胞的增殖、成骨分化以及成骨和破骨因子表达的影响。方法:原代分离培养人牙周膜细胞,实验组细胞加入0.01 mg/L淫羊藿苷溶液。采用MTT法检测牙周膜细胞的增殖速率,采用碱性磷酸酶活性检测试剂盒评估牙周膜细胞的成骨分化能力,分别通过real-time PCR和Western印迹检测成骨相关的OCN、Runx2、BMP2以及破骨相关的RANKL的基因和蛋白表达。结果:MTT结果揭示,淫羊藿苷显著提高了人牙周膜细胞的增殖速率(P0.05),也显著促进了碱性磷酸酶活性的提升(P0.05),揭示其对于牙周膜细胞的成骨分化具有促进作用;real-time PCR和Western印迹结果均表明,淫羊藿苷能够上调OCN、Runx2和BMP2的基因和蛋白表达(P0.05),并下调RANKL的基因和蛋白表达(P0.05)。结论:淫羊藿苷能够促进体外牙周膜细胞的增殖、成骨分化潜能和成骨功能活性。本研究有望为牙周炎治疗以及基于组织工程技术的牙周组织的损伤修复提供新思路。     

The physicochemical properties of the cytoplasmic androgen receptor in the kidneys of normal, carrier female (tfm/+) and androgen-insensitive (tfm/y) mice.

The physicochemical properties of the androgen-receptor complex in mouse kidney were determined. The sedimentation coefficient, 7.9S and Stokes radius of 82A, are compatible with an asymmetric protein [frictional ratio f/fo 1.98; axial ratio, assuming a prolate ellipsoid, 20] having a molecular weight of 270,000 daltons. The kidney receptor is a relatively acidic protein of esoelectric point (pI) 4.8 and readily precipitable with protamine sulfate or ammonium sulfate. Studies with protein-specific reagents suggest that both cysteine and tryptophan residues may be necessary for maintaining the functional configuration associated with androgen binding. The kidney receptor can promote the association of testosterone with purified DNA. These properties of the androgen receptor in mouse kidney are remarkably similar to those of male accessory sexual tissue. The receptor detected in carrier female mice (tfm/+ heterozygous for the gene for testicular feminization (tfm), has the same physical properties as that of normal mice. However, due to a decrease in receptor concentration, binding activity is only 69% that of normal. In cytosol from androgen-insensitive mice (tfm/+), a specific androgen receptor cannot be demonstrated by 5 different techniques.     

Long-term survival and production of testosterone by immobilized human embryonic testis fragments

To study a new approach of graft preparation for transplantation of Leydig cells to correct androgen abnormality we have cultivated immobilized human embryonic testis fragments. The cultures were viable for at least 17 days and continuously produced testosterone. The paraffin sections showed histotypic organization of immobilized tissues, but not of free tissues, with good preservation of cells. This indicates that immobilized testicle fragments can be considered as candidates for transplantation grafts.     

Specificity of androgen resistance inMus caroli kidney

Androgen controls the expression of beta-glucuronidase and several other proteins in the kidney of the standard laboratory mouse, Mus musculus. Other species within the genus Mus exhibit a variety of response patterns for kidney beta-glucuronidase and other markers of androgen action. We have investigated the mechanism of androgen action in M. caroli, a Mus species that does not produce beta-glucuronidase in response to testosterone. The failure of testosterone to induce beta-glucuronidase in M. caroli females cannot be overcome by treatment with dihydrotestosterone, with pharmacological doses of testosterone propionate or dihydrotestosterone propionate, or with a variety of potent androgen analogues. All of these compounds induce kidney beta-glucuronidase in M. musculus females and kidney ornithine decarboxylase, submandibular gland renin, and submandibular gland epidermal growth factor in both M. caroli and M. musculus females. Furthermore, kidney androgen receptor proteins from M. caroli and M. musculus animals have the same sedimentation characteristics on sucrose density gradients. These data indicate that androgen resistance in M. caroli is not due to deficient 5 alpha-reductase or aberrant hormone metabolism producing suboptimal levels of functional androgen and is not caused by a defective androgen receptor. They suggest that the resistance of beta-glucuronidase in M. caroli kidney to induction by androgen occurs at the level of the beta-glucuronidase gene.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Effect of antiandrogen cyproterone acetate on the action of testosterone on mouse kidney.

The loss of endogenous testosterone in castrated male mice leads to a marked decrease in seminal vesicle and kidney tissue weight. 21 days' administration of exogenous testosterone abolished the effect of castration on the seminal vesicles and kidney tissue. The antiandrogen cyproterone acetate produced significant changes in the target tissue for androgens, i.e. in the seminal vesicles. In every case it blocked the action of both exogenous and endogenous testosterone on the seminal vesicles, but failed to block the "renotropic" action of testosterone, expressed as relative kidney weight. Contrary to its effect on the seminal vesicles, it did not influence relative kidney weight in normal animals. It likewise did not block the effect of exogenous testosterone on kidney tissue. The mechanism of the action of cyproterone acetate in androgen-dependent tissues is known to consist in inhibition of androgen binding to specific cell receptors in the target tissues. Some of the specific androgen receptors in mouse kidney are evidently different in character from those in the accessary sex glands, that being the reason why cyproterone acetate has an antiandrogenic, but not an antirenotropic effect. In agreement with experiments on rats, adrenal weight also decreases in mice after the administration of cyproterone acetate.     

Winning agonistic encounters increases testosterone and androgen receptor expression in Syrian hamsters

Winning aggressive disputes is one of several experiences that can alter responses to future stressful events. We have previously tested dominant and subordinate male Syrian hamsters in a conditioned defeat model and found that dominant individuals show less change in behavior following social defeat stress compared to subordinates and controls, indicating a reduced conditioned defeat response. Resistance to the effects of social defeat in dominants is experience-dependent and requires the maintenance of dominance relationships for 14 days. For this study we investigated whether winning aggressive interactions increases plasma testosterone and whether repeatedly winning increases androgen receptor expression. First, male hamsters were paired in daily 10-min aggressive encounters and blood samples were collected immediately before and 15 min and 30 min after the formation of dominance relationships. Dominants showed an increase in plasma testosterone at 15 min post-interaction compared to their pre-interaction baseline, whereas subordinates and controls showed no change in plasma testosterone. Secondly, we investigated whether 14 days of dominant social status increased androgen or estrogen alpha-receptor immunoreactivity in brain regions that regulate the conditioned defeat response. Dominants showed more androgen, but not estrogen alpha, receptor immuno-positive cells in the dorsal medial amygdala (dMeA) and ventral lateral septum (vLS) compared to subordinates and controls. Finally, we showed that one day of dominant social status was insufficient to increase androgen receptor immunoreactivity compared to subordinates. These results suggest that elevated testosterone signaling at androgen receptors in the dMeA and vLS might contribute to the reduced conditioned defeat response exhibited by dominant hamsters.     

Androgen receptor expression and morphology of forebrain and neuromuscular systems in male green anoles displaying individual differences in sexual behavior

Investigating individual differences in sexual performance in unmanipulated males is important for understanding natural relationships between behavior and morphology, and the mechanisms regulating them. Among male green anole lizards, some court and copulate frequently (studs) and others do not (duds). To evaluate potential factors underlying differences in the level of these behaviors, morphology and androgen receptor expression in neuromuscular courtship and copulatory structures, as well as in the preoptic area and amygdala, were compared in males displaying varying degrees of sexual function. This study revealed that individual differences in behavior among unmanipulated males, in particular the extension of a throat fan (dewlap) used during courtship, were positively correlated with the size of fibers in the associated muscle and with soma size in the amygdala. The physiological response to testosterone, as indicated by the height of cells in an androgen-sensitive portion of the kidney, was also correlated with male sexual behavior, and predicted it better than plasma androgen levels. Androgen receptor expression was not related to the display of courtship or copulation in any of the tissues examined. The present data indicate that higher levels of male courtship behavior result in (or are the result of) enhanced courtship muscle and amygdala morphology, and that androgen-sensitive tissue in studs may be more responsive to testosterone than duds. However, some mechanism(s) other than androgen receptor expression likely confer this difference in responsiveness.     

Unique patterns of androgen regulation of the expression of two genes in murine kidney

The kidneys of androgen stimulated mice exhibit a hypertrophic response but no hyperplasia or concomitant DNA replication. Androgens increase the expression of several genes in mouse kidney. The response of the beta-glucuronidase gene to testosterone in this tissue is characterized by a 1-2 day lag and relatively slow induction kinetics. The gene coding for kidney androgen-regulated protein (KAP) exhibits quite a different response to the hormone when compared on the basis of initial response to a given dose, dose required to produce maximal response, and apparent sensitivity to low levels of androgen-receptor complexes in renal nuclei. The analysis of the accumulation of the mRNAs produced by these two genes suggests that gene-specific differential sensitivity to androgen receptor complexes governs the development of the cellular male phenotype in this tissue.     

Influence of testosterone and a novel SARM on gene expression in whole blood of Macaca fascicularis

Anabolic hormones, including testosterone, have been suggested as a therapy for aging-related conditions, such as osteoporosis and sarcopenia. These therapies are sometimes associated with severe androgenic side effects. A promising alternative to testosterone replacement therapy are selective androgen receptor modulators (SARMs). SARMs have the potential to mimic the desirable central and peripheral androgenic anabolic effects of testosterone without having its side effects.In this study we evaluated the effects of LGD2941, in comparison to testosterone, on mRNA expression of selected target genes in whole blood in an non-human model. The regulated genes can act as potential blood biomarker candidates in future studies with AR ligands.Cynomolgus monkeys (Macaca fascicularis) were treated either with testosterone or LGD2941 for 90 days in order to compare their effects on mRNA expression in blood. Blood samples were taken before SARM application, on day 16 and on day 90 of treatment.Gene expression of 37 candidate genes was measured using quantitative real-time RT-PCR (qRT-PCR) technology.Our study shows that both testosterone and LGD2941 influence mRNA expression of 6 selected genes out of 37 in whole blood. The apoptosis regulators CD30L, Fas, TNFR1 and TNFR2 and the interleukins IL-12B and IL-15 showed significant changes in gene expression between control and the treatment groups and represent potential biomarkers for androgen receptor ligands in whole blood.