【作者】 刘晓晖；

【导师】 陈虹； 张莉；

【作者基本信息】 河北医科大学 ， 药理学， 2009， 硕士

【摘要】 目的:研究红景天苷(salidroside)对大鼠睾丸间质细胞、吊尾应激小鼠睾酮分泌的影响,考察不同剂量红景天苷在大鼠体内的动力学过程。方法:(1)将雄性大鼠断头处死后,立即摘取睾丸,剥离包膜,用Ⅱ型胶原酶消化。取睾丸间质细胞混悬液或含垂体组织块间质细胞悬液分别加入不同浓度的红景天苷(终浓度10-4、10-5、10-6和10-7 mol/L)或/和氢化可的松(终浓度10-6 mol/L)体外培养3h,用放免法(RIA)测定培养基中睾酮含量;(2)昆明种小鼠60只,随机分为对照组、应激组、红景天苷高剂量组(ig. 60mg/kg)、中剂量组(ig. 30mg/kg)和低剂量组(ig. 15mg/kg)。采用悬吊应激模型,除对照组外各组小鼠连续重复悬吊应激11d,每天应激一次,并循序增加应激强度,每次应激前30min给予相应剂量的红景天苷或生理盐水,并于末次应激结束30min后眼眶取血,用RIA测定血清中睾酮含量。摘取脾脏、胸腺、肾上腺和睾丸,称重并计算脏器指数,其中小鼠左侧肾上腺和睾丸用4%多聚甲醛固定并制作组织切片,HE染色,于显微镜下观察形态学变化;(3)健康雄性SD大鼠尾静脉注射不同剂量红景天苷(15、30、60mg/kg)后,按规定时间从眼眶静脉丛取血0.50ml,离心取血清用高氯酸沉淀蛋白,取上清液进行HPLC测定。色谱条件:色谱柱为VP-ODS柱SHIMADZU C18(250mm×4.6mm,5μm),流动相为乙腈-水-三乙胺-冰醋酸(10:90:0.011:0.016,V:V:V:V),流速为1.5ml/min,紫外检测波长为277nm,柱温50℃,进样量10μl,内标:咖啡因。并对所建立的方法进行方法学确证。测得的红景天苷峰面积用标准曲线换算成血药浓度,绘制不同给药剂量下红景天苷的血药浓度-时间曲线图,并用3P97药物动力学程序计算药物在大鼠体内的动力学参数。结果:(1)体外研究结果表明,间质细胞用含1%乙醇的DMEM培养基孵育时,乙醇对睾酮的分泌无影响;各个浓度的红景天苷均能显著刺激间质细胞睾酮的基础分泌,各个浓度的红景天苷对睾酮分泌的影响无浓度依赖关系;浓度为10-5和10-6mol/L的氢化可的松均可促进间质细胞的基础睾酮分泌,但睾酮分泌水平无显著性差异;间质细胞经红景天苷预处理后,再加入氢化可的松共培养时,间质细胞睾酮分泌增加,但是与单用红景天苷相比,睾酮分泌水平无差异;间质细胞与垂体共培养后间质细胞睾酮基础分泌显著增加,加入不同浓度红景天苷后对睾酮分泌没有显著影响;垂体和间质细胞共培养条件下氢化可的松可显著抑制间质细胞睾酮的分泌,但细胞经红景天苷预处理后可显著逆转氢化可的松所引起的睾酮分泌下降。(2)在体研究显示,应激后小鼠体重增长程度明显受抑,脾脏指数下降,肾上腺指数上升,肾上腺皮质肥厚,髓质萎缩甚至消失,且应激组小鼠血浆睾酮水平显著低于对照组。实验发现每次应激前30min给予低剂量红景天苷可使应激导致的体重增加减缓、肾上腺指数上升、肾上腺皮质肥厚及髓质萎缩等病理性改变减轻,睾酮恢复至正常水平。但红景天苷中剂量组和高剂量组上述作用不显著。(3)高效液相色谱法测定红景天苷的标准曲线方程:A样/A标=0.1041C+0.0924(r=0.9991,n=7),红景天苷浓度在0.50～80.00μg/ml内线性关系良好,以信号/噪音=3:1计算,红景天苷的最低检测浓度为0.15μg/ml,其中浓度为5、20、80μg/ml血清样品的日内精密度分别4.82%、1.84%和1.70%,日间精密度分别为4.15%、1.83%和1.54%,血清样品的平均回收率为(98.54±4.12)%。所得血药浓度-时间数据经3P97药物动力学程序处理,根据药动学房室模型的判断方法,初步判定静脉注射后红景天苷在体内大鼠的过程符合二室模型。低、中、高剂量下红景天苷的分布半衰期(T1/2α)分别为4.96、2.01和4.36min,消除半衰期(T1/2β)分别为24.24、19.40和16.88min。AUC0-t和AUC0-∞随着剂量的增加均呈线性增加,其中AUC0-t和AUC0-∞分别为362.15、879.77、1421.48和383.09、884.52、1426.63μg·min/ml。结论:(1)氢化可的松可显著抑制垂体存在条件下间质细胞睾酮的分泌,提示应激所产生的高水平的GCs可能通过影响垂体促性腺激素如LH等的释放对间质细胞睾酮水平产生影响。红景天苷可促进间质细胞的基础睾酮分泌,且该作用不受氢化可的松影响;在垂体与睾丸间质细胞共孵育条件下,红景天苷对垂体刺激下间质细胞睾酮的分泌无影响;而当间质细胞用红景天苷预培养30min后再加入氢化可的松时,培养基中睾酮水平上升,提示红景天苷与垂体所分泌的促性腺激素无协同作用,且红景天苷可拮抗氢化可的松所引起的睾酮水平的下降,该作用可能是通过竞争性拮抗糖皮质激素或者同时增强11β-HSD活性从而加速糖皮质激素分解来实现的,其确切机制有待于进一步的研究。(2)低剂量红景天苷可显著改善应激小鼠体重增加减缓、肾上腺指数升高、肾上腺皮质肥厚、髓质萎缩和结构层次模糊等,且能显著抑制悬尾应激所引起的睾酮水平低下并使其恢复正常,提示红景天苷可能通过改善应激所致的睾酮水平降低及维持肾上腺的正常结构和正常生理功能发挥抗应激作用。(3)建立RP-HPLC内标法测定血清中红景天苷的方法简便、快速、准确,适用于红景天苷的血药浓度测定及动力学研究。且红景天苷给药剂量在15～60 mg/kg范围内,其体内药物动力学过程属于线性药物动力学,体内消除为一级速率过程,模型拟合二室为最匹配模型。更多还原

【Abstract】 Objective: To study the effects of salidroside on testosterone secretion in Leydig cells of rats and in tail-suspension stressed mice,and to study the pharmacokinetics features among different doses of salidroside in rats.Method:(1)The adult Wistar male rats were killed by decapitation , Leydig cells were isolated through typeⅡcollagenase enzymatic digestion.Then the culture containing Leydig cells or containing pituitary gland and Leydig cells were incubated with different concentration of salidroside or/and hydrocortisone in vitro for 3,respectively.Testosterone levels in incubates were measured by radioimmunoassy.(2)60 healthy male Kunming mice were randomly divided into five group including control group,stress group, low dose of salidroside(ig.15mg/kg)group,medium dose of salidroside(ig.30mg/kg)group,high dose of salidroside(ig.60mg/kg)group.The repeated tail-suspension stress model has been accepted in this experiment. Each group mice were stressed by hanging once daily(9:00am～2:00pm)for 11 days(1～3 day hanging for 2 hours,4～6 day hang for 3hours,7～9 day hang for 4hours,10～11day hang for 5 hours)except the control group. Each group was administrated with salidroside or saline 30min before the tail-suspension stress.On day 11,30min after stress the blood samples from the orbit were collected.Immediately,to select the relative tissue,such as spleen,thymus,adrenal and testis.And the organ indexes were calculated after weighing the spleen,thymus,adrenal and testis of rats.The left adrenal and testicle were fixed by 4% paraformaldehyde solution. And all tissue sections were taken HE staining. Testosterone level in the serum was measured by radioimmunoassy.(3)A series of doses of salidroside(15,30,60mg/kg)was given to rats intravenously. About 0.5ml of venous blood samples from orbital vein were collected at different time after administration.The serum was pretreated by precipitating with perchloric acid. SHIMADZU C18 column was used.The mobile phase was consisted of acetonitrile,water,triethylamine and acetic acid(10:90:0.011:0.016,V:V:V:V).The flow rate was 1.5ml/min,and the UV detection was set at 277nm.The column temperature was 50℃and the sample size was 10μl. Caffeine was used as internal standard. The peak area of salidrside was converted into concentration through the linear regression equation.The serum concentration of salidroside were analyzed through reversed-phase high-performance liquid chromatography ( RP-HPLC ) and the pharmacokinetics parameters were calculated by 3P97.Result :(1 )There was no effect on Leydig cells testosterone production by using DMEM which containing 1%alcohol.Each concentration of salidroside could significantly stimulated the testosterone production of Leydig cells,and there was no difference among all salidroside groups ; The testosterone production increased when Leydig cells incubated with hydrocortisone,and there was no significant change between the two concentrations; The testosterone production increased when Leydig cells was incubated with salidroside for 30min before hydrocortisone was added,but the testosterone level was not alteration compared with groups of single using salidroside;When the pituitary gland and Leydig cells were co-cultured , the testosterone level was significantly increased.But the level did not change when salidrside was added. Hydrocortisone could significantly inhibit the testosterone level when the pituitary gland and Leydig cells were co-cultured.But this case could be reversed if Leydig cells were preatreated with salidroside.(2)The experiment in vivo has showed that the weight gain and spleen index of mice were descreased,the adrenal index was increased,the adrenal cortex was diffuse hyperplastic and the medulla was atrophic compared with the control group after 11 days stress. At the same time,we found that 30min before stress administrated with low dose of salidroside(15mg/kg)can improve all the symptom.But we did not see the same effect in the other two groups.( 3 ) The linear regression equation was A样/A标 =0.1041C+0.0924(r=0.9991,n=7).The linear calibration curve was obtained in the concentration range from 0.5 to 80μg/ml. The LOD(S/N=3)of the method was 0.15μg/ml. The intra-day and inter-day precision were in the range from 1.70% to 4.82% and from 1.54% to 4.15%,respectively.And the range of the mean recovery in serum at concentrations of 5,20 and 80μg/ml was(98.54±4.12)%.The parameter which calculated by 3P97 shows the characteristics of a two-compartment model after iv injection of salidroside at three doses. The corresponding distribution half-lives(T1/2α)were 4.96,2.01,4.36min and the elimination half-lives(T1/2β)were 24.24,19.40,16.88min,respectively.There was a linear increase in AUC proportional to doses , AUC0-t and AUC0-∞were 362.15 , 879.77 ,1421.48μg·min/ml and 383.09,884.52,1426.63μg·min/ml,respectively.Conclusions:(1)In this experiment we found that the testosterone secretion of Leydig cells which was stimulated by pituitary gland can be inhibited by hydrocortisone , it is suggestion that the high level of GCs which induced by stress maybe affect the releasing of pituitary gonadotropic hormone of pituitary gland,such as LH. And finally this may suppress the testosterone secretion. Salidroside could increase the testosterone secretion of Leydig cells,and this action was not disturbed by hydrocortisone;When Leydig cells and pituitary gland were cultivated together,salidroside did not affect the testosterone production level which stimulated by pituitary gland,and it’s suggestion that between salidroside and pituitary gonadotropic hormone maybe not exist synergistic effect;And it is interesting that salidroside could inhibit the effect of hydrocortisone. This effect may be through the competitive antagonism to suppress the effect of hydrocortisone or/and enhance the activity of 11β-HSD to accelerate the decomposition of hydrocortisone,but the specific mechanism remains to be elucidated.(2)In repeated tail-suspension stressed mice,it was shown that the increasing of weight was descend,the adrenal index was ascendant,the adrenal cortex was diffuse hyperplastic and the medulla was atrophic,which may be caused by excessive secretion of corticocosterone in cortex and depletion of adrenalin vesicle in medulla. While adrenal gland of stressed mice treated with salidroside 15mg/kg was improved obviously,even to be normal,and the serum testosterone level showed significantly improved. It was indicated that salidroside may have the antistress effect through improve stress-induced the reduction of testosterone secretion,and maintain the normal structure and physiologic function of adrenal glands.(3)The method of RP-HPLC that we established is simple,rapid and suitable for the determination of the concentration of salidroside in rat serum and the study of pharmacokinetics.There was hardly difference of pharmacokinetics for salidroside within the range of doses used.The concentration-time curve of salidroside was fitted to a two-compartment model and the elimination is linear.更多还原