The Effects Of Salidroside On Testosterone Secretion In Leydig Cells Of Rats And In Tail-suspension Stressed Mice, And Its Pharmacokinetics In Rats

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.