| ObjectiveGlycosides of Tripterygium wilfordii(GTW)is the frequently-used Chinese patent medicine in clinic. Since its instant effect in clinic, GTW can be used for many diseases, such as Renal glomerular disease,rheumatic disease, SLE, psoriasis, tumour, organ transplantation, etc. and in many life science fields, including internal medicine, surgery, endocrine, dermatology, ophthalmology, gynecology and obstetrics, pediatrics, etc. However, some clinicians excessively worry about its gonad lesion. Its application in clinic greatly varies. In pediatric clinic especially in renal disease subject GTW is used more and more generally in recent years, but there are few studys on its gonad lesion in pediatric clinic, and empirical study objects are mature rats or mouse, no research report on juvenile rats. So how to scientifically appraise and reduce its side effect in pediatrics or juvenile rats is staring us in the face. Because of difficulty in clinical research on gonad lesion and too long time, so we will undertake this empirical study which bases on follow-up investigation of side effect caused by GTW, and the objects of this study will be the juvenile rats.This study will discuss the gonad lesion of the rat caused by the routine dosage of GTW in clinic and the preventive action of invigorating kidney Chinese medicine on gonad lesion, by vivo and vitro research on rat, and to providing with science basis to clinical medication.Method1. In Vivo Experiment(1)The research that invigorating kidney Chinese medicine and their extraction prevent the gonad lesion of male juvenil rat caused by GTWThe 60 male juvenil SD rats of 3-weeks were divided into 5 groups: normal group, GTW group,, Liuwei-Dihuang–Wan and GTW group(Liu Wei group) , TuSiZi -HuangJing granule and GTW group (Granule group), TuSiZi flavone and GTW group(Flavone group); There are 12 rats in each group. The normal group are fed with 0.5% CMC-Na daily(0.5ml/100g), the GTW group are fed with GTW(9mg/kg) daily, the Liu Wei group are fed with Liuwei-Dihuang–Wan(1g/kg) and GTW(9mg/kg) daily, the Granule group are fed with TuSiZi granule(1g/kg) and HuangJing granule(1g/kg) and GTW(9mg/kg) daily, the Flavone group are fed with TuSiZi flavone (0.01g/kg) and GTW(9mg/kg) daily. The dosage for above are all accounted according to crude drug. After 12 weeks, one hour post the last administration, the blood serum is collected from the rats for the determination of serum testosterone (T). one side testicle and epididymis are taken out, weighed and measured, accounted organ exponential, observed its change of histomorphology by the light microscope after fixation by Bouin's fluid, determined the expression of SCF by in situ hybridization and c-kit by immunohistochemistry, tested the apoptosis of sperm cell by TUNEL method.(2)The effect of invigorating kidney Chinese medicine and their extraction on reproductive activity of lesion of male juvenil rat caused by GTWThe 30 irty male juvenil SD rats of 3-weeks age are randomly divided in to 5 groups. The grouping method, dosage and period of treatment are all same to the first part. One hour post the last administration, all the rats are fed in separate cage, one rat per cage; put one adult female rat into one cage according the proportion of 1:1. After 15 days, the male rats are taken out for execution, write down in details that the matched female rats foaled or not, the time of foaling and the amount of the baby rat. The observing time last 26 days from the united male and female cage to the separation.2. In Vitro Experiment(1)Preparation for the medicated serum of the ratThe 15 male juvenil SD rats of 3-weeks age are randomly divided in to 5 groups. The grouping method and medicated dosage are all same to the in vivo experiment. After one week administration, we execute all rats 1 hour post the last administration, and take the serum from the armpit for cell culture.(2)The effect that invigorating kidney Chinese medicine combined with GTW effect the leydig cell cultured in vitro of male juvenil ratWe separate the testicle leydig cell of the 15-day age juvenil SD rat for original cell culture, and inoculate the well developed cell into the three plates with 96 holes and the one plate with 24 holes. Take the 30 holes of the plate with 96 holes, and divide them into 6 groups (the above 5 group added fetal bovine serum, viz FBS group), 5 holes in each group; after adding cellular suspension and culturing for 24h, we add in rat medicated serum to each pole according to the grouping in advance and continue culturing; take one plate separately when 12h, 24h and 48h, and take the supernatant liquor to determine the T concentration by Radioimmunoassay.We take the supernatant liquor separately when 12h and 24h ,then put MTT into cell culture box to continue incubating for 4h; take out the supernatant liquor and added to DMSO to dissolve completely the crystals; choose 490nm and 630 nm double wavelengths to measure the OD on the enzyme lablled equipment and record the results.Results1. In Vivo Experiment(1)The research that invigorating kidney Chinese medicine and their extraction prevent the gonad lesion of male juvenil rat caused by GTWSerum T(ST): The ST in GTW group, Liu Wei group and Granule group are all higher than the normal group, in which GTW tops first, Granule group takes second place and the Liu Wei group is close to the normal group. Statistic analysis: each group mean lies significant difference (P<0.01); two-group comparison indicate that significant difference lies in normal group, compared with GTW group (P<0.05). ST in Liu Wei group and Granule group are lower to a certain extent than it dose in GTW, yet no significant differences in statistics (P>0.05). However, ST in Flavone group is lower than it does in normal group and other groups, with significant difference compared with GTW group, Liu Wei group and Granule group (P<0.05).Rat weight, testicle weight and testicle weight ponderal index: Rat weight in normal group ranks first, lower to a certain extent in other groups, yet with no significant difference in statistics (P>0.05). Rat weight both in Liu Wei group and Flavone group increase compared with GTW group, yet no significant difference in statistics (P>0.05). Rat weight in Granule group slightly decrease compared with GTW group, yet with no significant difference in statistics (P>0.05). Order In terms of the testicle weight is follows: Flavone group >Sham group> Liu Wei group > Granule group > GTW group; significant difference lies in Flavone group and GTW group (P<0.05), yet no significant differences among other groups (P>0.05). From the comprehensive analysis of the testicle weight and ST, we can see that they are negative correlation, with tested correlation (r=-0.94093). In terms of testicle index, all the medicated GTW rat testicle index is higher than it dose in normal group, yet no significant differences in statistics (P>0.05).Rat epididymis weight and epididymis ponderal index: Rat epididymis weight and epididymis index in normal group is higher than it dose in other groups, with significant difference to Rat epididymis weight (P<0.05), yet no significant difference to Rat epididymis weight index (P>0.05). Epididymis weight in Chinese medicine medicated group are higher than it does in GTW group, epididymis weight index in Liu Wei group and Granule group is lower than it does in GTW group, yet no significant difference (P>0.05).Testicle and epididymal histomorphology change: The structure of the rat testicle seminiferous tubule in normal group is very distinct, and the spermatogenic cell is regular and excessive in amount; The structure in GTW group is relatively random, and the spermatogenic cell decrease. The pathological change in other groups is rather lighter than it is in GTW group, spermatogenic cell increases in amount. (see more detail in relative parts of the papers).Amount of testicle spermatogenic cell: In normal group, the amount of spermatogenium and primary spermatocyte in testicle seminiferous tubule is greater than it is in other groups, least in GTW group with significant difference (P<0.05), yet no significant difference among Liu Wei group, Granule group and Flavone group (P>0.05).Apoptosis of testicle spermatogenic cell: A few apoptotic cells of testicle spermatogenic cell can be seen in normal group. Apoptotic cells in GTW group are more than it is in normal group, with significant difference (P<0.05). Apoptotic cells of testicle spermatogenic cell in Liu Wei group, Granule group and Flavone group decrease markedly, with significant difference (P<0.05).OD determination of testicle tissue's SCF expression: The mean OD value of SCF expression in testicle testicle seminiferous tubule in GTW group visibly lower than it is in normal group, with significant difference (P<0.05). The mean OD value of SCF expression in testicle tissue in Chinese medicine group is markedly higher than it is in GTW group, ranking first in Granule group and Flavone group, with significant difference (P<0.05), yet no significant difference among intergroups (P>0.05).OD determination of testicle tissue's c-kit expression: The mean OD value of c-kit expression in testicle testicle seminiferous tubule in GTW group visibly lower than it is in normal group, yet no significant difference (P>0.05). C-kit expression of the testicle tissue in Chinese medicine group is markedly stronger than it is in GTW group, ranking first in Liu Wei group, with significant difference (P<0.05), yet no significant differences among intergroups (P>0.05).(2)The effect of invigorating kidney Chinese medicine and their extraction on reproductive activity of lesion of male juvenil rat caused by GTWComparison of the matched female rat pregnancy ratio: The matched female rat pregnancy ratio in normal group rank first(83.33%), slightly decrease in medicated GTW group, lowest in GTW group(but still to remain 20%). Increase to a certain extent in medicated Chinese medicine group, to achieve 38.89%. The significant difference lies between combined GTW group and normal group (P<0.05). There is no significant differences in medicated group compared with normal group and GTW group (P>0.05).Mean procreating number and date: Since only one procreating female rat in GTW group, the reliability can't be evaluated. The mean in combined with medicated Chinese medicine group is lower than it is in normal group, yet no significant differences (P>0.05). From the terms of the mean procreating date, except a little longer in GTW group, there is no difference in other groups, and no statistic difference neither (P>0.05).2. In Vitro Experiment(1)The effect of invigorating kidney Chinese medicine combined with GTW on the T secretion of the male juvenil rat leydig cell cultured in vitroT content in the supernatant liquor is very low in FBS group (fetal bovine serum group), but in other groups T content is higher than it is in FBS group. T secretion in normal group reaches its peak on 12h post added serum, and then gradually decreases. T secretion peak in other groups postpone on 24h, and then gradually decrease. Except Flavone group, T secretion in all medicated GTW groups lower than it does in normal group, with significant difference (P<0.05). Compared with GTW group, the significant difference lies between Liu Wei group and Granule group (P<0.05). T content in Liu Wei group are all higher than they are in Granule group at that three time point, no statistic difference at 12h point (P>0.05), with significant difference at 24h and 48h point(P<0.05).T content in the supernatant liquor in Flavone group reaches the peak at 12h, 24h and 48h point, higher than it is in normal group with significant differences (P<0.05).(2)The effect that invigorating kidney Chinese medicine combined with GTW impact the proliferation of the male juvenil rat leydig cell cultured in vitroThe mean OD value in FBS group and normal group reach its peak at 12h and 24h point; yet in other groups medicated GTW decrease to a certain extent, there is no significance differences (P>0.05). a little higher in medicated Chinese medicine groups than in GTW group, ranking first in Flavone group, yet no statistic difference between inter-groups (P>0.05).(3)The effect that invigorating kidney Chinese medicine combined with GTW impact the apoptosis of the male juvenil rat leydig cell cultured in vitroA few apoptotic leydig cell can be seen in normal group. The apoptosis ratio of leydig cell in medicated GTW group is visibly higher than it is in normal group, with significant difference (P<0.05); the apoptosis ratio of leydig cell in medicated Chinese medicine group obviously lower than it does in medicated GTW group, yet no statistic difference (P>0.05).Conclusions1. GTW has a certain effect on SD rat reproductive system, testicle and epididymis tissue. manifested in influencing ST contents, rat epididymal weight, leading to pathological changes of the testicle tissue, decreasing spermatogenic cell number, increasing apoptosis of the spermatogenic cell, weakening SCF/c-kit expression in testicle tissue.2. Liuwei-Dihuang–Wan, TuSiZi and HuangJing granule and TuSiZi flavone have a certain prevention on the above manifestation caused by GTW. The prevention manifests in influencing ST contents, alleviating the pathological changes of the testicle tissue, increasing spermatogenic cell number, decreasing apoptosis of the spermatogenic cell, strengthening SCF expression in testicle tissue; but no enough proof to prove the role of increasing rat weight and epididymal weight, strengthening c-kit expression in testicle tissue.3. GTW can lower procreative ability of male rat. The invigorating kidney Chinese medicine can properly advance procreative ability of rat.4. GTW has a certain effect on the original-generation leydig cell cultured in vitro of male juvenil rat, manifested in reducing T in the supernatant liquor, decreasing cell proliferative ability, and increasing cell apoptosis.Invigorating kidney Chinese medicine and their extraction can properly increase testicle Leydig cell T secretion and decrease the cell apoptosis.5. The active sites of the gonad lesion caused by GTW include the spermatogenium and primary spermatocyte.6. TuSiZi flavone , which indicates the unique action in many researches of this experiment, is worth more deep study.
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