Tripterygium Glycosides Caused The Female Pups Reproductive Damage And The Dodder Flavonoids Interfere With Their Injury In Rats

ObjectiveTripterygium wilfordii(GTW) with anti-inflammatory,immunosuppressive,anti-fertility, anti-tumor and other pharmacological effects can be frequently used for many diseases,such as Henoch-Schonlein Nephritis,Glomerulonephritis,Lupus erythematosus,Nephrotic syndrome,Rheumatoid arthritis,Rheumatoid arthritis,Uterine fibroids, Endometriosis,etc.GTW has significant efficacy.However,some clinicians excessively worry about its gonad lesion.Its application in clinic greatly varies.GTW on current reproductive damage in children are few and the majority of experimental studies reported on the mature rats,so how to scientifically appraise pediatric dose of GTW on reproductive damage in young rats,how to minimize or mitigate its toxicity side effects is staring us in the face.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 SD rats..This stud y 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(Cuscuta flavonoids, Liuweidihuangwan) on gonad lesion,to providing with science basis to clinical medicationMethods1 In vitro experiment1.1 Preparation for the medicated serum and normal serum of rat24 SD female young rats,3-week-old,were randomly divided into 4 groups:Control group (A group),GTW group(B group),GTW and Liuweidihuangwan group(C group),GTW and Cuscuta flavonoids group(D group).There are 6 rats in each group.A group were fed with 0.5%sodium carboxymethyl cellulose 0.5ml/100g,B group were fed with GTW0.9 mg/kg,C group were fed with GTW0.9 mg/100g and Liuweidihuangwan 0.1g/100g,and D Group were fed with GTW0.9 mg/100g and flavonoids 0.01g/100g daily for 7 days.The blood of rats were collected from abdominal aorta With 10%chloral hydrate intraperitoneal anesthesia in the last 1～2h after oral administration.The serum of the collected blood were obtained after being centrifugated 2500r/min for 20min.Then the prepared serum were used for cell culture after being inactivated in a water bath 56℃for 30min and sterilized through filter membrane(membrane pore size is 0.22um).1.2 the effect of the medicated serum on the secretion of estradiol and progesterone and cell proliferation of GC cultured in vitro of the SD young rats20 female SD rats of 3-week-old were executed in 48 hours after intraperitoneal injection of PMSG661U per rat.Separated from young rats,the granulosa cells were inoculated into 3 plates with 96 holes and 2 plates with 24 holes for original cell culture.After 48 hours 30 holes were selected per plate and divided into 5 groups(In addition to the above 4 groups a E group of fetal bovine serum is added.),6 holes in each group.The original cells were cultured continually by being added in the medicated serum and fetal bovine serum respectively,then one plate was took separately,supernatant liquor was drawn and the concentration of E2 and P were detected by radioimmunoassay after the 12,24 and 48 hours.After the 0h,12h,24h,48h of the medicated serum being added in and the supernatant liquor being drawn,20ul MTT were put into each hole of cell culture supernatant board and cultured continually for 4h.Then 150ul DMSO were put into each hole to dissolve the crystals fully,490nm and 630 nm double wavelengths were chosen to measure the light density values (OD) and the results were recorded.1.3 the effect of the medicated serum on the Bcl-2 expression of the granulosa cells in vitro of the SD impuberal ratsSingle cell suspension were inoculated into two 24-hole culture plate at 48h point and central selected 15 holes of each plate were divided into 5 groups,6 holes in each.The medicated serum and fetal bovine serum were added respectively culture for 24h,then the supernate were drawn and the granulosa cells were fixed by paraformaldehyde.The expression of Bcl-2 protein were detected by immunohistochemical SP method.2 In vivo experiment2.1 the effect of the Cuscuta flavonoids interfering in the ovary Bax expression of the reproductive damage of female young rats caused by GTW40 SD female rats,3-weeks-old,were randomly divided into 4 groups,10 in each.The dose of medicine was the same as above.After 12 weeks of continuous administration,fasting for 12 hours and 10%intraperitoneal chloral hydrate anesthesia the ovary were taken out and fixed by formaldehyde,then the apoptosis gene Bax of ovarian tissue were detected by immunohistochemical SP method.2.2 the effect of the Cuscuta flavonoids against the procreative damage of female young rats caused by GTW and the observation of the reversible damage to the reproduction100 SD female rats,3-week-old,were randomly divided into 4 groups,25 in each.The doses of medicine were the same as above.After 12 weeks of continuous administration all the female rats were fed alone,one per cage,then the adult male SD rats were put into the all cages in proportion of 1:1.Two weeks later the adult male SD rats were taken out.The birth time,the numbers and the weight in 0,1st,2nd,3rd week of the baby rats were recorded in detail.Non-pregnant females were continued to be together with male rats for two weeks.The same processes were observed 3 cycles.After three weeks of delivery and 12h fast,the female rats were weighed and anesthetized with 10%chloral hydrate,then the livers,kidneys, uteruses,ovaries,thymuses,adrenal glands and other organs of them were obtained and the organ weight coefficients were calculated.Results1 In vitro experiment1.1 the effect of the medicated serum on the secretion of estradiol and progesterone and cell proliferation of GC cultured in vitro of the young SD ratsThe cultured GC of young SD rats were added the medicated serum of GTW and Liuweidihuangwan.At 12h and 24h points density of E2 in supernatant were measured and there were not statistical significance(p＞0.05) compared to that of the control group.There was a very significant difference(p＜0.01) between control group and Cuscuta flavonoid group.At 48h point density of E2 in supernatant of GTW group was significantly lowered and compared with that of the control group was significant difference(p＜0.01).Two Chinese medicine groups and the fetal calf serum group,compared with the control group there were no significant difference(p＞0.05).At 2h,24h,48h points density of E2 in supernatant of two Chinese medicine groups were higher than those of GTW group,there were very significantly different(p＜0.01) at 12h and 24h points and was not significantly(p＞0.05) at 48h point,and Cuscuta flavonoid group was more higher than Liuweidihuangwan group..At 24h and 48h points density of E2 in supernatant in vitro GC of FBS group was the lowest.This might be related to that in vitro GC of SD rat needed to be stimulated by special contents of secreted P.the cultured that in vitro GC of SD rats were added to GTW group; Liuweidihuangwan group and Cuscuta flavonoids group,At 12h point all of the density of p in supernatant were higher than that of control group with significant difference(p＜0.05).At 24h pointdensity of p in supernatant of GTW group and Liuweidihuangwan group were significantly increased with significant difference(p＜0.05) compared to that of control group,but there wasn't significant difference between Cuscuta flavonoids group and control group(p＞0.05).At 48h point densityof p in supernatant all of the GTW groups were decreased.Compared with control group,GTW group were significantly different(p＜0.05),yet the other two groups were not significantly different(p＞0.05).At 12h and 24h points OD of Liuweidihuangwan group were the highest,that it was significantly different(p＜0.05) at 12h point,yet wasn't at 24h point compared with that of control group.After 24h OD of Liuweidihuangwan group was gradually decreased,however OD of fetal bovine serum group and control group were increased gradually.The highest OD was fetal bovine serum group followed by control group,At 12h,24h and 48h points control group had very significant difference compared with the GTW groups(p＜0.01),and at 48h point there was very significant difference(p＜0.01) compared with the Liuweidihuangwan group and significant difference(p＜0.05) compared with Cuscuta flavonoids group.At the three points OD of Chinese medicine groups were higher than those of GTW group,and Cuscuta flavonoids group was the most obvious with highly significant difference (p＜0.01).Cuscuta flavonoids group and Liuweidihuangwan group were significantly different (p＜0.05).1.2 the effect of the medicated serum on the Bcl-2 expression of the granulosa cells in vitro of the SD young ratsBcl-2 protein in normal control group and fetal calf serum group had the most expression, no significant difference between the two groups(p＞0.05),yet expression of Bcl-2 protein was the weakest in the GTW group with very significant difference compared to that in control group(p＜0.01).The expression of Bcl-2 protein in Chinese medicine groups were more obvious than that in the GTW group with very significant difference(p＜0.01). especially in Cuscuta flavonoids group,but there were no significant difference(p＞0.05)between the Chinese medicine groups.There was a very significant difference of the expression of Bcl-2 protein between control group and the Liuweidihuangwan group(p＜0.01)and a significant difference between control group and the Cuscuta flavonoids group (p＜0.05).2 In vivo experiment2.1 the effect of the Cuscuta flavonoids interfering in the ovary Bax expression of the reproductive damage of female young rats caused by GTWBax protein in ovarian tissue of SD rats in the control group was the lowest expression with very significant differences(p＜0.01)compared to GTW and Liuweidihuangwan groups and no significant difference(p＞0.05) compared to Cuscuta flavonoids group.Bax protein expression in GTW group was the highest,followed by Liuweidihuangwan group,and there was no significant difference(p＞0.05)between the two groups,yet a very significant difference(p＜0.01) compared to Cuscuta flavonoids group.There was a very significant difference(p＜0.01) between the two groups of Chinese medicine.2.2 the effect of the Cuscuta flavonoids against the reproductive damage of female young rats caused by GTW and the observation of the reversible damage to the reproduction The weights of female SD young rats of all experimental groups gradually increased along with the time of administration.At 4 and 12 week points,the mean weight was the highest in Cuscuta flavonoids group,and that of Liuweidihuangwan group is slightly lower than in the control group without significant difference(p＞0.05).At 1st cycle that male rats were togethe with female rats,control group pregnancy rate was the highest(95.8%),followed by GTW and Cuscuta flavonoids group(91.3%),GTW group was the lowest(90.0%),but there were no significant difference in each group(p＞0.05).The baby rats of GTW groups were not found the malformations of appearance and genitals and GTW had no effect on the 3-week survival rate of baby rats without significant difference(p＞0.05) in each group.At 2nd cycle control group pregnancy rate was 95.8%,and GTW groups were 100.0%.It can be seen that the damageto the reproduction caused by GTW had the reversibility.The3-week survival rate of baby rats were Cuscuta flavonoids group(97.9%), Liuweidihuangwan group(97.4%),GTW group(97.0%) and control group(96.7%), without significant difference(p＞0.05) among all the groups.It should be noted that one rat of the control group whose weight was 483g in 19 weeks had not conceived in 2 cycles because it's an obese;Another rat at 1st cyle bored seven baby rats that all died and at 2nd cycle it did not conceive.So the pregnancy rate and survival rate of control group were effected.The 0-week mean weight of rats descended from GTW and Cuscuta flavonoids group, control group,GTW group to GTW and Liuweidihuangwan group.The 3-week mean weight of control group was the maximum and that of GTW group was the minimum,but there were no significant difference(p＞0.05) among the three groups.After 7 weeks withdrawal,ovary,thymus,adrenal gland and kidney org an coefficient means of female rats descended from GTW and Cuscuta flavonoids group,control group, GTW and Liuweidihuangwan group to GTW group.There were very significant differences in ovary,thymus and adrenal gland organ coefficient means among the groups(p＜0.05,p＜0.01,p＜0.01).The kidney organ coefficient mean of GTW group was the minimum,yet that of the other two groups were higher than that of the control group without significant difference(p＞0.05).The liver organ coefficient of GTW groups were higher than that of the control group, whicn showed Cuscuta flavonoids and Liuweidihuangwan could increase the liver weight of SD rat.Cuscuta flavonoids group was better than Liuweidihuangwan group,but there was no significant difference(p＞0.05) between of them.Average uterus organ coefficient of GTW andLiuweidihuangwan group were lower than that of the control group.It was considered that two medicines administered simultaneously worsened the damage of the uterus probably though changing pharmacokinetics and/or pharmacodynamics.Those of other two groups were higher than that of the control group.It was considered that the uterus has been natural recovery,so histological examination,the results of hormone levels should be in combination with for further judgments. Conclusions1 GTW has a certain effect on the granulosa cellsin vitro of the SD young rats,that is shown in Estradiol concentration in the supernatant being lowered,the ability of cell proliferation being decreased and the expression of Bcl-2 protein being inhibited.Cuscuta flavonoids and Liuweidihuangwan can regulate steroid hormone secretion of ovarian granulosa cells and promote the expression of Bcl-2 protein,thereby adjusting reproductive hormones of young rats which was imbalanced caused by GTW and reducing the apoptosis of GC.Cuscuta flavonoid is superior to Liuweidihuangwan.2 GTW has a certain effect on reproductive system and ovarian tissue of SD female rat,which is manifested in decreasing ovary organ coefficient and increasing the expression of prooapoptotic protein Bax gene that causes ovarian tissue pathology injury.Cuscuta flavonoids and Liuweidihuangwan have a certain prevention on reproductive damage caused by GTW.The possible mechanisms are preventing the side effects of GTW and protecting Ovarian through r egulating the epidermal growth factor of ovarian tissue,inhibiting the expression of Bax protein and restoring the normal ratio of Bcl-2/Bax.Cuscuta flavonoid is superior to Liuweidihuangwan.3 The reproductive damage of young female SD rats caused by GTW is reversible.It is manifested in seven weeks after drug withdrawal SD rat organ coefficient of uterus returning to normal,4 weeks after drug withdrawal the birth rate of each group to 100.0%.If GTW is applied to young rats before sexual maturity it is no obvious effect on conception,pregnancy, birth process and growth and development of generation rats.4 GTW has a certain effect on the thymus and adrenal gland.The Effect has hysteresis;it is manifested in seven weeks after drug withdrawal SD rat thymus and adrenal organ coefficient yet not returning to normal.For the effect of opposing the thymus and adrenal injury of female rats caused by GTW,Cuscuta flavonoid is superior to Liuweidihuangwan.5 It is significant that the effect of the Cuscuta flavonoids against the reproductive damage of SD rats caused by GTW in the vitro and vivo experiment.This function is worth studying further.