The Pharmacodynamic Action On EMS And Toxicological Pathology Of "San Leng Pill"

Background: SLW is a traditional Chinese medicine compound which had evidenced to reach good therapeutic effect on endometriosis by our team. Our preliminary works carried out were the pharmacodynamic tests of the preparation on rat endometriosis. To develop the SLW to be a new drug as a Chinese traditional medicine, this article observed toxicological effects of SLW, and carried out further pharmacodynamic tests and its relevant mechanisms research.Objective:1. ToxicologyTo observe the toxicity of SLW in mice by one-time intragastric administration, reached the half lethal dose in mice (LD50) and its 95% onfident limit with probability; To detect animal hematology target, blood biochemistry target and organ coefficient and the effect on organ coefficient in the female rats in the 6 month course of long-term toxicity of SLW, and to find target organ toxicity of drugs and the organ's pathology changes.2. Pharmacodynamics and Mechanism of the drugTo investigate the mechanism of the reduce of the blood levels of estrogen by researching the impact to the rat's pituitary- ovary in the use of SLW. To observe the improved effects of SLW on morphological changes and the expression of ectopic endometrium adhesion molecule ICAM-1 and CD44.Methods:1. Acute toxicity test in miceWe determined the mice D0% (the dose with no dead animal) and D100% (the dose with all animals died) after administration by the modified Karber's method, which are used as the minimum dose and maximum dose respectively in the formal test. To convert the highest, lowest dose to common logarithms, and then figure out the positive difference between the common logarithms of highest and lowest dose. According to the number, the animals are randomly divide into eight group on the number of equidistant dose logarithms, 10 mice in each group, male and female half. After administration, at least two weeks to observe the mice nervous system, respiratory system, digestive system, reproductive system, skin, eyes, body weight, apastia or not and etc. every day, and have a detailed records of the time when the emergence and disappearance of all kinds of toxicity. All animals, including the dead because of SLW or not, should held autopsy to find abnormal organ, which should be fixed by 10% neutral formaldehyde for histological examination.2. Long term toxicity test in ratsThe 160 medical fitness rats were randomly assigned into four groups: the solvent control group and three administration groups which were given respectively distilled water, high-dose of SLW (1000mg/kg), moderate-dose of SLW (500mg/kg), low-dose of SLW (250mg/kg) for every 6 days a week continue 6 months. The rats form the solvent control group and three administration groups were killed in the end of 3 months and 6 months in the last 24 hours after the last administration to detection the examinations, the number of these groups of animals killed were 10 and 14 respectively. The rest of rats were observed the reversible toxicity and delayed toxicity in the next 30 days to recovery without administration, in the end of the 30th day kill all the rest rats to detection the examinations. The examinations include: the general target such as: the appearance of signs, behavioral activity, gland secretion, breath, feces, food intake and so on add target of hematology, blood biochemical parameters, the system autopsy and histopathological examination, organ weight and organ coefficient weight determination, estrogen and progesterone tests.3. The function of SLW in protein expression of estrogen receptor (ERα) in pituitary and ovarian in rats.72 female rats were randomly divided into four groups as follow: solvent control group, SLW 250mg/kg group, SLW 500mg/kg group, SLW 1000mg/kg group, 18 rats for each group, and were taken intragastric administration once a day for 6 monthes . The rats were killed in 3 bitches the number of each time is 6 rats. The time point is after administration 3monthes,6 monthes and the 1 month after the last administration. The rats'blood estrogen (E2) and progesterone (P) level were determined, and their pituitary and ovarian were cut off and conventional paraffin-embedded to explore the expression changes of ERαprotein in each group were observed by immunohistochemistry.4. Experiment of pharmacodynamics-the effect of SLW on volume of ectopic endometrial tissues and adhesion molecules expression in ectopic endometrium of rats.Built a model of endometriosis of rat through autologous transplantation, and ensured the successful modeling by observing the appearance, size and pathological examination of graft. In addition to the normal control group, the successful modeling rats were randomly divided into five group, such as SLW 80mg/kg/d group, 240mg/kg/d group, 240mg/kg/d group , 200mg/kg/dGestrinone group, model control group, the delivery methods was oral administration once a day for 4 weeks. 4 weeks later we ended the treatment, observed those rats'vaginal smear cytology, and then killed the rats, cut open their abdomen to observe the growth of ectopic endometrium. Their tissues of uterines and ectopic endometriums were cut off and sent to take a pathological morphology examination by HE staining, and detect the expression of adhesion molecules such as ICAM-1 and CD44 by immunohistochemical method.Results:1. Acute toxicity test in miceThe minimum dose and maximum dose of SLW for acute toxicity test in mice by intragastric administration is 2600mg/kg and 9800mg/kg, when the dose reaches to 2400mg/kg no animal die, and the medial lethal dose is 5414.58mg/kg, which equal to 22.56 times the amount of clinical daily usage, its 95% confident limit with probability is 5292.34~5536.82 mg/kg. These numerical value convert to human's were as follow: 303.60mg/kg,1239.7mg/kg,694.84mg/kg, respectively. The signs of toxicity were as follow: hypokinesia, depressed, apastia, climb cage, jump, oligopnea, dyspnea, Cheyne-Stokes respiration, ptosis, leg convulsion. Autopsy death mice, the main organs had unobviously pathological changes by both eyes observation and microscope except minimal lesion disease of mice's liver. Its tell us SLW's toxicity is little.2. Long term toxicity test in ratsAfter continuous administration of SLW for 3 months and 6 months, we detected hematology,blood biochemical parameters, organ weight organ coefficient weight determination, and compared these indicators with the ones of those rats with drug withdraw for a month. We found the SLW have not significantly affected on the general behavior of rats indicators, the general physiological indicators such as indicators of the blood, organs and organ coefficient of pathological examination. The toxicity of SLW was to reduce the number of reticulocyte and estrogen, and to elongate prothrombin time.3. The impact of SLW on hormone levels in rats.We determined the levels of blood estrogen (E2) and progesterone (P) at the time of the drug administration for 3 months, 6 months and drug withdrawal for 1 month respectively. And we found the levels of blood estrogen (E2) in the groups of SLW 500mg/kg and 1000mg/kg were much higher than the normal control group's(the P<0.05), while the levels of blood progesterone (P) in the groups of SLW 500mg/kg and 1000mg/kg were much lower than the normal control group's(the P<0.05). The positive cells of ERα, which were brown when they were total-cell-stained, were expressed in basophilic granulocyte of hypophysis, follicular cell and lutein cell of ovaries of rats. In the group of SLW 250mg/kg,500mg/kg and 1000mg/kg, the number of ERα, positive cells decreased markedly and the positive color of brown was lighten in hypophysis and ovaries of rats, and this effect can not be recoveried after stopping administration.4. The effect of SLW on adhesion molecules expression in ectopic endometrium of rats:SLW 240mg/kg,720mg/kg markedly improve the morphological characters and decreased the expression of ICAM-1 and CD44 in ectopic endometrial tissue.Conclusion:1. The medial lethal dose is 5414.58mg/kg in mice by one-time intragastric administration, which equal to 22.56 times the amount of rats'pharmacodynamics usage. Administration SLW for 180 days continuity, combinate with weight, the blood cells number value and the biochemistry number value of blood, relative organ weight and result of pathobiology , we find that SLW has obsolete effect of the indexes ,have no target of organs. It was demonstrated that SLW was a security traditional Chinese medicine compounds with low toxicity.2. SLW can significantly reduce the level of estrogen in blood, while significantly increas the level of progesterone, and these effects are relevant with the inhibition of SLW for bobbin of hypothalamus-pituitary- gonad.3. SLW can lessen the pathological changes of ectopic endometrium in the endometriosis rat model, and its mechanism might be associated with inhibiting the expression of ICAM-1 and CD44 protein.