Study On The Synergic Compatibility Law Of Sanleng-Ezhu Based On Qi Stagnation And Blood Stasis Syndrome Of Uterine Leiomyoma In Rats

Objective:Under the guidance of the basic theory of TCM,the model of uterine leiomyoma integrated with Qi stagnation and blood stasis was established by using the estrogen and progesterone load method combined with multifactor stimulation.By observing the morphology and pathology of the uterus,and analyzing estrogen,progesterone,estrogen receptor,progesterone receptor,fibroblast activation protein expression in the uterus,ovarian tissue and serum,we can explore the effect and Synergic Compatibility Law of Rhizoma Sparganic and Rhizoma Curcumae component compatibility on Uterine Leiomyoma in rats,basing on innovative drug discovery patterns‘prescription-disease-syndrome,Chinese Herbs-disease-syndrome,effect components-disease-syndrome,effective ingredients-diease-syndrome’.And the result will provide new methods and new ideas for the scientific research and prescription compatibility of the uterine fibroids,as well as the scientific basis for clinical application.Method:1.Group,modeling and medication.A total of 88 healthy Sprague Dawley rats were randomly divided into 11 groups:normal control group,model group,Gongliuxiao group,S-E component compatibility（1:1）high dose group,S-E component compatibility（1:1）middle dose group,S-E component compatibility（1:1）low dose group,S-E component compatibility（1:2）group,S-E component compatibility（2:1）group,S group,E group,S-E decoction group.Except for normal control group,the daily gavage of each group was 1.35mg/kg,and the lateral muscle injection of progesterone injection was 1.0mg,lasting for 5 weeks.Subcutaneous injection of adrenaline hydrochloride 0.9 mg/kg·d^-1 wad started from the fourth week,lasting for2 weeks.And giving chronic unpredictability stimulus four hours later:（1）60 db noise stimulation for 3 h,（2）day and night upside down,3.5¹0℃ice water bath swimming 4 min,（4）tail suspension 10 min,5 to 50℃oven to bake for 10 min,giving 1 kind of stimulation each day,and to ensure that each stimulus is not less than2 times during entire cycle),for 2 weeks.The normal control group was given a daily amount of distilled water 2ml.and every Monday,Wednesday,Friday,was injected by normal saline 0.05ml,for 5 weeks.Medicines were given in 2 hours after molding.Respectively are normal saline,9%of gongliuxiao capsule solution,66.7%,33.3%,16.7%of S-E（1:1）component compatibility group solution,100%of the S-E（1:2）component compatibility group solution,100%of S-E（2:1）component compatibility groups,66.7%of S group,66.7%of E group,and S-E decoction group,for 5 weeks.2.Observation and detection of all the indexes in rats.（1）General behavioral observation of rats.We observed the behaviors,appearances,diet and sleep quality three times a day（7am,12noon and 6pm）,and took notes.（2）Uterine morphology and histopathology.The rats were fasting for 12h after the last administration,and then we took out the tissues of the uterus,removed the fat,cleared the blood,and took photos to record the shape of the uterus.We selected uterus tissues which had obvious lesions（edema,nodules,cysts,vacuolar degeneration,etc.）in the uterus dividing angle.（3）Measurement of uterus,ovary coefficient and uterine transverse diameter.We took out the tissues of the uterus,removed the fat,cleared the blood,and weigh the wet weigh.And then we calculate uterus and ovary coefficient.（4）Detection of various indexes in rat serum.We collected blood from rat femoral artery,and collected serum after 3000r/min,centrifugation for 20min.The contents of E₂,ER,P and PR in serum were determined by enzyme-linked immunoassay kit as soon as possible.（5）Detection of indicators in ovary and uterine tissues.We cut the right amount of tissue,weigh it and put it in the homogenate tube;the homogenizer was to2000r/min,1min.serum should be collected carefully.The contents of E₂,ER,P and PR in the uterus and ovaries were detected by enzymo-elisa kit.The FAP content in uterine tissues of rats was determined by the Western Blot.Results:1.The effect of S-E decoction and its effective ingredients on the appearance,morphology and histopathology of rats basing on‘prescription-disease-sydrome and effective ingredients-disease-sydrom’Compared with the normal control group,the appearance of the rats in the other groups presented changes such as dark hair color,messy fur,peeling and unstable hair roots.Compared with normal control group,model group rats with bilateral uterus shape change,from a cyst,nodule,edema,bilateral uterine pathological changes such as size,shape,asymmetric,other groups in the rat uterus appeared different degree of edema,nodules,cysts,unequal texture pathological changes.Compared with the normal control group,the results of pathological sections of rats in other groups showed pathological manifestations such as disordered muscle fiber arrangement,thickening,myocyte hyperplasia and inflammatory cell infiltration.The appearance and uterine pathological changes of rats in the S-E decoction and S-E（1:1）high dose group were less than those in other groups.2.The effect of S-E decoction and its effective ingredients on uterus,ovary coefficient and uterine transverse diameter basing on‘prescription-disease-sydrome and effective ingredients-disease-sydrom’Compared with the normal control group,the uterus,ovary coefficient and vertical diameter of uterus were all increased in the model group.Compared with model group,the treatment group coefficient of uterus,ovary and uterus of rats bearing diameter reduced to some extent,the S-E decoction and the S-E（1:1）high dose group could reduce coefficient of of rats uterus,ovaries and uterus bearing diameter obviously.3.The effect of S-E decoction and its effective ingredients on the content of E₂,ER,P and PR in rat serum basing on‘prescription-disease-sydrome and effective ingredients-disease-sydrom’Compared with the normal control group,the content of E2,ER,P and PR in serum of the model group increased significantly.Compared with model group,the content of E2,ER,P,PR content decreases in the other rats’serum.The S-E decoction group rats serum E2,ER,P,PR content reduced obviously.The content of E2,P and PR in serum of rats in the S-E（1:1）high dose group was significantly decreased and the content of ER was also decreased.4.The effect of S-E decoction and its effective ingredients on the content of E₂,ER,P and PR in rat ovary basing on‘prescription-disease-sydrome and effective ingredients-disease-sydrom’Compared with the normal control group,the content of E2,ER,P and PR in the ovarian tissue of the model group increased significantly.Compared with model group,of the content of E2,ER,P,PR in other groups are reduced,the S-E decoction group can obviously inhibit ovarian tissue expression of ER and P,S-E（1:1）high dose group of ER in rat ovarian tissue,P,PR content reduce obviously,E2 levels are reduced.The results were better than those of other groups5.The effect of S-E decoction and its effective ingredients on the content of E₂,ER,P,PR and FAP in rat uterus basing on‘prescription-disease-sydrome and effective ingredients-disease-sydrom’Compared with the normal control group,the expression of E2,ER,P,PR and FAP in the uterine tissue of rats in the model group was significantly increased.Compared with model group,the expression of ER and P,PR,FAP were reduced in other groups,the S-E decoction group can obviously inhibit the expression of ER,P,PR,FAP,S-E（1:1）high dose group of uterus tissue E2,ER,P,PR,FAP were decreased obviously,and other drug effect is good.Conclusion:The above results indicated that S-E decoction could improve appearance,uterine morphology and pathologic change,and inhibited the expression of E₂,ER,P,PR and FAP in serum,uterus and ovary.The effect of other groups could act as S-E decoction;however,S-E component compatibility（1:1）high dose group had a better inhibitory effect on uterine fibroids.Its mechanism may be the inhibition effect of S-E component compatibility on E₂,ER,P,PR and FAP simultaneously.In addition,results also indicated the growth of estrogen in the uterine tissues of rats was positively correlated with the growth of fibroblast activated protein,and the high expression of fibroblast activated protein may be caused by the stimulation of oestrogen.