Study On The Inhibitory Effect Of Anti-tumor Proved Recipe Of Dai Medicine On Mouse Hysteromyoma

Objective:(1)Finding new drugs from ethnic drugs is recognized as an important way in the world.Dai drug has abundant and underexploited resources that can be utilized for preventing and curing incurable diseases and the anti-tumor proved recipe of Dai medicine is one of them.This project uses the method of modern pharmacological experiments to verify the feasibility and effectivity of this proved recipe and it can promote the development of new drugs.(2)Uterine leiomyoma is smooth muscle neoplasm caused by cell mutation and gene stimulation.The morbidity of uterine fibroids is about 70% among women of reproductive age.Moreover,uterine leiomyoma seriously impacts women's physical health and fertility.This proved recipe is an effective prescription for curing tumor and lump.It has been found that this proved recipe has a good effect on clinic,but so far it hasn't been reported.Therefore,this study uses mouse model of uterine fibroids and chooses indices which are uterine pathomorphology,estradiol,progestone,tumor necrosis factor-?,interleukin-2,the activity of nitric oxide synthase and the expression of estrogen receptor and progesterone receptor to study on the mechanism of this proved recipe.Methods:(1)Preparation of the test sampleUse the methods of ethanol extraction,pure water extraction and respective concentration and then combine concentrated liquor to make the experimental drug.(2)The screening and replication of mouse model of uterine fibroidsUse the method of loading the mouse with estrogen to make model.(3)The effect of folk remedies on uterine leiomyoma miceMake model,at the same time,and each group take intragastric administration once everyday.Measure the uterine pathomorphology and hormone level changes.(4)The effect of the original formula on pathological changes of uterus miceUse the method of hematoxylin-eosin staining to observe the pathological changes of the uterus and measure the thickness of the uterine muscle wall.(5)The effect of the original formula on estradiol,progestone,tumor necrosis factor-? and interleukin-2 in blood serum.Use the method of competitive ELISA to measure levels of estradiol and progestone in blood serum and double-antibody sandwich ELISA to measure levels of tumor necrosis factor-? and interleukin-2 in blood serum.(6)The effect of the original formula on nitric oxide synthase(NOS)activity in uterine leiomyoma miceUse the method of chemical colorimetry to measure the activity of NOS in mouse uterine tissue.(7)The effect of the original formula on expression of estrogen receptor(ER)and progesterone receptor(PR)in mouse uterine tissueUse the method of immunohistochemical technique to observe the positive expression of ER and PR in mouse uterine tissue and motic digital medical image analysis system to determine the positive target area.Result:1.In this experiment,there were two folk remedies from the Dai nation,including anti-tumor proved recipe of Dai drug(hereinafter referred to as original formula group)and anti-tumor proved recipe of cockroach(hereinafter referred to as cockroach group).Since the original formula group included seaweed and licorice and this combination is against eighteen incompatible medicaments,so added two experimental groups.One of the groups retained seaweed and got rid of licorice(hereinafter referred to as removing licorice group)and the other retained licorice and got rid of seaweed(hereinafter referred to as removing seaweed group).There were altogether four groups.The concentration of each group was 1.26 g crude drug/mL.The dosage of folk remedies suspension was 12.64 g crude drug/kg.After screening experiment,the original formula group suspension(high dose 25.45 g crude drug/kg,medium dose 12.64 g crude drug/kg,low dose 4.21 g crude drug/kg)was obtained.2.The model was successfully established.Normal mouse uterus was Y-shaped and threadlike.It had no swelling and nodule phenomenon.The texture was uniform and smooth.Its color was pale pink.In the model group,the uterine color of the mouse was sombre,the texture was irregular and some uteri showed abnormality and edema.Its pathological sections showed a local pharmaceutical hyperplasia in uterine smooth muscle.The cell nucleus was hyperchromatic and the shape of cell was oval.Endometrial stroma showed infiltration of the inflammatory cells and the thickness of membrane was thicker.Compared with the normal group,the uterine coefficient and the uterine wet weight of the model group were increased(P<0.01,P<0.01).3.The original formula group could improve the pathomorphologic of uterine leiomyoma mice and significantly reduce the level of E2 and P in the serum(P<0.01,P<0.01).The cockroach group and the original formula group had a similar inhibition effect to uterus mice,but the cockroach group didn't improve the situation of uterus edema.The inhibitory effect of the removing licorice group and the removing seaweed group was not obvious.4.All groups of original formula could improve the pathomorphologic of uterus mice.Under light microscope,the area of local pharmaceutical hyperplasia in uterine smooth muscle was obviously reduced.Most nucleus were spindle or rod.The muscle cells arranged regularly.The original formula could reduce the uterine coefficient,uterine wet weight and endometrial thickness with a significant difference(P <0.01).5.?The level of estrogen in model group was significantly higher than normal group(P<0.01).The level of estrogen in positive drug control group and other treatment groups was lower than model group.The difference between the two groups was statistically significant(P <0.01).?The level of progesterone in model group was significantly higher than normal group(P<0.01).The level of progesterone in positive drug control group,low-dose group and middle-dose group was lower than model group(P<0.01).The high-dose group showed statistically significant reduction(P<0.05).?The level of TNF-? in model group was significantly higher than normal group(P<0.05).The level of TNF-? in high-dose group and middle-dose group was lower than model group(P<0.05).Positive drug control group and low-dose group were statistically significant(P<0.01).?The level of IL-2 in model group was decreased significantly.Except for the high dose group,the level of IL-2 in other treatment groups was increased significantly(P<0.05).6.The activity of NOS in model group was significantly increased(P<0.01).The activity of NOS was significantly decreased in other treatment groups(P<0.01).7.The original group could decrease the expression of ER and PR.Conclusions:This study established an animal model of experimental uterine myoma successfully.After screening experiment,this result showed that the original formula group had better effect than others.As this original formula group was thoroughly researched,the result showed that this original formula group had an obvious effect of inhibiting hyperlasia of uterus smooth muscle,the mechanism of original formula group was related to following aspects including reducing the level of TNF-?,the activity of NOS,the level of hormone and the expression of corresponding receptor and increasing the level of IL-2 in the serum.