The Mechanism Research On Dabuyuanjian Decoction Affecting Rats Of Premature Ovarian Insufficiency Induced By VCD From MTOR Signal Pathway

Objective:With the changing of social structure and the concept of marriage and childbearing in China,more and more women delay their first pregnancy and childbirth,which leads to the increasingly serious problem of elderly childbearing.The incidence rate of premature ovarian insufficiency?POI?is increasing year by year as well as a young trend.Ovarian insufficiency may cause female fertility decline.Combined with the existing theoretical research,basic experiment and clinical research,Dabuyuanjian decoction can delay the aging.The purpose of this research is to establish a rat disease model of POI for further study,and then to use Dabuyuanjian decoction to intervene the animal model for observation about the effect of Dabuyuanjian decoction on the rat model of POI.In this study,positive control drugs estradiol valerate was selected.For the mechanism research on Dabuyuanjian decoction affecting rats of POI induced by VCD,the general condition?body shape,living habits,feeding and excretion?,weight change,estrus cycle,autonomic activity,weight and organ index of ovary and uterus,histopathology of ovary and uterus,serum sex hormone level,serum cytokine level and the expression status of key protein molecules of mTOR signal pathway such as PI3K,AKT,mTOR,PTEN were tested.Method:1.Methods of building disease model:two-months-old female SD rats with two normal estrous cycles were injected with Vinylcyclohexene dioxide?VCD??160mg/kg?intraperitoneally for 15 days.The rats in the blank group were intraperitoneally injected with equal volume of normal saline every day for same days.2.Administration method:from the 31st day after stopping the intraperitoneal injection of VCD,the control group was given estradiol valerate solution wihile the treatment group was given Dabuyuanjian Decoction with different concentration by the dosage shown in table 2.13,and the blank group and the model group were given distilled water of equal volume.The treatment lasted for 4 weeks.3.Method of specimen collection:the rats were fasted for 12 hours before the specimen collection.The rats were weighed before the anesthesia.Then the rats were anesthetized with 0.5ml/100g 4%chloral hydrate solution by intraperitoneal injection.The blood of abdominal aorta?at least 10ml of each rats?,uterus and both sides of ovarians were taken.Serum of abdominal aortic blood which was collected by putting the abdominal aorta blood into centrifuge at 4?3000r/min was stored in refrigerator at-80?.After weighing,one side of ovarian and uteru of each rats were put into 4%formaldehyde solution for making paraffin sections for microscopy observation,while the other side of ovarian was put into liquid nitrogen cryopreservation for the detection of relevant indexes.4.The levels of serum sex hormones?estradiol,follicle stimulating hormone,luteinizing hormone?and cytokines?anti Mullerian hormone,serum inhibin B?were detected by Enzyme-Linked Immunosorbent Assay?ELISA?.The relative expression quantity of PI3K mRNA,AKTmRNA,mTORmRNA and PTEN mRNA in ovarian tissues of rats in each group were detected by Real time PCR.The relative protein expression quantity of p-PI3K,p-AKT,p-mTOR and PTEN in ovarian tissues of rats in each group were detected by Western Blot.The expression of Bcl-2,Bax and caspase-3 protein in ovarian tissues of rats in each group were detected by immunohistochemistry.Result:1.General situation:the blank group rats were normal in appearance,physical signs,behavior,diet and defecation condition.In the model group,the fur of the rats was thinner and paler than healthy rats,and there was more fat which was distributed in abdomen and buttocks.The model rats'activities became weaker and some of them liked to huddle or push each other.A few rats were restless,aggressive and easily provoked.Most of the model rats ate less.The activities of daytime were increased while decreased at night.Those model rats had more urine and their stool was thinner and softer.In the control group and treatment group,the abnormal symptoms above were improved in different degrees.2.Weight change:compared with the blank group,the rate of weight gain of the model group increased rapidly?P<0.01?.Compared with the model group,the growth rate of weight in the control group and each treatment group of TCM was slower?P<0.01?.3.Estrus cycle:compared with the blank group,the duration of estrus cycle of the model group was prolonged?P<0.01?.Compared with the model group,the rats in the control group and each treatment group of TCM had shorter estrus cycle time?P<0.01?.4.Spontaneous activity:compared with the blank group,the total distance of activity in the model group was shorter?P<0.05?,the average speed was slower?P<0.05?,the rest time was longer?P<0.01?,and the central activity time was shorter?P<0.01?.Compared with the model group,the rest time of rats in the control group was shorter?P<0.01?,the central activity time was longer?P<0.01?,the total distance and average speed of activity had no statistical difference?P?0.05?;the total distance and average speed of activity of rats in the high dose group and the middle dose group of TCM were longer?P<0.05?,the average speed was faster?P<0.05?,the rest time was shorter?P<0.05?,and the central activity time was longer?P<0.05?In the low dose group of TCM,the total distance of activity was prolonged?P<0.05?,the rest time was shortened?P<0.01?,the central activity time was prolonged?P<0.05?,and the average speed of activity was not statistically different?P?0.05?.5.Ovary weight and organ index:compared with the blank group,the ovary weight and organ index of the model group were lower?P<0.01?.Compared with the model group,the ovary weight and organ index in the control group,the high dose group and the middle dose group of TCM were heavier?P<0.01?and higher?P<0.05?,while in the low dose group were not statistically different?P?0.05?.6.Uterus weight and organ index:compared with the blank group,the uterus weight and organ index of the model group were lower?P<0.05?.Compared with the model group,the uterus weight and organ index in the control group,the high dose group and the middle dose group of TCM were heavier?P<0.05?and higher?P<0.01?,while in the low dose group were not statistically different?P?0.05?.7.Histomorphology of ovary:in the blank group,the structure of cortex and medulla in the ovarian tissue was complete,and the developed follicles,mature follicles and luteal tissue could be seen.There were abundant blood vessels,in ovary and there was no lymphocyte infiltration and fibrosis in the intercellular substance.In the model group,the ovarian tissue structure was disordered,the number of developing follicles at different levels was reduced,the number of primordial follicles and primary follicles was significantly reduced,and there were more atretic follicles.The arrangement of granulosa cells in follicles was loose,and the number of granulosa cells was reduced while no luteal tissue was found in the tissues.Compared with the model group,ovary of the rats in the control group and the TCM groups has more follicles at different levels,the mature follicles and granulosa cells in the follicles were more than those in the model group,which were arranged in a radial and orderly manner.The change of follicles'number in the high dose group and the middle dose group of TCM were more obvious,there were still undeveloped follicles in the ovarian tissues in each group,and the luteal tissues could be seen in the ovarian tissues of each treatment group.8.Histomorphology of uterine:the uterine myometrium and endometrium of the blank group were thick,with a large number of glands.Some of the glands transformed into sacs,and the epithelial cells became double layer.The cells were columnar and without heteromorphism.The connective tissue was loose in uterine.In the model group,the uterine myometrium and endometrium became thinner,the number of glands decreased,the structure was disordered and irregular,the arrangement of epithelial cells was disordered,and the nuclear staining was deep.Compared with the model group,the endometrium of the control group and each treatment group of TCM was thickened,the glands and blood vessels were increased,the epithelial cells were arranged regularly,the morphology tended to be normal,and the connective tissue was loose.9.Serum sex hormone level:compared with the blank group,the rats in the model group had lower serum E₂ level?P<0.01?and higher serum FSH,LH level?P<0.01?.Compared with the model group,higher serum E₂ level and lower serum FSH,LH level was found in the control group and each treatment group of TCM?P<0.01?.10.Cytokine level:compared with the blank group,AMH and INHB decreased in the model group?P<0.01?.Compared with the model group,AMH and INHB in the control group,high dose group,middle dose group of TCM while low dose group increased significantly.11.The relative expression quantity of PI3KmRNA,AKTmRNA,mTORmRNA and PTENmRNA in ovarian of rats:compared with the blank group,the expression of PI3KmRNA,AktmRNA and mTORmRNA in model group was increased?P<0.05?,and the expression of PTEN mRNA was decreased?P<0.05?.Compared with the model group,the expression of PI3KmRNA,AKTmRNA,mTOR mRNA was increased?P<0.05?and PTENmRNA was decreased?P<0.05?in the control group and each treatment group of TCM.12.The relative protein expression quantity of p-PI3K,p-AKT,p-mTOR and PTEN in ovarian tissues of rats:compared with the blank group,the phosphorylation of PI3K,Akt and mTOR in model group decreased?P<0.01?,and the expression of PTEN increased?P<0.01?.Compared with the model group,the phosphorylation of PI3K,Akt and mTOR in the control group,high dose group and middle dose group of TCM increased?P<0.05?,while the expression of PTEN decreased?P<0.05?;there was no significant difference in the phosphorylation of PI3K,mTOR and PTEN expression in the low dose group of TCM?P?0.05?,but the phosphorylation of Akt increased?P<0.01?.13.The positive expression of Bcl-2,Bax and caspase-3 in ovarian tissues of rats:?1?compared with the blank group,the level of Bcl-2 IOD in the model group was lower?P<0.01?and Bax IOD was higher?P<0.01?.Compared with the model group,the rats in the control group,the high dose group and the middle dose group of TCM had higher Bcl-2 IOD?P<0.01?,lower Bax IOD?P<0.01?;the rats in the low-dose group had no statistical difference in Bcl-2?P?0.05?,but lower Bax IOD?P<0.01?.?2?Compared with the blank group,caspase-3 IOD in the model group was higher?P<0.01?.Compared with the model group,the caspase-3 IOD in the control group and each treatment group of TCM was lower?P<0.05?.Conclusion:1.Vinylcyclohexene dioxide can be used to establish the rat model of POI.The disease model has similar pathological characteristics with that of POI.It can be used as an ideal animal model for the further study of POI.2.With the treatment of Dabuyuanjian Decoction,the model rats had similar therapeutic effect with estradiol valerate in the aspects of behavior,histology,sex hormone and cytokine.It is speculated that Dabuyuanjian Decoction has estrogen like effect.3.Dabuyuanjian Decoction has a positive regulatory effect on mTOR signal pathway,at the same time,it can enhance the expression of Bcl-2 and inhibit the expression Bax and Caspase-3.Due to these functions,Dabuyuanjian Decoction can improve ovarian function by protecting the growthing of follicles from apoptosis of granulosa cell.This may be one of the molecular mechanisms of Dabuyuanjian Decoction in the treatment of POI.