Study On The Effect Of Qilin Pill On Ovarian Dysfunction Mice And The Mechanism Of Its Main Component Icariin In Ovarian Dysfunction

Objective: 1.Establish a mouse model of ovarian hypofunction(kidney deficiency syndrome)induced by CTX.2.Evaluate the pharmacodynamic effect of Qilin-Pill on CTX-induced ovarian dysfunction(kidney deficiency syndrome)mouse model.3.To explore the action concentration and time of CTX and icariin on human ovarian granulosa cells(KGN).4.Explore the molecular mechanism of icariin,one of the main components of Qilin Pill,on CTX-induced ovarian hypofunction model of KGN cells.Methods: 1.Animal experiments(1)to establish a Mouse model of CTX-induced ovarian dysfunction(kidney deficiency syndrome): 20 C57BL/6 female mice were divided into 2 groups on average by random number table method,normal control group and CTX modeling group,CTX modeling group was injected intraperitoneally,and normal control group was injected with saline of the same volume,a total of 15 days.During this period,the general condition,hair,weight,activity and estrous cycle of the mice were observed.After the mice were killed,organs such as uterus were taken for weighing and indexes of each organ were calculated.The organs such as uterus and ovary were taken for Hematoxylin-Eosin(HE)staining to observe their tissue structure characteristics.The contents of AMH,FSH,Prog,LH and E2 in serum were detected by ELISA.2.Animal experiments(2)the Qi Lin-pill on the ovarian function impairment induced by CTX(kidney deficiency)the efficacy of mouse model to explore: confirmed after the success of the building,then 60 C57 BL / 6 female mice by random number table method divided into 6 groups,normal control group,positive control group,CTX made module,Qi Lin-pill low,medium and high dose groups,There are 10 mice of each group.The other groups except the normal control group were treated with CTX intra-abdominal injection for 15 days.Then the positive control group was given Qi Lin-pill for 30 days,and the low,medium and high dose Qi Lin-pill for 30 days.The general condition,coat,body weight,activity and estrous cycle of mice were observed.After 45 days,all the anatomical materials were collected and the indexes of each organ were calculated.The uterus and ovaries were taken for HE staining to observe their tissue structure characteristics.Immunohistochemical(IHC)staining was used to detect the expression of estrogen receptor(ER),follicle-stimulating hormone receptor(FSHR),luteinizing hormone receptor(LHR)and progesterone receptor(Pg R)proteins in the ovaries and uterus of mice.Serum hormones AMH,FSH,Prog,LH and E2 were detected by ELISA.Western Blot was used to detect the protein expressions of ER,FSHR,LHR and Pg R in ovary and uterine tissues of mice.The m RNA expression levels of ER,FSHR,Pg R and LHR genes in ovary and uterine tissues of mice were detected by fluorescence quantitative PCR(SYBR Green method).3.Cell experiment(1)explore the CTX and Epimedium glycoside in KGN cell concentration and time: with KGN cells as the research object,its culture,batches,frozen storage,recovery,through the cell proliferation/toxicity experiment,the experimental cell apoptosis,cytokine secretion changes in selecting rational drug concentration and action time,the different concentration of Epimedium glycoside CCK8 testing or CTX under different time length of KGN cell proliferation,flow cytometry technique to detect KGN cell apoptosis,ELISA to detect cell supernatant of secretory expression of TGF-β and E2-β.4.Cell experiment(2)to explore the molecular mechanism of icariin’s effect on CTX-induced ovarian hypofunction model of KGN cells: The activator/blocker of SMAD /TGF-β pathway(recombinant protein TGF-β /IDT-1)was used to study the regulatory effects of icariin /CTX on KGN cell apoptosis,PI3K/AKT pathway and SMAD /TGF-β pathway under the effect of TGF-β /IDT-1.Flow cytometry was used to detect the apoptotic changes of KGN cells under the interaction of TGF-β,CTX and IDT-1.Western Blot was used to detect the expression changes of PI3 K,AKT,TGF-β and Smad1 under the interaction of CTX and IDT-1.The secretion of TGF-β and E2-β in the supernatant of KGN cells was detected by Elisa after being stimulated together with CTX.Results: 1.Animal experiments(1)Establishment of CTX-induced ovarian hypofunction(kidney deficiency syndrome)mouse model.1.1 Compared with normal control group,CTX made module mice general condition is poor,easy to appear the phenomenon such as irritability,anger,be Mao Pengsong lacklustre,late appear even depilation,and daily activity were significantly decreased,smear vaginal exfoliated cells show estrus cycle of mice is disorder,characterized by estrogen,extended,no estrus,etc.,between the late building appear even less vaginal epithelial cells in mice,cell volume small,middle and bottom cells increased,surface mature Angle of the epithelium decreased,no emotional cycle.1.2.Compared with the normal control group,the uterine weight and uterine coefficient of CTX model group were reduced(P < 0.05),and the differences were statistically significant.The gross and microscopic appearance of the uterus and ovary were obviously atrophic,the uterus was generally elongated and the diameter of the tube became smaller.The endometrial glands were short and straight.The epithelial cells of the glands were flat and secretory.The endometrial stromal cells were small,generally fusiform,with clear boundaries and few inflammatory response cells.The ovary presented polycystic development,follicular atreia,decreased number of follicles at all levels,increased number of follicles with cystic expansion,decreased number of granular layer cells,decreased levels,loss of oocytes or radiocarcinoma in follicles,decreased lutein number and incomplete luteinization,ovarian stromal hyperplasia and fibrosis.1.3 Compared with the normal control group,AMH and E2 in CTX model group were decreased(P<0.05)and FSH was increased(P<0.05),with statistically significant differences.2.Animal experiments(2)Effect of Qilin-pill on CTX-induced ovarian dysfunction(kidney deficiency syndrome)in mice.2.1 Compared with the normal control group,the mice in CTX model group were the same as those in animal experiment.2.2 Compared with the CTX model group,the mice in the low,medium and high dose Qilin-pill group generally improved in the later stage of administration,and the irritability and irritability were alleviated,the hair was neat and shiny,and the daily activity was increased.The vaginal smear showed that the estrous cycle of the mice returned to normal.2.3 Compared with the CTX model group,the uterine weight and uterine coefficient of mice in the positive control group and the low,medium and high dose Qilin-pill groups were increased,and the differences were statistically significant(P < 0.05).2.4 Compared with the CTX model group,the gross and endoscopic morphology of the uterus and ovaries of the mice in the low,medium and high dose Qilin-pill group were normal,the gross morphology of the uterus was shorter and thicker,and the diameter of the tube was larger.Under the microscope,the uterine wall was thickened,the number of endometrial glands increased,the glands were long and curved,the epithelial cells of the glands were cubic or columnar and secreted a lot,the endometrial stromal cells were large,oval,spindle or irregular,the boundary was stained clearly,and there were more inflammatory cells in them.The number of follicles at all levels increased,the number of follicles at all levels decreased,the number of granulosa layer cells increased,oocytes in follicles were often seen,and normal luteal formation was observed.2.5 Compared with the CTX model group,AMH and E2 of mice in the low,medium and high dose Qilin-pill groups were all increased(P<0.05),and the differences were statistically significant.The decrease of FSH in low,medium and high dose Qilin-pill group was statistically significant(P<0.05).2.6 Compared with the CTX model group,the immunohistochemistry and WB of mice in the low,medium and high dose Qilin-pill groups showed increased ER and Pg R proteins(P<0.05),and the difference was statistically significant.The m RNA levels of ER and Pg R in the uterus and ovary of mice were increased(P<0.05),and the difference was statistically significant.The m RNA levels of FSHR and LHR decreased(P<0.05),and the difference was statistically significant.2.7Compared with normal control group,Qilin-pill low,medium and high dose group mice normally,vaginal exfoliated cells smear,sex hormone tests have no difference,body weight,uterus,ovaries,spleen,kidney,pituitary,hind PAWS are no difference,the weight of the histological appearance indifference,normal mice ovary and uterus tissue protein expression,ER of FSHR and LHR,Pg R,no difference expression of ER,FSHR,Pg R,LHR gene m RNA level no difference. 2.8Compared with positive control group,Qilin-pill low,medium and high dose group mice normally,vaginal exfoliated cells smear,sex hormone tests have no difference,body weight,uterus,ovaries,spleen,kidney,pituitary,the weight of the hind PAWS are no difference,histological appearance indifference,mice ovary and uterus tissue protein expression,ER of FSHR and LHR,Pg R,no difference expression,ER of FSHR and LHR,Pg R gene m RNA level no difference.3.Cell experiment(1)Explore the concentration and time results of CTX and icariin on KGN cells.3.1 When CTX was higher than 10μM,it showed an obvious toxic effect on KGN cells,which was positively correlated with the concentration of the drug.At the same time,with the extension of time,different concentrations of CTX showed significant toxicity.1μM-25μM of Icariin could promote the proliferation of KGN cells.When the concentration was higher than 25μM,icariin could inhibit the proliferation of KGN cells to some extent,but the cells still proliferated normally.3.2 CTX can promote the increase of apoptosis,and the number of apoptosis increases with the increase of concentration.When CTX was greater than 10 μM,CTX concentration was positively correlated with apoptosis of KGN cells.There was no effect on apoptosis of KGN cells in the concentration range of 1μM-100μM.3.3 CTX increased TGF-β secretion with increasing concentration,which inhibited TGF-β secretion at 5μM;The secretion of E2-β was negatively correlated with the concentration of CTX,suggesting that CTX had certain regulatory capacity on E2-β.Glycosides can promote the secretion of E2-β and inhibit the secretion of TGF-β in KGN cells,and the secretion regulation of these three factors reached the maximum effect at 5 μM concentration.4.Cell experiment(2)Explore the molecular mechanism of the effect of icariin on CTX-induced KGN cell ovarian hypofunction model: 4.1The results showed that the apoptosis of KGN cells could be promoted by Icariin or CTXalone,but the apoptosis of KGN cells stimulated by Icariin was weaker than that of CTX alone.The apoptosis of KGN cells could be promoted by the combination of Icariin and CTX(P < 0.01),but the effect of the combination of Icariin and CTX on KGN cells was weaker than that of CTXalone(P < 0.01),The differences were statistically significant. 4.2The secretion of PI3 K and AKT were increased in KGN cells stimulated by Icariin alone;the secretion of PI3 K,Akt,TGF-β and Smad1 were increased in KGN cells stimulated by CTX alone or the combination of Icariin and CTX,but the increase of TGF-β and Smad1 was weaker than that of CTX alone.4.3The results showed that the secretion of TGF-β was decreased and E2-β was increased in KGN cells stimulated by Icariin alone(P < 0.05);TGF-β was increased and E2-β was decreased when KGN cells were stimulated by CTX alone or by the combination of Icariin and CTX,(P < 0.05);but the increase of TGF-β was more weaken than that of CTX alone,the decrease of E2-β was more weaken than that of CTX alone(P < 0.05),The differences were statistically significant.4.4When KGN cells were co stimulated with Icariin and TGF-β,the apoptosis of KGN cells was decreased compared with that of TGF-β alone and increased compared with that of Icariin alone(P < 0.01);when KGN cells were stimulated by CTX and IDT-1,the apoptosis of KGN cells was increased compared with that of IDT-1 alone and decreased with that of CTXalone(P < 0.01),the differences were statistically significant.4.5The secretion of PI3 K and AKT were increased in KGN cells treated with Icariin alone,while PI3 K,AKT,TGF-β and Smad1 secretion were increased when TGF-β were given alone.The PI3 K,AKT,TGF-β and Smad1 secretion were increased when Icariin and TGF-β were given at the same time,but the increase of TGF-β and Smad1 was weaker than that of TGF-β alone;when IDT-1 was given alone,PI3 K,AKT,TGF-β and Smad1 were not secreted,When CTX was given,the secretion of PI3 K,AKT,TGF-β and Smad1 was increased.At the same time,when CTX and IDT-1 were given,PI3 K and AKT were not secreted,but TGF-β and Smad1 were still secreted.Conclusion 1.The mouse model of ovarian dysfunction(kidney deficiency syndrome)can be formed by CTX modeling;2.Qilin Pill can be used to treat CTX-induced ovarian dysfunction(kidney deficiency syndrome)model mice;3.Qilin Pill showed no adverse reactions to CTX-induced ovarian dysfunction(kidney deficiency syndrome)in mouse model at the clinical equivalent dose;4.In vitro cell experiments,Icariin had no obvious apoptotic effect on KGN cells,but also promoted the secretion of E2-β,which was most obvious at 5 μM for 24 h.5.In vitro experiments,CTX showed an obvious toxic effect on KGN cells when the concentration was higher than 10μM,which was positively correlated with the concentration of the drug.The toxic effects on cells will accumulate with the prolongation of stimulation time.At the same time,CTX higher than 10μM had an obvious apoptotic effect on KGN cells,and inhibited the secretion of E2-β,but promoted the secretion of TGF-β.6.The apoptotic effect of CTX on KGN cells depends on the activation of SMAD /TGF-β pathway,while Icariin can effectively block the activation of SMAD /TGF-β pathway to reduce the apoptosis of KGN cells,thus achieving the role of protecting KGN cells.At the same time,Icariin can effectively promote the secretion of E2-β,a key cytokine,by KGN cells.