Research On The Mechanism Of Mongolian Medicine Sugemule-7 In The Treatment Of Ovarian Dysfunction

Objective:To explore the therapeutic effect and mechanism of Mongolian medicine Sugemule-7 on ovarian dysfunction(Explanation:Mongolian medicine Sugemule-7 is hereinafter referred to as Su-7,model plus Sugemule-7 low dose group is referred to as Su-7 low group,model plus Sugemule-7 medium dose group is referred to as Su-7 middle group,model plus Sugemule-7 high dose group is referred to as Su-7 high group.)Methods:1.90 cases of premature ovarian failure(POF)and physiological decline of ovarian function(premenopausal)and postmenopausal women were divided into three groups according to age and diagnosis:①POF group;②premenopausal women aged 40-50 years;③postmenopausal women aged 45-55 years.Su-7 was given for 2-3 months.The total clinical efficacy was evaluated by comparing the changes of menstruation,clinical symptoms and sex hormone levels before and after treatment.It will lay a foundation for the study of the later action mechanism.2.To explore the mechanism of Su-7 in the treatment of ovarian dysfunction based on serum proteomics.Methods:according to the diagnostic criteria and inclusion criteria of ovarian dysfunction,15 Mongolian medicine Sugemule-7 capsules were given to the patients with ovarian dysfunction of Heyi abnormally exuberant type or Heyibadakan abnormally exuberant type,twice a day,for 60 days.Before administration and on the 61st day of administration,5ml of venous blood was collected to separate serum.Protein was extracted from serum and detected by liquid chromatography-mass spectrometry(LC-MS).Protein was identified by Proteome Discoverer software.Serum protein was analyzed by Sieve software.GO annotation and enrichment analysis were performed by Kobas Database.Pathway annotation and enrichment analysis were performed by Keggpathway,React,Biocyc,Panther and PID databases,To find out the significant difference protein before and after treatment and participate in the enrichment pathway.3.To investigate the protective effect of Su-7 on ovarian function and its mechanism in rats with cyclophosphamide(CTX)-induced ovarian injury.Ninety six rats with normal sexual cycle were randomly divided into six groups:model group(CTX),model plus estradiol valerate group(CTX+progynova),model plus low-dose Sugemuler-7 group(CTX+Su-7low),model plus low-dose Sugemuler-7 group(CTX+Su-7low),m odel plus low-dose Sugemuler-7 group(CTX+Su-7low),model plus low-dose Sugemuler-7 group(CTX The rats in model group were treated with medium dose(CTX+Su-7 mid)and high dose(CTX+Su-7 high).In the normal group(NC),except for the normal group,the other 5 groups were given intraperitoneal injection of CTX 30 mg/kg.D for 7 days.The normal group and model group were given the same amount of normal saline by gavage,and the other groups were given Su-7 high(2.0g/kg.D),medium(1.0g/kg.D)and low(0.5g/kg.D)estradiol valerate(0.1 mg/kg.D)by gavage from the first day of modeling(at the same time of modeling)for 24-29 days,respectively.Vaginal smears of all rats were analyzed daily to observe the changes of estrous cycle;The male rats were mated at the ratio of 2:1 during estrus to monitor their reproductive capacity;Serum hormone levels and inflammatory factors were detected by ELISA;One side of the ovary was taken,and the histological changes and the number of follicles at different stages were observed under light microscope;The localization and expression of 8-OHdG,4-HNE,nty,BMP-15 and GDF-9 were detected by immunohistochemistry;Western blot was used to detect the expression of apoptosis related protein and antioxidant related protein Bax,Bci-2,SOD in ovarian tissue,and to verify the expression of serum proteomics differential proteins TSP-1,Cathepin D,bstl,CFH in patients with previous clinical trials.Results:1.Su-7 has a significant effect on menstruation and sex hormone level in the group of POF.There was no significant difference(P>0.05);The symptom score decreased significantly after treatment(P<0.05);The total clinical effect was 43.33%.For premenopausal women aged 40-50,menstruation was significantly improved,estrogen level was significantly increased(P<0.05),FSH was significantly decreased(P<0.01);It can significantly improve the clinical symptoms(P<0.01);The total clinical effect was 90%.For the postmenopausal group,the menstruation and sex hormone levels were not significantly improved(P>0.05),the symptom score was significantly decreased(P<0.01),and the total clinical effect was 93.33%.2.The results of serum proteomics of clinical patients before and after treatment showed that there were 15 differential proteins after treatment.These proteins are mainly immunoglobulin,complement factor H,complement factor C,complement factor C and complement factor C α-2-hs-glycoprotein,antitrypsin,cathepsin-D,platelet-reactive protein,ADP ribocyclase/cyclic ADP ribohydrolase-2,matrix metalloproteinase-9,etc.go analysis showed that they were involved in immune regulation,immune enhancement,complement activation,inflammatory response,external stimulus response and other biological processes;KEGG analysis showed that the above differential proteins were involved in 10 signaling pathways,including nicotinate and nicotinamide metabolism,salivorysecretion,pancreatic secret,metabolic pathways,Staphylococcus aureus infection,complex and coordination cascades,regulation of complex cascade,complex cascade,innateimmune system and immune system.3.The results of animal experiment and mechanism of action showed that Su-7 low and middle groups could improve the ovarian function of rats after CTX.At the end of the experiment,the body weight,ovarian weight and spleen weight of the model gruop decreased significantly(P<0.05);The weight of ovaries and spleens in low Su-7 group increased significantly(P<0.05).Sex hormone levels:①with the increase of age,AMH in normal group decreased.②At the age of 11-12 weeks(CTX+Su-7 treatment for 15-18 days),the level of AMH in CTX group decreased significantly(P<0.05).Compared with the model group,the level of AMH in Su-7 group was significantly higher(P<0.01),and compared with the estradiol valerate group,the level of AMH in Su-7 group was significantly higher(P<0.05).The level of AMH in Su-7 low group was significantly higher than that in model group(P<0.05);Su-7 low group can significantly increase E2 level.③There was no significant difference in AMH,E2 and FSH among the groups when the rats were 13-14 weeks old(CTX+Su-7 treatment for 24-29 days)(P>0.05),which may be related to the recovery of endocrine function and the passing of the best time of Su-7 efficacy.Compared with the model group,there was significant difference in the ratio of estrus recovery(normal sexual cycle)among the three groups(P<0.01).The number of antral follicles decreased significantly compared with the normal group(P<0.05),but the number of atresia follicles increased significantly(P<0.05).The number of antral follicles in Su-7 high and low groups increased significantly compared with the model group(P<0.05).The number of atresia follicles in Su-7 groups decreased significantly compared with the model group(P<0.05).Results of ovarian histomorphology:in CTX group,the ovaries were slightly atrophied,the number of follicles at all levels was less,the level of granulosa cells was reduced,some follicles were dysplasia,the number of cystic expanded follicles was increased,the oocytes disappeared,the cortex thickened,the medulla was less,the interstitial fibrous connective tissue proliferated and focal lymphocyte infiltrated;In the middle Su-7 group,the cortex was slightly thickened,the medulla was less,and there were infiltrations of lymphocytes in all levels of follicles,corpus luteum and stroma;In Su-7 high group,the cortex was slightly thickened,the medulla was less,the corpus luteum and all kinds of follicles were seen,and the sinus follicles were relatively increased or the sinus follicles were mainly proliferated;In Su-7 low group,the cortex was slightly thickened,the medulla was less,the interstitial blood vessels were congested,the corpus luteum and follicles at all levels were seen,the granulosa cell layer increased,and the sinus follicles proliferated;In bujiale group,the number of follicles at all levels was less,the cortex was thickened,the medulla was less,and the interstitial lymphocytes were infiltrated.Results:compared with the normal group,the fertility rate and the survival rate of newborn rats in CTX group decreased significantly(P<0.01);Compared with CTX group,the fertility rate and survival rate of newborn rats in three doses of Su-7 were significantly increased(P<0.01);The effect of Su-7 middle group was the strongest,compared with Su-7 high and low group and bujiale group(P<0.01).Effect of TNF-α on serum of rats-α、IL-6、TGF-(3 Compared with the normal group,CTX group had a higher level of TNF-α-α、IL-6 was significantly increased(P<0.05),TGF-β was significantly increased(P<0.05)-β1 decreased significantly;Compared with CTX group,the TNF level of Su-7 group was lower-α、IL-6 decreased significantly(P<0.05),TGF-β decreased significantly(P<0.05)-β1 was significantly increased(P<0.05).Western blot was used to detect the expression of Bax,Bci-2,SOD,TSP-1,Cathepin D,bstl and CFH in the ovaries of rats.Results:the expression of Bax in the ovaries of rats in CTX group was significantly increased,while the expression of Bcl-2 was significantly decreased(P<0.05);Bax and Bcl-2 were significantly decreased and increased in Su-7 low group(P<0.05),and the effect of Su-7 low group was the best,compared with Su-7 middle group(P<0.05).The expression of tps-1 and CFH protein in CTX group was significantly higher than that in normal group,while SOD was significantly lower(P<0.01);Tps-1 and CFH were significantly decreased and SOD was significantly increased in Su-7 group(P<0.05).There was no significant difference in immunohistochemical results among the groups.Conclusion:1.Su-7 has different clinical effects on premature ovarian failure and premenopausal and postmenopausal groups.Su-7 has good curative effect on premenopausal and postmenopausal patients.It can improve ovarian function,delay ovarian aging and improve perimenopausal symptoms.Therefore,premenopausal patients as the object,further study on the mechanism of action.2.Su-7 can delay ovarian aging and improve ovarian function through nicotinic acid nicotinamide metabolic pathway,pancreatic secretion pathway,salivary secretion pathway,regulation of innate immune system pathway,immune system pathway and complement cascade regulation pathway.3.Su-7 has protective effect on body weight,ovary and spleen of chemotherapy model rats.It can improve immunity,ovarian repair,follicle maturation and ovulation to restore sexual cycle,reproductive capacity and survival ability of newborn rats.The effect of low and middle dose Su-7 is better,so further study on the mechanism of this dose is needed.4.The protective effect of low and middle dose Su-7 on ovarian endocrine and reproductive function may be related to the inhibition of TNF-α、IL-6 can inhibit inflammatory injury and protect organ function;By up regulating TGF-β It can promote the growth and proliferation of granulosa cells,promote the development and maturation of follicles,increase the number of antral follicles,and promote ovulation,so as to improve the pregnancy rate of rats;On the one hand,it can improve the antioxidant capacity of ovarian tissue by increasing the expression of SOD,and inhibit ovarian granulosa cell apoptosis and follicular atresia by resisting chemotherapy to ovarian oxidative stress.On the other hand,it can prevent premature apoptosis and resist follicular atresia by increasing anti apoptotic factors and reducing Pro apoptotic factors,so as to increase the number of healthy follicles to protect the ovarian reserve and ovarian function of chemotherapy rats Improve ovarian function;On the other hand,it may improve ovarian microcirculation by reducing TSP-1,prevent ovarian fibrosis,provide favorable environment for follicular development and other mechanisms to protect endocrine and reproductive function of rats with chemotherapy-induced premature ovarian failure.5.Low dose Su-7 is suitable for the prevention and treatment of chemotherapy-induced premature ovarian failure.