Anti-aging Effects Of YFN And Its Mechanism On Rat Ovary

Ovarian aging starts or accelerates from reproductive maturity,which is the common features of females and is a universal, cumulative and progressive, endogenous processes of life. Perimenopausal women’s most prominent change is ovarian aging, reduction in weight and ovarian volume decreases, and harden the surface shrinkage uneven and texture, follicles used up or the rest of the follicle is not sensitive to gonadotropin stimulation, synthesis and secretion of sex hormones. At present, the pathogeny of ovarian aging mechanism is not fully clear,such as genetic factors, some external factors(radiation therapy, chemotherapy, toxins, ovarian trauma or surgery), body endocrine factors, growth factors, cytokines and other factors have impact on ovarian aging. Ovarian aging is not only related to the changes of nerve-endocrine-immune system, but also in its its inner aging mechanism.In the menopausal transition, ovaries is autonomous loss of organ function, normal follicular atresia process is accelerated, or eggs destroyed by various mechanisms, lead to ovarian follicles depletion, oocyte atrophy, ovarian granulosa cell apoptosis and so on. Western medicine for the treatment of ovarian aging mainly include hormone replacement therapy, stimulate ovulation treatment, immunosuppressants and donor eggs, etc., but which have no satisfactory curative effect; Hormone replacement therapy (HRT) due to long term therapy, side effects, and have a potential risk on the breast and estrogen target organs, or make the patients gain weight, increase heart pressure, fear of cancer, and so on. Therefore, more and more scholars turn their attention to traditional Chinese medicine research, prevention and treatment of this disease.Yifuning(YFN) as the traditional Chinese medicine is the treatment of women around the menopause syndrome, which is made up of Oviducts Ranae and Rhizoma Curcumae, the party of herbs the frog oil and Chen medicine Curcuma nourishing liver.It can nourish the kidney, coordinate qi and blood, remove stagnation and decrease muddy. Existing animal experiments and clinical studies have had shown that YFN had the square of antioxidant, anti-aging effects. It could increase level of serum E2in ovariectomized rat model, increase uterus, adrenal index, but also could modulate the immune system, the hypothalamus, pituitary and plasma B-endorphin,5-serotonin, norepinephrine epinephrine and dopamine content. In other words, the role of YFN, in the removal of ovarian animals, could be improve disorder of hormone secretion of hypothalamus-pituitary-gonadal axis, increase content of neurotransmitter, make interal environment stable and relieve menopausal syndrome,improve the hypothalamic-pituitary-gonadal axis neuroendocrine function, regulate peripheral blood hormone levels, and significantly improve the clinical women of perimenopausal symptoms.But the conclution of YFN could have direct impacted on ovarian function is no evidence.Objective:YFN is oviducts ranae compound, the existing experiment of cell biology and animal experiment showed that oviducts ranae possess an action of increased estradiol secretion and proliferation, also can improve the pathology of ovarian aging and decrease the negative factors of cell proliferation; therefore, the early experiment provide a experiment basis the treatment of YFN on ovarian aging. But at present, the study is still lack of effective components control of YFN,and the verification of the overall animal efficacy and mechanism. So this topic proposed the further research on effective constituents and overall efficacy study, combining with the previous cell research results to further explore its mechanism of action of rat ovarian senescence, offers a basis for the application of YFN on perimenopausal ovarian aging.Methods:1Research on the active ingredients of YFNIn this study, using Coomassie brilliant blue and ultraviolet spectrophotometry to determine the protein and amino acid content of YFN and OR, detecte the YFN and OR active ingredient of estradiol1-Methylhydantoin, linoleic acid, vitamin E and the curcumol content of YFN using high performance liquid chromatography.2Pharmacodynamic studies of anti-ovarian aging2.1Establishment of animal model, grouping and method of administration16-18month-old of the natural aging perimenopausal SD female rats(n=24) were separated into four groups at random:old control group (O-C), the positive control diethylstilbestrol tablets group (DT,0.05mg/kg), Yifuning high-dose group (YFN-H, which is equivalent with raw materials2.0g/kg), low-dose group (YFN-L group, equivalent to with raw materials1.0g/kg), six animals were in each group. The young control group (Y-C) were six3-4month-old SD rats. The rats were accepted intragastric administration for6weeks.2.2Specimen collection and research methods (1) To observe the food and drink, hair color and the body weight change of aged rats during taking YFN. The change of vaginal epithelial keratosis was observed in two weeks before rats were killed, to calculate days of the estrous cycle.(2) Serum were obtained after the rats undergoing experiment for42d.We detected level of sex hormone indicators estradiol (E2), testosterone (Te), progesterone (P) in serum by electrochemiluminescence.(3) Ovaries and uterus were weighed to calculate the organ index:the organ index= tissue wet weight (mg)/body weight (g) x100%.(3) Observed the pathological changes of rat ovary, uterus, vagina tissues with hematoxylin and eosin staining by microscope.3The mechanism of delaying ovarian senescence.3.1The mechanism of antioxidant effect(1) Using UV spectrophotometry to detect level of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and malondialdehyde (MDA), over hydrogen peroxide (H2O2) in serum, and measure level of SOD, CAT MDA and H2O2in liver.(2) Evaluated the expression of oxidative stress protein p53and DNA damage marker8-OHdG by IHC in ovarian tissue.(3) The antioxidant enzyme gene mRNA expression of Gpxl, Prdx3, Gls2and Mn-SOD were detected by real-time qRT-PCR in ovarian tissues.3.2The mechanism of promoter cell proliferation.Using qRT-PCR and Western blotting to detect the expression of cell proliferation negative regulatory factor p19, p53, p21, Rb gene and protein in ovarian tissue.Result1Yifuning active ingredient results showed that there are222.00±5.80mg/g of protein,146.17±5.30mg/g of amino acids,0.0031±0.0001mg/g of estradiol0.0528±0.0251mg/g of1-methylhydantoin,391.7333±2.7392mg/g of linoleic acid,0.1073±0.0096mg/g of vitamin E,1.1500±0.0014mg/g of curcumol in YFN and442.70±7.87mg/g of protein,287.07±5.99mg/g of amino acids,0.0071±0.0001mg/g of estradiol,0.1257±0.0060mg/g of1-methylhydantoin,362.8100±4.3827mg/g of linoleic acid,0.045±0.004mg/g of vitamin E in OR,Which was in line with the composition of the capsule:the principal drug OR is approximately42%, the content of Curcuma oil is10%. 2Effect of YFN on senescent signs, sex hormones index and histomorphology in natural aging.2.1General indications:Compared with Y-C group, the rats of O-C group look sickly, less activity, hair loss is obvious, hair color is dark and yellow; DT group and YFN groups rats compared with group O-C group, the signs were improved; And the effect of YFN-H, L group is better than DT group.2.2Weight results:The weight of Y-C rats increaseed with time; The weight of O-C group was in a stable trend, compared with group Y-C group, had significant difference (P<0.05); The weight of DT and YFN-H、L group had no difference with O-C group(P>0.05).2.3Estrous cycle results:The estrous cycle of O-C group is disorder, the estrous cycle of O-C group was significantly longer than Y-C group; DT group and YFN-H, L group can significantly shorten the aging rats estrous cycle (P<0.05), and the effect of YFN-H group is more significant.2.4Ovarian index results:The ovarian index of O-C group had no statistical significance with Y-C group(P>0.05); The ovarian index of DT group increased,but compared with O-C group,had no statistical significance (P>0.05), while YFN-H group marked increased in ovarian index of aging rats(P<0.05).2.5Uterus index results:The uterus index of O-C group was significantly reduced than Y-C group(P<0.05); DT and YFN-H group marked increased in aging rat uterus index (P<0.05).2.6Sex hormone indicators resultsThe level of serum E2in O-C group significantly decreased than Y-C group (P<0.05); the levels of serum E2in DT group and YFN-H, L group significantly increased (P<0.05).The level of serum Te in O-C group was significantly lower than Y-C group (P<0.05), DT group and YFN-H, L can marked increase in aging rat Te levels (P<0.05).The level of serum P in O-C group were reduced, but no statistical difference with Y-C group (P>0.05), the level of serum P in DT group were decreased, but no statistical difference (P>0.05); The level of serum P in DT group significantly declined than Y-C group(P<0.05), the level of serum P in YFN-H was higher than O-C group, but no statistical difference (P>0.05), the content of serum P in YFN-L was decreased compared with group O-Cgroup, but no statistical difference (P>0.05), but the levels of serum P in YFN-H, L were still elevated significantly than DT group (P<0.05).2.7Pathology results2.7.1Ovarian pathology results:Compare to Y-C group, ovarian cortex and medulla structure of O-C group was clear, while developmental ovarian follicle was blocked, small number of mature follicle and corpus luteum decreased, part of corpus luteum were observed,and a small amount of primary and secondary follicles were occasionally seen by microscopy. The structure of ovarian cortex and medulla in DT group and YFN-H, L groups was clear than O-C group, different levels ovarian follicle and mature follicle in DT group and YFN-H, L groups were seen by microscopy, multilayer granular cells in ovarian follicle were seen,and number of corpus luteum increased in DT and YFN groups. But DT group compared with the rest of the group, longitudinal section product significantly narrowed.2.7.2Uterus pathology results:Compared to Y-C group, the structure of uterus in O-C group was significantly changed, showed analogical atrophic pathplogy change.Its stromal cells became denser,glands in the intimal layer became small, round and distribution significantly reduced, glandular expanded into a bladder, glandular epithelium became flat; Compared to O-C group, the structure of uterus in DT group and YFN-H, L groups wsa clear, intimal thickening, increase the number of glands, glands increase, uterine serosa layer visible, rich capillaries, and the endometrial thickening of DT group is obviously than YFN-H, L group.2.7.3Vaginal pathology results:Compared to Y-C group, the vagina of O-C group was shrink,showed mucosal fold less, stratified squamous epithelium thiner, shallow hole cells smaller, mucous membrane layer thiner, lamina propria layer cytoplasm eosinophilic, connective tissue loosen, vaginal discharge reduced obviously.Compared with O-C group, the shrink of vadina of DT group and YFN-H, L groups was improved,showed mucosal fold increased, epithelial cells clear, cell number increased, epithelial shallow hole cell bigger, lamina propria became dense connective tissue, the secretion of the vaginal cavity significantly increased. The mucosal fold of YFN-L was more than YFN-H group, mucous membrane layer thicker, epithelial structure more clearly, the vaginal cavity more secretion, but the density of lamina propria of connective tissue in YFN-H group was thickening than YFN-L group, and the mucosa of YFN-H, L groups were thickened than DT group.3. The mechanism of YFN on defering ovarian senescence3.1Effect on antioxidant3.1.1Serum antioxidant indicators results:The activity of serum SOD in O-C group significantly decreased than Y-C group (P<0.05), DT and YFN-H, L group significantly increased the activity of serum SOD in aging rats (P<0.05), and the effect of YFN-H, L groups was superior to DT group (P<0.05).The activity of serum GSH-px in O-C group decreased significantly than Y-C group (P<0.05), the activity of serum GSH-px in DT and YFN-H, L groups was rised, but had no statistical significance (P>0.05).The activity of serum CAT was significantly decreased than Y-C group (P<0.05), the activity of serum CAT in DT group had no significant difference with O-C group (P>0.05), and the activity of serum CAT in YFN-H was significantly rised than O-C group (P<0.05).The level of serum MDA was significantly increased than Y-C group (P<0.05), the level of serum MDA in DT group had no significant difference with O-C group (P>0.05), YFN-H, L group can significantly reduce the level of serum MDA in aging rats (P<0.05), and YFN-H group was obviously reduced.The level of serum H2O2in O-C group was significantly increased than Y-C group (P<0.05), while DT group and YFN-H, L can significantly reduce the level of H2O2in serum (P<0.05), and YFN-H group was significantly better than the effect of DT group (P<0.05).3.1.2Liver tissue antioxidant results:The activity of liver tissue SOD in O-C group significantly decreased than Y-C group (P<0.05), the activity of liver tissue SOD in DT group was increased than O-C group,but had no statistical difference (P>0.05), while the activity of liver tissue SOD in YFN-H,L groups was significantly rised than O-C group (P<0.05), and the role of YFN-L group especially apparent.The activity of liver tissue CAT in O-C group was decreased significantly than Y-C group (P<0.05), DT group and YFN-H, L can rise the activity of CAT in aging rat (P<0.05), the effect of YFN-H group significantly better than YFN-L group, and they were significantly superior to DT group (P<0.05).The level of liver tissue MDA in O-C group was significantly rised than O-C group (P<0.05), the lecel of liver tissue MDA in DT group had no significantly difference with O-C group(P>0.05), while YFN-H, L group can significantly reduce the level of liver tissue MDA in the aging rats (P<0.05), and YFN-H group is better than YFN-L group. The level of liver tissue H2O2in O-C group was decreased significantly (P<0.05), the level of liver tissue H2O2in DT group was significant rised than O-C group (P<0.05), while the level of H2O2in YFN-H, L groups had no significant difference with O-C group (P>0.05), but significantly lower than DT group (P<0.05).3.1.3Real-time quantitative PCR (qRT-PCR) test resultsThe expression of ovarian tissue GPX1, Prdx3and Mn-SOD gene decreased significantly than Y-C group (P<0.05), but the expression of GLS2gene had no statistical difference with Y-C group(P>0.05), DT group and YFN-H, L can significantly rise the expression of GPX1, Prdx3, GLS2, Mn-SOD gene in aging rats (P<0.05), and the expression of GPX1H, L, Prdx3, GLS2gene in YFN--H,L groups was rised than DT group (P<0.05).3.1.4Immunohistochemical detection resultsImmunohistochemical results showed that the expression of p53positive staining in nucleus of all levels of follicular granulosa cells and oocytes and corpus luteum cells, corpus luteum expression was stronger than the expression of follicle;8-OHdG as a DNA damage markers, the expression of positive staining was mainly in stromal cell cytoplasm.IOD values of p53in O-C group ovarian tissue was significantly rised than Y-C group (P<0.05), DT and YFN-H, L groups can significantly reduce IOD values of p53than O-C group(P<0.05), YFN-H group is obviously better than YFN-L group, and were significantly superior to DT group (P<0.05).IOD values of8-OHdG in O-C group ovarian tissue was significantly rised than Y-C group (P<0.05), DT and YFN-H, L group can significantly reduce the expression of8-OHdG than O-C group (P<0.05), the role of YFN-L group especially apparent, and was significantly better than the DT group (P<0.05).3.2Effect on promoting cell proliferation 3.2.1qRT-PCR detection resultsThe expression of p19, p53, p21and Rb gene of ovarian tissue in O-C group was significantly higher than Y-C group (P<0.05), DT and YFN-H, L group can significantly reduce the expression of p19, p53, p21and Rb gene (P<0.05), the reduced expression of p19, p53, p21gene in YFN-H group significantly superior to the effect of YFN-L group (P<0.05). Compared with DT group, YFN-H group significantly reduced the expression of p21gene (P<0.05), while DT group reduce the expression of p19gene better than YFN-H group (P<0.05).3.2.2western blot method detection resultsThe expression of p19, p53, p21and Rb protein of ovarian tissue in O-C group was significantly higher than Y-C group (P<0.05), DT and YFN-H, L group can significantly reduce the expression of p19, p53, p21and Rb protein than O-C group (P<0.05), the effect of YFN-H and DT group was superior to that of YFN-L group (P<0.05), and the effect of YFN-H group on p19and p53protein was significantly better than DT group (P<0.05).Conclusion1. YFN is rich in the ingredient of enhancing immunity and anti-oxidation: protein, amino acid, estradiol, and1-methyl Maine, linoleic acid, vitamin E, curcumenol, etc., they may be the main effective ingredient in the treatment of perimenopausal syndrome and ovarian aging.2. YFN can improve general signs of aging rats, restore the estrous cycle, regulate peripheral blood sex hormone secretion, improve index of the ovary, uterus, delay the ovary, uterus, vagina and other reproductive organs degeneration,3. YFN have antioxidant and promoting cell proliferation effect. YFN defer ovarian aging may through the mechanism of antioxidant and promoting cell proliferation,and then to adjust the perimenopausal syndrome.