| Objective To observe the clinical effect and estrogen promoting effect of DKTP by the intervention of traditional Chinese medicine DKTP and estradiol western medicine group and the blank model group,and to simulate the body state of perimenopause in natural aging 12-month old rats.By detecting the expression of key enzymes in the pathway of synthesis,metabolism and operation of ovarian granule cells in perimenopause rats,the molecular mechanism of the effect of Decoction of Kidney-yin tonifying prescription on promoting estrogen was explored,providing a target for the clinical treatment of perimenopausal syndrome.Methods 1.Healthy and clean 12-month old Wistar rats were selected and 18 rats with longer estrous cycle were selected as the perimenopause model rats through the observation of vaginal exserted cells for 5 consecutive days.It was divided into the experimental group of nourishing Yin and nourishing kidney prescription,the estradiol control group and the blank control group in a completely random manner.After 4 weeks of treatment,the effects of vaginal exfoliated cells,observed peripheral blood E2 level and uterine morphology in rats.And rat tail skin temperature measurement is adopted,open field experiment and high cross maze test,the extraneous object recognition test observation model rats of hot flashes,mood disorders and memory disorders such as anxiety,Decoction of Kidney-yin tonifying prescription for the clinical symptoms of the menopausal transition rats improvement effect,promote estrogen and estrogen receptor function evaluation.2.Use completely random grouping of the menopausal transition model rats after surgery to remove the ovaries are divided into Decoction of Kidney-yin tonifying prescription in low dose group,middle dose group,high dose group(4 weeks),and Neil female alcohol group(4 weeks)and compared with blank control group,using q PCR,WB,ria method each rat estrogen key enzyme in the synthesis,transport,metabolism pathway(ER?,,CYP17,CYP19,3 ?HSD,17?HSD)activity and expression,investigate the molecular mechanism of Decoction of Kidney-yin tonifying prescription side effect on promoting the estrogen.Results 1.General situation of perimenopause rats: the skin temperature of the rats in the model control group was high and unstable,the state of anxiety was obvious,and the recognition ability of new things was poor,which was in line with the body state of perimenopause.Compared with the model control group,the skin temperature of rats in the experimental group of DKTP decreased slightly and the change range was not significant.There was no significant difference between the low-dose group and the medium-dose group,while the changes were significant in the high-dose group,but the improvement status was not significant compared with the E2 control group.According to the observation of the film results,the estrous cycle of the rats in the model control group showed different degrees of disorder(vaginal exfoliated cell keratinization index < 50%)and prolonging,among which interestrous period and anestrous period were the main ones.The estrous cycle of E2 control group rats was significantly shortened,especially the estrous period.The estrous period of rats in the experimental group of nourishing Yin and tonifying kidney formula was prolonged,which was more common in the early estrous period and estrous period.The morphological changes are as follows: proestrus:oval with increased nuclear epithelial cells,large volume,dark nuclear,white blood cells and fewer keratinocytes.estrus phase: flake horn changes epithelial cell more,much overlap accumulates,have nuclear epithelial cell and leucocyte are less.estrus: a large number of white blood cells,nuclear epithelial cells and keratinized epithelial cells reduced.Anestrus: white blood cells,nuclear epithelial cells and keratinocytes are present,but there is no significant proportion.According to the film results,the endometrium of the rats in the model group became thinner and atrophic during the estrous phase,with few glands and scattered single tubes,different sizes of glandular cavities,thinner muscular layer,irregular arrangement of muscle fibers,and less smooth serous layer.In E2 control group,the increase of glandular epithelial cells was more obvious,the number of glands was significantly increased,the arrangement of muscle fibers was more regular,the serosal layer was smoother,and papillary hyperplasia occurred in some individual endometrial epithelium.In the experimental group of DKTP,the endometrium of rats showed increased glandular epithelium,increased number of glands,slightly regular arrangement of muscular layer and smooth serous layer.Detection results of cell activity: there were statistically significant differences in cell activity and cell inhibition rate between the two groups(p < 0.05).Compared with KGN group,MPP group,5% drug-containing serum group and MPP+5% drug-containing serum group all had statistically significant differences in cell activity and inhibition rate(p < 0.05).2.Effects of DKTP on promoting estrogen and estrogen receptor: the percentage binding rate of serum E2 concentration in the low,medium and high dose DKTP group was significantly different from that in the blank control group(p < 0.05),and there was significant statistical difference compared with that in the E2 control group(p < 0.05).There were significant statistical differences between serum E2 with 2%,5% and 10% concentrations and serum E2 without drugs(p < 0.05),and there were significant statistical differences between serum E2 with different concentrations and serum E2 without drugs(p < 0.05).In terms of the m RNA expression of ER?,there were significant statistical differences in the low,medium and high dose DKTP groups compared with the blank control group(p < 0.01).Compared with E2 control group,there were significant statistical differences in DKTP among low,medium and high dose groups(p < 0.01).In terms of ER protein expression,there were significant statistical differences between the low,medium and high dose DKTP groups compared with the blank control group(p < 0.05).Compared with E2 control group,there were significant statistical differences in DKTP among low,medium and high dose groups(p < 0.05).There was no significant difference in DKTP protein expression between the lowdose group and the medium-dose group(p > 0.05).The m RNA expression of ER in serum was significantly different at different concentrations(p < 0.05).Compared with the blank serum group,the ER protein expression in 5% and 10% drug-containing serum group showed significant statistical significance(p < 0.05),while the protein expression in 2% drugcontaining serum group showed no statistical difference from the blank serum group(p > 0.05).3.Discussion on the molecular mechanism of estrogen promoting effect: compared with KGN group,m RNA expression of key enzymes(ER,CYP17,CYP19,3?HSD,17?HSD)in ovarian granulosa cells in MPP group,5% drug-containing serum group and MPP+5% group were significantly different(p < 0.05).Compared with KGN group,ER protein expression was significantly different(p < 0.05),and 17?HSD protein expression was significantly different between KGN group and control group(p < 0.05).Compared with KGN group,there were significant statistical differences in the percentage binding rate of serum concentration of ovarian granule cells between different groups(p < 0.05),and there were also statistical differences in the serum concentration binding rate between different groups.Conclusion 1.The rat model of perimenopause syndrome was successfully constructed,including hot flashes,memory loss,anxiety,decreased estrous cycle,and histopathological changes.2.DKTP can effectively improve the general symptoms of perimenopausal syndrome, such as hot flashes,memory loss and anxiety,and promote the recovery of ovarian and uterine functions.At the same time,it has fewer side effects than hormone therapy.3.Different doses of DKTP have significant up-regulation effect on the m RNA expression and protein expression levels of estrogen and estrogen receptor in serum,and meanwhile enhance the activity of ovarian granulosa cells and reduce the rate of cell inhibition.It is suggested that the low,medium and high dose group of DKTP has a clear role in promoting estrogen and estrogen receptor,and the stronger the dose increases,the stronger the effect.4.The expression level of 17?HSD in ovarian granule cells was significantly upregulated by DKTP,indicating whether perimenopausal syndrome can be treated with targeted drugs related to 17?HSD. |
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