| Objects1.The clinical therapeutic effects were observed to clarify the therapeutic use of the TCM method of fortifying the spleen and tonifying the kidney,activating blood and resolving stasis with a before-and-after trial by comparing the serum gonadal hormone,score of TCM symptoms and modified Kupperman score.2.To evaluate the differences between three commonly used methods for establishing mouse models of POI,and to determine the method that is most accordant with the clinical features of POI.3.The animal study is to explore the treatment effects of JJGSF on the treatment of POI triggered by 4-vinylcyclohexene diep-oxide(VCD),an endocrine-disrupting chemical(EDC),and clarify its potential mechanism of action.Methods1.Clinical research:A total of 26 patients with POI admitted to the gynecology department of Dongzhimen Hospital,Beijing University of Chinese medicine were selected and treated with JJGSF for three periods(three months for amenorrhea patients).The scores of TCM symptoms and modified Kupperman were contrasted and the serum level of follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2)were observed to evaluated the curative effects of JJGSF.2.Study of animal models:Ninety-six ICR mice with ordered estrous cycles were randomly divided into 4 groups:the control group(A),the tripterygium glycosides(TG)group(B),the 4-vinylcyclohexene diepoxide(VCD)group(C),and the D-(+)-galactose(D-gal)group(D).After intervention with the different chemicals,eight mice in each group were sacrificed at day 16,46 and 76,the serum sex hormone levels were measured,and the uterus and ovaries were examined.3.Medicines sensitivity experiment in vitro:A total of senventy-two eight-week-old ICR female mice were randomly divided into six groups,including the model group,the control group,three JJGSF groups,and the Progynova positive control group.After the establishment of the model by VCD,the Progynova group received Progynova by intragastric administration,and the three JJGSF groups(high-,medium-and low dose group)received JJGSF at doses of 9,4.5 and 2.25 g/kg by intragastric administration respectively for four weeks.The general growth and the estrous cycle of the mice were observed.The enzyme-linked immunosorbent assay(ELISA)was used to measure the concentrations of serum hormones such as anti-mullerian hormone(AMH),follicle-stimulating hormone(FSH),luteinizing hormone(LH),inhibin B(INHB),and estrogen(E2).The protein expressions of H3K9me3,HP1,and HMGA1/HMGA2,which were concerned with senescence-associated heterochromatic foci(SAHF),were determined by immunofluorescence and western blot analysis to explore the potential mechanism of action.Results1.Clinical trial:Score of TCM symptoms:Various degree of improvement of menstruation situation after treatment.The symptoms of soreness and weakness of waist and knees,sexual hypoactivity,tidal fever,night sweating,irritability,chest distress,hypochondriac pain,insomnia and dreaminess were improved and the differences were statistically significant(P<0.05).Aggregate efficiency is 73%for treatment POI by JJGSF The score of modified Kupperman:The symptoms scores of hot flashes sweating,paresthesia,skin ant line sense,anxiety,myalgia and sexual pain were decreased significantly(P<0.05).Laboratory indexes:the serum levels of FSH and LH were lower than those in prior treatment,the difference was statistically significant(P<0.05).The level of E2 increased but without statistically difference(P>0.05)2.Study of animal models:The study proved that the establishing method of TG via gavage could induce dysfunction of ovary at day 16,on account of that the body weight of ICR mice in TG group decreased significantly,the majority of animals had disordered estrous cycle,the levels of AMH and E2 decreased,the number of developing and mature follicles decreased and endometrium turned thinner.The body weight restored at day 46,the levels of E2 and the number of developing follicles and corpus luteum still decreased.At day 16,the ovarian index,the levels of AMH and E2 and the thickness of endometrium in D-gal group decreased showing that the D-gal establishing method could damage the ovarian function of ICR mice.The damage persisted after 46 days of administration appeared in low E2,the lower number of developing follicles and corpus luteum.At day 76,the low levels of AMH and E2,thin endometrium and disordered estrous cycle in more than half of mice of D-gal group indicated that the ovarian function was decreasing during drug administration and the POI establishing method of D-gal via subcutaneous injection at a dose of 500 mg/kg was an ideal method.The establishing method of VCD via intraperitoneal injection could induce dysfunction of ovary at day 16 with low ovarian weight and index,low levels of AMH and E2 and high levels of FSH and FSH/LH rate without disordered estrous cycle though.At day 46,the number of developing follicles and corpus luteum were further reduced and that indicated the damage of ovarian function persisted after a month of withdrawn.At day 76,the levels of AMH,LH and E2 were still lower,the number of developing and mature follicles and the thickness of endometrium and myometrious wall decreased,and the majority of ICR mice had disordered estrous cycle3.Medicines sensitivity experiment in vitro:The estrous cycle:After being treated,estrous cycles significantly recovered in the JJGSF groups and the Progynova group compared with the model group(P<0.05).The level of serum sexual hormone:Comparing with other groups,the concentrations of AMH,INHB and E2 decreased significantly,and the level of FSH and the ratio of FSH/LH in the model group(P<0.05).The serum concentrations of AMH and INHB in the JJGSF high dose group and the Progynova group were higher than those in the model group(P<0.05).The level of LH in all JJGSF groups,the Progynova group,and the control group showed no significant difference compared with the model group(p>0.05).SAHF formation and the expression of HP1/H3K9me3 in ovaries The fraction of ovarian cells which were SAHF-positive in the JJGSF groups(high and medium dose),and the Progynova group showed significant reduction compared with the model group(p<0.05).Although the fraction of SAHF-positivity in the low dose JJGSF group was lower than that in the model group,there was no significant difference between them(p>0.05).The expression of HP1/H3K9me3 was high in the model group and low in the control group.The expression was similar between the JJGSF high dose group and the Progynova group,and seemed to depend on the concentration of JJGSF.The expression of signature proteins of SAHF in ovaries:HMGA1 and HMGA2 in the model group were over-expressed compared with the control group(p<0.01).After treatment with JJGSF and Progynova,the expression of HMGA1 and HMGA2 was significantly lower than in the model group(p<0.01),but obviously higher than in the control group(p<0.01).From the WB analysis,it could be seen that the expression of the HMGA1 and HMGA2 in the control group,Progynova group,JJGSF high,medium and low groups and in the model group increased progressivelyConclusion1.JJGSF can reduce the levels of serum basic FSH and LH of patients with POI and improve the situation of menstruation,the score of TCM and perimenopausal symptoms,which indicates that JJGSF can improve the presenility of ovarian function2.Mouse models of POI can be successfully established with VCD via intraperitoneal injection.This method has the advantages of being an easier operation and having a higher success rate,as compared with the other two models.It is the ideal one of the three establishing methods and is suitable for popular use.3.JJGSF could improve the ovarian function of POI mice and the potential mechanism may be involved with suppressing SAHFs. |
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