Effect Of Estrogen Replacement Therapy On T Lymphocytes Apoptosis Of Surgically Menopausal Women And The ER Mechnism

BackgroundRecent studies have suggested that estrogen played an important role in immune modulation and immune-related disorders,perhaps with estrogen receptors(ERs) involved.A rapid decline in female sex hormone production after menopause may thus affect the immune response,possibly contributing to postmenopausal syndromes and senescence.People have paid more and more attention to the effect of estrogen replacement therapy(ERT) on immune function of postmenopausal women since ERT was widely used.However,there are few researches on the effect of ERT on immunocytes apoptosis of postmenopausal women and the complex mechanisms of ER pathways of estrogenic effect have not been clarified.Partâ… Effect of transdermal estrogen replacement therapy on T lymphocytes apoptosis and ER subtype expressions of surgically menopausal womenObjectiveTo investigate the effect of transdermal estradiol gel on T lymphocyte apoptosis and ER subtypes expressions of surgically postmenopausal women and its clinical effects.Methods15 healthy women aged 48 to 57 years old who have undergone total hysterectomy and bilateral salpingo-oopherectomy within 1 to 5 years were chosen as the experimental group;15 naturally menopausal women and 15 young women as control groups.Peripheral vein blood were collected and serum E₂ and FSH levels were assessed using ACCESS.T lymphocytes were separated,apoptosis rates were detected using Flow Cytometry,and the protein expressions of Fas,FasL and ER subtypesαandβwere determined by Westernblot.There were 13 women in the surgically menopausal group who finished 3 months of transdermal ERT(estradiol gel,containing 0.75mg 17β-estradiol per 1.25g gel,smearing every day).Their menopausal complaints and life quality were evaluated by "Improved Kupperman index" and "menopause-specific quality of life questionnaire" before and after ERT.The levels of E₂,FSH,T lymphocyte apoptosis,Fas,FasL,ERαand ERβwere assessed again after ERT and compared to those of before ERT.Results1,The serum E2 levels of surgically and naturally menopausal women were significantly lower than that of young group,and the FSH level significantly higher than young group(P＜0.01).The T lymphocyte apoptosis of two menopausal groups was significantly higher than that of young group(P＜0.05).2,The Fas and FasL protein expressions on T lymphocytes of surgically and naturally menopausal women were significantly higher than those of young group(P＜0.05); ERαand ERβprotein expressions significantly lower than those of young group(P＜0.05 or P＜0.01).3,After ERT,the E2 level of surgically menopausal group was significantly higher than that of before ERT(P＜0.05);the FSH level was decreased but the difference was not significant.4,After ERT,the T lymphocyte apoptosis and FasL protein expression of surgically menopausal women were decreased,but the difference was not significant.The ERαand ERβprotein expressions were significantly higher than those of before ERT(P＜0.05 and P＜0.01).5,After ERT,the "Improved Kupperman index" and "menopause-specific quality of life questionnaire" of surgically menopausal women scored significantly lower than before ERT(P＜0.01);APTT was significantly higher than before ERT(P＜0.05); triglyceride(TG) and low-density lipoprotein(LDL) levels were significantly lower than before ERT(P＜0.05).Conclusions1,After menopause,women undergo changed hormone levels,increased T lymphocyte apoptosis and Fas,FasL expressions,and decreased ERα,ERβexpressions.The increased T lymphocyte apoptosis may be related to menopausal complaints and senescence. 2,Transdermal ERT significantly upleveled the serum E2 level and ERαand ERβexpressions on T lymphocytes of surgically menopausal women,especially ERβ.3,Transdermal ERT could decrease the T lymphocyte apoptosis and FasL expression of surgically menopausal women,although the difference was not significant.It indicated that low dose ERT may have a protective effect on menopausal women's immune function.4,Transdermal estradiol gel significantly improved menopausal symptoms and life quality of surgically menopausal women,and improved to some extent the coagulation function and lipid metabolism.Partâ…¡Effects of different doses of estradiol on apoptosis of T lymphocytes of ovariectomized mice and possible mechanismsObjectiveTo investigate the effects of different doses of estradiol(E₂) on splenic T lymphocytes's apoptosis and ER subtypes expressions of ovariectomized mice and to explore the possible mechanisms.MethodsThe splenic T lymphocytes of female Balb/c mice(8 weeks old) were separated and divided into 10 groups:young group,sham-ovariectomized group,ovariectomized group,four ovariectomized plus E₂ groups(E₂ concentration:10^-11M,10^-10M,10^-8M,10^-6M),ovariectomized plus E₂(10^-10M) plus ICI182,780(10^-7M) group, ovariectomized plus E₂(10^-10M) plus MPP(10^-6M) group and ovariectomized plus E₂(10^-10M) plus PDTC(10^-5M) group.All cells were harvested after 72h of incubation. The apoptosis rates were determined by Flow Cytometry using Annexin V-FITC/PI, and the expressions of ERα,ERβ,Bax,Bcl-2 and P65 proteins were detected by Westernblot. Results1,The T lymphocytes apoptosis rate of ovariectomized group were significantly higher than that of young group and sham-ovariectomized group(P＜0.01);while Bcl-2 and nuclear P65 proteins expressions were lower than those of young group and sham-ovariectomized group(P＜0.01).2,The T cells apoptosis of ovariectomized plus E₂(10^-10M) group was lower than that of ovariectomized group(P＜0.05),and the Bcl-2 and nuclear P65 protein levels were higher than that of ovariectomized group(P＜0.01).The apoptosis of ovariectomized plus E₂(10^-8M,10^-6M)group were higher than that of ovariectomized group(P＜0.05 and P＜0.01,respectively).3,The ERαand ERβprotein levels of ovariectomized group were lower than those of young group and sham-ovariectomized group.The ERαand ERβprotein levels of ovariectomized plus E₂(10^-11M,10^-10M,10^-8M)group were higher than those of ovariectomized group,while those of ovariectomized plus E₂(10^-6M) were lower than those of ovariectomized group(P＜0.01).4,The apoptosis of ovariectomized plus E₂(10^-10M) plus ICI182,780 group or ovariectomized plus E₂(10^-10M) plus PDTC group was not significantly different from that of ovariectomized group.It was the same with ovariectomized plus E₂(10^-10M) plus MPP group and ovariectomized plus E₂(10^-10M) group.Conclusions1,The ovariectomy of mice led to increased apoptosis rates of splenic T lymphocytes, decreased protein levels of ERα,ERβ,bcl-2 and decreased transcription activity of NF-κB.2,The effects of E₂ on the apoptosis of ovariectomized mice T lymphocytes were dependent on doses:physiological doses of E₂ inhibited while supraphysiological doses of E₂ exacerbated the apoptosis of ovariectomized mice T lymphocytes.3,Physiological doses of E₂ might act on mice T lymphocytes via ERβ.and NFκB signaling. Partâ…¢Mechanism of estradiol regulating apoptosis of human T lymphocytesObjectiveTo investigate the possible mechanisms of E₂ regulating the apoptosis of human T lymphocyte.MethodsJurkat cells in their exponential growth phase were divided into 9 groups:control group,E₂10^-10M treating 24 hours,E₂10^-8M treating 24 hours,E₂10^-6M treating 24 hours,E₂10^-10M treating 48 hours,E₂10^-8M treating 48 hours,E₂10^-6M treating 48 hours,E₂10^-8M plus ICI182,780(10^-7M) treating 48 hours,and E₂10^-8M plus MPP(10^-6M) treating 48 hours.The apoptosis of each group was detected by Flow Cytometry using Annexin V/PI;the protein expressions of ERα,ERβ,Fas,FasL and nuclear P65 were determined by Westernblot.Co-immunoprecipitation was applied to detect the direct binding of ERαor ERβwith NF-κB.Results1,The Jurkat cell apoptosis and Fas,FasL expressions in each E₂-treating group were significantly higher than those of control group;apoptosis rate increased as doses of E2 increased and treating time prolonged.2,Their exist intrinsic ERαand ERβprotein expressions in Jurkat cells;their expressions were significantly upleveled by E₂ treatment,and positively related to the doses of E₂.3,The apoptosis of E₂(10^-8M) plus ICI182,780(10^-7M) treating 48h group was significantly lower than that of E₂(10^-8M) group,but not significantly different from that of control group.The apoptosis of E₂(10^-8M) plus MPP(10^-6M) treating 48h group was not significantly different from that of E₂(10^-8M) group,but significantly higher than that of control group.4,The nuclear P65 protein level of E2 plus TNFαtreatment groups was significantly higher than that of TNFαonly treatment group and control group;the level was positively related to the doses of E₂ and treatment duration.5,Co-IP was performed using anti-P65 polyclonal antibody and Protein A-agarose added to Jurkat nuclear extractions,and P65 and ERβproteins were detected by Westernblot to co-exist in the product.Conclusions1,E₂ may induce the apoptosis and Fas,FasL expressions of Jurkat cell,its effect positively related to the dose and treating time of E₂.2,There is intrinsic co-expression of ERαand ERβon Jurkat cell,which can be upregulated by E₂,positively related to E₂ doses.3,E₂ may act as an apoptosis inducer of Jurkat cell via ERβ.4,E₂ can promote the transcriptive activity of NF-κB in Jurkat cell.The increased NF-κB activity may lead to increased apoptosis of Jurkat cell.NF-κB and ERβare directly binding together in Jurkat nucleus.E₂ may affect the transcriptive activity of NF-κB via ERβand further affect the fate of Jurkat cell.