Effects Of Raloxifene On Estrogen Receptor α And β,PPARγ MRNA Of Bone And Uterus Of Ovariectomized Female Rats

Background:Selective estrogen receptor modulators(SERMs) win people's attention for their selective estrogenic agonist/antagonist effects on various estrogen target tissues depending on different cell types and hormonal status.Raloxifene(RAL),the second generation of SERM,is being used clinically by its agonist effect in bone and antagonist effects in uterus and breast.But its selective mechanisms on bone and uterus are still not well defined,such as the effects on the expressions of estrogen receptorαandβwhich are the study basis of tissue-specific actions of SERM,need to be clarified.In addition,effects of peroxisome proliferator activated receptor gamma(PPARγ) on the osteoporosis are becoming experimental focus during these years and the study on PPARγmay help to understand the tissue-specific actions of SERM.Objective:To study the selective actions of raloxifene on bone and uterus of ovariectomized female rats and its effects on the expressions of estrogen receptorαandβ,PPARγmRNA of bone and uterine endometrium,thus to explore the tissue-specific mechanisms of raloxifene.Methods:Forty four-month-old Sprague-Dawley female rats were randomised into four groups(n=10):sham-operated group(SHAM),ovariectomized group(OVX),estradiol valerate treated group(OVX+E₂),raloxifene treated group(OVX+RAL).The latter three groups were taken the bilateral ovariectomy operation and the SHAM group was subjected to the same general surgical procedure except that the ovaries were not excised.Four weeks after the operation,Sham group and control OVX were administrated with distilled water(1ml/d),whereas the other groups received estradiol valerate(800ug/kg·d) and raloxifene(1mg/kg·d) by gavage respectively for three months.3 months later,all rats were sacrificed by carotid artery exsangingnation. Vertebrae L₆ and right femur shaft were excised,stripped of fat and connective tissue, used for RT-PCR analysis for ER-α,ER-β,PPARγmRNA expression.Vertebrae L_1-5 and left femur were removed to take the bone mineral density analysis and L₄ was excised to make hematoxylin and eosin-stained sections for bone histomorphometry evaluation under light microscope(100×).The uterus was removed,stripped of fat and connective tissue.The uteri were divided,one half was attained to make pathological sections for endometrium pathomorphology and histomorphometry for assaying the thickness of uterine luminal epithelium,endometrial stroma,endometrium and cross-sectional tissue area;endometrium samples were scraped off from the other half to be used for RT-PCR analysis for ER-αand ER-βmRNA expression.Results:1.Bone mineral density(BMD):femur and lumbar vertebral BMD of the OVX group are lower than those of SHAM group(lumbar vertebrae:P＜0.01;femur:P＜0.05). Those of OVX+E₂ group and OVX+RAL group are higher than the OVX group(lumbar vertebrae:P＜0.01;femur:P＜0.05).2.Bone histomorphometry evaluation under light microscope:for OVX group,the bone trabecula lie spongic and the medullary cavity is infiltrated with great amount of adipocytes.The amount of bone trabecula of OVX+E₂ and OVX+RAL group is higher than OVX group and the amount of adipocytes in the medullary cavity is lower in these two groups than OVX group.The bone trabecula lie compact in OVX+E₂ and OVX+RAL group.3.The level of ER-α,ER-βmRNA on bone:the levels of the OVX group on femur and lumbar vertebrae are lower than those of SHAM group(ER-α:P＜0.05;ER-β:P＜0.01).The levels of OVX+E₂ group are higher than those of OVX group(P＜0.05).There are no significant differences of the levels of ER-αmRNA on femur and lumbar vertebrae between the OVX+RAL group and the OVX group,but the levels of ER-βmRNA of OVX+RAL group are higher than those of the OVX group(P＜0.05).4.The level of PPARγmRNA on bone:the levels of the OVX group on femur and lumbar vertebrae are higher than those of SHAM group(femur:P＜0.01;lumbar vertebrae:P＜0.05).The levels of OVX+E₂ and OVX+RAL groups are lower than the OVX group(P＜0.01).5.Uterine histomorphometry assay:all the results of OVX group are the lowest(compared with SHAM group:except for the cross-sectional tissue area:P＜0.05;the others:P＜0.01).All the results of OVX+E₂ group are higher than the OVX group(except for the cross-sectional tissue area:P＜0.05;the others:P＜0.01).All the results of the OVX+RAL group have no significant differences compared with the OVX group,and are lower than the OVX+E₂ group(P＜0.01).6.The level of ER-α,ER-βmRNA on endometrium:The level of ER-αmRNA of the OVX+E₂ group is the highest(compared with the OVX group:P＜0.01),and that of the OVX group is the lowest(compared with SHAM group:P＜0.01).That of the OVX+RAL group has no significant difference compared with the OVX group,and is lower than the OVX+E₂ group(P＜0.01).The level of ER-βmRNA only could be detected in the SHAM and the OVX+E₂ groups.Conclusions:1.Both ER-αand ER-βexpress on cortical and cancellous bone.Raloxifene and estrogen can increase the femur and lumbar vertebral BMD of the OVX female rats.Estrogen may exert its protective effects on bone by ER-αand ER-β. Raloxifene may exert its protective effects on bone mainly by ER-βand has little effect on ER-αon bone.2.The increase in marrow adipogenesis is associated with osteopenia in postmenopausal osteoporosis.The mechanisms may include PPARγpathway. Both raloxifene and estrogen can decrease the expression of PPARγmRNA on femur and vertebrae and bone marrow adipogenesis,that could be one of the mechanisms of their effects on increasing bone volume.3.ER-αis the main estrogen receptor on endometrium.Raloxifene performs anti-estrogen effects on the endometrium of the OVX rats mainly by ER-αand has little effect on the expression of ERβon the endometrium.