Effects of Ovariectomy and 17β-Estradiol Replacement on the Activity of Dopamine D2 Receptors in the Selection of Macronutrients Carbohydrates, Lipids and Proteins in Females Rats

17β-estradiol modulates the activity of D2 receptors in the regulation of food intake and body weight. The functional lack of 17β-estradiol in postmenopausal women could create a dietary imbalance and cause body weight gain. This study aimed to better understand the interferences that could exist between 17β-estradiol, D2 receptors and the selection of carbohydrate, fat and protein consumption, as well as their consequences on body weight gain by using an animal model of the menopause. Ovariectomy exacerbates the consumption of foods rich in lipids. Thus confirming an inhibitory action of 17β-estradiol (E2) on the consumption of these types of foods. This consumption stimulates body weight gain, which is promoted by the high caloric content of these foods and not by the amount consumed. Our results showed a direct involvement of D2 receptors in food choice. This choice would be made according to the two (2) isoforms of the D2 receptors. The D2/BR isoform directs towards a high carbohydrate consumption, without causing a gain in body weight. While D2/SUL, promotes high fat food consumption, causing an increase in body weight. In women, 17β-estradiol modulates the activity ratio between these two D2 receptor isoforms to ensure energy and homeostatic balance, stabilizing food intake and body weight.

Study on Relationship between Ovulation Inducing Effect of Drug-Acupuncture and Endometrial Contents of Estradiol Receptor and Progesterone Receptor

Objective: To study the effect of Chineseherbal medicine for replenishing Kidney combined with acupuncture in treating anovulationand infertility, and the relationship betweenits ovulation inducing effect and endometrialcontents of estradiol receptor (ER) and progesterone receptor (PR). Methods: Twentynine cases were treated with replenishing Kidney drugs combined with acupuncture for 1～3months. Patients' ER and PR were measuredby immunohistochemical method. And pa- tients were classified according to PR contentinto PR positive group and mild PR positivegroup. Results: Fifteen cases of PR positivegroup, completed treatment for 45 cycles, among them, 40 cycles showed ovulation, theovulation rate being 88. 89 %. Ten in 14 cases, who complicated with infertility, becamepregnant, the pregnant rate being 71. 43%.While in 11 cases of Ph mild positive group, 9complicated with in fertility, completed treatment for 33 cycle, 10 cycles showed ovulation(30. 30 % ), and pregnant rate 22. 22 % (2/9). The difference between the two groupswas significant (P < 0. 01 ). Conclusion: Thereplenishing Kidney drugs combined withacupuncture treatment could result a good effect in treating infertility due to anovulation,especially on those with high endometrial PRcontent.

17β-Estradiol Regulates Cultured Immature Boar Sertoli Cell Proliferation via the cAMP-ERK1/2 Pathway and the Estrogen Receptor β

Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells via the estrogen receptor β （ERβ） and the cAMP-extracellular signal-regulated kinase （ERK1/2） pathway. Low levels （10-10-10-8 mol L-1） of 17β-estradiol increased cell number, but high levels （10-7-10-6 mol L-1） decreased it （P〈0.05）. Sertoli cell number began to recover for an additional 24 h in the medium without 17β-estradiol （10-6 mol L-l） （P〉0.05）. The effects of 17β-estradiol （10-9 mol L-1） peaked at the first 24 h （P〈0.05）. 17β-estradiol activated ERK1/2 from 5 min to 24 h, but the activiy of ERK1/2 began to decrease after 4 h. Both PD98059 and U0126, two ERK inhibitors, blocked cell division （P〈0.05）. 17β-estradiol （10-10-10-6 mol L-1） dose-dependently increased cAMP production （P 〈 0.05）, and both 17β-estradiol （10-9 mol L-1） and forskolin, which increases cAMP levels, induced cell proliferation and activated ERK1/2 （P〈 0.05）. Rp-cAMP, an antagonist of cAMP, blocked this 17β-estradiol activity （P〈 0.05）. Two estrogen receptor antagonists, ICI 182780 and ERβ antagonist （ERβAnt）, reduced Sertoli cell number, cAMP production and ERK1/2 activation （P〈 0.05）, but ERaAnt did not （P〉 0.05）. Therefore, 17β- estradiol mainly promotes pig Sertoli cell proliferation via ERβ to induce cAMP production and ERK activation to promote cell proliferation.

Effects of Ovariectomy and 17<i>β</i>-Estradiol Replacement on Dopamine D2 Receptors in Female Rats: Consequences on Sucrose, Alcohol, Water Intakes and Body Weight

Background: Mechanisms underlying overeating-induced obesity in post-menopausal woman include functional lack of 17β-estradiol dysregulating dopamine D2 receptors, thereby inducing food addiction, glucose craving or alcohol dependence through reward circuitry. This study aimed at further understanding 17β-estradiol and dopamine D2 receptors interferences in the etiology of woman obesity. Method: Seventy-two Wistar female rats weighing 200 - 205 g, individually-housed, were divided into non-ovariectomized control (C = 6 groups) and ovariectomized rats (OVX = 6 groups) which were concurrently subjected to the following treatments: Non-drug-treated (DMSO vehicle), 17β-estradiol (E2, 5 μg/kg, s.c.), sulpiride (SUL, 20 mg/kg, i.p.), bromocriptine (BR, 0.1 mg/kg, i.p.), E2 + SUL or E2 + BR, designating the 6 constitutive groups of either control or ovariectomy. Within each experimental group, consumption of different solutions (10% alcohol, 10% sucrose and water) as well as food intake and body weight were daily measured, for 10 consecutive days. Results: This study indicated that D2S was a specific inducer of alcohol and food intakes, but reduced sugar consumption. In addition, 17β- estradiol regulated the body weight set point, modulating D2S functions towards increased food intake at lower weights and decreased food intake at higher weights. D2S met the slow genomic actions induced by 17β-estradiol. Conversely, D2L inhibited alcohol and food intakes, but induced specifically sugar consumption, thereby regulating blood glucose levels and promoting energy expenditure in reducing body weight. Indeed, 17β-estradiol exerted a tonic inhibition on D2L which was released by OVX, exacerbating sugar intake and increasing body weight. D2L mediated the rapid metabolic effects of 17β-estradiol. Conclusion: Our results supported physiological data reporting that activation of the mostly expressed presynaptically D2S-class autoreceptors decreased dopamine release stimulating food intake, whereas activation of the predominantly postsynaptic isoform D2L receptors increased dopamine activity inhibiting food intake. Our studies indicated that 17β-estradiol acted on the two types of D2 receptors showing opposite functions to equilibrate energy intake vs. expenditure for weight set point regulation. Our data also supported biochemical findings reporting that 17β-estradiol induced D2 genes transcriptional regulation, thereby involving both types of D2 receptors in the etiology of obesity. The combined dysregulated effects of D2L and D2S receptors, as 17β-estradiol was lacking, would be causal factors underlying the etiology of obesity.

Increased Cellular Invasion and Proliferation via Estrogen Receptor after 17-<i>β</i>-Estradiol Treatment in Breast Cancer Cells Using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC)

17-β-estradiol (estrogen) is a steroid hormone important to human development;however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and activate the estrogen receptor (ER) and initiate gene transcription. Currently, estrogen mechanisms of action are classified as genomic and non-genomic and occur in an ER-dependent and ER-independent manner. In this study, we examine estrogen signaling pathways, by measuring changes in protein expression as a function of time of exposure to estrogen in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. Using a robust experimental design utilizing isotopic labeling, two-dimensional LC-MS, and bioinformatics analysis, we report genomic and non-genomic ER regulated estrogen responsive proteins. We find a little over 200 proteins differentially expressed after estrogen treatment. Cell proliferation, transcription, actin filament capping and cell to cell signaling are significantly enriched in the MCF-7 cell line alone. Translational elongation and proteolysis are enriched in both cell lines. Subsets of the proteins presented in this study are for the first time directly associated with estrogen signaling in mammary carcinoma cells. We find that estrogen affected the expression of proteins involved in numerous processes that are related to tumorigenesis such as increased cellular division and invasion in an ER-dependent manner. Moreover, we identified negative regulation of apoptosis as a non-genomic process of estrogen. This study complements gene expression studies and highlights the need for both genomic and proteomic analyses in unraveling the complex mechanisms by which estrogen affects progression of breast cancer.

Collagen types in relation to expression of estradiol and progesterone receptors in equine endometrial fibrosis

The aim of this study was to determine the influence of collagen I and III on the expression of estrogen and progesterone receptors in equine endometrial fibrosis. A total of 25 crossbred mares were studies. Two endometrial samples were collected from each mare,1 inthe estrus and1 inthe diestrus phase. The samples were classified according to histological changes. Collagen was typed and quantified using the picrosirius red histochemical technique, and steroid receptors were identified by immunohistochemistry. The results showed a predominance of collagen type III in all the endometrial samples. The expression of estrogen (RE2) and progesterone (RP4) receptors varied according to the estrous cycle. RE2 and RP4 expression varied in the estrus and diestrus phases;there was no influence of collagen I or II on receptor expression.

Effects of 17<i>β</i>-Estradiol on Dopamine D2 Receptors in Thiamine-Deficient Female Rats: Consequences on Sucrose, Alcohol, Water Intakes and Body Weight

Our previous studies showed that 17β-estradiol (E2) modulated dopamine D2 receptor in regulating body weight set-point. The aim of this study was to understand whether thiamine deficiency influenced the E2 modulation on dopamine D2 receptors, using bromocriptine mesylate (BR) and sulpiride (SUL) as selective central dopamine-D2 receptors agonist and antagonist respectively. We studied the E2-dopamine D2 receptors interferences in a 10-day thiamine-deficient female rats for which consumptions of water, sugar, alcohol and food were daily-recorded and their consequences on body weights assessed. Our results showed that the volume of water daily ingested doubled in thiamine-deficient female rats (OXT), while sugar and alcohol consumptions collapsed with decreased weight and food consumption. On the one hand, thiamine potentiated D2/BR activity (bromocriptine-activated D2 receptors) to induce sugar intake and inhibited the same D2/BR receptors to induce water intake. On the other hand, thiamine promoted D2/SUL receptors (sulpiride-inhibited D2 receptors) for enhanced alcohol intake, increased food consumption and weight gain. Taking together, thiamine modulated the actions of 17β-estradiol on both D2/BR and D2/SUL receptors activities.

17β-Estradiol Regulates SKP2 Expression in Cultured Immature Boar Sertoli Cells Mainly via Estrogen Receptor β,cAMP-PKA and ERK1/2

Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 （SKP2） plays a central role in mammalian cell cycle progression. The objective of this study was to determine whether 17β-estradiol can regulate the expression of SKP2, and the Sertoli cell cycle, via estrogen receptor β （ERβ）, the cyclic adenosine monophosphate （cAMP）-protein kinase A （PKA） and extracellular signal-regulated kinase （ERK1/2） pathway. When cultured immature boar Sertoli cells were treated with 17β-estradiol, a time-dependent increase in SKP2 mRNA and protein level was observed by real-time PCR and Western blot, and 17β-estradiol activity peaked at 30 min. Treatment with ICI182780 and ERβ antagonist reduced 17β-estradiol-induced expression of SKP2 and proliferating cell nuclear antigen （PCNA）, while increasing the protein concentration of p27kip1. However, the effect of ERa antagonist on these parameters was lower than that of ICI 182780 and ERβ. Forskolin had a similar effect as 17β-estradiol on the expression of SKP2, PCNA and p27kip1, Rp-cAMP, H-89 and U0126 treatment reduced 17β-estradiol-induced changes, while H-89 also inhibited ERK1/2 activation. Therefore, 17β-estradiol mainly regulates SKP2 mRNA and protein expression via ERβ-cAMP-PKA and ERK1/2 activation. SKP2 and PCNA expression were positively correlated, while increased SKP2 expression likely resulted in p27kip1 degradation.

槲皮素通过雌激素受体下调长非编码RNA MALAT-1并发挥抗乳腺癌的作用机制

目的探索槲皮素抑制乳腺癌细胞恶性生物学行为的分子机制。方法以乳腺癌细胞系MCF-7和MB231作为研究对象,用慢病毒LV-ERα、LV-MALAT-1转染乳腺癌MB231细胞和MCF7细胞,RT-qPCR检测MALAT-1表达,Western blot检测肿瘤细胞中ERα蛋白表达,CCK-8细胞实验、平板克隆形成实验检测细胞增殖能力,PI染色法检测细胞周期,通过mRFP-GFP-LC3荧光双标腺病毒转染检测自噬水平,观察槲皮素和雌二醇对乳腺癌细胞增殖能力的影响。结果17β-雌二醇(E2)可以促进乳腺癌细胞MCF-7的增殖,而5μmol·L^(-1)槲皮素可以明显逆转E2对增殖的促进作用(P<0.05)。槲皮素对不表达雌激素受体α(estrogen receptor-α,ERα)的乳腺癌细胞MB231不表现抑制作用;而过表达ERα后,槲皮素则抑制了E2对MB231-ERα的促进作用。同时槲皮素可以抑制E2激活的MALAT-1表达;并且其抑制作用被过表达MALAT-1所逆转,包括细胞增殖,细胞周期进展以及克隆形成能力。结论槲皮素依赖于ERα的表达对乳腺癌的增殖等恶性行为起抑制作用,并且很可能是通过抑制MALAT-1的表达来发挥作用。

柴胡四物汤对围绝经期综合征模型大鼠血清E_(2)水平、子宫形态及ERα表达的影响

目的:通过观察柴胡四物汤对围绝经期综合征模型大鼠血清E_(2)水平、子宫形态及子宫、下丘脑ERα表达的影响,初步探讨柴胡四物汤治疗围绝经期综合征的作用机制。方法:将75只雌性SD大鼠随机分为假手术组、模型组、西药组、柴胡四物汤高、中、低剂量组,采用双侧卵巢切除法造模,选取符合条件的60只大鼠纳入实验,每组10只。从术后第14天开始,连续给药28 d。给药结束后,观察各组大鼠子宫指数及形态学的改变;采用ELISA法检测血清E_(2)水平;采用RT-PCR法及Western Blot法检测子宫、下丘脑ERα的表达水平。结果:与假手术组比较,模型组血清E_(2)水平明显下降(P<0.01),子宫湿重、子宫指数显著下降(P<0.01),子宫体积明显缩小,宫腔变窄,腺体数量较少,内膜及肌层萎缩,子宫ERαmRNA及蛋白的表达水平明显下降(P<0.01),下丘脑ERαmRNA及蛋白的表达水平显著升高(P<0.01)。与模型组比较,西药组、柴胡四物汤高、中剂量组血清E_(2)水平明显升高(P<0.01),西药组、柴胡四物汤高、中、低剂量组的子宫指数明显上升(P<0.01),子宫腺体数量增加,内膜及肌层的萎缩程度有所缓解,西药组、柴胡四物汤高、中剂量组子宫ERαmRNA表达水平增加(P<0.01),西药组、柴胡四物汤高剂量组子宫ERα蛋白表达水平增加(P<0.01),西药组、柴胡四物汤高、中、低剂量组下丘脑ERαmRNA及蛋白的表达水平降低(P<0.01)。结论:柴胡四物汤可改善围绝经期的各类症状,其作用机制可能与升高围绝经期综合征模型大鼠血清E_(2)水平,改善子宫指数及萎缩程度,提高子宫ERα的表达并同时下调下丘脑ERα的表达有关。

卵形鲳鲹促性腺激素β亚基的克隆鉴定及其受雌二醇的调控

促性腺激素(GtH,gonadotropin hormone)是垂体合成和分泌的一类重要糖蛋白激素,包括促卵泡生成素(Fsh,follicle stimulating hormone)和促黄体生成素(Lh,luteinizing hormone),在促进性腺发育和成熟过程中发挥重要作用。本研究成功克隆了卵形鲳鲹(Trachinotus ovatus)fshβ和lhβ基因的开放阅读框(ORF,open reading frame)序列,fshβORF长度为363 bp,lhβORF长度为447 bp。通过荧光定量PCR技术(qPCR,quantitative realtime PCR)检测,发现fshβ和lhβ基因在卵形鲳鲹的下丘脑-垂体-性腺轴(HPG,hypothalamus-pituitary-gonadal axis)显著表达,在垂体中的表达水平最高,其次是下丘脑和性腺。此外,通过体外实验评估17β-雌二醇(E2,17β-estradiol)对卵形鲳鲹垂体组织中fshβ和lhβ基因表达的影响。结果显示,10μmol/L E2处理6 h后,GtHβ亚基基因的表达被极显著抑制。最后,使用雌激素受体(ER,estrogen receptor)拮抗剂体外孵育卵形鲳鲹垂体组织,发现广谱性拮抗剂Fulvestrant、ERβ拮抗剂Cyclofenil和ERα拮抗剂MPP(MPP dihydrochloride)均能够消除E2对GtHβ亚基基因表达的抑制作用。综上所述,研究结果表明E2对卵形鲳鲹GtH的分泌具有负反馈调节作用,为卵形鲳鲹生殖调控机制提供了有价值的见解。

The protective effect of 17 beta-estradiol on oxygen-induced retinopathy and its relation with the changes of malondialdehyde

Objective： Retinopathy of prematurity is becoming obvious with the improvement of neonatal ambulance. However there is still not a good treatment. The present study is to observe the effect of 17 beta-estradiol （E2） on oxygen-induced retinopathy （OIR）, and explore the relationship between the changes of avascular area and malondialdehyde （MDA） in retina. Methods： Newborn oxygen-exposed mice underwent subcutaneous injections of different dose of E2 （0.1 μg, 1.0 μg, 10.0 μg ）, tamoxifen or phosphate buffered saline （PBS; controls）everyday from post-natal day （p）7 to p17. At p17, retinal flat mounts were scored for the percentage of avascular/total retinal area, and pathological changes during revascularization. The MDA concentration in the retina was determined also. In the most efficacious E2 group （10.0 μg）, 100.0 μg tamoxifen was also administered, and the percentage of capillary-free/total retinal area determined, and the retinal malondialdehyde concentration assayed. Results： The mean percentage of capillary-free area over total retinal area was 0（PBS, in room air）, 34.197±1.301（PBS, in hyperoxia）, 23.685±0.407 （0.1 μg E2）, 14.648±0.355 （1.0 μg E2）, 4.693±0.450 （10.0 μg E2） and 32.240±0.654 （10.0 μg E2 ＋100.0 μg tamoxifen）. The difference was significant （F = 2778.759, P 〈 0.01）, and the difference between any two groups were also significant （all P value were less than 0.01）. The predilection of tufts and clusters during revascularization was mainly aggregated in zones 2 and 3, but the difference of retinal neovascular clusters and tufts in fourth zone among different groups were significant [clusters （F = 44.719, P 〈 0.01） vs tufts （F = 39.997,P 〈 0.01）]. The mean MDA concentration were 0.711 ±0.037（PBS, in room air）, 2.084±0.066 （PBS, in hyperoxia）, 1.829±0.091（0.1 μg E2）, 1.152± 0.067（1.0 μg E2）, 0.796 ±0.027（10.0 μg E2）, 1.988 ± 0.049（10.0μg E2 ＋100.0 μg tamoxifen） （F = 628.103, P 〈 0.01）. The difference between any two groups were also significant （all P value were less than 0.05）. The close relation between the percentage of avascular/total retinal area and MDA concentration was also verified （r = 0.981, P 〈 0.01）. Conclusion： Oxidative stress responses play a pivotal role in OIR, by means of receptor pathway. E2 can alleviate oxidative stress reaction, and thus ameliorate the severity of oxygen induced retinopathy.

Xenoestrogens challenge 17β-estradiol protective effects in colon cancer

Several epidemiological,cellular,and molecular studies demonstrate the role of environmental chemicals with endocrine disrupting activities,typical of Westernized societies,in the pathogenesis of numerous diseases including cancer.Nonetheless this information,the design and execution of studies on endocrine disruptors are not yet cognizant that the specific actions of individual hormones often change with development and ageing,they may be different in males and females and may be mediated by different receptors isoforms expressed in different tissues or at different life stages.These statements are particularly true when assessing the hazard of endocrine disruptors against 17β-estradiol(E2)actions in that this hormone is crucial determinant of sexrelated differences in anatomical,physiological,and behavioral traits which characterize male and female physiology.Moreover,E2 is also involved in carcinogenesis.The oncogenic effects of E2 have been investigated extensively in breast and ovarian cancers where hormone-receptor modulators are now an integral part of targeted treatment.Little is known about the E2preventive signalling in colorectal cancer,although this disease is more common in men than women,the difference being more striking amongst pre-menopausal women and age-matched men.This review aims to dissect the role and action mechanisms of E2 in colorectal cancer evaluating the ability of estrogen disruptors(i.e.,xenoestrogens)in impair these E2 actions.Data discussed here lead to define the possible role of xenoestrogens in the impairment and/or activation of E2signals important for colorectal cancer prevention.

Effects of estrogen on growth of breast cancer cells and expression of p185 and EGF receptor

In order to study the mechanism of estrogen and other events involved in the development of breast cancer,we observed the cell growth as well as the expression of p185 and epidermal growth factor(EGF) receptor in human breast cancer cell line SK-BR-3 in the presence of estradiol(E2) using flow cytometry. The results showed that E2 could act to expedite the proliferation of breast cancer cells and promote DNA synthesis , and that the percentage in S phase rose significantly (E2 group 26. 7?2. 5,control group 21. 2?2.1, P<0. 05). Under E2 action,p185 presented a reduced expression (E2 35?5. 6, control 61?13.1, P<0. 05) while the EGF receptor expression was greatly enhanced (E, 39?6. 9, control 21?5. 4,P<0. 05),suggesting that EGF may play a more important role in early development of breast cancer.

Studies of the Expression of Estrogen Receptor Gene in the Rat Uterus during the Estrous Cycle and Periimplantational Period

The correlation or serum estradiol concentrstion and uterine estrogen receptor (ER) gene expression (ERn and ERc quantitated by Dextrsn Coat Charcoal assay and ER mRNA by Northern blotting) was studied during the rat estrous cycle and early Pregnant stage (dl-d10). The ER gone expression was up - regulated by estrogen and the levels of ER mRNA synchronized with the changes of ER protein, suggesting that estrogen influenced the trsnscriPtional step of the ER gene. Post-coitum ER expression increased with the serum estrsdiol progressively, reached a peak on d4-ds (Just before implantation), but drastically dropped to the nadir on d6-d7 (during implantation) and then recovered. It was of interest to discover that ER mRNA level in the nonimplantstion sites (NIS) of uterus was much higher than that in the implantstion sites (IS).

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1（NMDAR1） plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D（-）-2-amino-5-phosphonopentanoic acid（AP-5）,or both once daily for 15 days.Compared with injured drug na？ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity（as assessed by immunohistochemistry） and protein（as determined by western blot assay） in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

雌二醇通过Calpain 2有限水解integrinβ4促进乳腺癌SK-Br3细胞迁移浸润的相关机制

目的研究雌二醇通过calpain 2/integrinβ4促进人表皮生长因子受体(Her2)阳性的乳腺癌SK-Br3细胞迁移和浸润的相关机制。方法常规培养乳腺癌SK-Br3细胞,calpain 2的特异性抑制剂或雌二醇(E2)处理细胞,并对汇合度为60%(低密度)和90%(高密度)的细胞进行培养;通过细胞划痕实验检测SK-Br3细胞的迁移能力,Transwell-Matrigel实验检测SK-Br3细胞的浸润能力,免疫细胞化学技术和激光共聚焦扫描显微镜观察integrinβ4和Her2的表达与共定位,免疫共沉淀技术检测integrinβ4和Her2的相互作用,Western blot技术检测相关蛋白或蛋白片段的表达及蛋白磷酸化水平。结果划痕和浸润实验显示,相对于对照组,Calpain2抑制剂处理的SK-Br3细胞迁移和浸润能力减弱(P<0.05);免疫细胞化学成像显示,Her2和integrinβ4的共定位主要在细胞膜上,在胞浆也有少量共定位,用calpain inhibitorⅣ处理后,Her2和integrinβ4的表达减少,共定位也明显减少;免疫共沉淀实验发现,在Her2免疫沉淀物中可检测到integrinβ4,在integrinβ4免疫沉淀物中可检测到Her2;给予calpain inhibitorⅣ处理,可以减少Her2和integrinβ4之间的免疫共沉淀;Western blot检测发现,高密度培养乳腺癌SK-Br3细胞中的integrinβ4比低密度培养的水解程度高;在高密度培养条件下,E2可以促进这些integrinβ4水解片段的产生,上调calpain 2的表达,也伴随着integrinβ4水解程度的增加,同时使Src的Y418位点的磷酸化水平增高。结论高密度培养条件下,E2可通过上调和激活calpain2,促进integrinβ4的有限水解,促进integrinβ4和Her2的相互作用,磷酸化激活Src,促进SK-Br3细胞的迁移和浸润。

雌二醇经雌激素受体调控分拣蛋白相关受体A对小鼠海马神经元树突棘密度和突触蛋白表达的影响

目的探讨雌二醇(E_(2))调控分拣蛋白相关受体A(SorLA)表达对小鼠海马神经元树突棘密度和突触蛋白表达的影响及机制。方法32只C57BL/6雌性小鼠随机分为假手术(sham)组、卵巢切除(OVX)组、卵巢切除+E_(2)组(OVX+E_(2))组、卵巢切除+氟维司琼(Fu)+E_(2)组(OVX+Fu+E_(2))组,通过Western blotting和免疫组织化学染色检测小鼠海马CA1区SorLA表达;40只C57BL/6雌性小鼠随机分为假手术+空白病毒(sham+NC-sgRNA)组、卵巢切除+空白病毒(OVX+NC-sgRNA)组、卵巢切除+空白病毒+E_(2)(OVX+NC-sgRNA+E_(2))组、卵巢切除+SorLA敲降病毒+E_(2)组(OVX+Sorl1-sgRNA+E_(2))组,通过高尔基染色检测小鼠海马CA1区神经元树突棘密度;通过Western blotting和免疫组织化学染色检测小鼠海马CA1区突触后致密蛋白95(PSD-95)和突触蛋白1(SYN1)表达。结果与sham组相比,和OVX组和OVX+Fu+E_(2)组SorLA蛋白表达显著降低(P<0.05),和OVX+E_(2)组差异无显著性(P>0.05);与sham+NC-sgRNA组相比,OVX+NC-sgRNA组和OVX+Sorl1-sgRNA+E_(2)组树突棘密度和PSD-95、SYN1表达显著降低(P<0.05),与OVX+NC-sgRNA+E_(2)组差异无显著性(P>0.05)。结论E_(2)可通过雌激素受体上调SorLA表达,进而增加小鼠海马神经元树突棘密度和突触蛋白表达。

循经通络推拿手法结合药物对乳腺增生大鼠雌孕激素及其受体水平的调节作用

目的观察循经通络推拿手法对乳腺增生(MGH)模型大鼠的治疗作用,并对比单纯推拿手法、药物治疗、手法联合药物治疗的疗效,探讨其调节大鼠性激素水平发挥治疗作用的机制。方法将50只SD大鼠随机分为对照组、模型组、手法组、药物组、药物加手法组共5组,每组10只。除对照组外,其余各组连续肌肉注射苯甲酸雌二醇(0.5 mL/kg)25 d,随后连续肌肉注射黄体酮(5 mg/kg)5 d建立乳腺增生模型。手法组予以循经通络推拿手法治疗,药物组给予三苯氧胺(0.5 mg/kg)灌胃治疗,药物加手法组同时给予循经通络推拿和药物灌胃,连续治疗30 d。观察各组大鼠一般体征,测量大鼠第2对左右乳头直径和乳头高度,HE染色观察乳腺组织病理变化,ELISA法检测血清中雌二醇(E_(2))和孕酮(P)含量,Western-blot检测乳腺组织中雌激素受体α(ERα)、孕激素受体(PR)的蛋白表达。结果与空白组比较,模型组大鼠乳头高度和直径显著增大(P<0.05),乳腺组织增生病变明显,血清中E_(2)含量显著升高,P含量显著降低(P<0.05),明显上调了乳腺组织中ERα的蛋白表达,下调了PR的表达(P<0.05)。手法组、药物组、药物加手法组大鼠乳头直径、乳头高度均较模型组显著减小(P<0.05);药物加手法组大鼠乳腺组织形态明显改善,增生程度明显减轻,手法组和药物组大鼠乳腺组织病理形态较模型组改善不明显;药物组和药物加手法组大鼠E_(2)含量及ERα的蛋白表达明显降低,P含量和PR的表达显著增高(P<0.05),其中药物加手法组的改善效果更佳,手法组大鼠血清和乳腺组织中E_(2)、P的含量及ERα、PR的蛋白表达较模型组无明显差异(P>0.05)。结论循经通络推拿手法结合三苯氧胺对乳腺增生大鼠的治疗作用优于单纯手法和单纯药物治疗,其作用机制可能与调节大鼠体内雌孕激素及其受体水平相关。

化瘀复膜方在宫腔粘连术后患者中的应用效果及对雌孕激素受体及血流参数的影响

目的:探讨化瘀复膜方对宫腔粘连术后患者雌孕激素受体及血流参数的影响。方法:选取2018年1月-2020年8月于河南省中医院治疗的84例宫腔粘连术后患者,按随机数字表法分为对照组和观察组,每组42例。两组均行宫腔镜下粘连电切术,术后对照组口服戊酸雌二醇片、黄体酮胶囊,观察组在对照组基础上联合化瘀复膜方(灌肠)。观察两组总有效率与月经恢复率、雌孕激素受体表达情况、子宫内膜厚度与动脉血流。结果:观察组总有效率、月经恢复率高于对照组,雌激素受体、孕激素受体、细胞增殖蛋白Ki67水平均高于对照组(P<0.05),子宫内膜厚度大于对照组,动脉血流收缩末期峰值与舒张末期峰值比值、搏动指数、阻力指数均低于对照组(P<0.05)。结论:化瘀复膜方灌肠可改善宫腔粘连术后患者子宫血流情况并促进子宫内膜增厚,有助于促进月经恢复和雌孕激素受体的表达,提高治疗效果。