Behavioural Brain Research 359 (2019) 845–852 Contents lists available at ScienceDirect Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr G-1 exhibit antidepressant effect, increase of hippocampal ERs expression and improve hippocampal redox status in aged female rats T Jing Wanga, Rui Yua,1, Qiu-Qin Hana,1, Hui-Jie Huanga,1, Ya-Lin Wanga,1, Hao-Yuan Lia,b, ⁎ ⁎ Hui-Mei Wanga,b, Xiao-Rong Chena,b, Shu-Lan Mab, , Jin Yua, a Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Shanghai Medical College, Fudan University, Shanghai, China b Experimental Teaching Center, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China ARTICLE INFO ABSTRACT Keywords: Oestradiol Depression Estrogen receptors GPER Mitochondrial protection Postmenopausal depression has been shown to be related to the reduction of ovarian hormones produced as a woman transitions from a menopausal to a post-menopausal stage. What remains to be known is which type of estrogen receptor plays a key role in estrogen neuroprotection, a process that may be mediated by potentiating brain mitochondrial function and inhibiting mitochondria-associated apoptosis. In order to better imitate the condition of postmenopause, we conducted our research on aged female rats. Plasma estrogen levels declined significantly in ovariectomized rats and 16-month-old female rats, while anxiety and depression-like behavior increase. Moreover, ERα, ERβ, GPER, Bcl2 and UCP2 expression decreased significantly in hippocampus in female rats following ovariectomy. In our study, the anxiety and depression-like behavior in aged female rats were significantly relieved after the treatment of G-1, the GPER agonist. Furthermore, G-1 could reverse the reduction of ERα, ERβ, GPER, Bcl2 and UCP2 expression within the hippocampus. Mitochondrial JC-1 staining indicated that mitochondrial membrane potential increased after G-1 treatment. In addition, total antioxidant capacity (TAC) and superoxide dismutase activity (SOD) were found to be elevated in aged female rats following G-1 treatment. Taken together, estrogen receptors, especially GPER, may activate anti-apoptotic signaling and accelerate mitochondrial function. Therefore, GPER could be the potential therapeutic target for estrogen deficiency-related affective disorders. 1. Introduction Menopausal and postmenopausal women have an increased frequency of developing mood and/or sleep disorders. They exhibit deterioration of mood, psychomotor retardation, marked decrease in interests, uneasiness, anxiety, sleep disorders, impaired concentration and memory [1–3]. These changes can be observed in both aging and estrogen deficiency. A recent study confirmed the serious risk of major depression during the menopausal transition. Researchers concluded that the risk of major depression is greater in women during and after the menopausal transition than when they are pre-menopausal [4]. Clinical hormone replacement therapy (HRT) has been prescribed for menopausal/climacteric syndromes, and putative antidepressant effects of HRT have been reported [5–13]. For these reasons, a number of women who experience depressive symptoms or are diagnosed with major depressive disorder during or after menopause may experience reprieve from these symptoms by using HRT therapy [14]. However, the use of female sex hormones has limitations due to the risk of developing serious side effects, including blood clots, embolism, heart attack, stroke and cancer [15–20]. Due to the severity of these side effects, the clinical hormone replacement therapy is severely handcuffed in being implemented as a treatment to help improve global cognitive function in postmenopausal women aged 65 years or older [15]. Studies have hinted however, that estrogen may exhibit a protective effect on cognition when initiated immediately following menopause, but the effect appeared to be lost in older women. Due to these limitations of HRT, researchers are looking for other effective treatment regimens for postmenopausal depression with less global influence on the body system. As a novel estrogen receptor, G protein-coupled estrogen receptor (GPER, formerly known as GPR30) is widely distributed and has numerous physiological and pathophysiological functions in central Corresponding authors. E-mail addresses: slma@fudan.edu.cn (S.-L. Ma), yujin@shmu.edu.cn (J. Yu). 1 These author contributed equally to this work. ⁎ https://doi.org/10.1016/j.bbr.2018.07.017 Received 22 April 2018; Received in revised form 9 July 2018; Accepted 20 July 2018 Available online 21 July 2018 0166-4328/ © 2018 Elsevier B.V. All rights reserved. Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. nervous system, especially in the hippocampus and cortex [21–24]. In cellular stress models and ovariectomy animal experiments, GPER agonist G-1 exhibited protective effects against vascular injury, cardiotoxicity and white matter injury by altering the oxidative and antioxidative parameters [25,26]. Researchers have found that G-1 inhibits the opening of mitochondrial permeability transition pore and improves cardiac function via actions in the MAPK pathway [27]. In addition, a recent study found the similar results in that GPER agonists enhanced cardiac mitochondrial function and biogenesis that had been impaired by ovariectomy [28]. Another recent study also suggested that G-1 could induce a cardioprotective effect in aged OVX female animals [29]. Taken together, GPER might be a potential therapeutic target for neuroprotection and oxidative stress [30]. Besides, the activation of GPER was found to exert anxiolytic and neuroprotective effects in ovariectomized mice [31–34]. A study recently published in Journal of Neuroscience showed that GPER in the dorsal hippocampus has an effect on regulating object recognition and spatial memory through novel pathways from normal estrogen receptors such as ERα and ERβ [35]. In conclusion, the literature hints that GPER could be a potential target in postmenopausal or elderly women and may help ameliorate depressive and anxiolytic symptoms. As we previously declared, hormone replacement therapy (HRT) has become routine in abrogating the negative effects associated with the decline in circulating 17β-estradiol (E2) in women postmenopause. Many studies agree that the neuroprotective effect of HRT is not as strong in elderly females compared to younger females [36–39]. The lack of protective response to HRT in older females may be due to the extended period of estrogen deficiency causing a less than favorable response to subsequent HRT. However, a link between pathological changes in brain and a lack of response to HRT has not yet been verified. It is also unknown whether estrogen receptor expression is altered in elderly individuals. Several studies have suggested that the expression of ERα decreases in correlation to age in rodents and estradiol has no effect changing the expression of ERα in the brain, which may be related to the lack of neuroprotection effect to HRT observed in elderly women [40–42]. Likewise, researchers found the expression and alternative slicing of ERβ to be altered with increased age. Unlike ERα, E2 could reverse the ERβ expression in aging rodents [43–47]. So far, agerelated difference in GPER expression in the brain has no yet been defined. Some studies have hinted that E2′s antidepressant-like effects may involve actions at ERβ, and that positive modulation of ERβ function may provide a novel treatment for affective disorders [48–51]. Another interesting study also revealed that targeting ERβ or G-protein coupled receptor 30 may be a strategy to permit beneficial effects of estrogen without its deleterious effect on selective serotonin reuptake inhibitor efficacy [51]. Therefore, in this study, we focus on the role of GPER in regulating expression of ERs including itself, as well as the potential protective effects of E2, the GPER-selective agonist G-1, and ERβ-selective agonist DPN on hippocampal mitochondria and postmenopausal depression behavior disorder in aged female rats. Animals. 2.2. Ovariecomy Ovariectomy surgeries on 7 months female rats were proceeded after 2-weeks’ environment adaptation. Female rats were anesthetized with isoflurane. Make a 1.5 cm incision at the lower abdominal midline, pulled both fallopian tubes and removed both ovaries with tweezers (surgical instruments were under high temperature and high pressure disinfection) then fed for 4 more weeks. 2.3. Drugs and treatment 16-months-aged SD female rats were divided randomly into four groups and treated differently with: β-Estradiol (Sigma-Aldrich,USA), Diarylpropionitrile (DPN, Sigma-Aldrich,USA), 1-[4-(6-bromo-1,3-benzodioxol-5-yl)-3aR,4S,5,9bS-tetrahydro-3H-cyclopenta[c]quinolin-8-yl (G-1, CAYMAN CHEMICAL, JAPAN) were dissolved in DMSO and redissolved in sesame oil then injected subcutaneously (i.p.) 10 μg/kg for two weeks. We also injected the DMSO saline solution subcutaneously on the 16-month-aged group as control. 2.4. Behavioral tests 2.4.1. Forced swimming test (FST) Forced swimming test was applied on rats based on the classic method described by Porsolt et al. Each rat was placed in a glass cylinder (45 cm × 35 cm×60 cm) filled with water of 30 cm height and temperature of (22 ± 1)℃. Rats were pre-swum for 15 min a day before the real test then dried the animals with air blower. 24 h later, the test session began. Rats were forced to swim for 5 min in the cylinder and the whole process was recorded by a video camera. Immobility is defined as floating in water and making only the minimal movements required to keep the head above the water. The immobility time could reflect the animals’ helplessness, as the floating is assumed to indicate the rat has given up attempting to escape. 2.4.2. Elevated plus-maze (EPM) The apparatus was consisting of four cross arms (30 cm × 15 cm×8 cm) 80 cm above the ground. Two arms were enclosed by acrylic walls and make it quite dark inside while the other two arms were flat with light. Put each rat into the middle of the apparatus with its head towards the open arm. Make sure each rat was put in the same position. Each trial last for 5 min and the whole process was recorded by video camera. The maze was cleaned after each rat with glacial acetic acid during the test in order to eliminate the odour of the former rat. We use a activity analysis software (Yishu Technology Shanghai China) to record and analyze the time that rat spent in the closed arms and open arms. The time spent in the open and closed arms may indicate the anxiety behavior level. 2.4.3. Open field test (OFT) The open field test was carried out in an acrylic quadratic box (100 cm × 100 cm×40 cm). Put each rat in the corner and watch it moving. Count the time when its body inclined vertically on the wall and stand upright with hindpaws. The process was recorded by video camera and analyzed by activity analysis software (Yishu Technology Shanghai China). The bottom of the box was divided into 16 squares. The motion and rearing information were measured using the software. The box was cleaned with glacial acetic acid after each rat with glacial acetic acid during the test in order to eliminate the odour of the former rat. The rearing time and center time may reflect their anxiety level. 2. Materials and methods 2.1. Animals Young Sprague-Dawley female rats (200–220 g, 2–3months old) were purchased from Experimental Animal Center of the Chinese Academy and acclimatized for 2 weeks before the experiment. The aged Sprague-Dawley female rats (9 months old) were from Vital River Laboratory Animal Technology Beijing and fed for 7 more months in our lab. Animals were caged in polycarbonate cages of four. Room temperature was controlled at (22 ± 1) °C with 12-hour light-dark cycle (7:00–19:00). All animal experiments were performed under the National Institutes of Health Guide for the Care and Use of Laboratory 2.5. Plasma estrogen detection by radioimmunoassay (RIA) All animals were sacrificed immediately after the behavior tests. 846 Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. The plasma samples of rats were achieved from eyes when sacrificing the animals. Centrifuge the whole blood at 3000RPM at 4 °C for 20 min carefully remove the supernatant. Estradiol (E2) Radioimmunoassay KIT with 125I (Beijing north institute of biological technology) was used in this experiment. The experiment was carried out under the assay protocol. Sigma-Aldrich USA) according to the manufacturer’s guidelines. Before measure the absorbance of sample, we make standards for colorimetric detection and dilute samples with proper fold to make sure the absorbance values of samples are within the linear range of the standard curve. Measure the absorbance at 570 nm with Molecular Devices SpectraMax® Paradigm. All samples and standards were measured in duplicate. 2.6. Hippocampus mitochondria isolation 2.10. Protein isolation and western blotting Isolate the mitochondria of the old rats with Mitochondria Isolation Kit (MITOISO1, Sigma-Aldrich USA). Operate the whole process according to the manufacture’s recommendations. Briefly, the hippocampus was carefully removed from the whole brain. The left hippocampus will be processing mitochondria Isolation immediately and the right hippocampus will be used on western blot (2.10 below). Wash the sample twice with 2 volumes of Extraction Buffer (isotonic solution, 10 mM HEPES, pH 7.5, containing 200 mM mannitol, 70 mM sucrose, and 1 mM EGTA). Cut small portions of the tissue (50–100 mg) and weigh in an eppendorf tube and then cut into smaller pieces. Homogenize the sample with 10 volumes of Extraction Buffer containing 2 mg/ml albumin. Pestle the tissue completely with Dounce Tissue Grinders. Keep the homogenate on ice. Centrifuge the sample at 600×g for 5 min. Keep supernatant liquid and then centrifuge it at 11,000×g for 10 min. Repeat the former two centrifuge steps and then suspend the pellet in Storage Buffer (10 mM HEPES, pH 7.4, containing 250 mM sucrose, 1 mM ATP, 0.08 mM ADP, 5 mM sodium succinate, 2 mM K2HPO4, and 1 mM DTT). The isolated mitochondrial was divided into three groups for the mitochondrial function detection below. After the behavior test, the brains were collected rapidly and frozen the right hippocampus in liquid nitrogen. The protein sample was prepared according to the protocol. Protein sample concentration was measured using a BCA protein assay (Pierce). Total protein sample was run on a 10% Tris-glycine SDS-PAGE gel (Bio-Rad Technology USA) and then transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore Germany). The membrane was blocked in 5% fat-free milk/ TBST for at least 2 h at room temperature. Incubate the membrane with primary antibody at 4 °C overnight. The primary antibodies were rabbit anti-ERα (1:1000,ab32063 Abcam), rabbit anti-ERβ (1:1000,ab3576 Abcam), rabbit anti-GPER (1:1000,492637606 Sigma), mouse anti-Bcl2 (1:1000,sc-23960 Santa Cruz Biotechnology), rabbit anti-UCP2 (1:1000,11081-1-AP Proteintech), β-actin (1:10000,12262 Cell Signaling Technology.1:10000,HRP-60008 Proteintech). Wash the membrane three times every 10 min with TBST then incubate with goat anti-rabbit IgG-HRP conjugate for 2 h at room temperature. Wash the membrane with TBST for three times. Detect the signal with western blot enhanced chemiluminiscence (ECL Millipore, Germany). GE ImageQuant LAS4000 mini was used for imaging. Stripe the blots with striping buffer (Thermo-Scintific, USA) then block the membrane and incubated the membrane with other antibodies. Analyze the densitometry with ImageJ software. 2.7. Determination of the mitochondrial membrane potential (ΔΨm) The decrease of mitochondrial membrane potential is a landmark event in the early stage of apoptosis. The change of cell membrane potential can be easily detected by JC-1 from red fluorescence to green fluorescence. Thus, the fluorescent intensity for both aggregates and monomeric forms of JC-1 was measured with a 96-well plate reader (JC-1 aggregates: excitation/emission = 525/590 nm; JC-1 monomers: excitation/emission = 490/530 nm). The fluorescent intensity ratio (590 nm/530 nm) is a simple and convenient indicator of the Δψ. Mix the mitochondrial suspensionwith Assay Buffer (20 mM MOPS, pH 7.5, containing 110 mM KCl, 10 mM ATP, 10 mM MgCl2, 10 mM sodium succinate, and 1 mM EGTA). Start the reaction by addition of 1 μl of JC1 Stain and mix by inversion. Keep the plate in dark at room temperature for 7 min. Read the plate at 490 nm with Molecular Devices SpectraMax® Paradigm. All samples and standards were measured in duplicate. 2.11. Statistical analysis All analyses were carried out using SPSS16.0 (SPSS Inc., Chicago, USA). Data are presented as means ± SEM. Student’s t-test, paired ttests, one-way ANOVA according to the factors introduced in the experimental design or followed by post hoc Student–Newmann–Keuls test when appropriate in order to identify significant differences. P < 0.05 was considered statistically significant. 3. Results 3.1. G-1 exhibited antidepressant and anxiolytic-like effect in aged female rats In order to explore whether treatment of E2, DPN and G-1 could improve the age-related mood symptoms, first we detected the potential antidepressant or anxiolytic effects of E2 and DPN, G-1 in aged female rats (16-months-old) via the behavior tests. The aged mice were injected with E2, DPN, G-1 or vehicle. In FST, E2, DPN and G-1 treatment significantly decreased the immobility time of aged female rats (F(3,16) =7.124, P < 0.01; Fig. 1A) which means the activation of estrogen receptors could release the depression condition in estrogen deficiency rats. In OFT, G-1 and E2-treated rats showed more rearing frequency (F (3,16) =10.4, P < 0.001; Fig. 1B) and increased central locomotor activity versus aged female rats (F(3,16) = 6.072, P < 0.01; Fig. 1C), whereas DPN did not exhibit significant effects. But, no difference was found in total locomotor activity among all four groups (F(3,16) =0.1945,P > 0.05; Fig. 1D). In EPM, G-1-treated rats spent more time in open arms (F(3,16) =3.616, P < 0.05; Fig. 1E) and less time in closed arms (F(3,16) = 3.655,P < 0.05; Fig. 1F) in comparing with the aged female ones. However, E2 and DPN could not cause significant behavior changes in EPM. Based on these results, among the agonists of estrogen receptors, the GPER agonist, G-1 exhibited the most significant 2.8. Superoxide dismutase enzyme activity levels Superoxide dismutase is a series of the most important antioxidative enzymes. It was measured in the mitochondria isolated from hippocampus with SOD determination kit (19160, Sigma-Aldrich USA) following the manufacturer’s guidelines. The assay was performed in a 96well microplate. Read the absorbance at 450 nm after incubating the plate for 20 min at 37 °C with Molecular Devices SpectraMax® Paradigm then calculate the SOD activity. All samples and standards were measured in duplicate. 2.9. Detection of the total antioxidant capacity Measurement of the total antioxidant capacity (TAC) indicates the ability to counteract oxidative stress-induced damage and may be a precise index of the treating efficiency on oxidative-stress related disorders. We make use of the change of Cu2+ ion to analyze the TAC level. Measured the TAC with the mitochondria isolated from hippocampus in old rats with Total Antioxidant Capacity Assay Kit (MAK187, 847 Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. Fig. 1. GPER agonist G-1 exhibited antidepressant- and/or anxiolytic-like effect on aged female rats. (A) G-1, DPN and E2 treatment significantly decreased the duration of immobility time in aged female rats in forced swimming test. G-1 and E2, but not DPN significantly increased rearing frequency (B) and distance travelled in central area (C) but not in the total distance (D) in aged female rats. Rats in G-1 group spent more time in open arms (E) and less time in closed arms (F) in elevated plus maze but not in E2 and DPN groups. Representative track of different groups in open field test (G) and in elevated plus maze (H). Data represent as mean ± S.E.M (n = 5 per group) and analyzed by one-way ANOVA.*p < 0.05;**p < 0.01;***p < 0.001. antidepressant- and anxiolytic-like effects on aged female rats. 3.3. G-1 upregulated the expression of ERs in hippocampus of aged female rats 3.2. G-1 improve hippocampal mitochondrial membrane potential and redox status of aged female rats Studies have suggested that E2 could not reverse the ERα decrease in aging or extended estrogen deprivation, which is related with the loss response to HRT in postmenopausal or older women. Meanwhile, the change of ERβ is paradoxical with aging. However, the change of GPER with aging has not been clarified yet. According to the results exhibited above hinted that G-1 might take effect in relief the emotional disorder in elderly females. And the effects of G-1 are better than E2 and DPN. To verify the influence of G-1 on ERs in aged female rats, we further compared the expression of ERα, ERβ, and GPER in the young intact female rats, OVX rats as well as aged female rats with or without G-1 treatment. Nevertheless, the results exhibited that the hippocampal protein levels of all three ERs, including ERα, ERβ and GPER remarkably decreased in young OVX rats (t(6) = 2.471 p < 0.05, Fig. 3A; t(6) = 3.794 p < 0.01, Fig. 3B, t(6) = 3.576 p < 0.05, Fig.3C) and aged female rats (t(6) = 2.768 p < 0.05, Fig. 3A; t (6) = 4.907 p < 0.01, Fig. 3B, t(6) = 4.077 p < 0.01, Fig. 3C). Interestingly, G-1 significantly up-regulated the protein levels of ERs in aged female rats (t(6) =3.887, P < 0.01,Fig. 3A; t(6) =3.795, P < 0.01,Fig. 3B; t(6) =3.726, P < 0.01,Fig. 3C) which may mediated the antidepressant-like effect of G-1. And we also detected the plasma 17β-estradiol level to preliminarily evaluate whether G-1 regulated ovary function and further make a positive impact on mood. The results exhibited that young rats had the highest plasma 17β-estradiol level and declined with aging (t(6) = 5.590 p < 0.01, Fig. 3D). It is well known that mitochondrial dysfunction and oxidative stress have been implicated in the pathogenesis of a series of affective neurological disorders. In this case we further detected the hippocampal mitochondrial function after the treatment of several agonists of estrogen receptors to figure out whether the estrogen agonist take its antidepressant- and anxiolytic-like effect via mitochondrial protection. After 2-weeks’ treatment of E2, DPN and G-1, hippocampal mitochondria were separated and utilized in detecting mitochondrial membrane potential with JC-1 stain dying. The results showed that the ratio of JC1 aggregates fluorescence intensities at 525/590 nm (Ex/Em) to JC-1 monomers fluorescence intensities at 490/530 nm significantly increased in G-1 group compared with the aged female rats (F(3,16) =4.063, P < 0.05; Fig. 2A), which indicated G-1 might protect the hippocampal mitochondrial membrane potential further maintain the mitochondrial condition. Likewise, G-1 and DPN treatment significantly increased Superoxide dismutase (SOD) activity in hippocampus compared with the aged female rats (F(3,16) =10.98, P < 0.001; Fig. 2B). Total antioxidant capacity (TAC) in G-1 group was also higher than the aged female ones (F(3,16) =3.244, P < 0.05; Fig. 2C). All these data showed that G-1 might exhibit the antidepressant- and anxiolytic-like effect through alleviating the hippocampal oxidative stress and improving mitochondrial function. 848 Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. Fig. 2. Hippocampal mitochondrial function was detected after E2, G-1 and DPN treatment. G-1 significantly enhanced the Mitochondria JC-1 fluorescence ratio (590/530 nm) (A), up-regulate Superoxide dismutase activity (B) and Total antioxidant capability (C) of aged female rats but not E2, while DPN only up-regulated SOD activity (B) to a less extent. All data represent as mean ± S.E.M (n = 5 per group) and analyzed by one-way ANOVA.* p < 0.05; *** p < 0.001. Ovariectomy also led to the drop of the plasma estrogen levels in young female rats (8-month-old) (t(6) = 5.514 p < 0.01, Fig. 3D). However, G-1 could not make any changes in the plasma estrogen levels in aged female rats (t(6) = 0.6236, p > 0.05; Fig. 3D), which suggested the antidepressant- and anxiolytic-like effect on G-1 is not associated with regulating ovary function in aged females. Based on the evidence above, we speculate that G-1 may take effect by regulating ERs expression in central nervous system. 3.4. G-1 increase hippocampal Bcl2 and UCP2 protein levels of aged female rats We have shown that G-1 could increase the mitochondrial function by increasing the membrane potential, superoxide dismutase activity and total non-enzymatic antioxidant capacity. For further evaluated the redox status of hippocampus in aged female rats, we further detected the protein levels of Bcl2, an anti-apoptotic marker, and UCP2, a predictive marker of cellular antioxidant capacity. The results showed that Fig. 3. G-1 increased protein levels of estrogen receptors in hippocampus but not plasma estrogen level in aged female rats. G-1 treatment significantly increased the expression levels of ERα (A), ERβ (B) and GPER (C) which was decreased in the OVX and aged female rats. G-1 treatment could not increase the 17β-estradiol level in plasma (D) of aged female rats, which was decreased in the OVX and aged female rats. Data represent mean ± S.E.M. (n = 5 per group) and analyzed by one-way ANOVA.*p < 0.05;**p < 0.01. 849 Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. Fig. 4. G-1 elevated protein levels of Bcl-2 and UCP-2 in hippocampus of aged female rats. The protein levels of Bcl2 (A) and UCP-2 (B) in hippocampus decreased significantly in aged and OVX female rats as compared to young female rats without ovariectomy. G-1 treatment significantly increased the protein levels of Bcl2 (A) and UCP-2 (B) in hippocampus of aged female rats. Data represent mean ± S.E.M. (n = 5 per group) and analyzed by one-way ANOVA.*p < 0.05; **p < 0.01. aging cause a remarkable decrease in protein levels of UCP2 (t (6) = 2.980 p < 0.05, Fig. 4A) and Bcl2 (t(6) = 5.514 p < 0.01, Fig. 4B) as well as UCP2 (t(6) = 3.007 p < 0.05, Fig. 4A) and Bcl2 (t (6) = 5.718 p < 0.01, Fig. 4B) in ovariectomy. In addition, G-1 significantly increased the protein levels of UCP2 (t(6) =3.177, P < 0.05; Fig. 4A) and Bcl2 (t(6) = 4.078, P < 0.01; Fig. 4B) in hippocampus in aged female rats. These results further confirmed that G-1 alleviated the antidepressant- and anxiolytic-like symptoms through improving oxidative status of hippocampus. the antioxidant effect of E2 and DPN are less obvious than G-1, which was consistent with previous findings that G-1 exhibited significant cardioprotective effects through alleviating cardiac oxidant stress induced by OVX [24]. A recent study concluded that 17β-estradiol and GPER independently regulate hippocampal memory, and suggested that hippocampal GPER may not function just as an estrogen receptor in the dorsal hippocampus [35]. These findings provide novel insights about the molecular mechanisms through which estrogen or GPER agonists modulate hippocampal function. On the other hand, aging is considered as a risk factor to menopausal or postmenopausal depression independent from estrogen decline. Many studies provide the compelling evidence, with no beneficial effects or increased adverse effects of HRT observed in women who were older [36,49,53–56,19,57,58]. In addition, estrogen presents beneficial effects in younger women with therapy initiated at the time of surgery. Expression of both ER subtypes (ERα and ERβ) have been proven to decline in the hippocampus in aged rodent models, which suggests that the loss of HRT response in aged females may be due to decreased activity of ERα and/or ERβ, and increased receptor expression in the hippocampus could rejuvenate estrogen responsiveness [59–61]. Furthermore, this view is corroborated by a recent study showing the protein expression of ERα and ERβ in the cerebral cortex to be significantly decreased within aged mice when compared to younger mice, and this decrease could not be rescued by E2 treatment. These results indicate that the down-regulation of ERα and ERβ in the cerebral cortex may contribute to the loss of estrogen efficacy against ischemic injury in aged females and may point to new therapies for ischemic stroke in aged postmenopausal women [42]. Similar to these findings, our results indicated that aging and extended estrogen deprivation caused a remarkable diminishment of ERα as well as ERβ and GPER. In these cases, why did G-1 still exhibit an antidepressant-, anxiolytic and antioxidant effect in aged female rats? This question remains perplexing and unanswered. Results showing that G-1 rescues the expression of ERs in aged female rats partially answer this question, but much remains unknown. In addition, similar to the findings in the present study, Bean and Kumar demonstrated that E2-mediated beneficial effects could be reinstated by up-regulation of ERα, but not ERβ in the hippocampus [62]. 4. Discussion In this study, we observed that GPER agonist G-1 presented antidepressant- and anxiolytic-like effects in aged female rats. Moreover, G1 remarkably improved antioxidant capacity in the hippocampus of aged female rats. We also confirmed that the protein levels of ERs in the hippocampus decreased significantly with aging and extended estrogen deprivation. Furthermore, G-1 can notably reverse the diminishment of ERs in aged female rats. Similar with the diminishment of ERs in the hippocampus, Bcl2, an anti-apoptotic marker, also was reduced significantly in the hippocampus in aged females and young OVX rats. However, G-1 was able to rescue the protein levels of Bcl2 as well as the expression of UCP2 in hippocampus in aged female rats. These results suggest that the increase of ER expression, including GPER, as well as antioxidant protein expression, might be involved in the antidepressant-, anxiolytic, and antioxidant effect observed from GPER agonist, G-1. 4.1. GPER’s antidepressant-, anxiolytic-like as well as antioxidant effects may be not related with E2 neuroprotection Although clinical data and animal research has hinted that HRT loses its neuroprotective and antioxidant effect in the aged brain, the benefits of HRT on mood in postmenopausal women is still controversial. Such treatments have been suggested to be prescribed for managing depressive symptoms in perimenopause, but not in the subsequent postmenopausal period [52]. Therefore, we utilized aged female rats to investigate the effect of E2 and special agonists of ERβ and GPER on postmenopausal depression. However, the results were beyond expectation. E2 exhibited some antidepressant- but little anxiolytic-like effects in aged female rats. Interestingly, G-1 presented significant antidepressant- and anxiolytic effects and its effect was significantly better than E2 and DPN, which only improved behavioral despair in forced swimming test. These results suggest that HRT maybe still have some beneficial effects on mood in postmenopausal women, and the anxiolytic-like effect of GPER activation may not be related to E2, which was further underlined by the results on the antioxidant function of G-1 in the hippocampus of aged female rats. We found that 4.2. GPER-related signaling pathway It is known that there are several intracellular signaling pathways that mediate the effects of GPER. GPER, as a member of the GPCR superfamily, couples to heterotrimeric G proteins, which subsequently regulate a multitude of downstream effectors within the cell. A growing body of evidence exists suggesting GPER’s coupling to both Gi/o and Gs proteins. Ligand-activated GPER triggers the rapid activation of ERK1/ 2, indicating the involvement of Gi/o proteins [1]. Additionally, agonist 850 Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. binding on GPER can effectively activate adenylyl cyclase, stimulating the production of cAMP, which is involved in the attenuation of ERK1/2 and CREB signal pathway [2]. GPER could also stimulate the PI3K/Akt axis in response to E2 and further interact with EGFR [3]. In cancer cells, researchers found that GPER accelerates sphingosine kinase to yield sphingosine 1-phosphate [4] as well as eNOS, resulting in the production ofnitric oxide within the vasculature [5,6]. Recently, studies have revealed GPER is involved in calcium mobilization [3,7] and potassium channels [8,9]. Consequently, these rapid signaling events show GPER to be a remarkable influence on gene expression [9]. GPER is known to regulate the expression of genes c-fos, cyclin A and D1 [10,11] as well as those for connective tissue growth factor [12,13], fatty acid synthase [14], and vascular endothelial growth function [15]. Phosphatidylinositol 3, 4, 5 trisphosphate (PIP3) production resulting from GPER activated PI3K further modifies the transcription factor SF-1 which could boost aromatase Cyp19a1 expression and as well as estrodiol production and diffusion [16]. Together, varied GPER activated pathways regulate diverse cellular functions including proliferation, metabolism, migration and secretion, which indicate the profound implications of GPER in normal conditions and in disease physiology. in enhancing mitochondrial biogenesis, improve its function and rescue Bcl2 expression. Former studies show that GPER activation might be related with the antioxidant action and cardioprotective effect [27,79]. We showed that GPER activation could not only up-regulate the membrane potential in mitochondria within aged female rats, but also raise the activity of SOD and total antioxidant capacity. Further analysis discovered the restoration of Bcl2 and UCP2 protein expression in the hippocampus of aged female mice. This matches the antioxidant effect of G-1 observed in our former study. Taken together, we show that GPER activation could not only enhance the expression of UCP2, but also raise the activity of SOD and TAC. In conclusion, GPER activation can rescue the redox status and regulate mood disorder in aged hippocampus. The effects may be correlated with the restoration of ERs and anti-apoptotic, antioxidant protein expression that decline with aging and extended estrogen deprivation. Acknowledgments The project was fund by State's Key Project of Research and Development Plan (2017YFB0403803) the National Key Basic Research Program of China (2013CB531906). The National Natural Science Fund of China (81671349, 81473437, 31371083 and 81271500), Development Project of Shanghai Peak Disciplines-Integrated Chinese and Western Medicine, and Natural Science Fund of Shanghai (14ZR1405200). We offer special acknowledgement to Prof. Yan -Qing Wang and Technician Jian-Wei Jiang for their selfless technical support, encouragement, and guidance during this work. We also offer our grateful acknowledgement to Nathaniel Ghena and Isabella Perone for their careful revisions to eliminate possible spelling or grammatical errors and to conform to correct scientific English for this manuscript. 4.3. The potential signaling mechanism of G-1 in up-regulating ERa, ERβ As mentioned above, GPER promotes the activation of adenylyl cyclase that stimulates the production of cAMP, which will ultimately influence the transcription factor cAMP response element-binding protein (CREB). A recent report demonstrated that the Eastern Bluebird estrogen receptor α gene carries two cAMP response element (CRE) consensus sequences in its promoter region [63] suggesting that the increased expression of ERα by G-1 might be mediated by CREB-CRE transcriptional pathway. However, there is still more to be known to better understand the regulatory elements in the mammalian ERα promoter region [64]. It has been reported that there is a relevant interaction of estrogens with GHR-JAK2-STAT5 signaling (FernandezPerez et al. 2013). STAT5, in turn, has the consensus sequence on the ERα 0/B promoter region [65]. Activation or inhibition of STAT5 DNA binding would result in decreasing or increasing ERα gene expression [66,67]. Taken together, G-1 may participate in the regulation of ERs; however, the specific underlying mechanisms require further investigation. This is particularly necessary in order to enhance our understanding of the regulation of ERs expression in a brain-region specific manner. References [1] E.J. Filardo, et al., Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF, Mol. Endocrinol. 14 (10) (2000) 1649–1660. [2] E.J. Filardo, et al., Estrogen action via the G protein-coupled receptor, GPR30: stimulation of adenylyl cyclase and cAMP-mediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis, Mol. Endocrinol. 16 (1) (2002) 70–84. [3] C.M. Revankar, et al., A transmembrane intracellular estrogen receptor mediates rapid cell signaling, Science 307 (5715) (2005) 1625–1630. [4] O. Sukocheva, et al., Estrogen transactivates EGFR via the sphingosine 1-phosphate receptor Edg-3: the role of sphingosine kinase-1, J. Cell Biol. 173 (2) (2006) 301–310. [5] M.R. Meyer, et al., Deletion of G protein-coupled estrogen receptor increases endothelial vasoconstriction, Hypertension 59 (2) (2012) 507–512. [6] S.H. Lindsey, et al., Vasodilation by GPER in mesenteric arteries involves both endothelial nitric oxide and smooth muscle cAMP signaling, Steroids 81 (2014) 99–102. [7] E. Haas, et al., Regulatory role of G protein-coupled estrogen receptor for vascular function and obesity, Circ. Res. 104 (3) (2009) 288–291. [8] X. Yu, et al., Activation of G protein-coupled estrogen receptor induces endothelium-independent relaxation of coronary artery smooth muscle, Am. J. Physiol. Endocrinol. Metab. 301 (5) (2011) E882–E888. [9] W.H. Dong, et al., Resveratrol inhibits K(v)2. 2 currents through the estrogen receptor GPR30-mediated PKC pathway, Am. J. Physiol. Cell Physiol. 305 (5) (2013) C547–C557. [10] A. Vivacqua, et al., 17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the G protein-coupled receptor GPR30, Mol. Pharmacol. 70 (4) (2006) 1414–1423. [11] L. Albanito, et al., Effects of atrazine on estrogen receptor α- and G protein-coupled receptor 30-mediated signaling and proliferation in cancer cells and cancer-associated fibroblasts, Environ. Health Perspect. 28 (5) (2015) 493–499. [12] D.P. Pandey, et al., Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF, EMBO J. 28 (5) (2009) 523–532. [13] A. Madeo, et al., Nuclear alternate estrogen receptor GPR30 mediates 17beta-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts, Cancer Res. 70 (14) (2010) 6036–6046. [14] M.F. Santolla, et al., G protein-coupled estrogen receptor mediates the up-regulation of fatty acid synthase induced by 17β-estradiol in cancer cells and cancerassociated fibroblasts, J. Biol. Chem. 287 (52) (2012) 43234–43245. 4.4. G-1 takes antidepressant effect by up-regulating ERs and maintenance mitochondria functions The previous studies have indicated that ERs, especially ERα shows neuroprotective effects by interacting with E2 in aged females [43,68–72]. G-1′s antidepressant effect may be due to the up-regulation of ERα and ERβ expression. However, numerous studies over the last decade have revealed that the maintenance of mitochondrial functions could significantly enhance neuroprotective effects by antioxidants and protective effects associated with GPER activation [27,73–78]. Therefore, we hypothesized that the up-regulation of ERs may be one of the key intermediates which will, in conjunction with the modulation of other direct intracellular signaling pathways, ultimately induce the maintenance of mitochondrial functions mediating G-1′s neuroprotective and antidepressant effect. G-1 antidepressant- and anxiolytic-like effect may be related with the improvement of the antioxidant capacity in hippocampus, including Bcl2 and UCP2 restoration. As mentioned above, the antioxidant action of G-1 perhaps mediated the antidepressant-, anxiolytic-like and neuroprotective effects in aged females. However, little is known regarding the related signaling pathways initiated by GPER that ultimately influence mitochondrial function. Although, some studies have hinted that GPER may take part 851 Behavioural Brain Research 359 (2019) 845–852 J. Wang et al. [15] E.M. De Francesco, et al., HIF-1alpha/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs), Breast Cancer Res. 15 (4) (2013) 64. [16] C. Lin, et al., Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation, Cancer Res. 69 (13) (2009) 5415–5423. [17] J.E. Rossouw, et al., Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause, JAMA 297 (13) (2007) 1465–1477. [18] L. Speroff, Transdermal hormone therapy and the risk of stroke and venous thrombosis, Climacteric 13 (5) (2010) 429–432. [19] D.F. Archer, E. Oger, Estrogen and progestogen effect on venous thromboembolism in menopausal women, Climacteric 15 (3) (2012) 235–240. [20] C. Antoine, et al., Treatment of climacteric symptoms in breast cancer patients: a retrospective study from a medication databank, Maturitas 78 (3) (2014) 228–232. [21] G.G. Hazell, et al., Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues, J. Endocrinol. 202 (2) (2009) 223–236. [22] R. Hammond, et al., GPR30 co-localizes with cholinergic neurons in the basal forebrain and enhances potassium-stimulated acetylcholine release in the hippocampus, Psychoneuroendocrinology 36 (2) (2011) 182–192. [23] E.M. Waters, et al., G-protein-coupled estrogen receptor 1 is anatomically positioned to modulate synaptic plasticity in the mouse hippocampus, J. Neurosci. 35 (6) (2015) 2384–2397. [24] E.M. De Francesco, et al., Protective role of GPER agonist G-1 on cardiotoxicity induced by doxorubicin, J. Cell Physiol. 232 (7) (2017) 1640–1649. [25] E. Kilanczyk, et al., Antioxidant protection of NADPH-depleted oligodendrocyte precursor cells is dependent on supply of reduced glutathione, ASN Neuro 8 (4) (2016). [26] L. Liu, et al., GPER activation ameliorates aortic remodeling induced by salt-sensitive hypertension, Am. J. Physiol. Heart Circ. Physiol. 310 (8) (2016) H953–961. [27] J.C. Bopassa, et al., A novel estrogen receptor GPER inhibits mitochondria permeability transition pore opening and protects the heart against ischemia-reperfusion injury, Am. J. Physiol. Heart Circ. Physiol. 298 (1) (2010) H16–23. [28] M. Sbert-Roig, et al., GPER mediates the effects of 17beta-estradiol in cardiac mitochondrial biogenesis and function, Mol. Cell Endocrinol. 420 (2016) 116–124. [29] A.K. Alencar, et al., Effect of age, estrogen status, and late-life GPER activation on cardiac structure and function in the Fischer344xBrown Norway female rat, J. Gerontol. A Biol. Sci. Med. Sci. 72 (2) (2017) 152–162. [30] S.B. Liu, et al., Neuroprotective effects of oestrogen against oxidative toxicity through activation of G-protein-coupled receptor 30 receptor, Clin. Exp. Pharmacol. Physiol. 38 (9) (2011) 577–585. [31] Z. Tian, et al., Estrogen receptor GPR30 exerts anxiolytic effects by maintaining the balance between GABAergic and glutamatergic transmission in the basolateral amygdala of ovariectomized mice after stress, Psychoneuroendocrinology 38 (10) (2013) 2218–2233. [32] B.R. Broughton, et al., Sex-dependent effects of G protein-coupled estrogen receptor activity on outcome after ischemic stroke, Stroke 45 (3) (2014) 835–841. [33] S.B. Liu, et al., Activation of GPR30 attenuates chronic pain-related anxiety in ovariectomized mice, Psychoneuroendocrinology 53 (2015) 94–107. [34] D. Lu, et al., Activation of G protein-coupled estrogen receptor 1 (GPER-1) ameliorates blood-brain barrier permeability after global cerebral ischemia in ovariectomized rats, Biochem. Biophys. Res. Commun. 477 (2) (2016) 209–214. [35] J. Kim, et al., 17beta-estradiol and agonism of G-protein-coupled estrogen receptor enhance hippocampal memory via different cell-signaling mechanisms, J. Neurosci. 36 (11) (2016) 3309–3321. [36] S.R. Salpeter, et al., Mortality associated with hormone replacement therapy in younger and older women: a meta-analysis, J. Gen. Intern. Med. 19 (7) (2004) 791–804. [37] W.A. Rocca, et al., Oophorectomy, menopause, estrogen, and cognitive aging: the timing hypothesis, Neurodegener. Dis. 7 (1–3) (2010) 163–166. [38] W.A. Rocca, et al., Oophorectomy, menopause, estrogen treatment, and cognitive aging: clinical evidence for a window of opportunity, Brain Res. 1379 (2011) 188–198. [39] P.M. Maki, Critical window hypothesis of hormone therapy and cognition: a scientific update on clinical studies, Menopause 20 (6) (2013) 695–709. [40] M.M. Adams, et al., Estrogen and aging affect the subcellular distribution of estrogen receptor-alpha in the hippocampus of female rats, J. Neurosci. 22 (9) (2002) 3608–3614. [41] M.E. Wilson, et al., Estrogen receptor-alpha gene expression in the cortex: sex differences during development and in adulthood, Horm. Behav. 59 (3) (2011) 353–357. [42] M. Cai, et al., The loss of estrogen efficacy against cerebral ischemia in aged postmenopausal female mice, Neurosci. Lett. 558 (2014) 115–119. [43] M.E. Wilson, et al., Age differentially influences estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) gene expression in specific regions of the rat brain, Mech. Ageing Dev. 123 (6) (2002) 593–601. [44] P.K. Sharma, M.K. Thakur, Expression of estrogen receptor (ER) alpha and beta in mouse cerebral cortex: effect of age, sex and gonadal steroids, Neurobiol. Aging 27 (6) (2006) 880–887. [45] N. Yamaguchi-Shima, K. Yuri, Age-related changes in the expression of ER-beta mRNA in the female rat brain, Brain Res. 1155 (2007) 34–41. [46] E.M. Waters, et al., Estrogen and aging affect the synaptic distribution of estrogen receptor beta-immunoreactivity in the CA1 region of female rat hippocampus, Brain Res. 1379 (2011) 86–97. [47] C.L. Shults, et al., Aging and loss of circulating 17beta-estradiol alters the [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] 852 alternative splicing of ERbeta in the female rat brain, Endocrinology 156 (11) (2015) 4187–4199. B.A. Rocha, et al., 17 Beta-estradiol-induced antidepressant-like effect in the forced swim test is absent in estrogen receptor-beta knockout (BERKO) mice, Psychopharmacology (Berl) 179 (3) (2005) 637–643. O.T. Wolf, et al., Estradiol or estradiol/progesterone treatment in older women: no strong effects on cognition, Neurobiol. Aging 26 (7) (2005) 1029–1033. Z.A. Hughes, et al., WAY-200070, a selective agonist of estrogen receptor beta as a potential novel anxiolytic/antidepressant agent, Neuropharmacology 54 (7) (2008) 1136–1142. S. Benmansour, et al., Comparison of the effects of estradiol and progesterone on serotonergic function, Biol. Psychiatry 71 (7) (2012) 633–641. A.R. Genazzani, et al., Hormone replacement therapy in climacteric and aging brain. International Menopause Society Expert Workshop, 15–18 March 2003, Pisa, Italy, Climacteric 6 (3) (2003) 188–203. E.F. Binder, et al., Effects of hormone replacement therapy on cognitive performance in elderly women, Maturitas 38 (2) (2001) 137–146. G.L. Anderson, et al., Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women’s Health Initiative randomized controlled trial, JAMA 291 (14) (2004) 1701–1712. M.A. Espeland, et al., Conjugated equine estrogens and global cognitive function in postmenopausal women: women’s health initiative memory study, JAMA 291 (24) (2004) 2959–2968. S.A. Shumaker, et al., Conjugated equine estrogens and incidence of probable dementia and mild cognitive impairment in postmenopausal women: women’s health initiative memory study, JAMA 291 (24) (2004) 2947–2958. J.M. Daniel, Estrogens, estrogen receptors, and female cognitive aging: the impact of timing, Horm. Behav. 63 (2) (2013) 231–237. C.L. Shufelt, et al., Hormone therapy dose, formulation, route of delivery, and risk of cardiovascular events in women: findings from the women’s health initiative observational study, Menopause 21 (3) (2014) 260–266. T.C. Foster, Role of estrogen receptor alpha and beta expression and signaling on cognitive function during aging, Hippocampus 22 (4) (2012) 656–669. N.N. Mott, T.R. Pak, Estrogen signaling and the aging brain: context-dependent considerations for postmenopausal hormone therapy, ISRN Endocrinol. 2013 (2013) 814690. Y. Hara, et al., Estrogen effects on cognitive and synaptic health over the lifecourse, Physiol. Rev. 95 (3) (2015) 785–807. L.A. Bean, et al., Re-opening the critical window for estrogen therapy, J. Neurosci. 35 (49) (2015) 16077–16093. A.B. Bentz, et al., Relationship between maternal environment and DNA methylation patterns of estrogen receptor alpha in wild Eastern Bluebird (Sialia sialis) nestlings: a pilot study, Ecol. Evol. 6 (14) (2016) 4741–4752. J.J. Pinzone, et al., Molecular and cellular determinants of estrogen receptor alpha expression, Mol. Cell. Biol. 24 (11) (2004) 4605–4612. J. Frasor, G. Gibori, Prolactin regulation of estrogen receptor expression, Trends Endocrinol. Metab. 14 (3) (2003) 118–123. J. Frasor, et al., PRL-induced ER alpha gene expression is mediated by janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase, Mol. Endocrinol. 15 (11) (2001) 1941–1952. F.A. Champagne, et al., Maternal care associated with methylation of the estrogen receptor-alpha 1b promoter and estrogen receptor-alpha expression in the medial preoptic area of female offspring, Endocrinology 147 (6) (2006) 2909–2915. M.C. Craig, D.G. Murphy, Estrogen: effects on normal brain function and neuropsychiatric disorders, Climacteric 10 (2007) 97–104. M.K. Thakur, P.K. Sharma, Transcription of estrogen receptor alpha and beta in mouse cerebral cortex: effect of age, sex, 17 beta-estradiol and testosterone, Neurochem. Int. 50 (2) (2007) 314–321. Q.G. Zhang, et al., C terminus of Hsc70-interacting protein (CHIP)-mediated degradation of hippocampal estrogen receptor-alpha and the critical period hypothesis of estrogen neuroprotection, Proc. Natl. Acad. Sci. U. S. A. 108 (35) (2011) E617–E624. D. Brann, et al., Oestrogen signalling and neuroprotection in cerebral ischaemia, J. Neuroendocrinol. 24 (1) (2012) 34–47. L.A. Bean, et al., Estrogen Receptors, the Hippocampus, and Memory, Neuroscientist 20 (5) (2014) 534–545. J.W. Simpkins, et al., Mitochondria play a central role in estrogen-induced neuroprotection, Curr. Drug Targets CNS Neurol. Disord. 4 (1) (2005) 69–83. C.M. Klinge, Estrogenic control of mitochondrial function and biogenesis, J. Cell. Biochem. 105 (6) (2008) 1342–1351. A. Razmara, et al., Mitochondrial effects of estrogen are mediated by estrogen receptor alpha in brain endothelial cells, J. Pharmacol. Exp. Ther. 325 (3) (2008) 782–790. M. Fiocchetti, et al., Neuroprotective effects of 17beta-estradiol rely on estrogen receptor membrane initiated signals, Front. Physiol. 3 (2012) 73. W.L. Li, et al., Activation of novel estrogen receptor GPER results in inhibition of cardiocyte apoptosis and cardioprotection, Mol. Med. Rep. 12 (2) (2015) 2425–2430. A.H. Kurt, et al., The protective role of G protein-coupled estrogen receptor 1 (GPER-1) on methotrexate-induced nephrotoxicity in human renal epithelium cells, Ren. Fail. 38 (5) (2016) 686–692. N. Kanda, S. Watanabe, 17beta-estradiol inhibits oxidative stress-induced apoptosis in keratinocytes by promoting Bcl-2 expression, J. Invest. Dermatol. 121 (6) (2003) 1500–1509.
Purchase answer to see full attachment