Sildenafil promotes the anti-amnesic activity of estrogen receptor alpha agonist in animals with estrogen insufficiency

Neurochemistry International 132 (2020) 104609

Contents lists available at ScienceDirect

Neurochemistry International journal homepage: www.elsevier.com/locate/neuint

Sildenafil promotes the anti-amnesic activity of estrogen receptor alpha agonist in animals with estrogen insufficiency

T

Ahsas Goyal, Debapriya Garabadu∗ Division of Pharmacology, Institute of Pharmaceutical Research, GLA University, Mathura, Uttar Pradesh, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Brain Estrogen insufficiency Sildenafil Cyclic nucleotides Memory

The cognitive function in the females is observed to modulate with the fluctuation in plasma estrogen level. The specific estrogen receptor alpha (ERα) agonist, (4,4′,4″-(4-propyl-[1H] pyrazole-1,3,5-triyl) tris phenol (PPT), exerts similar therapeutic activity to that of estrogen replacement therapy. It can also exert cyclic adenosine monophosphate (cAMP)-dependent carcinogenic activity in the uterus of the ovariectomized animals. However, there is no report of cGMP on the ERα-mediated phosphorylation of Akt in the experimental condition. Sildenafil increases the level of cGMP in most of the tissues including brain. Hence, the present study evaluated the therapeutic effect of Sildenafil with or without PPT in rats with experimentally-induced estrogen insufficiency. The condition of estrogen insufficiency was induced in female rats through bilateral ovariectomy on day-1 (D-1) of the experimental schedule. Sildenafil (1.0 and 10.0 mg/kg) and PPT attenuated ovariectomy-induced cognitive deficits in behavioural tests and increase in body weight in the rodents. Sildenafil and PPT increased the cholinergic function and the ratio of cGMP/cAMP in the hippocampus, pre-frontal cortex and amygdala of the animals. Further, the ovariectomy-induced decrease in the extent of phosphorylation of ERα in all the brain regions was attenuated with the monotherapy of either Sildenafil or PPT. Interestingly, the combination of Sildenafil and PPT exhibited better therapeutic effectiveness than their monotherapy. However, Sildenafil attenuated the PPT-induced increase in the level of expression of phosphorylated protein kinase-B (Akt) in the discrete brain regions and the weight of uterus of these rodents. Hence, it can be assumed that the combination could be a better therapeutic alternative with minimal side effect in the management of estrogen insufficiencyinduced disorders.

1. Introduction The age-associated diseases such as Alzheimer's disease (AD) prevails over other illness throughout the World as the life expectancy of the individuals are increasing, and therefore these are big challenges to the biomedical research (Andersen et al., 2016). The incidence of dementia is observed to be high in women than their counter sex while considering the global population (Carter et al., 2012; Maki and Henderson, 2012). The fluctuation in the level of plasma estrogen is one of the predisposing factors in the cognitive function decline during dementia and body weight gain for women (Proietto, 2017; Yonker et al., 2003). In support of the above fact, it is reported that hippocampal dendritic spine density was significantly decreased in adult ovariectomized female rats and it was restored with estrogen replacement therapy (Gould et al., 1990). It is also observed that apical dendritic spine density fluctuated with the estrous cycle in adult female rats (Woolley et al., 1990). Thus, the fluctuation in the estrogen level is

∗

considered as one of the predisposing factors in the genesis of several neurodegenerative disorders, including AD (Daniel and Bohacek, 2010; Kelly et al., 2008; Schupf et al., 2008). Therefore, considering the postmenopausal estrogen therapy, hormone down-regulation manipulations or comparing menstrual cycle phases, the estrogen insufficiency can be assumed to influence the cognitive function and body weight in females (Poromaa and Gingnell, 2014; Proietto, 2017). The replacement therapy with estrogen is recommended for the restoration of many physiological functions in the female during insufficiency, especially in the case of menopause. Estrogen can also regulate cognitive function and metabolism through its receptor (Choleris et al., 2006; Foster, 2012). Due to polymorphism in the estrogen receptors, there are several distinct types of estrogen receptors in the mammalian physiological system. In this context, it is well reported that estrogen exhibits its action through membrane-bound G-protein coupled and/or cytoplasmic liganded estrogen receptor in different cells including neurons (Brailoiu et al., 2007; Milner et al., 2001; Mitra

Corresponding author. Division of Pharmacology, Institute of Pharmaceutical Research, GLA University, Mathura, 281406, India. E-mail addresses: [email protected], [email protected] (D. Garabadu).

https://doi.org/10.1016/j.neuint.2019.104609 Received 29 September 2019; Received in revised form 21 November 2019; Accepted 22 November 2019 Available online 25 November 2019 0197-0186/ © 2019 Elsevier Ltd. All rights reserved.

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

et al., 2003; Mitterling et al., 2010). It is also reported that the activation of membrane-bound estrogen receptor can phosphorylate unliganded estrogen receptors which are subsequently translocated in to the nucleus for the transcription in different cells including neurons similar to that of other estrogen receptors (Hall et al., 2001; Kushner et al., 2000a, 2000b; Saville et al., 2000). Interestingly, the use of estrogen replacement therapy is restricted due to carcinogenic effect on several tissues including uterus (Daniel et al., 2006; Wang et al., 2017) which is considered as a consequence to increased extent of phosphorylation of Akt in these tissues. It is also reported that the specific ERα agonist, (4,4′,4″-(4-propyl-[1H] pyrazole-1,3,5-triyl) tris phenol (PPT), exhibits therapeutic activity similar to that of estrogen replacement therapy and thus ERα-mediated mechanism can be considered as a substituent to non-specific estrogen replacement therapy (Qu et al., 2016, 2013). However, PPT also increases the phosphorylation of Akt through cyclic adenosine monophosphate (cAMP)-dependent mechanism and thus exerts carcinogenic activity in the uterus of the ovariectomized animals (Liao et al., 2010; Steagall et al., 2017). On the other hand, cyclic guanosine monophosphate (cGMP) inhibits the phosphorylation of Akt and thus exhibits anti-proliferative activity in human glioma cell lines (Swartling et al., 2009). The cyclic nucleotides such as cAMP and cGMP are considered as one of the downstream molecules of membrane-bound ERα-receptor activity in several tissues including memory-sensitive brain regions (Greengard, 2001; Lesch and Lerer, 1991; Neve et al., 2004; Sharma et al., 2013; Szego, 2006). Further, there is a decrease in the level(s) of either cAMP or cGMP or both in experimental animals with the decreased expression of ERα during estrogen insufficiency (Cao et al., 2015; Sagredo et al., 2013). However, there is no report of cGMP on the ERα-mediated phosphorylation of Akt in the experimental condition. Sildenafil, a phosphodiesterase-V inhibitor, increases the level of cGMP in most of the tissues including brain (Zhang et al., 2002). The neuroprotective activity of sildenafil is well established (Ding et al., 2008; Farooq et al., 2008; Puzzo et al., 2009). Recently, it has been demonstrated that sildenafil exhibits anti-amnesic activity against intracerebroventricular streptozotocin-induced memory loss and vascular dementia in the experimental animals (Venkat et al., 2019; Zhu et al., 2019). In addition, sildenafil improves cognitive function in animals with hepatic encephalopathy or AD-like behavioural manifestations (Cuadrado-Tejedor et al., 2011; Hernandez-Rabaza et al., 2015). However, there is no report on the effect of sildenafil on estrogen receptor-mediated cognitive function. Hence, the present study evaluated the therapeutic effect of sildenafil with or without PPT against ovariectomy-induced dementia in experimental rodents. In addition, the extent of phosphorylation of ERα and cholinergic dysfunction in the memory-sensitive brain regions such as hippocampus (HIP), pre-frontal cortex (PFC) and amygdale (AMY) of the ovariectomized animals were evaluated to elaborate the effect of sildenafil on ERα-mediated action in these tissues with or without the presence of PPT. Moreover, the effect of sildenafil on the extent of phosphorylation of Akt in the discrete brain regions was evaluated with or without the presence of PPT in the animals with estrogen insufficiency.

Fig. 1. Diagrammatic representation of experimental schedule.

Committee (IAEC; GLAIPR/CPCSEA/IAEC/P'Col/2015/01). 2.2. Chemicals Sildenafil and PPT were purchased from Tocris Bioscience (Tocris House, IO Centre Moorend Farm Ave., Bristol, BS11 0QL, UK). All the chemicals and reagents were of analytical grade and purchased from local suppliers. 2.3. Experimental design The diagrammatic representation of the experimental schedule was depicted in Fig. 1. Briefly, the rats were randomly grouped into eight of six animals each, named as Control, Sham (subjected to bilateral incisions without removing the ovaries), Ovariectomy (OVX), OVX+Sildenafil-0.1 (S-0.1), OVX+S-1.0, OVX+S-10.0, OVX+ PPT and OVX +PPT+S-10.0. Animals of all groups except the control group were anesthetized using pentobarbitone (45 mg/kg, i.p.). The procedure of ovariectomy was performed under aseptic condition in all anesthetized animals of the groups except Control and Sham group rats (Qu et al., 2016, 2013). Cleaning of the cages and wounds disinfection was performed on a daily basis. Sildenafil (0.1, 1.0 and 10.0 mg/kg, i.p.; Shafiei et al., 2006) was administered to OVX+S-0.1, OVX+S-1.0 and OVX +S-10.0 group animals for 60 consecutive days, respectively. Further, PPT (333 μg/kg; i.p.; Pisani et al., 2016) was administrated to OVX +PPT group animals for 60 consecutive days. Moreover, PPT and sildenafil (10.0 mg/kg) were administered to the OVX+PPT+S-10.0 group animals at a time lag of 30 min for 60 consecutive days. The Control and Sham group animals received intraperitoneal injection of vehicle (30 μl dimethyl sulfoxide (DMSO), 90 μl Tween 80 and 0.88 ml of saline). The volume of administration of Sildenafil, PPT, Sildenafil and PPT, or vehicle was ≤1.0 ml/kg to the respective animals in the experimental design. The whole experimental protocol was followed for 60 days. The rats were exposed to Morris Water Maze (MWM) test paradigm for 5 consecutive days, i.e., from Day (D)-56 to D-60 of the experimental schedule. Subsequently, on D-60 the animals were exposed to Y-maze test after 30 min to MWM test. The behavioural activities were recorded and measured in ANY-maze™ (Version-4.96, USA) video-tracking system. The serum level of estradiol of the animals was estimated on D-1 before ovariectomy and on D-60 of the experimental protocol using the manufacturer's instruction of standard assay kit (Abcam Pvt. Ltd., ab108667). All the animals were sacrificed by decapitation immediately after the successful behavioural performance. The uterus and brains of all animals were collected and the brain of each animal was further micro dissected into the HIP, PFC and AMY for estimating the biochemical parameters (Palkovits and Brownstein, 1988).

2. Material and methods 2.1. Animals Female Wistar rats (250–280 g) were collected from the animal house of Institute of Pharmaceutical Research, GLA University, Mathura. The animals were grouped and housed in optimum condition of 22–26 °C temperature, 45–55% relative humidity, and 12 h light: 12 h dark cycle in polyacrylic cages lined with husk. Animals were allowed to feed their standard soya-free chow diet and water ad libitum. All the experimental protocols were carried out under the regulations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted Institutional Animal Ethics

2.4. Procedure of bilateral ovariectomy The female anesthetized wistar rats were shaved and clean with ethanol on their dorsal surface. A transverse peritoneal incision of 0.4–0.6 cm was made using surgical scalpel blade on the middle part of the left and right dorsal side of flanks, and the ovaries were removed from both sides to perform bilateral ovariectomy. The uteri on both sides were pushed back, and incisions were sutured using absorbable 2

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Fig. 2. Effect of S (0.1, 1.0 and 10.0 mg/ kg), PPT or the combination of S (10.0 mg/ kg) and PPT on ovariectomy (OVX)-induced changes in body weight (A), estradiol level (B) and uterus weight (C) of the animals. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, b P < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, eP < 0.05 compared to OVX +S-1.0, fP < 0.05 compared to OVX+S10.0 and gP < 0.05 compared to OVX +PPT (Repeated measures of Two-way ANOVA followed by Bonferroni's multiple comparison test for body weight and estradiol level and one-way ANOVA followed by Student-Newmann-Keuls post hoc test for uterus weight among groups).

Briefly, the animal was placed in a large water pool (160 cm in diameter and 60 cm in depth) separated into four equal quadrants. The tendency of the animal was to find out a hidden platform (10 × 10 cm), submerged 1.5 cm below the surface of the water, to escape. Four consecutive days of practice (with a gap of 5 min in between), each animal was subject to a quest for a hidden platform for four consecutive days. Temperature of experimental room and tank water was maintained at 27 ± 1 °C. The escape latency in each session in four consecutive days (time required to reach the platform as a goal; D-56 to D59) and a mean time spent by the animals in target quadrant, percentage of total distance travelled in target quadrant and swimming speed on D-60 of each animal was recorded and analyzed in ANY-maze video

suture (Ethicon chromic sutures, Johnson and Johnson Ltd., India). Neomycin antibiotic powder was applied twice daily on wounds for one week. Throughout the operation, the high degree of aseptic procedure was followed, and the animals were permitted to recover. The rats were housed separately in cages after surgery, provided for a period of one week with clean and dry bedding sets (Qu et al., 2013).

2.5. Evaluation of cognitive deficits in different behavioural test paradigms 2.5.1. Morris Water Maze (MWM) test for assessing learning and memory The MWM test is frequently used to evaluate learning and memory functions of the animals (Morris, 1984; Sharma and Singh, 2012). 3

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Fig. 3. The tracking plots of representative animal of each group during the learning phase (day 56–59; searching submerged platform) and probe trial (day 60; searching removed platform) in Morris water maze test protocol are depicted. Further, the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in escape latency from Day-56 to Day-59 (A), time spent in target quadrants (B), percentage of total distance travelled in the target quadrant (C) and swimming speed (D) of the animals in Day-60 in MWM test paradigm. All values are expressed in mean ± SEM (n = 6). a P < 0.05 compared to Control, bP < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, e P < 0.05 compared to OVX+S-1.0, f P < 0.05 compared to OVX+S-10.0 and g P < 0.05 compared to OVX+PPT (Repeated measures of Two-way ANOVA followed by Bonferroni's multiple comparison test for escape latency and one-way ANOVA followed by Student-NewmannKeuls post hoc test for the time spent in target quadrants and percentage of total distance travelled in target quadrant).

50 μl of 4 M potassium acetate was mixed to modify the pH to 4.0 which was followed by centrifugation at 4000g for 15 min (Muthuraju et al., 2009).

tracking system (Stoelting Co., Version-4.96, USA). Each rodent was allowed to explore the pool for 120 s. The mean time spent in all the quadrants in search of the hidden platform was recorded. The experiments were performed at the same place with appropriate care that includes the relative position of other artefacts in the laboratory in order to avoid any disturbance in prominent visual indications.

2.6.2. Assay of ChAT activity The levels of ChAT were determined using spectrophotometer (BioTek Instruments Inc., Epoch®, USA) at 450 nm using an enzymelinked immunosorbent assay kit (SEB929Mu; Wuhan, Hubei, China) according to the manufacturer's instructions.

2.5.2. Y-maze test for assessing spontaneous alteration behaviour On Day-60, working memory in Y-Maze for spontaneous alteration behaviour (SAB) was assessed (Mouri et al., 2007). The device was a black painted wooded horizontal labyrinth (40 × 3 × 12 cm3) with three arms (A, B and C labeled) arranged at an angle of 120° to each other. Each animal was placed in the middle of the apparatus. The animal was allowed to move freely for 8 min through the maze. The number of alterations (i.e., consecutive sequences of entry of ABC, CAB or BCA, but not BAB) and total arm entries were recorded. The arms were thoroughly cleaned with water spray to remove residual odour in between the tests. The percentage alteration was calculated according to the following equation: percentage alteration = [(number of alterations)/(total arm entries-2)] x 100.

2.6.3. Evaluation of ACh level The quantity of ACh in brain tissue was estimated with the use of Amplex red assay kit (Molecular Probes, Inc., USA) following the procedure of Zoukhri and Kublin (2001). The fluorescence was recorded at 530 nm excitation and 590 nm wavelengths with the help of spectrofluorometer (Hitachi, F-2500, Japan). A standard protocol was used to determine protein content (Lowry et al., 1951). 2.6.4. Evaluation of activity of AChE The increase in the activity of AChE is regarded as an indicator of the loss of cholinergic neurons in brain tissue. The activity of AChE was evaluated in assay kit of Amplex red AChE (Molecular Probes, Inc., USA). The fluorescence was determined at 530 nm excitation wavelength and 590 nm emission wavelength with the assistance of spectrofluorometer (Hitachi, F-2500, Japan). The Lowry method was used to determine protein content (Lowry et al., 1951).

2.6. Assessment of the cholinergic dysfunction 2.6.1. The method of sample preparation The brain tissue was homogenized with a homogenizer containing 1 ml of 0.1 M perchloric acid. Homogenate was stored and thereafter 4

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

binding, aliquots of 200 μl of the tissue preparation, containing 200–250 μg protein/tube, were incubated with 10 concentrations (0.1–2.5 nM) of [3H]QNB (48 Ci/mmol) and buffer (pH 7.4) to a final volume of 250 μl. Non-specific binding was defined as that binding inhibited by 100 μM Pirenzepine. After the radioligand had been added, the homogenate was mixed thoroughly using a vortex apparatus and incubated in a warm bath at 37 °C for 15 min. After incubation, the assay was terminated by rapid vacuum filtration through Whatman GF/ C filters pre-soaked in Tris-buffer. The filters were washed rapidly with 2 × 5 ml ice-cold Tris. The filters were placed in polypropylene counting tubes containing scintillation fluid. The tubes were left for 3 h in the scintillation counter (Model Wallac 1409, Turku, Finland) where after counting commenced. Radioactivity trapped on the filters was determined by liquid scintillation. Specific binding was calculated as the total minus nonspecific binding. Bmax and Kd were calculated from Scatchard plots by using SigmaPlot Systat Software (version 13, USA). The concentration of protein was determined using bovine serum albumin as standard (Lowry et al., 1951). 2.7. Estimation of cyclic AMP (cAMP) and cyclic GMP (cGMP) levels Intracellular levels of cyclic nucleotides (cAMP and cGMP) in tissue were determined in microplate reader (BioTek Instruments Inc., Epoch®, USA) using direct cAMP (ab133051; Abcam Plc., Cambridge, USA) and cGMP (ab133052; Abcam Plc., Cambridge, USA) enzyme immunoassay kit according to the manufacturer's instruction. Results were expressed as pmol/mg protein. Fig. 4. The tracking plots of representative animal of each group during working memory formation in Y-maze test protocol are depicted. Further, the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in spatial memory in terms of spontaneous alteration behaviour of rats in the Y-maze test. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, bP < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, e P < 0.05 compared to OVX+S-1.0, fP < 0.05 compared to OVX+S-10.0 and g P < 0.05 compared to OVX+PPT (One-way ANOVA followed by StudentNewmann-Keuls post hoc test).

2.8. Immunoblotting The tissues were lysed in buffer containing protease inhibitor cocktail for protein analysis. Subsequently, tissues were subjected to homogenization in a Potter-Elvehjem homogenizer and thereafter the homogenate was centrifuged at 1500 g for 15 min. The post-nuclear fraction was then centrifuged at 100000 g for 60 min. The resulting supernatant was considered as the cytosolic fraction. Concentrations of proteins were determined by the standard technique in each fraction (Bradford, 1976). A standard plot was made using bovine serum albumin. An aliquot of each cytoplasmic samples were electrophoresed on 10% SDS-PAGE gels for M1, ERα, phosphorylated ERα, Akt and phosphorylated Akt proteins. It was then transferred to polyvinylidene fluoride membranes and probed with specific antibodies. Similarly, an equal aliquot of each nuclear sample was electrophoresed on 10% SDSPAGE gels for phosphorylated ERα protein, transferred to polyvinylidene fluoride membranes and probed with the specific antibody. The membrane was incubated overnight with rabbit anti-M1 receptor (Abcam Plc., Cambridge, USA; ab180636), anti-ERα (Abcam Plc., Cambridge, USA; ab3575), anti-Akt (Abcam Plc., Cambridge, USA; ab18785), anti-phosphorylated ERα (Abcam Plc., Cambridge, USA; ab131111) and anti-phosphorylated Akt (Cell Signaling Technology, USA; 9271) polyclonal primary antibody at a dilution of 1:1000, 1:1000, 1:1000, 1:100 and 1:1000 respectively. The membrane was stripped with stripping buffer (25 mM Glycine pH 2.0, 2% SDS for 30 min at room temperature) after treatment with the secondary antibodies of respective proteins. Thereafter, it was again probed with rabbit anti-β-actin (Abcam Plc., Cambridge, USA; ab8227) and antihistone-3 (H3; Abcam Plc., Cambridge, USA; ab1791) polyclonal primary antibody at a dilution of 1:500 and 1:1000 to confirm equal loading of protein in cytoplasmic and nuclear fraction respectively. The secondary antibodies of either β-actin or H3 were used to probe the membrane. The enhanced chemiluminescence (ECL) reagents (Amersham Bioscience, USA) were used to detect the Immunoreactive band of proteins. The quantitative analysis was estimated by a densitometric scan of films. The densitometric analysis was used to calculate the area of immunoreactive band using Biovis gel documentation software.

2.6.5. The M1 receptor binding studies in discrete brain regions In saturation binding assays, receptor density (Bmax) and dissociation constant (Kd) was evaluated and expressed as fmol/mg of protein and nM, respectively. On the last day of the experimental protocol, animals were decapitated, brains were quickly removed, and the brain tissues were dissected on ice. Tissue was stored at −80 °C until the assays were performed. On the day of assay, brain tissues were thawed at room temperature, weighed and were then suspended in 20 ml of icecold 50 mM Tris–HCl buffer containing 4 mM CaCl2 with a pH of 7.7. Then they were homogenized in a polytron homogenizer (setting 6, 5s) and centrifuged (18, 000×g, 10 min, 4 °C). The pellets were reconstituted with 20 ml of fresh buffer and homogenized for 5s and centrifuged (washed) to eliminate unwanted endogenous substances that may interfere with the radioligand binding procedures. The ‘washing’ procedure was performed by resuspending the pelleted membrane fraction after which it was centrifuged. The procedure was repeated twice. The resultant pellet was reconstituted in 20 ml of Tris–HCl buffer, homogenized for 5 s and incubated in a shaking water bath for 10 min at 37 °C. The incubation and shaking furthermore eliminate unwanted endogenous substance. After incubation the homogenate was centrifuged for the last time (18, 000×g, 10 min, 4 °C). The final pellet was re-suspended in 20 vol of ice-cold Tris–HCl buffer containing the 4 mM CaCl2 and homogenized for the last time (setting 6, 5s). The assay of [3H] QNB binding to muscarinic M1 receptor was performed according to standard protocol (Garabadu and Sharma, 2019; Yamamura and Snyder, 1974). For determination of total 5

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Fig. 5. Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in cholinergic activity in terms of level of ACh (A), activity of AChE (B), and activity of ChAT (C) in rat HIP, PFC and AMY. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, b P < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, eP < 0.05 compared to OVX+S-1.0, fP < 0.05 compared to OVX+S-10.0 and gP < 0.05 compared to OVX+PPT (One-way ANOVA followed by Student-Newmann-Keuls post hoc test).

2.9. Analysis of data

3. Results

All the data were represented as mean ± standard error of the mean (SEM). The data were analyzed with GraphPad Prism 6.0 (San Diego, CA). Repeated measures of two-way analysis of variance (ANOVA) followed by Bonferroni Post hoc test was used for statistical analysis for body weight, estradiol level and escape latency of the animals. All other statistical analysis was done using one-way ANOVA followed by Student Newman-keuls Post-hoc test. P < 0.05 was considered significant.

3.1. Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in body weight, estradiol level and uterus weight of the animals Fig. 2 depicts the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the body weight (A), estradiol level (B) and uterus weight (C) of the rats. Statistical analysis revealed that there were significant differences in the body weight and plasma level of estradiol of the animals among group ([F (7, 80) = 15.3, P < 0.05] and [F (7, 80) = 11.4, P < 0.05] respectively) and day ([F (1, 80) = 246.3, P < 0.05] and [F (1, 80) = 240.2, P < 0.05] respectively). Further, there was a significant 6

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

interaction between group and day in both of these parameters of the animals ([F (7, 80) = 13.2, P < 0.05] and [F (7, 80) = 14.1, P < 0.05] respectively). Post-hoc test showed that there were no significant differences in body weight and the plasma level of estradiol of the animals among the groups on D-1 of the experimental schedule. On D-60, the monotherapy of S (1.0 and 10.0 mg/kg) and PPT caused a significant attenuation in OVX-induced increase in the body weight of the animals. Further, the combination of S (10.0 mg/kg) and PPT exhibited higher decrease in the OVX-induced increase in the body weight of the animals compared to their monotherapy in the animals. In contrast, none of the treated animals showed a significant change in the OVX-induced decrease in the estradiol level. One-way ANOVA revealed that there were significant differences in uterus weight of animals among the groups [F (7, 40) = 47.8, P < 0.05] on D-60 of the experimental schedule. Post-hoc test showed that none of the dose level of sildenafil was able to alter the OVX-induced decrease in the weight of uterus of the animals. However, PPT caused a significant increase in the OVX-induced decrease in the weight of uterus of the rodents. Interestingly, S (10.0 mg/kg) attenuated the effect of PPT on OVX-induced decrease in the weight of rat uterus.

3.2. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in the learning and memory formation of the animals in MWM test

Fig. 6. Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in level of expression M1 in rat HIP, PFC and AMY. The blots (A) are representative of M1 in rat HIP, PFC and AMY. The results are in the histogram of M1 (B) are expressed as the ratio of relative intensity of level of expression of M1 to β-actin. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, bP < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S0.1, eP < 0.05 compared to OVX+S-1.0, fP < 0.05 compared to OVX+S-10.0 and gP < 0.05 compared to OVX+PPT (One-way ANOVA followed by StudentNewmann-Keuls post hoc test).

Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the period of escape latency from D-56 to D-59 (A), and the time spent in the target quadrant (B), percentage of total distance travelled in target quadrant (C) and swimming speed of the animals in D-60 (D) during MWM test paradigm is depicted in Fig. 3. Statistical analysis revealed that there were significant differences in the escape latency among group [F (7, 160) = 93.31, P < 0.05] and day [F (3, 160) = 418.4, P < 0.05]. Further, there was a significant interaction between group and day in the escape latency of the animals [F (21, 160) = 9.06, P < 0.05]. Bonferroni test showed that there were no significant differences in the escape latency of the animals between groups on D-56. OVX caused a significant increase in the escape latency of the animals on D-57 of the experimental schedule. This effect was persistent on D-58 and D-59 of the schedule. S (1.0 and 10.0 mg/kg) and PPT significantly attenuated OVX-induced increase in the escape latency of the rats on D-57 to D-59. Further, the combination of S (10.0 mg/kg) and PPT exhibited higher attenuation in the escape latency of the animals on D-57 to D-59 compared to the OVX+S-1.0, OVX+S-10.0 and OVX+PPT group animals. Statistical analysis revealed that there were significant differences in the time spent in the target quadrant [F (7, 40) = 33.2, P < 0.05] and percentage of total distance travelled in target quadrant [F (7, 40) = 25.0, P < 0.05] of the animals in D-60 during MWM test paradigm. However, there were no significant differences in swimming speed [F (7, 40) = 0.31, P > 0.05] of the animals among groups. Posthoc test showed that OVX significantly reduced the amount of time spent and percentage of total distance travelled in target quadrant of the animals in the MWM test protocol compared to Control and Sham group rodents indicating that OVX significantly caused a loss in memory formation of the animals in MWM test protocol. S (1.0 and 10.0 mg/kg) and PPT significantly attenuated OVX-induced decrease in the time spent and percentage of total distance travelled in target quadrant in D-60 during the MWM test protocol. Furthermore, the combination of S (10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in the time spent and percentage of total distance travelled in target quadrant of animals compared to OVX+S-1.0, OVX+S-10.0 and OVX+PPT group animals.

Fig. 7. Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in Bmax (A) and Kd (B) for M1 receptor in HIP, PFC and AMY. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, bP < 0.05 compared to Sham, c P < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, eP < 0.05 compared to OVX+S-1.0, fP < 0.05 compared to OVX+S-10.0 and gP < 0.05 compared to OVX+PPT (One-way ANOVA followed by Student-NewmannKeuls post hoc test).

7

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Fig. 8. Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the extent of phosphorylation of ERα receptor in cytoplasm and nucleus of rat HIP, PFC and AMY tissues. Blots are representative of cytoplasmic ERα (A), cytoplasmic p-ERα (B) and nucleus p-ERα (C) of rat HIP, PFC and AMY. The results in the histogram (D) are expressed as the ratio of the relative intensity of cytoplasmic p-ERα/cytoplasmic ERα and (E) are expressed as the ratio of the relative intensity of nuclear p-ERα/cytoplasmic p-ERα. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, bP < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, eP < 0.05 compared to OVX+S-1.0, fP < 0.05 compared to OVX+S-10.0 and gP < 0.05 compared to OVX+PPT (One-way ANOVA followed by Student-Newmann-Keuls post hoc test).

therapeutic effectiveness in terms of increased ACh level and activity of ChAT and decreased in the AChE activity in OVX rats compared to OVX +S-1.0, OVX+S-10.0 and OVX+PPT group animals.

3.3. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in the spatial memory in Y-maze test Fig. 4 depicts the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in spatial memory in terms of SAB in Y-maze test protocol. Statistical analysis revealed that there were significant differences in the SAB [F (7, 40) = 18.95, P < 0.05] of the animals among groups. Post-hoc test showed that OVX challenged rats exhibited significant decrease in the SAB during Y-maze test compared to Control and Sham group animals. S (1.0 and 10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in the SAB of the animals during Y-maze test. Moreover, the combination of S (10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in the SAB of the animals compared to OVX+S1.0, OVX+S-10.0 and OVX+PPT group animals.

3.5. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in the level of expression of M1 in discrete brain regions Fig. 6 depicts the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the level of expression of M1 (B) in discrete brain regions. Statistical analysis revealed that there were significant differences in level of expression of M1 in HIP [F (7, 40) = 74.1, P < 0.05], PFC [F (7, 40) = 60.4, P < 0.05] and AMY [F (7, 40) = 44.5, P < 0.05] among groups. Post-hoc test showed that OVX significantly reduced the level of expression of M1 in all the rat brain regions compared to Control and Sham group animals. S (1.0 and 10.0 mg/kg) and PPT significantly attenuated OVX-induced decrease in the level of expression of M1 in all rat brain regions. Furthermore, the combination of S (10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in level of expression of M1 in all the rat brain regions compared to OVX+S-1.0, OVX+S-10.0 and OVX+PPT group animals.

3.4. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in cholinergic activity in discrete brain regions Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the level of ACh (A) and activities of AChE (B) and ChAT (C) in discrete brain regions is depicted in Fig. 5. Statistical analysis revealed that there were significant differences in the level of ACh and activities of AChE and ChAT in HIP ([F (7, 40) = 28.9, P < 0.05], [F (7, 40) = 7.3, P < 0.05] and [F (7, 40) = 34.6, P < 0.05] respectively), PFC ([F (7, 40) = 29.4, P < 0.05], [F (7, 40) = 8.1, P < 0.05] and [F (7, 40) = 26.4, P < 0.05] respectively) and AMY ([F (7, 40) = 47.2, P < 0.05], [F (7, 40) = 7.9, P < 0.05] and [F (7, 40) = 34.0, P < 0.05] respectively) among groups. Post-hoc test showed that OVX caused significant decrease in cholinergic activity in terms of decrease in ACh level and activity of ChAT, and increase in the activity of AChE in all the brain regions of the rats compared to Control and Sham group animals. S (1.0 and 10.0 mg/kg) and PPT significantly attenuated OVX-induced decrease in the level of ACh and activity of ChAT, and increase in the activity of AChE in all the rat brain regions. Furthermore, the combination of S (10.0 mg/kg) and PPT significantly increased the

3.6. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in density and affinity of M1 receptor Fig. 7 illustrates the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in levels of Bmax (A) and Kd (B) of M1 receptor in rat HIP, PFC and AMY. Statistical analysis revealed that there were significant differences in the levels of Bmax and Kd of M1 receptor in HIP ([F (7, 40) = 39.2, P < 0.05] and [F (7, 40) = 53.7, P < 0.05] respectively), PFC ([F (7, 40) = 36.2, P < 0.05] and [F (7, 40) = 63.7, P < 0.05] respectively) and AMY ([F (7, 40) = 40.3, P < 0.05] and [F (7, 40) = 73.2, P < 0.05] respectively) among groups. Post-hoc test showed that OVX significantly reduced the density and affinity of M1 receptor in rat HIP, PFC and AMY compared to Control and Sham group animals. S (1.0 and 10.0 mg/kg) and PPT significantly increased OVX-induced decrease in 8

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Fig. 9. Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the level of cGMP (A), cAMP (B) and their ratio cGMP/cAMP (C) in rat HIP, PFC and AMY. All values are expressed in mean ± SEM (n = 6). aP < 0.05 compared to Control, bP < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, eP < 0.05 compared to OVX+S-1.0, fP < 0.05 compared to OVX+S-10.0 and g P < 0.05 compared to OVX+PPT (One-way ANOVA followed by Student-Newmann-Keuls post hoc test).

that there were significant differences in the extent of phosphorylation of ERα in cytoplasm and nucleus of HIP ([F (7, 40) = 52.6, P < 0.05] and [F (7, 40) = 18.2, P < 0.05] respectively), PFC ([F (7, 40) = 43.2, P < 0.05] and [F (7, 40) = 16.2, P < 0.05] respectively) and AMY ([F (7, 40) = 32.5, P < 0.05] and [F (7, 40) = 12.3, P < 0.05] respectively) among groups. Post-hoc test revealed that OVX significantly reduced the extent of phosphorylation of ERα in cytoplasm and nucleus of all the rat brain regions compared to Control and Sham group animals. S (1.0 and 10.0 mg/kg) and PPT significantly attenuated OVXinduced decrease in the extent of phosphorylation of ERα in cytoplasm and nucleus of all the rat brain regions. Furthermore, the combination of S (10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in the extent of phosphorylation of ERα in cytoplasm and nucleus of all the rat brain regions compared to OVX+S-1.0, OVX+S-

the density and affinity of M1 receptor in all rat brain regions. Furthermore, the combination of S (10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in density and affinity of M1 receptor in rat brain regions compared to OVX+S-1.0, OVX+S-10.0 and OVX+PPT group animals.

3.7. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in the extent of phosphorylation of ERα receptor in discrete brain regions Effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the extent of phosphorylation of ERα receptor in cytoplasm (D) and nucleus (E) in discrete brain regions is illustrated in Fig. 8. Statistical analysis showed 9

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Fig. 10. Effect of S (0.1, 1.0 and 10.0 mg/ kg), PPT or the combination of S (10.0 mg/ kg) and PPT on OVX-induced changes in the extent of phosphorylation of Akt in rat HIP, PFC and AMY. Blots are representative of Akt (A) and pAkt (B) of rat HIP, PFC and AMY. The results are in the histogram (C) are expressed as the ratio of relative intensity of pAkt/Akt. All values are expressed in mean ± SEM (n = 6). a P < 0.05 compared to Control, bP < 0.05 compared to Sham, cP < 0.05 compared to OVX, dP < 0.05 compared to OVX+S-0.1, e P < 0.05 compared to OVX+S-1.0, f P < 0.05 compared to OVX+S-10.0 and g P < 0.05 compared to OVX+PPT (Oneway ANOVA followed by StudentNewmann-Keuls post hoc test).

in all the rat brain regions compared to Control and Sham group animals. None of the dose level of sildinafil caused any significant change on OVX-induced decrease in the ratio of p-Akt/Akt in all the rat brain regions. In contrast, PPT significantly increased OVX-induced decrease in the ratio of p-Akt/Akt in all rat brain regions. Interestingly, the combination of S (10.0 mg/kg) and PPT significantly decreased the PPT-induced increase in the ratio of p-Akt/Akt in OVX-challenged animals.

10.0 and OVX+PPT group animals. 3.8. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in the levels of cyclic nucleotides in discrete brain regions Fig. 9 illustrates the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the levels of cGMP (A), cAMP (B) and their ratio of cGMP/cAMP (C) in rat HIP, PFC and AMY. Statistical analysis revealed that there were significant differences in the levels of cGMP and cAMP, and ratio of cGMP/cAMP in HIP ([F (7, 40) = 35.0, P < 0.05], [F (7, 40) = 13.1, P < 0.05], and [F (7, 40) = 14.9, P < 0.05] respectively), PFC ([F (7, 40) = 35.7, P < 0.05], [F (7, 40) = 12.0, P < 0.05], and [F (7, 40) = 18.2, P < 0.05] respectively) and AMY ([F (7, 40 = 24.1, P < 0.05], [F (7, 40) = 8.5, P < 0.05], and [F (7, 40) = 14.9, P < 0.05] respectively) among groups. Post-hoc test revealed that OVX significantly decreased the levels of cGMP and cAMP, and ratio of cGMP/cAMP in all the brain regions of rats compared to Control and Sham group animals. S (1.0 and 10.0 mg/kg) significantly increased OVX-induced decrease in the level of cGMP but the treatment did not affect the level of cAMP in all the rat brain regions. However, treatment with PPT significantly increased OVX-induced decrease in level of cAMP as well as cGMP. Further, the ratio of cGMP/cAMP was found to be increased in all the brain regions. Moreover, the combination of S (10.0 mg/kg) and PPT significantly increased the OVX-induced decrease in levels of cGMP as compared to their monotherapy but their combination did not affect the level of cAMP as compared to monotherapy of PPT. Further the ratio of cGMP/cAMP was found to be increased as compare to monotherapy of OVX+S-10.0 and OVX+PPT.

4. Discussion The present study for the first time demonstrated the protective activity of Sildenafil (1.0 and 10.0 mg/kg) similar to that of PPT against ovariectomy-induced cognitive deficits and increase in body weight in the rodents. The Sildenafil increased the cholinergic function and the ratio of cGMP/cAMP in hippocampus, PFC and amygdala of the ovariectomized animals similar to that of PPT. Further, the ovariectomyinduced decrease in the extent of phosphorylation of ERα was attenuated with the monotherapy of either Sildenafil or PPT. Interestingly, the therapeutic effectiveness of either monotherapy was further pronounced while their combination was administered in these ovariectomized animals, suggesting that Sildenafil strengthens the action of PPT in these animals. However, the Sildenafil attenuated the PPT-induced increase in the level of expression of Akt in discrete brain regions and the weight of uterus of these rodents. Hence, it can be assumed that the combination could be a better therapeutic alternative with minimal side effect in the management of estrogen insufficiency-induced disorders. In the present study, sildenafil (1.0 and 10.0 mg/kg) attenuated the ovariectomy-induced increase in body weight in the animals. Similar to our results, it has been reported the anti-obesity activity of sildenafil in the experimental and clinical studies (Johann et al., 2018; Li et al., 2018) The PPT attenuated the ovariectomy-induced increase in the body weight of the animals in the present study similar to that of sildenafil. Earlier report also documents the anti-obesity activity of PPT in the experimental animals and they proposed the fact that the ERαmediated activity on galaninergic system is responsible in the pituitary of the animals with estrogen insufficiency (Gajewska et al., 2016). When sildenafil was administered along with PPT, the extent of effect of either monotherapy was further enhanced in ovariectomy-induced increase in the body weight in the animals. Thus, it can be assumed that sildenafil may facilitate the ERα-mediated action on the body weight of

3.9. S (1.0 and 10.0 mg/kg), PPT or the combination of S (10.0 mg/kg) and PPT attenuated OVX-induced decrease in the extent of phosphorylation of Akt in discrete brain regions Fig. 10 depicts the effect of S (0.1, 1.0 and 10.0 mg/kg), PPT and the combination of S (10.0 mg/kg) and PPT on OVX-induced changes in the ratio of p-Akt/Akt (C) in discrete rat brain regions. Statistical analysis showed that there were significant differences in the ratio of p-Akt/Akt in HIP [F (7, 40) = 100.1, P < 0.05], PFC [F (7, 40) = 96.5, P < 0.05] and AMY [F (7, 40) = 67.2, P < 0.05] among groups. Posthoc test revealed that OVX significantly reduced the ratio of p-Akt/Akt 10

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

2005; Thomas et al., 2005). However, sildenafil did not cause any change in the ovariectomy-induced decrease in the weight of uterus and phosphorylation of Akt in the brain regions of the animals. Interestingly, sildenafil significantly attenuated the PPT-induced increase in the uterus weight and the level of expression of phosphorylated Akt in the ovariectomized animals. It is well established that cGMP inhibits cAMPmediated action (Polito et al., 2013) and the phosphorylation of Akt is regulated by cAMP (Liao et al., 2010) in the neuronal cells. Thus, it can be assumed that sildenafil exhibits cGMP-dependent action which could be responsible to inhibit cAMP-dependent phosphorylation of Akt in the ovariectomized animals that is subject to future clarification. Moreover, cGMP can stimulate the phosphorylation of several cytoplasmic proteins including Erk 1/2 which can phosphorylate the unliganded ERα at S118 to execute subsequent genomic action (Duplessis et al., 2011; Li et al., 2006). Therefore, the exaggerated action of sildenafil could be due to the dominant activity of cGMP-mediated activity which is again subject to further experimentations. In conclusion, sildenafil exhibited protective activity against ovariectomy-induced cognitive deficits and increase in body weight in the rodents. The sildenafil increased the cholinergic function and the ratio of cGMP/cAMP in hippocampus, PFC and amygdala of the ovariectomized animals. Further, sildenafil attenuated ovariectomy-induced decrease in the extent of phosphorylation of ERα. Interestingly, sildenafil pronounced the therapeutic effectiveness of PPT in these ovariectomized animals, indicating that sildenafil facilitates ERα-mediated action. Hence, it can be assumed that the combination could be a better therapeutic alternative with minimal side effect in the management of estrogen insufficiency-induced disorders.

these rodents. The sildenafil exhibits anti-amnesic activity in the experimental animals (Venkat et al., 2019; Zhu et al., 2019). It also attenuated ovariectomy-induced decrease in the learning and memory formation, and the spatial memory function of the animals in the MWM and Ymaze test in the present study respectively. At the sub-cellular level, sildenafil improved the cholinergic function that was deteriorated in terms of decrease in the ACh level and ChAT activity, and increase in the AChE activity in all the brain regions of the ovariectomized animals. Further, Sildenafil treatment also improved the level of expression and density of M1 receptor and its affinity those were significantly reduced in all the brain regions of the ovariectomized animals. Therefore, sildenafil perhaps attenuates the cholinergic dysfunction possibly through increased synthesis and release, and decreased metabolism of ACh and increased receptor activity in all these brain regions of the animals. In support to the above fact, it is well suggested that estrogen promotes cholinergic function through estrogen receptor-mediated action (Cardoso et al., 2010). Moreover, the PPT improved the cholinergic function in vascular tissue of the animals with estrogen insufficiency (Bansal and Chopra, 2014). Similar to our studies, it has been well reported that sildenafil can improve the cholinergic function in hippocampus of the animals with cognitive deficits (Hosseini-Sharifabad et al., 2012). In another study, it is also documented that sildenafil can improve the activity of cortex and amygdale in experimental animals (Nieoczym et al., 2010; Venkat et al., 2019). Interestingly, the combination of sildenafil and PPT exhibited better therapeutic effectiveness on cognitive deficits and cholinergic dysfunction in the ovariectomized female rats than their monotherapy in the present study. Hence, it can be presumed that Sildenafil may facilitate the ERα-mediated effect on cognitive function and cholinergic activity in addition to the body weight in the ovariectomized animals. The sildenafil elevated the ovariectomy-induced decrease in the level of cGMP and the ratio of cGMP/cAMP in all the brain regions of the animals in the present study. These observations indicate the fact that cGMP-mediated mechanism is probably responsible in the course of action of sildenafil in all the rodent brain regions (Zhang et al., 2002). PPT also exhibited similar therapeutic effect as of sildenafil on the ovariectomy-induced decrease in the levels of cyclic nucleotides in all the rat brain regions suggesting the activation of cytosolic estrogen receptor. The monotherapy of sildenafil and PPT increased the extent of phosphorylation of ERα in all the rat brain regions in the present study. Interestingly, when sildenafil was administered along with PPT there was a further increase in the cGMP-mediated mechanism in terms of the ratio of cGMP/cAMP, phosphorylation of ERα in all the brain regions of the animals. It is well reported that both of these cyclic nucleotides enhance the phosphorylation of ERα and facilitate the genomic action of unliganded ERα (Ince et al., 1994; Rowan et al., 2000). It is also documented that cAMP elicits the phosphorylated Akt-dependent phosphorylation at serine-167 (S167) site of the unliganded ERα (Liao et al., 2010; Martin et al., 2000; Steagall et al., 2017). Moreover, it is also reported that the phosphorylation of unliganded ERα at S118 can exhibit similar genomic action (Duplessis et al., 2011). Thus, it can be assumed that sildenafil may facilitate the cGMP-dependent ERα-mediated action on the anti-obesity, anti-amnesic and cholinergic activity in the ovariectomized animals. The carcinogenic activity in different tissues is well established in the course of estrogen replacement therapy. In the present study, PPT significantly increased the weight of uterus in the ovariectomized animals similar to that of earlier finding (Qu et al., 2013). It is well established that the increase in the level of expression of phosphorylated Akt is responsible for the carcinogenic activity of PPT in the rodent (Qu et al., 2016; Saegusa et al., 2009). In addition, PPT also increased the level of expression of phosphorylated Akt in all analyzed brain regions. In support of our results, it has been suggested that the activation of membrane-bound estrogen receptor increases the levels of cyclic nucleotides which subsequently phosphorylates Akt (Revankar et al.,

CRediT authorship contribution statement Ahsas Goyal: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Debapriya Garabadu: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Declaration of competing interest The authors declare that there is no conflict of interest regarding publication of this article. Acknowledgments AG is thankful to GLA University, Mathura, Uttar Pradesh, India for the financial assistantship. References Andersen, O.M., Rudolph, I.-M., Willnow, T.E., 2016. Risk factor SORL1: from genetic association to functional validation in Alzheimer's disease. Acta Neuropathol. 132, 653–665. Bansal, S., Chopra, K., 2014. Distinct role of estrogen receptor-alpha and beta on postmenopausal diabetes-induced vascular dysfunction. Gen. Comp. Endocrinol. 206, 51–59. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brailoiu, E., Dun, S.L., Brailoiu, G.C., Mizuo, K., Sklar, L.A., Oprea, T.I., Prossnitz, E.R., Dun, N.J., 2007. Distribution and characterization of estrogen receptor G proteincoupled receptor 30 in the rat central nervous system. J. Endocrinol. 193, 311–321. Cao, X., Zhou, C., Chong, J., Fu, L., Zhang, L., Sun, D., Hou, H., Zhang, Y., Li, D., Sun, H., 2015. Estrogen resisted stress-induced cardiomyopathy through increasing the activity of β2AR–Gαs signal pathway in female rats. Int. J. Cardiol. 187, 377–386. Cardoso, C.C., Ricardo, V.P., Frussa-Filho, R., Porto, C.S., Abdalla, F.M.F., 2010. Effects of 17β-estradiol on expression of muscarinic acetylcholine receptor subtypes and estrogen receptor α in rat hippocampus. Eur. J. Pharmacol. 634, 192–200. Carter, C.L., Resnick, E.M., Mallampalli, M., Kalbarczyk, A., 2012. Sex and gender

11

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

extranuclear sites. J. Comp. Neurol. 429, 355–371. Mitra, S.W., Hoskin, E., Yudkovitz, J., Pear, L., Wilkinson, H.A., Hayashi, S., Pfaff, D.W., Ogawa, S., Rohrer, S.P., Schaeffer, J.M., McEwen, B.S., Alves, S.E., 2003. Immunolocalization of estrogen receptor β in the mouse brain: comparison with estrogen receptor α. Endocrinology 144, 2055–2067. Mitterling, K.L., Spencer, J.L., Dziedzic, N., Shenoy, S., McCarthy, K., Waters, E.M., McEwen, B.S., Milner, T.A., 2010. Cellular and subcellular localization of estrogen and progestin receptor immunoreactivities in the mouse hippocampus. J. Comp. Neurol. 518, 2729–2743. Morris, R., 1984. Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60. Mouri, A., Noda, Y., Hara, H., Mizoguchi, H., Tabira, T., Nabeshima, T., 2007. Oral vaccination with a viral vector containing A cDNA attenuates age-related A accumulation and memory deficits without causing inflammation in a mouse Alzheimer model. FASEB J. 21, 2135–2148. Muthuraju, S., Maiti, P., Solanki, P., Sharma, A.K., Amitabh, Singh, S.B., Prasad, D., Ilavazhagan, G., 2009. Acetylcholinesterase inhibitors enhance cognitive functions in rats following hypobaric hypoxia. Behav. Brain Res. 203, 1–14. Neve, K.A., Seamans, J.K., Trantham-Davidson, H., 2004. Dopamine receptor signaling. J. Recept. Signal Transduct. Res. 24, 165–205. Nieoczym, D., Socała, K., Rundfeldt, C., Wlaź, P., 2010. Effects of sildenafil on pentylenetetrazol-induced convulsions in mice and amygdala-kindled seizures in rats. Pharmacol. Rep. 62, 383–391. Palkovits, M., Brownstein, M., 1988. Maps and guide to microdissection of the rat brain. In: Bruyn, G.W. (Ed.), Journal of the Neurological Sciences. Elsevier, New York, pp. 116. Pisani, S.L., Neese, S.L., Katzenellenbogen, J.A., Schantz, S.L., Korol, D.L., 2016. Estrogen receptor-selective agonists modulate learning in female rats in a dose- and taskspecific manner. Endocrinology 157, 292–303. Polito, M., Klarenbeek, J., Jalink, K., Paupardin-Tritsch, D., Vincent, P., Castro, L.R.V., 2013. The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons. Front. Cell. Neurosci. 6, 1–10. Poromaa, S.I., Gingnell, M., 2014. Menstrual cycle influence on cognitive function and emotion processing-from a reproductive perspective. Front. Neurosci. 8, 1–16. Proietto, J., 2017. Obesity and weight management at menopause. Aust. Fam. Physician 46, 368–370. Puzzo, D., Staniszewski, A., XianDeng, S., Privitera, L., Leznik, E., Liu, S., Zhang, H., Feng, Y., Palmeri, A., Landry, D.W., Arancio, O., 2009. Phosphodiesterase 5 inhibition improves synaptic function, memory, and amyloid-β load in an Alzheimer's disease mouse model. J. Neurosci. 29, 8075–8086. Qu, N., Wang, L., Liu, Z.-C., Tian, Q., Zhang, Q., 2013. Oestrogen receptor α agonist improved long-term ovariectomy-induced spatial cognition deficit in young rats. Int. J. Neuropsychopharmacol. 16, 1071–1082. Qu, N., Zhou, X.-Y., Han, L., Wang, L., Xu, J.-X., Zhang, T., Chu, J., Chen, Q., Wang, J.-Z., Zhang, Q., Tian, Q., 2016. Combination of PPT with LiCl treatment prevented bilateral ovariectomy-induced hippocampal-dependent cognition deficit in rats. Mol. Neurobiol. 53, 894–904. Revankar, C.M., Cimino, D.F., Sklar, L.A., Arterburn, J.B., Prossnitz, E.R., 2005. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307, 1625–1630. Rowan, B.G., Garrison, N., Weigel, N.L., O'Malley, B.W., 2000. 8-Bromo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein. Mol. Cell. Biol. 20, 8720–8730. Saegusa, M., Hashimura, M., Kuwata, T., Okayasu, I., 2009. Requirement of the Akt/βcatenin pathway for uterine carcinosarcoma genesis, modulating E-cadherin expression through the transactivation of Slug. Am. J. Pathol. 174, 2107–2115. Sagredo, A., del Campo, L., Martorell, A., Navarro, R., Martín, M.C., Blanco-Rivero, J., Ferrer, M., 2013. Ovariectomy increases the participation of hyperpolarizing mechanisms in the relaxation of rat aorta. PLoS One 8, e73474. Saville, B., Wormke, M., Wang, F., Nguyen, T., Enmark, E., Kuiper, G., Gustafsson, J.-åke, Safe, S., 2000. Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. J. Biol. Chem. 275, 5379–5387. Schupf, N., Lee, J.H., Wei, M., Pang, D., Chace, C., Cheng, R., Zigman, W.B., Tycko, B., Silverman, W., 2008. Estrogen receptor-α variants increase risk of Alzheimer's disease in women with down syndrome. Dement. Geriatr. Cognit. Disord. 25, 476–482. Shafiei, M., Mahmoudian, M., Rostami, P., Nemati, F., 2006. Effect of sildenafil (viagra) on memory retention of a passive avoidance response in rats. Acta Physiol. Hung. 93, 53–59. Sharma, B., Singh, N., 2012. Defensive effect of natrium diethyldithiocarbamate trihydrate (NDDCT) and lisinopril in DOCA–salt hypertension-induced vascular dementia in rats. Psychopharmacology (Berlin) 223, 307–317. Sharma, S., Kumar, K., Deshmukh, R., Sharma, P.L., 2013. Phosphodiesterases: regulators of cyclic nucleotide signals and novel molecular target for movement disorders. Eur. J. Pharmacol. 714, 486–497. Steagall, R.J., Yao, F., Shaikh, S.R., Abdel-Rahman, A.A., 2017. Estrogen receptor α activation enhances its cell surface localization and improves myocardial redox status in ovariectomized rats. Life Sci. 182, 41–49. Swartling, F.J., Ferletta, M., Kastemar, M., Weiss, W.A., Westermark, B., 2009. Cyclic GMP-dependent protein kinase II inhibits cell proliferation, Sox9 expression and Akt phosphorylation in human glioma cell lines. Oncogene 28, 3121–3131. Szego, E.M., 2006. Estrogen induces estrogen receptor α-dependent cAMP response element-binding protein phosphorylation via mitogen activated protein kinase pathway in basal forebrain cholinergic neurons in vivo. J. Neurosci. 26, 4104–4110. Thomas, P., Pang, Y., Filardo, E.J., Dong, J., 2005. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146,

differences in Alzheimer's disease: recommendations for future research. J. Women’s Health 21, 1018–1023. Choleris, E., Ogawa, S., Kavaliers, M., Gustafsson, J.-Å., Korach, K.S., Muglia, L.J., Pfaff, D.W., 2006. Involvement of estrogen receptor α, β and oxytocin in social discrimination: a detailed behavioral analysis with knockout female mice. Genes Brain Behav. 5, 528–539. Cuadrado-Tejedor, M., Hervias, I., Ricobaraza, A., Puerta, E., Pérez-Roldán, J.M., GarcíaBarroso, C., Franco, R., Aguirre, N., García-Osta, A., 2011. Sildenafil restores cognitive function without affecting β-amyloid burden in a mouse model of Alzheimer's disease. Br. J. Pharmacol. 164, 2029–2041. Daniel, J.M., Bohacek, J., 2010. The critical period hypothesis of estrogen effects on cognition: insights from basic research. Biochim. Biophys. Acta Gen. Subj. 1800, 1068–1076. Daniel, J.M., Hulst, J.L., Berbling, J.L., 2006. Estradiol replacement enhances working memory in middle-aged rats when initiated immediately after ovariectomy but not after a long-term period of ovarian hormone deprivation. Endocrinology 147, 607–614. Ding, G., Jiang, Q., Li, L., Zhang, L., Zhang, Z.G., Ledbetter, K.A., Panda, S., Davarani, S.P.N., Athiraman, H., Li, Q., Ewing, J.R., Chopp, M., 2008. Magnetic resonance imaging investigation of axonal remodeling and angiogenesis after embolic stroke in sildenafil-treated rats. J. Cereb. Blood Flow Metab. 28, 1440–1448. Duplessis, T.T., Williams, C.C., Hill, S.M., Rowan, B.G., 2011. Phosphorylation of estrogen receptor α at serine 118 directs recruitment of promoter complexes and gene-specific transcription. Endocrinology 152, 2517–2526. Farooq, M.U., Naravetla, B., Moore, P.W., Majid, A., Gupta, R., Kassab, M.Y., 2008. Role of sildenafil in neurological disorders. Clin. Neuropharmacol. 31, 353–362. Foster, T.C., 2012. Role of estrogen receptor alpha and beta expression and signaling on cognitive function during aging. Hippocampus 22, 656–669. Gajewska, A., Zielinska-Gorska, M., Wasilewska-Dziubinska, E., Baran, M., Kotarba, G., Gorski, K., 2016. Pituitary galaninergic system activity in female rats: the regulatory role of gonadal steroids. J. Physiol. Pharmacol. 67, 423–429. Garabadu, D., Sharma, M., 2019. Eugenol attenuates scopolamine-induced hippocampal cholinergic, glutamatergic, and mitochondrial toxicity in experimental rats. Neurotox. Res. 35, 848–859. Gould, E., Woolley, C.S., Frankfurt, M., McEwen, B.S., 1990. Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. J. Neurosci. 10, 1286–1291. Greengard, P., 2001. The neurobiology of slow synaptic transmission. Science 294, 1024–1030. Hall, J.M., Couse, J.F., Korach, K.S., 2001. The Multifaceted mechanisms of estradiol and estrogen receptor signaling. J. Biol. Chem. 276, 36869–36872. Hernandez-Rabaza, V., Agusti, A., Cabrera-Pastor, A., Fustero, S., Delgado, O., TaoroGonzalez, L., Montoliu, C., Llansola, M., Felipo, V., 2015. Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: underlying mechanisms. J. Neuroinflammation 12, 195. Hosseini-Sharifabad, A., Ghahremani, M.H., Sabzevari, O., Naghdi, N., Abdollahi, M., Beyer, C., Bollen, E., Prickaerts, J., Roghani, A., Sharifzadeh, M., 2012. Effects of protein kinase A and G inhibitors on hippocampal cholinergic markers expressions in rolipram- and sildenafil-induced spatial memory improvement. Pharmacol. Biochem. Behav. 101, 311–319. Ince, B.A., Montano, M.M., Katzenellenbogen, B.S., 1994. Activation of transcriptionally inactive human estrogen receptors by cyclic adenosine 3’,5’-monophosphate and ligands including antiestrogens. Mol. Endocrinol. 8, 1397–1406. Johann, K., Reis, M.C., Harder, L., Herrmann, B., Gachkar, S., Mittag, J., Oelkrug, R., 2018. Effects of sildenafil treatment on thermogenesis and glucose homeostasis in diet-induced obese mice. Nutr. Diabetes 8, 9. Kelly, J.F., Bienias, J.L., Shah, A., Meeke, K.A., Schneider, J.A., Soriano, E., Bennett, D.A., 2008. Levels of estrogen receptors alpha and beta in frontal cortex of patients with Alzheimer's disease: relationship to mini-mental state examination scores. Curr. Alzheimer Res. 5, 45–51. Kushner, P.J., Agard, D., Feng, W.-J., Lopez, G., Schiau, A., Uht, R., Webb, P., Greene, G., 2000. Neuronal and Cognitive Effects of Oestrogens, Novartis Found Symposium. Novartis Foundation Symposia. John Wiley & Sons, Ltd, Chichester, UK. Kushner, P.J., Agard, D.A., Greene, G.L., Scanlan, T.S., Shiau, A.K., Uht, R.M., Webb, P., 2000. Estrogen receptor pathways to AP-1. J. Steroid Biochem. Mol. Biol. 74, 311–317. Lesch, K.P., Lerer, B., 1991. The 5-HT receptor- G-protein- effector system complex in depression I. Effect of glucocorticoids. J. Neural Transm. Gen. Sect. 84, 3–18. Li, S., Li, Y., Xiang, L., Dong, J., Liu, M., Xiang, G., 2018. Sildenafil induces browning of subcutaneous white adipose tissue in overweight adults. Metabolism 78, 106–117. Li, Z., Zhang, G., Feil, R., Han, J., Du, X., 2006. Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin alphaIIb beta3. Blood 107, 965–972. Liao, H., Hyman, M.C., Baek, A.E., Fukase, K., Pinsky, D.J., 2010. cAMP/CREB-mediated transcriptional regulation of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) expression. J. Biol. Chem. 285, 14791–14805. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Maki, P.M., Henderson, V.W., 2012. Hormone therapy, dementia, and cognition: the Women's Health Initiative 10 years on. Climacteric 15, 256–262. Martin, M.B., Franke, T.F., Stoica, G.E., Chambon, P., Katzenellenbogen, B.S., Stoica, B.A., McLemore, M.S., Olivo, S.E., Stoica, A., 2000. A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I. Endocrinology 141, 4503–4511. Milner, T.A., McEwen, B.S., Hayashi, S., Li, C.J., Reagan, L.P., Alves, S.E., 2001. Ultrastructural evidence that hippocampal alpha estrogen receptors are located at

12

Neurochemistry International 132 (2020) 104609

A. Goyal and D. Garabadu

Natl. Acad. Sci. U. S. A 71, 1725–1729. Yonker, J.E., Eriksson, E., Nilsson, L.-G., Herlitz, A., 2003. Sex differences in episodic memory: minimal influence of estradiol. Brain Cogn. 52, 231–238. Zhang, R., Wang, Y., Zhang, Li, Zhang, Z., Tsang, W., Lu, M., Zhang, Lijie, Chopp, M., 2002. Sildenafil (Viagra) induces neurogenesis and promotes functional recovery after stroke in rats. Stroke 33, 2675–2680. Zhu, L., Zhang, Z., Hou, X., Wang, Y., Yang, J., Wu, C., 2019. Inhibition of PDE5 attenuates streptozotocin-induced neuroinflammation and tau hyperphosphorylation in a streptozotocin-treated rat model. Brain Res. 1722, 146344. Zoukhri, D., Kublin, C.L., 2001. Impaired neurotransmitter release from lacrimal and salivary gland nerves of a murine model of Sjögren’s syndrome. Investig. Ophthalmol. Vis. Sci. 42, 925–932.

624–632. Venkat, P., Chopp, M., Zacharek, A., Cui, C., Landschoot-Ward, J., Qian, Y., Chen, Z., Chen, J., 2019. Sildenafil treatment of vascular dementia in aged rats. Neurochem. Int. 127, 103–112. Wang, K., Li, F., Chen, L., Lai, Y., Zhang, X., Li, H., 2017. Change in risk of breast cancer after receiving hormone replacement therapy by considering effect-modifiers : a systematic review and dose-response meta-analysis of prospective studies. Oncotarget 8, 81109–81124. Woolley, C.S., Gould, E., Frankfurt, M., McEwen, B.S., 1990. Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons. J. Neurosci. 10, 4035–4039. Yamamura, H.I., Snyder, S.H., 1974. Muscarinic cholinergic binding in rat brain. Proc.

13